阅读说明：本技术 一种青蒿鳖甲颗粒及其制备方法 (Artemisia apiacea and turtle shell granules and preparation method thereof ) 是由 田纪祥 张东 赵晓昂 曹春雨 于 2021-09-14 设计创作，主要内容包括：本发明公开了一种青蒿鳖甲颗粒及其制备方法,属于中药制备技术领域。该青蒿鳖甲颗粒包括以下重量配比的原料；青蒿9份,知母6份,桑叶6份,鳖甲15份,牡丹皮6份和天花粉6份；制备方法为：将原料加水进行回流提取,将提取液浓缩,然后经干燥、制粒,得到青蒿鳖甲颗粒。本发明在传统青蒿鳖甲汤方剂基础上进行调整,采用简单的制备工艺制备得到了治疗效果好、安全性高的青蒿鳖甲颗粒,该青蒿鳖甲颗粒可用于解热以及治疗营热阴伤证等,同时制备方法简单,颗粒剂型便于携带,服用更加方便。(The invention discloses sweet wormwood and turtle shell granules and a preparation method thereof, and belongs to the technical field of traditional Chinese medicine preparation. The sweet wormwood and turtle shell granules comprise the following raw materials in parts by weight; 9 parts of sweet wormwood, 6 parts of rhizoma anemarrhenae, 6 parts of mulberry leaf, 15 parts of turtle shell, 6 parts of tree peony bark and 6 parts of trichosanthes root; the preparation method comprises the following steps: adding water into the raw materials, carrying out reflux extraction, concentrating the extracting solution, drying, and granulating to obtain the sweet wormwood herb and turtle shell granules. The sweet wormwood and turtle shell granules are prepared by a simple preparation process on the basis of a traditional sweet wormwood and turtle shell decoction formula, have good treatment effect and high safety, can be used for relieving fever and treating yin injury syndrome due to heat in ying and yang, and are simple in preparation method, convenient to carry and more convenient to take.)

1. The sweet wormwood and turtle shell granules are characterized by comprising the following raw materials in parts by weight:

9 parts of sweet wormwood, 6 parts of rhizoma anemarrhenae, 6 parts of mulberry leaf, 15 parts of turtle shell, 6 parts of tree peony bark and 6 parts of trichosanthes root.

2. The method for preparing the granules of sweet wormwood herb and turtle shell according to claim 1, comprising the following steps:

adding water into the raw materials for reflux extraction, concentrating an extracting solution, drying and granulating to obtain the sweet wormwood herb and turtle shell granules.

3. The method of claim 2, wherein the reflux extraction is performed 3 times with 1h extraction time.

4. The method according to claim 3, wherein the reflux extraction is carried out with a first amount of water added being 14 times the total mass of the starting materials and the remaining two amounts of water added being 12 times the total mass of the starting materials.

5. The preparation method according to claim 2, wherein the concentration temperature is 60-70 ℃, and the density of the concentrated extract is 1.05-1.20 g/mL.

6. The method of claim 2, wherein the concentration is performed at a temperature of 60 ℃ and the density after concentration is 1.10 g/mL.

7. The method of claim 2, wherein the drying is spray drying.

8. The preparation method according to claim 2, wherein dextrin is added in the granulation process, and the mass ratio of the dextrin to the dry extract obtained after drying is 1: 1.

9. The preparation method according to claim 2, further comprising a step of adding a flavoring agent after drying, wherein the flavoring agent is sucralose, and the addition amount of the sucralose is 0.1-0.5% of the mass of the dry extract obtained after drying.

10. The preparation method according to claim 9, wherein the addition amount of the sucralose is 0.3% of the mass of the dry extract obtained after drying.

Technical Field

The invention relates to the technical field of traditional Chinese medicine preparation, in particular to sweet wormwood herb and turtle shell granules and a preparation method thereof.

Background

The sweet wormwood herb and turtle shell decoction is derived from Wen Bing tiao Bian (differentiation of Wen Bing), and the prescription comprises 6g of sweet wormwood herb, 15g of turtle shell, 12 g of dried rehmannia root, 6g of rhizoma anemarrhenae and 9g of cortex moutan. The pharmacological actions of the sweet wormwood and turtle shell soup mainly comprise antipyretic, anti-inflammatory, sedative, pathogenic microorganism resistant and the like.

The sweet wormwood herb and turtle shell soup has obvious antipyretic effect clinically, can inhibit organism autoimmunity and allergic inflammation, has the effects of inhibiting experimental arthritis and relieving spasm, and has different degrees of inhibition effects on typhoid bacillus, dysentery bacillus, diphtheria bacillus, staphylococcus, pneumococcus and the like.

The addition and subtraction treatment is carried out on the basis of the formula of the sweet wormwood and turtle shell decoction, the symptomatic treatment effect and the safety and effectiveness can be ensured, meanwhile, the enrichment of the medicament dosage form also becomes a new medicament research, the backward preparation level not only influences the exertion of the medicament curative effect, but also causes low additional value of the medicament, directly influences the market competition and the industrial economic benefit, in addition, the diversification of the dosage form can lead the medicament taking selectivity of patients to be more, and the granule is a very convenient choice.

Therefore, the provided sweet wormwood and turtle shell particles with good treatment effect and the corresponding preparation method can efficiently retain the effective ingredients of the medicine and realize excellent treatment effect, which is a technical problem to be solved at present.

Disclosure of Invention

The invention aims to provide a sweet wormwood and turtle shell particle and a preparation method thereof, and aims to solve the problems in the prior art.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides sweet wormwood and turtle shell granules which comprise the following raw materials in parts by weight:

9 parts of sweet wormwood, 6 parts of rhizoma anemarrhenae, 6 parts of mulberry leaf, 15 parts of turtle shell, 6 parts of tree peony bark and 6 parts of trichosanthes root.

The invention also provides a preparation method of the sweet wormwood and turtle shell granules, which comprises the following steps:

adding water into the raw materials for reflux extraction, concentrating an extracting solution, drying and granulating to obtain the sweet wormwood herb and turtle shell granules.

Preferably, the reflux extraction is performed 3 times, each extraction time being 1 h.

Preferably, when the reflux extraction is carried out, the water addition amount of the first time is 14 times of the total mass of the raw materials, and the water addition amounts of the other two times are 12 times of the total mass of the raw materials.

Preferably, the concentration temperature is 60-70 ℃, and the density of the concentrated extract is 1.05-1.20 g/mL.

Preferably, the density of the concentrated extract is 1.08-1.13 g/mL.

Preferably, the temperature of the concentration is 60 ℃ and the density after the concentration is 1.10 g/mL.

Preferably, the drying is spray drying.

Preferably, dextrin is added in the granulation process, and the mass ratio of the dextrin to the dry extract obtained after drying is 1: 1.

Preferably, the method further comprises a step of adding a flavoring agent after drying, wherein the flavoring agent is sucralose, and the adding amount of the sucralose is 0.1-0.5% of the mass of the dry extract obtained after drying.

Preferably, the addition amount of the sucralose is 0.3 percent of the mass of the dry extract obtained after drying.

1g of the sweet wormwood and turtle shell granules are equivalent to 2.40g of crude drugs, and 48g of the crude drugs are taken orally/day. The weight of the adult is calculated according to 70kg, and the clinical dose is 0.69g crude drug/kg d.

The invention discloses the following technical effects:

the invention is adjusted on the basis of the traditional sweet wormwood and turtle shell decoction formula, and the sweet wormwood and turtle shell granules with good treatment effect and high safety are prepared by adopting a simple preparation process, so that the sweet wormwood and turtle shell granules have good antipyretic effect. Toxicity tests and acute toxicity tests of orally-administered rats for 3 months show that the prepared sweet wormwood and turtle shell granules are safe and nontoxic.

The sweet wormwood and turtle shell granules can be used for relieving fever and treating ying-heat yin injury and the like, and are simple in preparation method, convenient to carry and more convenient to take.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a liquid chromatogram of mangiferin in the extractive solution of herba Artemisiae Annuae and carapax Trionycis;

FIG. 2 is a liquid chromatogram of a mangiferin reference;

FIG. 3 is a liquid chromatogram of timosaponin BII in the extractive solution of herba Artemisiae Annuae and carapax Trionycis;

FIG. 4 is a liquid chromatogram of timosaponin BII control.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

The "parts" in the present invention are in parts by weight unless otherwise specified.

Example 1

The sweet wormwood and turtle shell granules comprise the following raw materials in parts by weight:

sweet wormwood 9g, anemarrhena rhizome 6g, mulberry leaf 6g, turtle shell 15g, moutan bark 6g and trichosanthes root 6 g.

The preparation method comprises the following steps:

adding water into the raw materials, refluxing and extracting for 3 times, wherein the extraction time is 1h each time: adding 14 times of water by mass for the first time, and adding 12 times of water by mass for the other two times; filtering the extracting solutions, combining filtrates, concentrating the obtained filtrate at 60 ℃ to obtain a thick extract with the density of 1.10g/mL, then performing spray drying on the thick extract to obtain a dry extract (dry powder), adding sucralose with the mass of 0.3% of that of the dry extract, then adding dextrin into the thick extract by adopting a one-step granulation method to perform granulation, wherein the mass ratio of the dextrin to the dry extract is 1:1, keeping the material temperature at 45 ℃ in the granulation process and the atomization pressure at 0.2Pa, and thus obtaining the sweet wormwood and turtle shell particles.

Example 2 Pilot plant preparation of Artemisia apiacea turtle Shell granules

The sweet wormwood and turtle shell granules comprise the following raw materials in parts by weight:

18kg of sweet wormwood herb, 12kg of rhizoma anemarrhenae, 12kg of mulberry leaf, 30kg of turtle shell, 12kg of tree peony bark and 12kg of Mongolian snakegourd root.

The preparation method comprises the following steps:

adding water into the raw materials, refluxing and extracting for 3 times, wherein the extraction time is 1h each time: adding 14 times of water by mass for the first time, and adding 12 times of water by mass for the other two times; filtering the extracting solutions, combining filtrates, concentrating the obtained filtrate at 70 ℃ to obtain a thick extract with the density of 1.08g/mL, then performing spray drying on the thick extract to obtain a dry extract (dry powder), adding sucralose with the mass of 0.1% of that of the dry extract, then adding dextrin into the thick extract by adopting a one-step granulation method to perform granulation, wherein the mass ratio of the dextrin to the dry extract is 1:1, keeping the material temperature at 50 ℃ in the granulation process and the atomization pressure at 0.2Pa, and thus obtaining the sweet wormwood and turtle shell particles.

Example 3 Pilot plant preparation of Artemisia apiacea turtle Shell granules

The sweet wormwood and turtle shell granules comprise the following raw materials in parts by weight:

6kg of sweet wormwood, 4kg of rhizoma anemarrhenae, 4kg of mulberry leaf, 10kg of turtle shell, 4kg of tree peony bark and 4kg of trichosanthes root.

The preparation method comprises the following steps:

adding water into the raw materials, refluxing and extracting for 3 times, wherein the extraction time is 1h each time: adding 14 times of water by mass for the first time, and adding 12 times of water by mass for the other two times; filtering the extracting solutions, combining filtrates, concentrating the obtained filtrate at 65 ℃ to obtain a thick extract with the density of 1.13g/mL, then performing spray drying on the thick extract to obtain a dry extract (dry powder), adding sucralose with the mass of 0.5% of that of the dry extract, then adding dextrin into the thick extract by adopting a one-step granulation method to perform granulation, wherein the mass ratio of the dextrin to the dry extract is 1:1, keeping the material temperature at 48 ℃ in the granulation process and the atomization pressure at 0.2Pa, and thus obtaining the sweet wormwood and turtle shell particles.

Example 4 Normal temperature Water absorption and high temperature Water absorption of the raw Material

(1) Water absorption capacity of raw material at normal temperature

Weighing the medicinal materials (9 g of sweet wormwood herb, 6g of rhizoma anemarrhenae, 6g of folium mori, 15g of turtle shell, 6g of moutan bark and 6g of radix trichosanthis) according to the prescription, adding 480ml of water, standing at normal temperature, and measuring the volume of the liquid medicine every 1h, wherein the results are shown in table 1.

TABLE 1 Water absorption at Normal temperature results

(2) High temperature water absorption of raw material

Weighing the medicinal materials (9 g of sweet wormwood herb, 6g of rhizoma anemarrhenae, 6g of mulberry leaf, 15g of turtle shell, 6g of moutan bark and 6g of trichosanthes root) according to the prescription, adding 480ml of water, stopping heating after heating to boil, and measuring the volume of the liquid medicine every 0.5h, wherein the results are shown in table 2.

TABLE 2 Water absorption at elevated temperature results

According to the results, the water absorption capacity of the prescription of the sweet wormwood herb and turtle shell is twice, so that twice as much water is added in the first decoction in the preparation process.

Example 5 Effect of extraction Process

(1) Design of extraction process

Selecting mangiferin and timosaponin BII in rhizoma anemarrhenae as evaluation indexes, and adopting L₉(3⁴) Orthogonal table, examine the solvent dosage, extraction time and extraction times affecting the water extraction process.

120g of raw medicinal materials are weighed according to the mass ratio of sweet wormwood herb, common anemarrhena rhizome, mulberry leaf, turtle shell, tree peony bark and mongolian snakegourd root of 9:6:6:15:6, the raw medicinal materials are evenly divided into 18 parts, water extraction process research is carried out, and orthogonal experiment factors are shown in a table 3. According to L₉(3⁴) And (3) extracting by using an orthogonal table, wherein the orthogonal table is shown in table 4, each group of experiments are performed in parallel by using two experiments, extracting solution is filtered, filtrates are combined and mixed uniformly, and the volume is accurately measured for later use.

TABLE 3 level table of factors of orthogonal experiment of extraction process

TABLE 4 orthogonal experimental chart of extraction process

(2) Extracting cream yield results of orthogonal experiments

The appropriate amount of the extract from the above test was removed precisely, placed in an evaporating dish dried to a constant weight, dried in a water bath, dried at 105 ℃ for 3 hours, placed in a desiccator for 30 minutes, and precisely weighed quickly, and the results are shown in Table 5.

TABLE 5 results of cream yield from orthogonal experiments

(3) Extracting content results of orthogonal experiments

a. The chromatographic conditions for measuring the mangiferin content are as follows:

detection wavelength: 258 nm; flow rate: 1.0 mL/min; column temperature: 30 ℃, mobile phase: acetonitrile (a) -0.2% glacial acetic acid (B) isocratically eluted (10: 90).

Preparation of a test solution:

precisely measuring appropriate amount of 18 groups of orthogonal water extraction extractive solutions, placing in a 25ml volumetric flask, adding anhydrous ethanol to scale, performing ultrasonic treatment for 30min, standing at room temperature, adding 50% ethanol to scale, shaking, filtering, and collecting the filtrate.

Preparation of control solutions:

taking 1.30mg of mangiferin reference substance, adding into a 10ml volumetric flask, and diluting with methanol to constant volume to obtain 0.130mg/ml mangiferin reference substance solution.

The determination method comprises the following steps:

precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

The liquid chromatogram of Mangiferin content in herba Artemisiae Annuae carapax Trionycis extractive solution is shown in figure 1; the liquid chromatogram of mangiferin reference is shown in FIG. 2.

The results of the transfer rate of mangiferin content in the orthogonal experiment are shown in Table 6.

TABLE 6 results of transfer rate of mangiferin content from orthogonal experiments

b. Chromatographic conditions for determining timosaponin BII glycoside content:

flow rate: 1.0 mL/min; column temperature: 30 ℃, the evaporable light drift tube temperature is 105 ℃, the gas flow rate is 2.7ml/min, and the acetonitrile (A) -water (B)22:78 isocratic elution is carried out.

Preparation of a test solution:

precisely measuring the extractive solution, evaporating the extractive solution with crude drug amount of 0.2g at 80 deg.C, re-dissolving with 30% acetone, transferring into 10ml measuring flask, adding 30% acetone to scale, shaking, centrifuging the filtrate at 6000r/min for 5min, and collecting supernatant.

Preparation of control solutions:

taking 4.21mg of timosaponin BII reference substance, adding into a 10ml volumetric flask, and diluting with methanol to constant volume to obtain 0.421mg/ml timosaponin BII reference substance solution.

The determination method comprises the following steps:

precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

The liquid chromatogram of timosaponin BII content determination in herba Artemisiae Annuae carapax Trionycis extractive solution is shown in FIG. 3; the liquid chromatogram of timosaponin BII reference substance is shown in FIG. 4.

Results of the content transfer rate of timosaponin BII in the orthogonal experiment are shown in Table 7.

TABLE 7 results of content transfer rates of timosaponin BII

(4) Analysis of results of orthogonal experiments

The water extraction orthogonal experiment was analyzed by the mangiferin content transfer rate and timosaponin BII content transfer rate, and the results of the anova analysis are shown in tables 8-9.

TABLE 8 analysis of mangiferin variance Table

TABLE 9 ANOVA TABLE OF ANOVASTIFICIDE BII

As can be seen from the table, the results of the 9 sets of orthogonal data show that the optimal extraction process of mangiferin is A3B1C3, the optimal extraction process of timosaponin is A3B2C3, and no significant difference exists, and the orthogonal results of the process 1(A3B1C3) and the process 2(A3B2C3) are verified by using the transfer rates of mangiferin and timosaponin biii as indexes.

(5) Validation of results of orthogonal experiments

Experimental verification was performed based on the above orthogonal results analysis, with three parallel experiments performed per process. The mangiferin transfer rate and timosaponin BII are used as evaluation indexes, and the experimental results are shown in tables 10 and 11.

TABLE 10 Experimental validation results of mangiferin transfer rate

TABLE 11 Experimental verification results of timosaponin BII transfer rate

As can be seen from the above, the transfer rate is highest in the experiment of the mangiferin process verification 1, and the extraction rate of the mangiferin in the process 1(12 times of water, 1 hour for each extraction, 3 times of extraction) is highest; the transfer rates of timosaponin BII under the two groups of conditions of the process 1 and the process 2 are 57.68 percent and 56.18 percent respectively, and no significant difference exists in the test.

Example 6 concentration Process Effect

(1) Temperature of concentration

Weighing the medicinal materials according to the proportion of the prescription, extracting by the optimal extraction process 1 in the embodiment 5, merging the extracting solution, equally dividing into 6 parts, carrying out reduced pressure concentration on the extracting solution, setting the concentration temperature at 60 ℃, 70 ℃ and 80 ℃, and investigating the influence of concentration on effective components under different temperature conditions.

The chromatographic conditions for measuring the mangiferin content are as follows:

detection wavelength: 258 nm; flow rate: 1.0 mL/min; column temperature: 30 ℃, mobile phase: acetonitrile (a) -0.2% glacial acetic acid (B) isocratically eluted (10: 90).

Preparation of a test solution:

precisely measuring an appropriate amount of the extractive solution in a 25ml volumetric flask, adding ethanol to scale, weighing, ultrasonically treating for 30min, standing at room temperature, weighing, supplementing weight, shaking, filtering, and collecting the filtrate.

Preparation of control solutions:

weighing mangiferin, and preparing into control solution of about 0.15mg/ml for use.

Assay method

Precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

The content transfer rate of mangiferin at different concentration temperatures is shown in Table 12.

TABLE 12 transfer rate of mangiferin content at different concentration temperatures

Chromatographic conditions for determining timosaponin BII glycoside content:

flow rate: 1.0 mL/min; column temperature: 30 ℃, the evaporable light drift tube temperature is 105 ℃, the gas flow rate is 2.7ml/min, and the acetonitrile (A) -water (B)22:78 isocratic elution is carried out.

Preparation of a test solution:

precisely measuring appropriate amount of extractive solution, evaporating to dryness at 80 deg.C in evaporating dish, re-dissolving with 30% acetone, transferring into 10ml measuring flask, adding 30% acetone to scale, shaking, filtering to obtain filtrate 6000r/min, centrifuging for 5min, and collecting supernatant.

Preparation of control solutions:

taking a proper amount of timosaponin BII reference substance, adding methanol to a constant volume to prepare a timosaponin BII reference substance solution with the concentration of about 0.4 mg/ml.

The determination method comprises the following steps:

precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

The content transfer rate of timosaponin BII at different concentration temperatures is shown in Table 13.

TABLE 13 results of timosaponin BII content transfer rate at different concentration temperatures

As can be seen from the concentration temperature data, the mangiferin and timosaponin BII have low loss rate at 60 deg.C and 70 deg.C, and high loss rate at 80 deg.C, so 60-70 deg.C is selected for concentration.

(2) Density of concentration

Weighing the medicinal materials according to the proportion of the prescription, extracting by the optimal extraction process 1 of the embodiment 5, combining the extracting solutions, equally dividing into 8 parts, and carrying out reduced pressure concentration at 60-70 ℃, wherein the concentration density is set to be 1.05g/mL, 1.10g/mL, 1.15g/mL and 1.20 g/mL.

The content determination method of mangiferin and timosaponin BII is the same as that in the section (1) of example 6.

The content transfer rate of mangiferin and timosaponin BII at different concentration densities are shown in Table 14 and Table 15, respectively.

TABLE 14 results of mangiferin content transfer rate at different concentration densities

TABLE 15 results of timosaponin BII content transfer rate at different concentration densities

From the concentration density data, the mangiferin and timosaponin BII are in the range of 1.05-1.20, the transfer rates are basically consistent, and the stability is good in the density range.

Example 7 drying Process Effect

The medicinal materials are weighed according to the proportion of the prescription, extracted by the best extraction process 1 in the example 5, decompressed and concentrated to a certain relative density, and the influence of microwave drying, vacuum drying and spray drying on the main components is examined (two groups of parallel experiments are carried out under each condition).

Microwave drying: and (3) placing the concentrated thick extract into a microwave dryer for microwave drying, wherein the microwave power is 500W, taking out the dry extract after drying for 4h, and obtaining 45.13g and 44.29g of dry extract for later use.

And (3) vacuum drying: and putting the concentrated thick extract into a vacuum drying oven, carrying out vacuum drying at 60 ℃ for 18h, and taking out the dry extract to obtain 49.38g and 49.79g of dry extract for later use.

Spray drying: spray drying the two concentrated extracts, heating the extracts, sieving with 100 mesh sieve, measuring the density to be 1.08(40 deg.C), air inlet temperature to be 160 deg.C, air outlet temperature to be 80 deg.C, and air flow rate to be 4m/s, and keeping.

HPLC is adopted to test the content of the active ingredients in different drying modes. The content results of mangiferin and timosaponin BII are shown in Table 16.

TABLE 16 content of mangiferin and timosaponin BII in different drying modes

From the drying mode data, the mangiferin and timosaponin BII have large loss under the conditions of microwave drying and vacuum drying, and the content of active ingredients is high under the spray drying condition.

Example 8 granulation Process Effect

The liquid medicine of the prescription of the invention has bitter taste and is difficult to swallow, and the tongue has obvious sour and bitter taste after being swallowed, so a proper amount of flavoring agent needs to be added to improve the mouth feel of the preparation and improve the medication compliance of patients. Sucralose and aspartame were considered for the selection of the taste modifier, and the aspartame was discarded because the liquid medicine added with aspartame was highly greasy, and the sucralose dosage was screened, and the results are shown in table 17.

TABLE 17 screening of sweetener type and amount

As can be seen from table 18, sample No. 2 received the most acceptable level, and the taste-modifying regimen was determined to be the addition of sucralose at a particle size of 0.3% for a suitable sweetness level.

Example 9 granulation Process Effect

One-step granulation parameter setting: and adjusting the temperature of the air inlet to be 80-100 ℃, controlling the temperature of the material to be about 50 ℃, blowing the material by the fan at the frequency of 24-30 Hz, and keeping the material in good fluidity. And (4) agglomeration occurs, the frequency of the fan needs to be increased, and the fluidity of the material is recovered. The liquid supply speed is high at the initial stage, particles are easy to form, and the liquid supply speed is slow at the later stage. Material (dextrin): dry extract (dry powder obtained by evaporating the thick extract) is 1: 1.

Experiments are carried out according to the parameters, and after granulation is finished, the granule state is better.

In conclusion, the parameters of the one-step granulation of the sweet wormwood herb and turtle shell granules are as follows: the air inlet temperature is 80-100 ℃, the material temperature is 45-50 ℃, and the atomization pressure is 0.2 Pa.

Example 10 pharmacodynamic experiment of Artemisia apiacea turtle Shell granules

A. Antipyretic effect of artemisia apiacea and turtle shell granules on rat fever caused by 2, 4-dinitrophenol

1. Experimental animals: SD rats, SPF grade, 48, male and female halves, 6-8 weeks old, body weight (221 + -18) g. Purchased from the experimental animals center of Chongqing university of medicine, the production license number of experimental animals: SCXK (Yu) 2018-.

2. Animal breeding environment: the environment is shielded, the temperature is 20-21 ℃, the relative humidity is 46-58%, and the light and the shade alternate every day for 12h/12 h. License number for experimental animals: SYXK 2020 (Chuan) 225.

Feed: complete rat feed purchased from certified animals Co., Ltd.

Drinking water: sterilizing water, and putting into drinking water box for animal to drink freely.

3. After the start of the experiment, 48 animals were randomly stratified by body temperature as follows:

normal Control group (Control group, C, NS gavage + NS subcutaneous injection): 8 are provided.

Model control (Model group, Mo, NS gavage + Model): 8 are provided.

Positive control group (LBQ group, L, chlorpromazine gavage + model): 8 are provided.

High dose group of artemisia apiacea turtle shell granules (QH-H, QH, high dose gavage + model of artemisia apiacea turtle shell granules): 8 are provided.

Mid-range dose group of artemisia apiacea turtle shell granules (QH-M, QM, mid-range dose of artemisia apiacea turtle shell granules gavage + model): 8 are provided.

Low dose group of granules of fresh water turtle shell of Artemisia apiacea (QH-L, QL, low dose intragastric lavage of fresh water turtle shell granules + model): 8 are provided.

4. Rat fever model caused by subcutaneous injection of 2, 4-dinitrophenol

Preparing a2, 4-dinitrophenol solution for molding: 2, 4-dinitrophenol was dissolved in physiological saline to give a solution of 3.4 mg/mL.

The method comprises the following steps: at the 5 th day, except the normal control group, rats in each group were injected with 3.4mg/mL of 2, 4-dinitrophenol solution subcutaneously into the back at a dose of 5mL/kg under a state of natural consciousness, and the whole process was performed aseptically. The normal control group was injected with the same amount of physiological saline.

5. Dosage and course of treatment

Rats in each group were gavaged with different drugs according to Table 18 for 1 time/day for 5 consecutive days, where the 5 th day was gavaged after molding.

TABLE 18 animal administration dosage table

6. Observation index

a. General observations in rats: the general state of the animals (respiration, voluntary movement, salivation, hair erection, urination, defecation and skin) was observed daily, and no abnormality occurred in each group of rats during the experiment.

b. And (3) measuring the body weight:

rat body weights were measured and recorded for the 1d and last dose (5 d dose) and compared for each group. The changes in body weight of the rats are shown in Table 19.

Table 19 rat body weight change table (g,)

compared with the normal control group, the body weight of the rats in the model control group has no obvious difference (P > 0.05). Compared with the model control group, the weight of rats in each group is not obviously different (P > 0.05).

c. Body temperature measurement

The anal temperature was measured 1 time a day for 3 consecutive days before the experiment, and the average value was taken as the basal anal temperature. Anal temperatures of animals were measured 1, 2, 3, 4, 5h after injection of 2, 4-dinitrophenol, and the body temperature rise values of each group of animals at different times after administration were calculated and compared for each group of differences, and the results are shown in table 20.

Table 20 rat thermometer change table (c,)

1h after molding, the body temperature of rats in the model control group is increased compared with that in the normal control group (P < 0.0000). The body temperature of rats in the positive control group is reduced compared with that in the model control group (P < 0.001); the body temperature of rats in the high-dose group of the artemisia apiacea turtle shell particles is reduced (P is less than 0.001); no significant difference was observed in the body temperature of the rats in the other groups (P > 0.05).

2h after molding, the body temperature of rats in the model control group is increased compared with that in the normal control group (P < 0.05). The body temperature of rats in the positive control group is reduced compared with that in the model control group (P < 0.001); the body temperature of rats in the high-dose group of the artemisia apiacea turtle shell particles is reduced (P is less than 0.05); no significant difference was observed in the body temperature of the rats in the other groups (P > 0.05).

3h after molding, the body temperature of the model control group rats is increased compared with that of the normal control group (P < 0.01). The body temperature of rats in the positive control group is reduced compared with that in the model control group (P < 0.001); the body temperature of rats in the high-dose group of the artemisia apiacea turtle shell particles is reduced (P is less than 0.01); the body temperature of rats in the middle dose group of the artemisia apiacea turtle shell particles is reduced (P is less than 0.05); no significant difference was observed in the body temperature of the rats in the other groups (P > 0.05).

4h after molding, the body temperature of the model control group rats is increased compared with that of the normal control group (P < 0.05). The body temperature of rats in the positive control group is reduced compared with that in the model control group (P < 0.001); the body temperature of rats in the high-dose group of the artemisia apiacea turtle shell particles is reduced (P is less than 0.05); no significant difference was observed in the body temperature of the rats in the other groups (P > 0.05).

5h after molding, compared with a normal control group, the body temperature of the rats in the model control group has no obvious difference (P is more than 0.05). Compared with the model control group, the body temperature of rats in each group is not obviously different (P > 0.05).

7. Content detection of TNF alpha, IL-1 beta and PGE2 in serum

After the last temperature measurement, the abdominal aorta of the rat is used for blood sampling at 3000 r.min^-1Centrifuging for 10min, collecting supernatant, and storing at-20 deg.C. Respectively measuring the contents of TNF alpha, IL-1 beta and PGE2 in serum according to a kit methodThe results are shown in Table 21.

Table 21 table of TNF α, IL-1 β, PGE2 content changes in rat serum (pg/mL,)

compared with the normal control group, the serum contents of TNF alpha, IL-1 beta and PGE2 of the rats in the model control group have no obvious difference (P is more than 0.05). Compared with the model control group, the contents of TNF alpha, IL-1 beta and PGE2 in the serum of rats in each group have no obvious difference (P is more than 0.05).

Example 11 toxicity test of orally administered granules of Artemisia annua and Trionyx sinensis Wiegmann in rats for 3 months

1. Laboratory animal

SD rat 120, license number: SCXK (Jing) 2016. 0006, 70-90g (4-5 weeks old).

2. Grouping animals

4 groups were set, a blank control group (gavage given equal volume of experimental animal drinking water) and 3 dose groups (low, medium, high). The day of the end of the acclimation period was called the weight without fasting, and 120 rats included in the test were classified by sex according to the weight per 5g by a hierarchical random method, which is referred to as "random classification of SOP-1-TQ030 experimental animals". 120 rats were randomly divided into 4 groups of 30 rats each, 15 rats each. In order to facilitate the evaluation of toxicity test, the sample is dry extract (dry extract powder) without adjuvant, and each g of dry extract powder is equivalent to 5.03g of crude drug.

Wherein, 3 administration doses, preparation concentration, administration volume, dose and clinical dose multiple are shown in a table 22.

TABLE 22

3. Route of administration and method

The administration route is as follows: administered orally (gavage). Consistent with the clinical route of administration.

The administration concentration is as follows: the low, medium and high dosage groups are respectively given with 0.114, 0.229 and 0.457g dry extract powder/ml, and the control group is given with equal volume of experimental animal drinking water.

The administration volume is 15 ml/kg/time. The following day after each weighing of the fasting-free body weight, the administration period was to administer a corresponding volume of liquid medicine/experimental animal drinking water in accordance with the new body weight.

The administration frequency was 1 time per day and 7 days per week.

The administration time is morning (at least 1h after dispensing for administration).

The administration period is as follows: after 8 days of quarantine adaptation, weighing and grouping. Divided into groups and administered the next day.

The first day of administration is day 1 (D1) of the administration period, the male and female animals are continuously administered for 91 days, and the male and female animals are continuously administered for 92 days.

A recovery period: after the administration, some of the male and female rats are selected for autopsy, and the rest of the animals enter the recovery period the next day. No drug was administered during the recovery period, which lasted 28 days.

The deadline selection basis is as follows: the clinical course of treatment of the artemisia apiacea and turtle shell granules is 15 days, the efficacy of nourishing yin and clearing heat is achieved, and the indications are the late stage of warm disease, syndrome of pathogenic factors hidden in yin, night fever, early cooling, fever reduction, no sweat, red tongue with little coating and thready and rapid pulse. According to the requirements of relevant guidelines, 3-month long-term toxicity tests in rats are carried out to support drug marketing applications.

4. General state observation:

animals were observed after each administration during the dosing period and the general condition, gross color, activity, gait, attitude, stool, urine, etc. of the animals were recorded. Observing whether toxic symptoms occur, and the occurrence time, duration and recovery condition, and recording dead or dying animals. The recovery period was observed once a day.

During the whole test period, the male and female animals of each group have no obvious change, the animal activity, gait and the like are basically normal, abnormal secretions, dyspnea and other abnormal changes are not seen in the mouth, the nose and the eyes, gastrointestinal reactions such as vomit and diarrhea are not seen, and no rat dies.

5. Body weight and food intake observations

The determination method comprises the following steps: quantitatively adding feed in each cage on the first day of administration; weighing for 2 times per week in the first 4 weeks, and quantitatively adding feed in each cage; and in the later administration period and the recovery period, weighing the residual feed amount of each cage once a week, quantitatively supplementing the feed in time according to the feeding condition of the animals, calculating the feed consumption g (total feed addition amount-residual feed amount) of each cage, and dividing the feed consumption g by the number of animals and the number of days of each cage to obtain the average feed intake (g feed/mouse day) of the cage.

Measuring frequency: the administration period is as follows: d1, D3, D7, D10, D14, D17, D21, D24, D28, D35, D42, D49, D56, D63, D70, D77, D84, D91; a recovery period: d7, D14, D21 and D28.

In the administration period and the recovery period, the body weight of female rats and male rats of each dose group of the artemisia apiacea turtle shell particles shows different growth trends, and the negative growth phenomenon does not occur.

After the drug is administered for 3 months and in the recovery period, the weight of female and male rats of each dose group of the artemisia apiacea turtle shell particles is not obviously different compared with that of a control group. Compared with a control group, the food intake of female and male rats of each dose group of the artemisia apiacea turtle shell particles is not obviously different after 3 months of administration and a recovery period.

6. Ophthalmic examination

Detection indexes are as follows: eye appearance inspection: including the eyelids, orbit, eyeball morphology, and lacrimal gland area. Checking the anterior segment: including conjunctiva, cornea, pupil, anterior chamber, and aqueous humor.

The results of the ophthalmic examination were not abnormal by a person in the department of pathology before the animals were sacrificed at 3 months of administration and at the recovery period.

7. Examination of urine

Collecting urine samples: the administration period D91 (male parent) and D92 (female parent) and the recovery period D28 are that the rats are conveyed to a metabolism room (fifth animal laboratory), and are placed in a metabolism cage alone, fasted for 15-16h, and freely drunk to collect 15-16h urine of the rats.

The urine collection time is 16:00-17: 00-7: 00-8:00 in the next day.

Detection indexes are as follows: amount of urine (ml): (post-metabolization urine cup weight-empty urine cup weight before metabolization)/urine specific gravity. The full-automatic urine analysis workstation adopts a refraction photometry to determine the urine specific gravity, and the urine specific gravity refers to the detection value.

Water intake (ml): post-metabolization bottle weight-the weight of the water bottle to which water was added quantitatively before metabolization (specific gravity of water calculated as 1 g/ml).

③ properties of urine: the color and clarity of the urine was observed and recorded for each rat.

(iv) urine test (dry chemistry analysis): bilirubin (BIL), Urobilinogen (UBG), ketone bodies (KET), vitamin C (ASC), Glucose (GLU), Protein (PRO), occult Blood (BLD), pH (pH), Nitrite (NIT), white blood cells (LEU), and Specific Gravity (SG).

In the recovery period of the artemisia apiacea turtle shell granules, the urine volume and the water intake of male rats in the high-dose group are obviously increased compared with those in the control group, but the urine volume and the water intake of female rats in the 3-dose group are not obviously different from those in the control group in the administration period. No pathological changes of relevant organs such as kidney and the like are seen in the administration period and the recovery period, and the numerical values of urine volume and water intake are in the background value range of animals of the same species and the same sex.

The urine color of male and female rats in 3 dosage groups is deepened to a certain degree in 3 months after administration, the male and female rats are changed from red to brown, the deepening of the high dosage groups is obvious and possibly related to the fact that some colored water-soluble small molecular substances in a test object are dissolved in the urine, the male and female rats are basically recovered to be the normal urine color in the recovery period, and clear toxicological significance is not considered. Other indicators of changes in urine, KET and BLD, decreased without toxicological implications, and pH increase was still within the normal range (mean 6.8), with no clear toxicological implications.

8. Hematology test (hemogram)

Sample type: EDTA-2K anticoagulation, determined on the same day.

Detection indexes are as follows: red blood cell count (RBC), red blood cell specific volume (HCT), mean red blood cell volume (MCV), red blood cell volume distribution width (RDW), Hemoglobin (HGB), mean red blood cell hemoglobin (MCH), mean red blood cell hemoglobin concentration (MCHC), platelet count (PLT), platelet specific volume (PCT), Mean Platelet Volume (MPV), Platelet Distribution Width (PDW), white blood cell count (WBC), white blood cell classification (where LYM is lymphocytes, NEUT is neutrophils, MONO is monocytes), reticulocyte percentage (RET%), reticulocyte count (RET).

9. Hematology test (blood coagulation index)

Sample type: plasma, day of measurement.

Detection indexes are as follows: prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT).

The high-dose group of the artemisia apiacea and turtle shell particles has certain rising influence on the RET percent and the RET in the peripheral blood of female rats after being administrated for 3 months, but the analysis result of the bone marrow elephant shows that only the female rats can see the obvious reduction of the erythrocyte of the early larvae, the variation of the erythrocyte of the middle/late larvae is not obvious, and the variation of the erythrocyte of the female rats is not obvious. The RET index in peripheral blood is not changed to be consistent with the bone marrow image, and the related indexes of red blood cells and hemoglobin in peripheral blood are not obviously anemic. Comprehensive analysis shows that no bone marrow picture change with clear toxicological significance is found after the artemisia apiacea turtle shell particles are administrated for 3 months. After the recovery period, the above-mentioned changed indexes all recovered, and no delayed toxic reaction was observed.

10. Blood biochemistry assay (conventional index)

Sample type: serum, measured on the day.

Detection indexes are as follows: serum Total Protein (TP), Albumin (ALB), aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), UREA (UREA), Creatinine (CRE), blood Glucose (GLU), Total Bilirubin (TBIL), total Cholesterol (CHO), Triglycerides (TG), Creatine Kinase (CK).

Compared with a control group, after the artemisia apiacea turtle shell particles are gastric-administrated to rats for 3 months, the reduction (-8%) of TP and the increase (+ 93%) of GGT of a rat high-dose group are seen, but no clear dose-effect relation exists, the reduction of TP is small, the increase amplitude of GGT is large, and other related biochemical indexes such as ALT, AST, TBIL, ALP and the like are not changed. No changes in the pathomorphology of the liver were observed. The toxicological significance is considered ambiguous.

Recovery period of female rat K⁺Decrease in non-excretion and increase in urine volumeAnd off. Increased body water intake leads to increased blood volume, decreased tubular reabsorption leads to increased urine volume to maintain osmotic pressure balance, Na in distal tubules⁺Ions are increased to pass Na⁺-K⁺The exchange increases the concentration of K + in urine and K in blood⁺The concentration is reduced. But the increase value of the water intake is still within the central background value, the urine volume is also within the range of the normal value, K⁺The content of (A) is reduced without obvious dose-effect relationship. The toxicological significance is considered ambiguous.

Other indexes with statistical significance, such as the reduction of female rat TBIL and CHO, the reduction of female rat TBIL and the like, have no clear clinical significance and no toxicological significance.

11. Pathological examination

Systematic necropsy (gross pathological observation) after the animals are sacrificed by bleeding, the body surface of the animals is examined for the constitution, development and nutritional status, etc. The animals were visually observed for abnormalities in their external appearance, all orifices, cranium, pleuroperitoneal cavity and internal organs including, but not limited to, brain, salivary glands, thyroid gland, thymus gland, lung and bronchus, heart, liver, spleen, kidney, adrenal gland, testis, epididymis, ovary, uterus, stomach, duodenum, small intestine (jejunum, ileum), large intestine (colon, rectum), cecum, bladder, sternum, femur, eyeball, optic nerve, pancreas, etc.

Organ weights and coefficients for the following organs: heart, liver, spleen, lung, kidney, adrenal gland, thymus, brain, stomach, testis, epididymis, ovary and uterus. A weight/body weight coefficient (body weight coefficient, g organ/100 g body weight) was calculated from the fasting body weight of each animal; the weight of the viscera/weight of the brain (number of viscera/g of brain) was calculated according to the weight of the brain of each animal:

the sweet wormwood and turtle shell granules are administrated for 3 months, the increase of the gastric body coefficient can be seen in a male rat high dose group, and the increase of the gastric weight, the gastric body coefficient and the gastrointestinal coefficient can be seen in a female rat high dose group and a male rat high dose group. The change of the weight and the coefficient of the stomach viscera can be related to the larger administration volume (15ml/kg) and the thicker concentration in the experiment, and the indexes are recovered in the recovery period without clear toxicological significance.

Rats are repeatedly administered the granules of Artemisia apiacea and carapax Trionycis for 3 months by mouth, and the gross examination of naked eyes and the histopathological examination under microscope are carried out on recovery animals of 3 months after administration and 1 month after cessation of administration under the condition of 3 doses of 8.6, 17.3 and 34.5g crude drug/kg body weight, and no obvious toxic injury effect is seen.

Example 12 acute toxicity test of Artemisia apiacea and Trionycis granules

1. Laboratory animal

120 ICR mice were 60 male.

2. Grouping animals

And 6 groups are set: control group (gavage given equal volume of experimental animal drinking water) and 5 doses of the group, 5 doses of the concentration, volume, dose and clinical dose fold are shown in table 23.

TABLE 23

Group body weights are as in table 24(g, mean ± standard deviation):

watch 24

3. Route of administration and method

The administration route is as follows: administered orally (gavage). Consistent with the clinical route of administration.

The administration concentration is as follows: the drug administration groups of the sweet wormwood herb and turtle shell particles are respectively provided with liquid medicine with corresponding concentration, and the control group is provided with equal volume of experimental animal drinking water.

Administration volume: 40 ml/kg/time.

The administration frequency is as follows: 1 administration/day.

The administration time is as follows: fasting body weights were administered on the day after group.

The test stage and the time limit are selected according to the following steps: with reference to the "guidelines for single drug dose toxicity study techniques" (release CFDA 2014-5-13), observations were continued for 2 weeks after a single dose. D14 was observed with D0 on the day of administration and D1 on the next day, and live animals were necropsied at D15.

4. General State Observation

After administration, the animals were closely observed for toxic reactions, and close observation was performed for at least 2h after administration. The administration was observed in the morning and afternoon of the day, and thereafter the animals were observed 1 time per day, and the general condition, hair color, activity, gait, attitude, stool, urine, etc. of the animals were recorded. Observing whether toxic symptoms occur, and the occurrence time, duration and recovery condition, and recording dead or dying animals.

(1) No toxic reaction was observed in the control group during the observation period after administration.

(2) Symptoms of toxicity reaction of the test article after administration: the sweet wormwood and turtle shell granules are prepared by 134.40, 107.52, 86.02, 68.81 and 55.05g crude drugs/kg and 40ml/kg administration volume, and are orally administered to mice for 1 time/day, and the main toxic reaction of animals after the first administration is diarrhea, and symptoms such as nausea, vomit, salivation, lacrimation, convulsion, respiratory acceleration or dyspnea and the like are not seen. The diarrhea symptoms appear in each group of animals 40min after administration, the diarrhea incidence rates of the 5 dose groups are respectively 50% (male 6/10 and female 4/10), 10% (2/10 and 0/10), 10% (1/10 and 1/10), 0% and 0%, except 134.40g crude drug/kg dose group of 1 female animal, the diarrhea animals recover to normal when observed at the 2 nd noon (about 4.5h after administration) on the day of administration, and the animals which do not recover on the day of administration recover to normal when D1 is administered.

(3) Death: 134.40g crude drug/kg dose group 1 female died within 1.5h after administration, and no animal death occurred in the remaining groups. The dead animals are mainly characterized by little movement, lying still to prostrate, disappearance of righting reflex to death, no symptoms such as excitation, convulsion, tremor and the like before death, and the occurrence of the symptoms can have certain correlation with the central nervous system. The dead mice are subjected to the autopsy in time, the pink color of the two lungs and the liquid medicine are observed in the stomach on the whole, the esophageal and tracheal rupture and the like are not found, and the death caused by the gastric lavage accident can be eliminated; the color, texture, size and position of the other major organs are not obviously abnormal. After that, the general conditions, activities, gaits, breaths, meals, drinking water, stool and skin and fur of the surviving animals are not abnormal until the observation period is finished for 14 days.

5. Body weight

Non-fasting body weights were regularly noted during the post-dose observation period.

Animals were weighed against fasted body weights of D1, D2, D3, D5, D8, D11, D14 after dosing.

The method comprises the following steps: on the 1 st day after administration, compared with the control group, the weight of animals in the group with 134.40g crude drug/kg dose of the sweet wormwood turtle shell granules is obviously reduced (P is less than 0.01), and the statistical significance is achieved; at other time points, the body weight of animals in each dose group and the body weight of a comparison control group slightly increase (the amplitude is within +/-6 percent), and the difference has no statistical significance (P > 0.05).

Male parent: the body weight of each administration group was slightly different (within + -6% of the range) from that of the control group at all time points, and the difference was not statistically significant (P > 0.05).

6. Food intake

The determination method comprises the following steps: adding a certain amount of feed (200 g/cage) into each cage, weighing the rest feed after a certain time, and adding a certain amount of feed (200 g/cage) after weighing the rest feed each time. The average feed intake (g feed/rat day) for each cage was calculated by dividing the feed consumption by the number of animals per cage and the number of days in between.

Measuring frequency:

the feed was dosed 1.5 hours after the end of dosing on the day of dosing (D0).

After the administration, D1, D2, D3, D5, D8 and D11 are weighed to obtain the residual feed amount, and then the feed is quantitatively added.

After the administration, D14 refers to the residual feed and does not need to be added with feed quantitatively.

The comprehensive analysis of the weight and the food intake of the mice shows that the weight of animals in the group of the southernwood turtle shell particles of 134.40g crude drug/kg dose is obviously reduced by D1 after the animals are administrated (P is less than 0.05 or P is less than 0.01), and the food intake has no significant difference and can be related to diarrhea after the animals are administrated, so that the weight is rapidly reduced. No obvious abnormality is found in various states of the animals after D2. Thus, the reduction in body weight of the animal is attributed to the transient response at the initial stage of administration.

7. Treatment at the end of the observation period

Fasting: d14 weighing the weight and the rest feed, stopping feeding in afternoon without stopping water supply.

Fasting body weight: the day after food withdrawal (fasting time 16h), the fasting weight was called and the animals were transferred to an anatomical chamber for sacrifice and the fasting time was 17.5h at the end of the necropsy.

The sacrifice mode is as follows: taking off the cervical vertebra and killing.

Examination after sacrifice: gross pathological observation was performed, changes in organs (including but not limited to heart, liver, spleen, lung, kidney, adrenal gland, brain, stomach, intestine, testis, prostate, ovary, uterus) were observed, and if changes in organ volume, color, texture, etc. were found, the changed organs were fixed with 10% formalin, embedded with paraffin, sectioned, stained with HE (haematxylin & Eosin), and examined histopathologically with a microscope.

All surviving animals were visually observed without obvious abnormalities, and tissue fixation and histopathological examination were omitted.

8. Examination of dead and moribund animals during the test

After administration, animals were found dead and examined immediately for gross pathology.

After the observation period of 14 days is finished, the thoracic cavity and the abdominal cavity of the animal are visually observed after dissection, and the heart, the liver, the spleen, the lung, the kidney, the adrenal gland, the stomach and intestine, the bladder, the testis, the epididymis and the like do not have macroscopic lesions such as congestion, blood stasis, bleeding, effusion, adhesion, erosion, ulcer and the like.

After administration, 1 female died and no abnormalities were detected by autopsy.

The maximum tolerance dose of the sweet wormwood and turtle shell granules to mice is 107.52g of crude drugs/kg, and the maximum nontoxic dose is 68.81g of crude drugs/kg.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.