1. A method for detecting toxicity of a live heart pill, the method comprising detecting a pathological condition of the heart and/or liver to determine the toxicity of the live heart pill.

2. The detection method according to claim 1, wherein the detection of the heart is in particular detection of the epicardium.

3. The detection method according to claim 2, wherein the detection of epicardium is in particular a detection of the presence of inflammation, preferably inflammatory cell infiltration, of the epicardium.

4. The method according to claim 1, wherein the liver is detected by detecting inflammation around central veins of the liver, preferably focal inflammatory cell infiltration, or by detecting vacuolization of liver cells.

5. The method according to any one of claims 3 to 4, wherein the inflammation is when the tissue is localized and/or dispersed and is in the form of round or oval inflammatory cells.

6. The method of claim 4, wherein the vacuoles are circular vacuoles of varying sizes that appear in the tissue cytoplasm and exhibit focal and/or mass-dispersed distribution.

7. The method according to any one of claims 1 to 6, wherein the method is specifically for detecting a heart and/or liver lesion in a mouse.

8. The method for detecting according to claim 7, wherein the Huoxing pill comprises the following components: ganoderma, artificial Moschus, fel Ursi, Carthami flos, in vitro cultured calculus bovis, Margarita, Ginseng radix, Bufonis venenum, radix Aconiti lateralis Preparata, and Borneolum Syntheticum.

9. The detection method according to claim 7, wherein the daily dose of the Huoxing pill is 0-3.5 g/kg.

10. The detection method according to claim 7, wherein the toxicity detection is in particular an early toxicity detection.

Background

The heart-activating pill is prepared from ten medicines of lucid ganoderma, artificial musk, bear gall, safflower, in-vitro cultured bezoar, pearl, ginseng, toad venom, monkshood and borneol, has the effects of tonifying qi and activating blood, warming channels and invigorating pulse, is mainly used for treating chest stuffiness and cardiodynia, is used for treating coronary heart disease, angina and other diseases, and is currently collected in eighteenth volume of Chinese medicinal prescription preparations (WS3-B-3452-98) in the drug standards of Ministry of public health of the people's republic of China.

Coronary heart disease is a clinical syndrome caused by coronary insufficiency, and the main symptoms comprise angina pectoris and acute myocardial infarction, and the angina pectoris and the acute myocardial infarction are myocardial damage caused by myocardial ischemia and oxygen supply. With the aging population architecture, coronary heart disease has become the fastest rising disease in the component of death.

Compared with other similar products, the heart invigorating pill has obvious heart strengthening effect and obvious curative effect on unstable angina patients with syndrome differentiation of qi deficiency and blood stasis, yang deficiency and congealing cold and heart blood stasis in traditional Chinese medicine. Clinical data show that the Huoxinwan pill has the effects of relieving clinical symptoms of mixed angina and improving myocardial ischemia equivalent to those of long-acting nitroglycerin tablets, and has better total effective rate and electrocardiogram improvement rate; in the aspect of treating stable angina pectoris, the western medicine basic drug combined with the Huoxinwan treatment can improve the cardiac function of a patient and reduce the systolic pressure, and has more advantages compared with the conventional treatment of pure western medicines. Therefore, the pill for invigorating heart has a wide market in the aspect of cardiovascular diseases. However, the existing heart-invigorating pill has less and shallow toxicity research reports, and still needs to be deeply researched, so that a theoretical basis is provided for clinical reasonable medication.

Disclosure of Invention

The invention aims to provide a more convenient method for detecting toxicity of live heart pills.

The technical scheme adopted by the invention is as follows:

the invention provides a method for detecting toxicity of a live heart pill, which comprises detecting pathological conditions of heart and/or liver to determine the toxicity of the live heart pill.

In some embodiments of the invention, the detecting a heart is in particular detecting an epicardium.

In some embodiments of the invention, the epicardium is specifically detected for the presence of inflammation.

In some preferred embodiments of the invention, the inflammation is inflammatory cell infiltration.

In some embodiments of the invention, the liver is specifically detected whether inflammation occurs around the central vein of the liver or whether vacuolization of hepatocytes occurs.

In some embodiments of the invention, the inflammation is focal inflammatory cell infiltration.

In some embodiments of the invention, the inflammation is when the tissue is localized and/or dispersed and is in the form of round or oval inflammatory cells.

In some embodiments of the invention, the vacuoles are circular vacuoles of varying sizes that appear in the tissue cytoplasm and exhibit a focal and/or a largely dispersed distribution.

In some embodiments of the invention, the detection method is a pathological detection of heart and/or liver tissue.

In some embodiments of the invention, the method of pathology detection is histopathology detection.

In some embodiments of the invention, the histopathological examination comprises the steps of:

(1) dyeing after manufacturing pathological sections;

(2) and (5) observing pathological changes of the tissue section by using a microscope.

In some embodiments of the invention, the staining method is Masson trichrome staining, HE staining, immunohistochemical staining, immunofluorescence staining, or special staining.

In some preferred embodiments of the invention, the staining method is HE staining.

In some embodiments of the invention, the heart-invigorating pill comprises the following ingredients: ganoderma, artificial Moschus, fel Ursi, Carthami flos, in vitro cultured calculus bovis, Margarita, Ginseng radix, Bufonis venenum, radix Aconiti lateralis Preparata, and Borneolum Syntheticum.

In some embodiments of the invention, the detection is in particular a detection of a heart and/or liver pathology condition in a mouse.

In some embodiments of the invention, the daily dose of the heart-invigorating pill is 0-3.5 g/kg.

In some embodiments of the invention, the toxicity test is an early toxicity test.

In some embodiments of the invention, the early phase is specifically 1 to 14 days after administration.

In some embodiments of the invention, the mouse has a body weight of 20 ± 2 g.

In some embodiments of the invention, the mode of administration is intragastric administration.

The invention has the beneficial effects that:

the invention provides a method for detecting acute toxicity of a live heart pill, which only needs to detect epicardium and liver of heart. According to the invention, a large number of experiments detect that the mental state, the activity, the food intake, the water drinking, the hair and the like of each group of mice are normal from the 2 nd day after the administration. Compared with a control group, the mice body quality and 16 blood routine indexes of the administration group have no significant influence, and 5 blood biochemical indexes, namely alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), alkaline phosphatase (ALP), urea nitrogen (BUN), creatinine (Crea) and organ indexes of the administration group have no obvious change in the same time, and the difference has no statistical significance. And the histopathology shows that mild inflammatory cell infiltration appears on the epicardium of the heart, focal inflammatory cell infiltration appears around the central vein of the liver and a small amount of liver cells vacuole in the mice of the administration group, so that the subsequent toxicity detection of the live heart pill can mainly detect the epicardium of the heart and the liver, and the toxic target organs of the live heart pill on the mice can be the heart and the liver, so that the clinical medication can be evaluated in an auxiliary way. In addition, clinical medication needs to be controlled by taking the medicine dosage, and the medicine should be taken under reasonable dosage.

Drawings

FIG. 1 shows the effect of Huoxinwan on the body mass of mice.

FIG. 2 is the effect of 1d Huoxinwan administration on the blood index of mice.

FIG. 3 shows the effect of 14d Huoxing pill administration on the blood index of mice.

FIG. 4 shows the effect of 1d Huoxinwan administration on the biochemical indicators of blood in mice.

FIG. 5 shows the effect of 14d Huoxinwan administration on the biochemical indicators of blood in mice.

FIG. 6 shows the effect of 1d Huoxing pill administration on the organ index of mice.

FIG. 7 shows the effect of 14d Huoxing pill administration on the organ index of mice.

FIG. 8 is the effect of Huoxing pills on mouse histopathology (200X).

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

Medicine preparation: huoxing pills (batch No. 1190001) sold by Baiyun pharmaceutical general factory, Guangzhou Baiyun pharmaceutical group GmbH. Glutamic-pyruvic transaminase kit (lot No. 2019004), glutamic-oxalacetic transaminase kit (lot No. 2019004), alkaline phosphatase kit (lot No. 2019001), creatinine kit (lot No. 2019003), and urea nitrogen kit (lot No. 2019003) sold by changchun hui biotechnology limited.

An experimental instrument: enzyme labeling instrument (BioTek, model: Epoch), full-automatic biochemical analyzer (Shenzhen Redu Life sciences, Inc., model: Chemray 240), full-automatic blood cell analyzer for veterinary use (Shenzhen Meyer biomedical electronics, Inc., model: BC-2800vet), and high-speed refrigerated centrifuge (SCILOGEX, model: D3024R).

Experimental animals: SPF-grade KM mice sold by Beijing Witonglihua laboratory animal technology Limited are half female and half male, are raised in animal room barrier environment of south China acupuncture research center of Guangzhou Chinese medicinal university, room temperature (23 +/-3) DEG C, relative humidity of 40-70%, indoor illumination adopts day-night ratio of 12h/12h, and are fed by conventional feed and freely drink water.

SPSS 22.0 software is adopted for experimental data statistics, data are expressed as mean values +/-standard deviation, One-way ANOVA (One-way ANOVA) is adopted for comparison among groups, LDS method statistics is adopted when the homogeneity of the variance is met, and Dunett's T3 method is adopted when the homogeneity of the variance is not met. The plots were made using Graphpad Prism 6.0 software.

Example 1

1. Preparing the medicine: grinding the Huoxing pill in a mortar into powder, and preparing the Huoxing pill liquid medicine with the required concentration by using normal saline. The No. 12 gastric perfusion needle is connected with a 1mL injection syringe to extract the liquid medicine without obvious blockage, and the liquid medicine can smoothly pass through the needle, and the concentration of the liquid medicine is the maximum administration concentration which is 0.175 g/mL.

2. Grouping and administration: after adaptive breeding for 3 days, 80 SPF-grade KM mice are randomly divided into a blank control group and an administration group according to sex and weight, wherein each group comprises 40 mice, and the mice are male and female. According to the maximum administration concentration (0.175g/mL) and the administration volume (20mL/kg) of the live heart pills, the single oral administration is carried out within 24h, the fasting is carried out for 12h before the administration without water prohibition, the medicine liquid of the live heart pills is administered in 3.5g/kg in the administration group, and the equal volume of normal saline is administered in the blank control group.

3. General observations: observing each group of mice for 1 time every 10min within 30min after the administration by gavage; observing for 1 time every 15min within 30 min-2 h after medicine application; observing every 30min within 2-4 h after medicine application; observing for 1 time every 1h within 4-8 h after medicine application; observing for 1 time 12h after the medicine is taken and 24h after the medicine is taken respectively; the observation was performed 1 time per day from 2d after the administration. Observation indexes are as follows: the mouse takes food, drinks, moves, mental states, hairs, excrement and the like, the poisoning and death of the mouse are paid attention to, and the dead mouse is dissected and observed in time.

Example 2

The general performance of the mice after administration in example 1 was recorded by close observation and the results indicated that:

within 24h after administration, compared with the blank control group, mice in the administration group have poor mental state, obviously reduced activity, accelerated respiration and hair erection, wherein 7 mice have poisoning phenomenon within 30min after administration, which is manifested as dyskinesia, obviously weakened activity, tachypnea, listlessness, convulsion, black stool and recovery of poisoning mice after 1 h. From 2d after administration, the mental state, activity, food intake, drinking water, hair and the like of the mice in each group were normal.

Furthermore, no death phenomenon occurs in mice in the administration group, which indicates that the maximum dose of the live heart pill tolerance of the mice is 3.5 g/kg. Calculating formula according to the maximum tolerance multiple of the mice: the maximum tolerance multiple of the mice is [ the tolerance drug quantity (mg) of each mouse/the average body weight (g) of the mice x the average body weight (kg) of adults/the daily drug quantity (g) of adults is (70/20 x 60/0.12) ], and the dosage of the administration group is 1750 times of the daily dose of adults.

Example 3

Before and after administration, 1d, 3d, 5d, 8d and 14d, the body mass of each group of mice in example 1 was recorded and statistically analyzed, and the results are shown in fig. 1, as can be seen from fig. 1, the body mass of each group of mice increases with time, and the body mass of the mice in the administration group does not change significantly and has no significant difference compared with the control group at the same time.

Example 4

The general blood index of each group of mice in example 1 was measured after 1d administration and 14d administration, 20 mice were drawn from each group, and the mice were half female and half male, fasted and water-deprived for 12h, and the eyeballs were removed from the mice to collect blood, and a part of the blood was collected by an anticoagulation blood collection tube and measured by a fully automatic blood cell analyzer, the main measurement index: neutrophil number, neutrophil percentage, erythrocyte hematocrit, hemoglobin, lymphocyte number, lymphocyte percentage, mean erythrocyte hemoglobin concentration, mean erythrocyte hemoglobin content, mean erythrocyte volume, monocyte number, monocyte percentage, platelet distribution width, platelet number, erythrocyte distribution width variation coefficient, leukocyte number.

The effect on the conventional index of blood in mice after administration of 1D is shown in FIG. 2, wherein A in FIG. 2 is the effect on the number of neutrophils, B in FIG. 2 is the effect on the percentage of neutrophils, C in FIG. 2 is the effect on the hematocrit, D in FIG. 2 is the effect on hemoglobin, E in FIG. 2 is the effect on the number of lymphocytes, F in FIG. 2 is the effect on the percentage of lymphocytes, G in FIG. 2 is the effect on the mean hemoglobin concentration, H in FIG. 2 is the effect on the mean hemoglobin content, I in FIG. 2 is the effect on the mean erythrocyte volume, J in FIG. 2 is the effect on the number of monocytes, K in FIG. 2 is the effect on the percentage of monocytes, L in FIG. 2 is the effect on the width of the platelet distribution, M in FIG. 2 is the effect on the number of platelets, N in FIG. 2 is the effect on the number of erythrocytes, the influence of the variation coefficient of the distribution width of erythrocytes on the O-plot in FIG. 2 and the influence of the number of leukocytes on the P-plot in FIG. 2 were shown.

The effect on the conventional index of the blood of mice after administration of 14D is shown in FIG. 3, wherein A in FIG. 3 is the effect on the number of neutrophils, B in FIG. 3 is the effect on the percentage of neutrophils, C in FIG. 3 is the effect on the hematocrit, D in FIG. 3 is the effect on hemoglobin, E in FIG. 3 is the effect on the number of lymphocytes, F in FIG. 3 is the effect on the percentage of lymphocytes, G in FIG. 3 is the effect on the mean hemoglobin concentration, H in FIG. 3 is the effect on the mean hemoglobin content, I in FIG. 3 is the effect on the mean erythrocyte volume, J in FIG. 3 is the effect on the number of monocytes, K in FIG. 3 is the effect on the percentage of monocytes, L in FIG. 3 is the effect on the width of the platelet distribution, M in FIG. 3 is the effect on the number of platelets, N in FIG. 3 is the effect on the number of erythrocytes, the influence of the variation coefficient of the distribution width of erythrocytes on the O-plot in FIG. 3 and the influence of the number of leukocytes on the P-plot in FIG. 3 were shown.

As can be seen from fig. 2 and 3, the heart-invigorating pill had no significant effect on the conventional index of 16 blood samples in mice after the administration of 1d and 14d in the administration group, and had no significant difference compared with the blank group.

Example 5

And (3) determining the influence of the live heart pills on the biochemical indexes of the blood of the mice after administration of 1d and administration of 14d, and analyzing the possible toxic effect of the live heart pills on the blood indexes of the mice.

Another part of the blood obtained in example 4 was put into a centrifuge tube without anticoagulant, centrifuged at 3000r for 10min in a centrifuge, and then the upper serum was collected and stored in a refrigerator at-20 ℃ and tested by a full-automatic biochemical analyzer for alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), alkaline phosphatase (ALP), urea nitrogen (BUN), and creatinine (Crea).

The effect of 1d Huoxinwan administration on biochemical indicators of blood in mice is shown in FIG. 4. Wherein the graph A in FIG. 4 shows the effect on alkaline phosphatase, the graph B in FIG. 4 shows the effect on glutamic-pyruvic transaminase, the graph C in FIG. 4 shows the effect on glutamic-oxaloacetic transaminase, the graph D in FIG. 4 shows the effect on urinary nitrogen, and the graph E in FIG. 4 shows the effect on creatinine.

The effect of 14d Huoxinwan administration on biochemical indicators of blood in mice is shown in FIG. 5. Wherein the graph A in FIG. 5 shows the effect on alkaline phosphatase, the graph B in FIG. 5 shows the effect on glutamic-pyruvic transaminase, the graph C in FIG. 5 shows the effect on glutamic-oxaloacetic transaminase, the graph D in FIG. 5 shows the effect on urinary nitrogen, and the graph E in FIG. 5 shows the effect on creatinine.

As can be seen from fig. 4 and 5, the values of 5 blood biochemical indicators, glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST), alkaline phosphatase (ALP), urea nitrogen (BUN), and creatinine (Crea), in the mice in the administration group were not significantly changed and were not significantly different from those in the blank group.

Example 6

The mice in example 1 were taken for anatomical observation and pathological examination: after blood is collected from eyeballs of all groups of mice, the mice are dislocated, killed and dissected, and pathological changes of organs such as intestines, stomach, heart, liver, spleen, lung, kidney, thymus, testis, ovary and the like are observed by naked eyes; taking a heart, a liver, a spleen, a lung and a kidney, removing tissues such as fat, fascia and the like, washing the heart, the liver, the spleen, the lung and the kidney by using normal saline at 4 ℃, weighing the weight of the viscera after the water is absorbed by filter paper, and calculating the viscera index which is the mass/the body mass of the viscera; after weighing, fixing the powder by using 4% paraformaldehyde to prepare pathological sections, and observing pathological changes of organs under a microscope. After 1d and 14d of administration, all mice were dissected and no significant pathological changes were observed in the vital organs by naked eye. Five organs of heart, liver, spleen, lung and kidney were weighed after dissection and organ index was calculated.

The effect of 1D Huoxing pill on mouse organ index is shown in FIG. 6, wherein panel A in FIG. 6 is the effect on heart index, panel B in FIG. 6 is the effect on liver index, panel C in FIG. 6 is the effect on spleen index, panel D in FIG. 6 is the effect on lung index, and panel E in FIG. 6 is the effect on kidney index.

The effect of 14D Huoxing pill on mouse organ index is shown in FIG. 7, where panel A in FIG. 7 is the effect on heart index, panel B in FIG. 7 is the effect on liver index, panel C in FIG. 7 is the effect on spleen index, panel D in FIG. 7 is the effect on lung index, and panel E in FIG. 7 is the effect on kidney index.

As can be seen from fig. 6 and 7, the organ index of the administered group was not significantly changed and the difference was not statistically significant, as compared with the blank control group.

The results of the pathological histological examination of five organs of the heart, the liver, the spleen, the lung and the kidney of the mice are also carried out after the administration of 1d and 14d, and the results are shown in figure 8, and it can be seen that compared with the blank group, the heart and the liver of the administration group have obvious pathological changes, and the central outer membrane of the administration group has mild inflammatory cell infiltration, but no pathological changes such as vacuolation, necrosis, myocardial hypertrophy and the like; local inflammatory cell infiltration around the central vein of the liver of the administration group occurs, and a small amount of vacuolization of liver cells occurs. No obvious pathological changes are found in the other organs after administration, and no abnormality is found compared with a blank group.

Therefore, after administration, some mice have toxic symptoms but do not die, the maximum tolerated dose of the live heart pills to the mice is 3.5g/kg, and the dose of the live heart pills is 1750 times of the daily dose of adults, so that the live heart pills are proved to have low oral toxicity and to be safe and effective when taken according to the clinically specified dose.

In conclusion, the present embodiment studies the maximum tolerance of the live heart pill on the mouse, detects the change of the body mass of the mouse after administration, the change of the blood routine and the blood biochemical index, anatomically observes the pathological changes of each organ of the mouse, analyzes the organ index and performs the pathological histological detection, finds that the live heart pill has no obvious influence on the body mass of the mouse, the blood routine index and the blood biochemical index, and the pathological histology shows that the heart epicardium of the mice in the administration group has mild inflammatory cell infiltration, the focal inflammatory cell infiltration around the central vein of the liver and a small amount of vacuole of the liver cells, and the subsequent toxicity detection on the live heart pill can mainly detect the heart epicardium and the liver. The toxic target organs of the live heart pill on mice can be heart and liver, and clinical medication needs to be controlled by taking the pill under a reasonable dose.

The present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.