CN112386635A

CN112386635A - Application of Jingfang preparation in preparing medicine for treating new coronary sequelae and preparation method thereof

Info

Publication number: CN112386635A
Application number: CN202011011605.2A
Authority: CN
Inventors: 张贵民; 关永霞; 程国良; 李冰冰
Original assignee: Lunan Pharmaceutical Group Corp
Current assignee: Lunan Pharmaceutical Group Corp
Priority date: 2020-09-23
Filing date: 2020-09-23
Publication date: 2021-02-23

Abstract

The invention discloses an application of a Jingfang preparation in preparing a medicine for treating new coronary sequelae and a preparation method thereof, belonging to the field of medicines. The Jingfang preparation is prepared from eleven raw material medicines, such as schizonepeta, divaricate saposhnikovia root, incised notopterygium rhizome, doubleteeth pubescent angilica root, Chinese thorowax root, whiteflower hogfennel root, Szechuan lovage rhizome, bitter orange, Indian buead, platycodon root, liquoric root and the like, and has the effects of sweating, relieving exterior syndrome. The Jingfang preparation is an effective prescription for treating the new coronary sequelae, can effectively treat the viral myocarditis of a new coronary patient in the convalescent period, and reduce the serum concentrations of liver function markers ALT and AST and kidney function markers Cre and BUN, thereby effectively protecting the liver and kidney functions, effectively inhibiting the inflammatory reaction of acute respiratory distress syndrome, simultaneously effectively treating diarrhea, treating both symptoms and root causes, and having exact treatment effect on the new coronary sequelae.

Description

Application of Jingfang preparation in preparing medicine for treating new coronary sequelae and preparation method thereof

Technical Field

The invention relates to application of a Jingfang preparation and a preparation method thereof, in particular to application of the Jingfang preparation in preparing a medicine for treating new coronary sequelae and a preparation method thereof, and belongs to the field of medicines.

Background

The new coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) is called new coronavirus pneumonia for short, and refers to acute infectious pneumonia caused by 2019 new coronavirus infection. The most common clinical symptoms of infected patients are fever, hypodynamia and dry cough, and a few patients are accompanied with upper respiratory tract and digestive tract symptoms such as nasal obstruction, watery nasal discharge, diarrhea and the like. In severe cases, dyspnea often occurs after 1 week, and severe cases further develop acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis, hemorrhagic blood coagulation dysfunction, and the like. The new coronary pneumonia is a multi-system disease, and the affected area of the disease is not only limited to the lung, but also causes damage to the kidney, liver, heart, brain, nerve and other systems of the patient to different degrees. Most critically ill patients are reported to be accompanied by organ function impairment, including cardiac injury, acute renal injury, liver dysfunction, pneumothorax, and the like. Until now, no targeted effective medicine exists for treating the new coronary pneumonia, and the isolated treatment and the symptomatic support treatment are mainly used.

Along with the enhancement of epidemic prevention and control strength, a large number of patients are cured and discharged. However, some patients who are discharged from hospital are negative in nucleic acid detection, have reduced or obvious pneumonia absorption and normal body temperature, but may have symptoms such as weakness, shortness of breath, appetite reduction, diarrhea, anxiety and the like, and some patients may have pulmonary fibrosis caused by poor lung inflammation absorption to influence the quality of life, especially the prognosis of the elderly and the patients with chronic basic diseases is poor. In addition, the cured patient suffers from various organ injury sequelae caused by new coronary pneumonia, such as heart injury, acute kidney injury, liver dysfunction, nervous system injury, digestive system injury, etc. According to a recent study published by frankfurter university hospitals, the majority of human hearts recovered from the new crown were damaged, and even if the new crown test was negative, 78% of the participants had heart problems, with myocarditis being the most common long-term damaging factor. Therefore, the treatment of new coronary sequelae needs to attract more attention and attention.

The traditional Chinese medicine plays an important role in epidemic situation diagnosis and treatment, wherein the traditional Chinese medicine preparation Jingfang granules are recommended to be used for treating cold-dampness stagnation lung syndrome type new coronary pneumonia in a scheme for preventing new coronary pneumonia in various provinces. The Jingfang preparation is derived from ancient prescription Jingfang toxin-vanquishing powder, is prepared from eleven raw material medicines of schizonepeta, divaricate saposhnikovia root, incised notopterygium rhizome, doubleteeth pubescent angelica root, Chinese thorowax root, whiteflower hogfennel root, Szechuan lovage rhizome, bitter orange, Indian buead, platycodon root, liquoric root and the like, has the effects of inducing sweat, relieving exterior syndrome, dispelling wind and eliminating dampness, and is clinically used for treating symptoms of wind-cold type cold, headache. The monarch drugs of the prescription, namely the schizonepeta and the divaricate saposhnikovia root, which are pungent and warm in nature, can induce sweat, release exterior and dissipate wind evil, and eliminate the first disease of all diseases; the ministerial drugs of notopterygium root and pubescent angelica root, pungent in flavor and warm in property, are used for dispelling wind-cold-dampness in the whole body, the ligusticum wallichii is used for promoting blood circulation and dispelling wind, the radix bupleuri is used for dispelling pathogenic factors from muscles, the notopterygium root and the pubescent angelica root are used for dispelling exogenous pathogenic factors and relieving pain. Fructus Aurantii is used for depressing qi, radix Platycodi is used for opening lung, radix Peucedani is used for eliminating phlegm, Poria is used for eliminating dampness, and radix Glycyrrhizae is used as adjuvant drug and guiding drug. The combination of the medicines can cure the disease by sweating and fever abatement, blood circulation and wind pain elimination, lung qi promotion, phlegm dampness elimination, cough elimination and cold elimination. In recent years, with the deep development of the research of the traditional Chinese medicine preparation, more and more effects of the Jingfang preparation are discovered.

The inventor finds that the Jingfang preparation has obvious effect on treating the new coronary sequelae in the process of upgrading the Jingfang preparation. Pharmacodynamic tests show that the Jingfang preparation is an effective formula for treating the sequelae of the new coronary pneumonia: the traditional Chinese medicine composition can effectively treat viral myocarditis, and reduce the serum concentration of liver function markers ALT and AST and kidney function markers Cre and BUN, so that the liver and kidney functions are effectively protected, the inflammatory response of acute respiratory distress syndrome is effectively inhibited, diarrhea is effectively treated, both symptoms and root causes are treated, and the traditional Chinese medicine composition has an exact and obvious treatment effect on new coronary sequelae.

Disclosure of Invention

The invention aims to provide application of a Jingfang preparation in preparation of a medicine for treating new coronary sequelae. The prescription Jingfang preparation of the invention is derived from ancient prescription Jingfang antiphlogistic powder, and is prepared into Jingfang granules, Jingfang tablets and other series Jingfang preparations by relying on the prior art. The application of the invention is discovered in the clinical application process of the company medicine, and has larger commercial value after being verified by related pharmacodynamic tests.

The application of the Jingfang preparation in preparing the medicine for treating the new coronary sequelae is characterized in that the Jingfang preparation is prepared from the following traditional Chinese medicine components:

preferably:

the test result shows that the Jingfang preparation can effectively treat viral myocarditis, reduce the serum concentration of liver function markers ALT and AST and kidney function markers Cre and BUN, thereby effectively protecting liver and kidney functions, effectively inhibiting the inflammatory reaction of acute respiratory distress syndrome, simultaneously effectively treating diarrhea, treating both symptoms and root causes, and having exact and obvious treatment effect on new coronary sequelae.

The invention also aims to provide a traditional Chinese medicine oral preparation containing the traditional Chinese medicine composition and a preparation method thereof, wherein the traditional Chinese medicine oral preparation is one of granules, tablets, capsules and microcapsules.

The preparation method of the traditional Chinese medicine oral preparation comprises the following steps:

A. extracting volatile oil from 7 medicinal materials of schizonepeta, divaricate saposhnikovia root, incised notopterygium rhizome, doubleteeth pubescent angilica root, whiteflower hogfennel root, szechuan lovage rhizome and bitter orange respectively to obtain the volatile oil of the 7 medicinal materials respectively, and distilling the residue, the szechuan lovage rhizome and the bitter orange to obtain aqueous solution for later use;

B. b, adding the volatile oil obtained in the step A into beta-cyclodextrin respectively to prepare an inclusion compound;

C. b, preparing the water solution of the distilled rhizoma ligustici wallichii and the fructus aurantii obtained in the step A into a 20-30% ethanol solution as a solvent, and percolating the residues of the rhizoma ligustici wallichii and the fructus aurantii obtained in the step A and the poria cocos to obtain percolate for later use;

D. b, decocting the residues of the schizonepeta, the divaricate saposhnikovia root, the incised notopterygium rhizome, the doubleteeth pubescent angilica root and the whiteflower hogfennel root obtained in the step A and the rest three medicines of the Chinese thorowax root, the platycodon root and the liquoric root in water for 2-3 times, wherein each time lasts for 1.5-2.5 hours, merging decoction, filtering, and concentrating the filtrate to an extract with the relative density of 1.18-1.;

E. mixing the percolate obtained in the step C and the extract obtained in the step D, uniformly mixing, standing, filtering, and concentrating the filtrate to obtain an extract with the relative density of 1.22-1.28 at 50-60 ℃;

F. and D, taking the extract obtained in the step E and the beta-cyclodextrin inclusion compound obtained in the step B, and preparing an oral medicinal preparation directly or by adding a pharmaceutically acceptable excipient through a conventional process.

The preferred dosage form of the present invention is a granule, and the preparation method of the granule comprises the following steps:

C. b, preparing a 25% ethanol solution of the distilled water solution of the ligusticum wallichii and the fructus aurantii obtained in the step A as a solvent, and percolating the residues of the ligusticum wallichii and the fructus aurantii obtained in the step A and the poria cocos to obtain a percolate for later use;

D. decocting the residues of the schizonepeta, the divaricate saposhnikovia root, the incised notopterygium rhizome, the doubleteeth pubescent angilica root and the whiteflower hogfennel root obtained in the step A and the rest three medicines of the Chinese thorowax root, the platycodon root and the liquoric root in water for 3 times, 2 hours for each time, combining decoction, filtering, and concentrating the filtrate to an extract with the relative density of 1.23 at the;

E. c, combining the percolated liquid obtained in the step C and the extract obtained in the step D, uniformly mixing, standing, filtering, and concentrating the filtrate to an extract with the relative density of 1.26 at 50-60 ℃;

F. and E, belt-type vacuum drying the extract obtained in the step E under the conditions of vacuum degree of-0.08 MPa to-0.10 MPa and drying temperature of 63 ℃, crushing the extract into fine powder, sieving the fine powder, adding the beta-cyclodextrin inclusion compound obtained in the step B, and uniformly mixing the fine powder to obtain the Jingfang extract fine powder, wherein the weight ratio of the extract to the sucrose powder in the formula is as follows: hydroxypropyl starch: mannitol 3: 2: 0.5 excipient, mixing, granulating, drying, and grading.

Another preferred dosage form of the present invention is a pellet formulation, and the method for preparing the microcapsule formulation comprises the following steps:

C. b, preparing a 28% ethanol solution of the distilled water solution of the ligusticum wallichii and the fructus aurantii obtained in the step A as a solvent, and percolating the residues of the ligusticum wallichii and the fructus aurantii obtained in the step A and the poria cocos to obtain a percolate for later use;

D. decocting the residues of the schizonepeta, the divaricate saposhnikovia root, the incised notopterygium rhizome, the doubleteeth pubescent angilica root and the whiteflower hogfennel root obtained in the step A and the rest three medicines of the Chinese thorowax root, the platycodon root, the liquoric root and the like in water for 2 times, 2.5 hours for each time, combining the decoctions, filtering, and concentrating the filtrate to an extract with the relative density of 1.;

E. c, combining the percolated liquid obtained in the step C and the extract obtained in the step D, uniformly mixing, standing, filtering, and concentrating the filtrate to an extract with the relative density of 1.25 at 50-60 ℃;

F. e, adding the extract obtained in the step E into the cyclodextrin inclusion compound obtained in the step B, uniformly mixing, preparing into granules, carrying out belt type vacuum drying, and crushing to obtain fine powder of the Jingfang extract for later use;

G. weighing the fine powder of the Jingfang extract, the capsule wall material, the anti-sticking agent and the plasticizer in the step F according to the formula, adding the capsule wall material, the anti-sticking agent and the plasticizer into purified water, heating and stirring at 54 ℃ to dissolve the capsule wall material, preparing a capsule wall material solution with the mass fraction of 33%, cooling to room temperature, adding the fine powder of the Jingfang extract and the emulsifier in a stirring state, homogenizing and emulsifying to obtain an emulsion for later use;

H. and G, carrying out spray drying on the emulsion in the step G under the conditions of air inlet temperature of 165 ℃, spray pressure of 0.40MPa and feeding speed of 21.5ml/min, collecting the microcapsules, and cooling to obtain the microcapsule.

Preferably, the belt type vacuum drying condition in the step F is vacuum degree of-0.08 MPa-0.10 MPa, and the drying temperature is 56 ℃.

Preferably, the capsule material in the step G is sodium complexing protein: maltodextrin 3:2, anti-tack agent octadecanol: titanium dioxide 3: 1, the plasticizer is polyethylene glycol: citric acid ═ 3: 1.

preferably, the emulsifier of step C is a sucrose fatty acid ester: the soybean lecithin is 8: 5 of the composite emulsifier, and the dosage of the composite emulsifier is 1.18 percent of the total amount of the preparation formula according to the mass fraction.

In order to verify the effect of the Jingfang preparation on treating the new crown sequelae, the inventor carries out corresponding animal test research. It should be noted that the animal experimental study is selected from the representative formula and the preparation method thereof, and the other formulas and products obtained by the preparation method thereof involved in the invention relate to the experiment and the result thereof, which are not exhaustive due to space limitation.

Experimental example 1 Effect of Jingfang preparation on mouse myocarditis

1 Material

1.1 experimental animal and feed SPF grade Balb/c male mice, 6-8 weeks old, 60 mice, weight 16-18g, provided by Lunan pharmaceutical group, Inc., the license number of experimental animal: SYXK (lu) 20180008. The mice are adaptively raised in a clean-grade animal laboratory for 1 week before experiments, the male and female parts are separated, the room temperature is 20-25 ℃, the relative humidity is 40% -60%, the mice are naturally illuminated, and the mice can freely eat and drink water.

1.2 Instrument, reagent and drug AG285 model electronic analytical balance (Mettler-Toledo, Switzerland), SE-LECTRA-E model fully automatic biochemical analyzer (the Netherlands Scientific Co.), Thermo Scientific Medifuge small bench centrifuge (Sammer fly, USA); eagle culture solution (Winsent biotechnology, Inc.), mouse TNF-alpha, cTnI enzyme-linked immunosorbent assay kit (Nanjing institute of biological research); the test drug was the Jingfang granules of example 5 (manufactured by Luonanpu pharmaceutical Co., Ltd.) and the positive control drug was ribavirin granules (lot No. 20190701, Chenxin pharmaceutical Co., Ltd.).

2 method

2.1 grouping and modeling to obtain 60 healthy Balb/c male mice, randomly dividing into 6 groups, and dividing each group into 10 mice, namely a normal control group, a model control group, a ribavirin positive control group (short for a positive control group), a Jingfang granule high dose group (short for a test high dose group), a Jingfang granule medium dose group (short for a test medium dose group) and a Jingfang granule low dose group (short for a test low dose group). Culture of CVB Using Eagle Medium₃Virus, except for normal control group, the abdominal cavity injection of other mice is 1X 10⁴/ml CVB₃0.2ml of the diluent, and 0.2ml of Eagle culture solution is injected into the abdominal cavity of a normal control group.

2.2 after the virus is inoculated for 2 hours, each group is administered by gastric perfusion, dose reduction is carried out according to dose reduction coefficients of different animals in annexes in the guideline of clinical research of new traditional Chinese medicines, a positive control group is administered by ribavirin of 0.10g/kg, a test high dose group, a test medium dose group and a test low dose group are administered by Jingfang granules of 9g/kg, 4.5g/kg and 2.25g/kg respectively, which are equivalent to 2 times, 1 time and 0.5 time of equivalent dose for human, the administration volume is 10ml/kg, a normal control group and a model control group are administered by normal saline with equal volume, and the administration is carried out for 1 time every day and 7 days continuously.

2.3 detection indexes and method administration for 7 days, taking blood from mouse eyeballs, centrifuging at 4000r/min for 10min, separating serum, and measuring the levels of cTnI (cardiac troponin I) and TNF-alpha (tumor necrosis factor alpha) in the serum by adopting an ELISA kit.

2.4 statistical processing SPSS19.0 statistical software was used for analysis, and experimental data were expressed as mean. + -. standard deviation

"is expressed in terms of form. The comparison among groups was performed by one-way anova, with P < 0.05 indicating that the difference was statistically significant.

3 results

Compared with a normal control group, the levels of cTnI and TNF-alpha in the serum of the mouse of the model control group are obviously increased, and the differences have statistical significance (P is less than 0.05), which indicates that the modeling of the experiment is successful. Compared with a model control group, the cTnI and the TNF-alpha in the serum of mice in a positive control group and a test high, medium and low dose group are obviously reduced, and the difference has statistical significance (P is less than 0.05). The above results suggest that Jingfang granules can effectively treat viral myocarditis. The results are shown in Table 1.

TABLE 1 Effect of Jingfang granules on viral myocarditis in mice: (

n＝10)

Note: comparison with normal control group: p < 0.05 for "^Δ"means; comparison with model control group: p < 0.05 is indicated by ". times..

Experimental example 2 Effect of Jingfang preparation on acute liver injury of mouse

1 Material

1.1 experimental animals and healthy-fed KM mice, each half of male and female, 60 mice, body weight (20 +/-2) g, provided by Lunan pharmaceutical group, Inc., and the license number of experimental animals: SYXK (lu) 20180008. The method is characterized in that the animal is bred adaptively for 1 week in a clean-grade animal laboratory before experiment, male and female parts are separated, the room temperature is 20-25 ℃, the relative humidity is 40% -60%, natural illumination is carried out, and free food intake and drinking are carried out.

1.2 Instrument, reagents and drug Thermo Scientific Medifuge Small bench centrifuge (Sammer fly, USA), AG285 electronic analytical balance (Mettler-Toledo, Switzerland); mouse ALT, AST enzyme linked immunosorbent assay kit (Sigma, USA), carbon tetrachloride (Shanghai Allantin Biotechnology Co., Ltd.); the test drug was the Jingfang granule (manufactured by Lunan Kappan pharmaceutical Co., Ltd.) of example 5, and the positive control drug was biphenyldicarboxylate (batch No. 19058003, Shandong health pharmaceutical Co., Ltd.).

2 method

2.1 healthy KM mice were divided into groups and molded into 6 groups, i.e., a normal control group, a model control group, a biphenyldicarboxylate positive control group (referred to as "positive control group"), a Jingfang granule high dose group (referred to as "test high dose group"), a Jingfang granule medium dose group (referred to as "test medium dose group"), and a Jingfang granule low dose group (referred to as "test low dose group"), each group consisting of 10 mice each having half male and female. Except for the normal control group, the mice of other groups are subjected to intraperitoneal injection of 0.2% carbon tetrachloride by 0.1ml/10g, an acute liver injury model is formed after 24 hours, and the mice of the normal control group are subjected to intraperitoneal injection of peanut oil with equal dosage. Blood samples of 0.5mL are collected (5 mice are randomly taken in each group, blood is taken from the orbit), ALT (glutamic-pyruvic transaminase) and AST (glutamic-oxalacetic transaminase) in serum are measured, and each molding group is obviously increased compared with a normal control group, which indicates that the molding is successful.

2.2 after the successful model building, on the 3 rd day, each group is administered by gastric perfusion, dose reduction is carried out according to the dose reduction coefficients of different animals in the appendix of the guideline for clinical research of new traditional Chinese medicines, the positive control group is administered by bifendate 0.15g/kg, the test high dose group, the test medium dose group and the test low dose group are administered by Jingfang granules 9g/kg, 4.5g/kg and 2.25g/kg respectively, which are equivalent to 2 times, 1 time and 0.5 time of equivalent dose for human, the administration volume is 10ml/kg, the model control group is administered by normal saline with equal volume, 1 time every day, and the administration is continuously carried out for 8 days.

2.3 detection indexes and method 1 hour after administration on day 8, the mice are bled through the eye sockets, 1.5mL of the blood is sampled to be put in a centrifuge tube, the centrifuge tube is kept stand for 30min, and serum is separated after centrifugation is carried out for 20min at 10000 r/min. ALT and AST levels were determined as required by ELISA kit instructions.

3 results

Compared with a normal control group, the ALT and AST levels in the serum of the mouse of the model control group are obviously increased, and the difference has statistical significance (P is less than 0.05), which indicates that the modeling of the experiment is successful. Compared with the model control group, the ALT and AST levels in the serum of the mice of the positive control group and the test high, medium and low dose groups are obviously reduced, and the difference has statistical significance (P is less than 0.05). The above results suggest that Jingfang granule pairs CCl₄The induced liver injury has protective effect. The results are shown in Table 2.

TABLE 2 Effect of Jingfang granules on liver injury in mice: (

n＝10)

Experimental example 3 Effect of drugs on acute Kidney injury in mice

1 Material

1.2 Instrument, reagent and drug AG285 model electronic analytical balance (Mettler-Toledo, Switzerland), SE-LECTRA-E model fully automatic biochemical analyzer (the science of the Williams, the Netherlands), Thermo Scientific Medifuge Small bench centrifuge (Sammer fly, USA); cisplatin (Sigma, usa), pentobarbital sodium (chemical limited, hong bo, china), creatinine assay kit (Sigma, usa), urea nitrogen kit (bio-technology limited, moral, quanza); the test drug was the Jingfang granule (manufactured by Lunan Kappan pharmaceutical Co., Ltd.) of example 5, and the positive control drug was resveratrol (batch No. 19067002, Guangdong Bilde Biotech Co., Ltd.).

2 method

2.1 healthy KM mice were divided into groups and molded into 6 groups, i.e., a normal control group, a model control group, a resveratrol positive control group (referred to as "positive control group"), a Jingfang granule high dose group (referred to as "test high dose group"), a Jingfang granule medium dose group (referred to as "test medium dose group") and a Jingfang granule low dose group (referred to as "test low dose group"), 10 mice each with half male and female groups. Except for the normal control group, the mice of other groups are subjected to intraperitoneal injection of 20mg/kg cis-platinum on the 1 st day of the experiment to form an acute kidney injury model of the mice, and the normal control group is subjected to intraperitoneal injection of the same amount of normal saline. Blood samples of 0.5mL are collected (5 mice are randomly taken in each group, blood is taken from the orbit), Cre (creatinine) and BUN (urea nitrogen) in the serum are measured, and each model group is obviously increased compared with a normal control group, which indicates that the model building is successful.

2.2 after the successful model building, on the 2 nd day, each group is administered by gastric perfusion, dose reduction is carried out according to dose reduction coefficients of different animals in annexes in the clinical research guiding principle of new traditional Chinese medicines, the positive control group is administered with resveratrol of 0.20g/kg, the test high dose group, the test medium dose group and the test low dose group are administered with Jingfang granules of 9g/kg, 4.5g/kg and 2.25g/kg respectively, which are equivalent to 2 times, 1 time and 0.5 time of equivalent dose for human, the administration volume is 10ml/kg, and the model control group is administered with physiological saline of equal volume for continuous administration for 7 days, 1 time every day.

2.3 detection of indexes and methods Each group of mice fasted for 12 hours before last gavage administration, 1 hour after last gavage, mice were anesthetized by intraperitoneal injection of pentobarbital sodium (60mg/kg), blood was taken after eye picking, centrifuged at 3000r/min for 10min, serum was separated, and serum creatinine (Cre) and urea nitrogen (BUN) contents were measured by a full-automatic biochemical analyzer.

3 results

Compared with a normal control group, the Cre and BUN levels in the serum of the mouse of the model control group are obviously increased, and the difference has statistical significance (P is less than 0.05), which indicates that the modeling of the experiment is successful. Compared with a model control group, the Cre and BUN levels in the serum of mice in a positive control group and a test high, medium and low dose group are obviously reduced, and the difference has statistical significance (P is less than 0.05). The above results suggest that Jingfang granules have protective effect on cisplatin-induced renal injury. The results are shown in Table 3.

TABLE 3 Effect of Jingfang granules on Kidney injury in mice: (

n＝10)

Experimental example 4 Effect of drugs on rat Acute Respiratory Distress Syndrome (ARDS)

1 Material

1.1 experimental animals and feed healthy male SD rats, 60, body weight (200 + -20) g, provided by Lunan pharmaceutical group, Inc., and license number of experimental animals: SYXK (lu) 20180008. The method is characterized in that the animal is bred adaptively for 1 week in a clean-grade animal laboratory before experiment, male and female parts are separated, the room temperature is 20-25 ℃, the relative humidity is 40% -60%, natural illumination is carried out, and free food intake and drinking are carried out.

1.2 instruments, reagents and drugs Thermo Scientific medicase small bench centrifuge (sammerfly usa), -80 ℃ cryogenic refrigerator (special electric appliances ltd, hail, qingdao), AG285 type electronic analytical balance (Mettler-Toledo, switzerland); rat TNF-alpha and IL-1 beta enzyme-linked immunosorbent assay kit (R & D company, USA), lipopolysaccharide LPS (Sigma company, USA), sodium pentobarbital (J.M. Hongbo chemical Co., Ltd.); the test drug was the Jingfang granule of example 5 (manufactured by Shandong Thick pharmaceutical Co., Ltd.), and the positive control drug was ambroxol hydrochloride (batch No. 19049001, Shanghai Boringer Yigaohn pharmaceutical Co., Ltd.).

2 method

2.1 healthy male SD rats were divided into 6 groups at random, and 10 rats in each group were selected from a normal control group, a model control group, an ambroxol hydrochloride positive control group (referred to as "positive control group"), a Jingfang granule high dose group (referred to as "test high dose group"), a Jingfang granule medium dose group (referred to as "test medium dose group"), and a Jingfang granule low dose group (referred to as "test low dose group"). Except for the normal control group, 7.5mg/kg LPS (prepared by using sterile physiological saline) is instilled into the trachea of the rats of other groups, then the rats are overturned, the liquid medicine is uniformly distributed in the two lungs of the rats, an ARDS model is constructed, and the same amount of sterile physiological saline is instilled into the trachea of the rats of the normal control group. If the rat has symptoms of listlessness, hypokinesia, anorexia, tachypnea, diarrhea and the like, the successful model building is proved.

2.2 after the model is formed for 6 hours by administration, the dosage is converted according to the dosage conversion coefficients of different animals in the appendix of the clinical research guiding principles of new traditional Chinese medicines, the positive control group rat is injected with 0.04g/kg of ambroxol hydrochloride injection in the abdominal cavity, the experimental high-dosage group rat, the experimental medium-dosage group rat and the experimental low-dosage group rat are respectively administrated with 9g/kg, 4.5g/kg and 2.25g/kg of Jingfang granules, which are equivalent to 2 times, 1 time and 0.5 time of equivalent dosage for human, the administration volume is 10ml/kg, and the normal control group and the model control group are administrated with normal saline with equal volume and are administrated 1 time a day for 21 days by continuous administration.

2.3 detection indexes and methods after the last administration of rats, 30mg/kg of sodium pentobarbital is injected into the abdominal cavity. After anesthesia, the rat was tracheotomy, the hollow catheter was inserted, the right bronchus was ligated, and the left lung was lavaged to the left bronchus with 5ml of cold physiological saline, and the procedure was repeated 3 times with a recovery rate of more than 90%. The collected BALF (bronchoalveolar lavage fluid) was centrifuged at 3000r/min at 4 ℃ for 10min, and the supernatant was stored in an ultra-low temperature freezer at-80 ℃. TNF-alpha and IL-1 beta ELISA kits are adopted to detect the content of TNF-alpha and IL-1 beta, and the operation is strictly carried out according to the kit instructions.

3 results

Compared with a normal control group, the levels of TNF-alpha and IL-1 beta in the BALF of the model control group rat are obviously increased, and the differences have statistical significance (P is less than 0.05), which indicates that the modeling of the experiment is successful. Compared with the model control group, the positive control group and the experimental high, medium and low dose groups have obviously reduced TNF-alpha and IL-1 beta levels in the serum of rats, and the difference has statistical significance (P is less than 0.05). The above results suggest that Jingfang granules have a certain inhibitory effect on ARDS inflammatory response. The results are shown in Table 4.

TABLE 4 Effect of Jingfang granules on rat acute respiratory distress syndrome ((R))

n＝10)

Experimental example 5 Effect of drugs on diarrhea in mice

1 Material

1.1 experimental animals and feed SPF-grade KM mice, male and female half, 60 mice, body weight (20 + -2) g, provided by Lunan pharmaceutical group, Inc., and the license number of experimental animals: SYXK (lu) 20180008. The method is characterized in that the animal is bred adaptively for 1 week in a clean-grade animal laboratory before experiment, male and female parts are separated, the room temperature is 20-25 ℃, the relative humidity is 40% -60%, natural illumination is carried out, and free food intake and drinking are carried out.

1.2 full-automatic colony counter of instrument, reagent and medicine (Shanghai Hengelectromechanics instruments Co., Ltd.), AG285 type electronic analytical balance (Mettler-Toledo, Switzerland), Thermo Scientific Medifuge small-sized desk centrifuge (Sammer fly, USA), -80 ℃ low temperature refrigerator (Qingdao Heier Special appliances Co., Ltd.), bacterial incubator (Shanghai Jinghong laboratory equipments Co., Ltd.), anaerobic bacteria incubator (Shanghai Xin instruments Co., Ltd.), selective medium (Qingdao Heibo biology Co., Ltd.); mouse diamine oxidase (DAO) ELISA kit (xiamen huijia biotechnology limited), D-lactic acid detection assay kit (colorimetric method) (wuhan eimei science and technology limited), SIgA kit (china atomic energy science research institute isotope research institute), lincomycin (shandong ruiyang pharmaceutical limited), PBS (american sermer fly); the test drug was the Jingfang granule (manufactured by Lunan Kappan pharmaceutical Co., Ltd.) of example 5, and the positive control drug was a live Bifidobacterium triple cell tablet (trade name Pefeikon, lot No. 19075002, Shanghai Xinyi pharmaceutical factory Co., Ltd.).

2 method

2.1 grouping and modeling 60 SPF-level KM mice are taken, wherein, 0.4 mL/time of gavage lincomycin hydrochloride is given to 50 mice 2 times per day for 4 consecutive days, and the mice are observed to have listlessness, reduced activity, reduced body mass, reddish anus and diarrhea; meanwhile, the defecation condition of the mouse is observed, yellow brown, thin and rotten excrement appears, and the number of bifidobacteria and lactobacilli cultured in fresh excrement is reduced, so that the molding success is shown. The 50 mice successfully molded are randomly divided into a model control group, a bifidobacterium triple viable cell control group (called as a positive control group for short), a Jingfang granule high dose group (called as a test high dose group for short), a Jingfang granule medium dose group (called as a test medium dose group for short) and a Jingfang granule low dose group (called as a test low dose group for short), wherein 10 mice in each group have half of male and female. The other 10 mice were gavaged with the same amount of physiological saline and set as normal control groups.

2.2 beginning on the 5 th day of administration, each group is administered by intragastric administration, dose reduction is carried out according to dose reduction coefficients of different animals in annexes in the clinical research guiding principle of new traditional Chinese medicines, a positive control group is administered with 0.2g/kg of bifidobacterium triple viable tablets, a test high dose group, a test middle dose group and a test low dose group are administered with 9g/kg, 4.5g/kg and 2.25g/kg of Jingfang granules respectively, which are equivalent to 2 times, 1 time and 0.5 time of equivalent doses for human, the administration volume is 10ml/kg, a normal control group and a model control group are administered with equal volume of physiological saline, 1 time per day and 14 days of continuous administration.

2.3 detection index and method

2.3.1 detection of intestinal flora after continuous administration for 14 days, mice were sacrificed by cervical dislocation, 100 μ g of cecal content of each group of mice was aseptically collected, 800 μ L of diluent was added and mixed thoroughly, and diluted again, 1 × 10 was selected^-3-1×10^-8Inoculating the dilution suspension to a selective culture medium, respectively placing in an incubator at 37 ℃ and an anaerobe incubator for culturing for 24 hours, and identifying enterobacteria, enterococci, bifidobacteria and lactobacilli by colony morphology, biochemical reaction and gram staining. The identified bacteria were counted and the number of viable dominant bacteria (lgCFU/g) was represented by a log value.

2.3.2 measurement of diamine oxidase and D-lactic acid content after continuous administration for 14 days, mice were sacrificed by cervical dislocation, blood was taken from the heart at 5mL for 10min after centrifugation at 3000r/min, and the contents of diamine oxidase (DAO) and D-lactic acid in the serum were measured.

2.3.3 detection of intestinal mucosa SIGA (secretory immunoglobulin A) from 10cm under pylorus toward 15cm cecal section, spreading on filter paper, splitting along longitudinal section, washing with sterile PBS buffer solution to remove intestinal mucosa surface substance, spreading slide glass on intestinal mucosa surface, scraping part of mucus, and freezing in 1.5mL sterile EP tube. The level of SIgA in the intestinal mucosa was analyzed by biotin-avidin labeled enzyme-linked immunoassay according to the kit instructions.

3 results

3.1 comparison of intestinal flora compared with normal control group, the model control group has reduced lactobacillus number and bifidobacterium number, increased enterobacteria number and enterococcus number, and the difference has statistical significance (P is less than 0.05). Compared with the model control group, the positive control group and the test high, medium and low dose groups have more lactobacillus and bifidobacterium, the enterobacter and enterococcus are reduced, and the difference has statistical significance (P is less than 0.05). The results are shown in Table 5. The results suggest that Jingfang granules have protection and repair effects on the mycoderm barrier of intestinal mucosa of mice with antibiotic-associated diarrhea.

3.2 comparing the content measurement of DAO and D-lactic acid in serum with that of normal control group, the content of DAO and D-lactic acid in serum of mice in model control group is obviously increased, and the difference has statistical significance (P is less than 0.05). Compared with a model control group, the content of the serum DAO and the content of the D-lactic acid of the positive control group and the mice of the high, medium and low dose groups are obviously reduced, and the difference has statistical significance (P is less than 0.05). The results suggest that Jingfang granules can effectively improve the intestinal barrier function of mice with antibiotic-associated diarrhea.

3.3 intestinal mucus SIGA content determination compared with normal control group, intestinal mucosa SIGA content of model control group mouse is significantly reduced, and difference has statistical significance (P is less than 0.05). Compared with a model control group, the intestinal mucosa SIgA content of mice in a positive control group and a test high, medium and low dose group is obviously increased, and the difference has statistical significance (P is less than 0.05). The results indicate that the Jingfang granules have positive promotion effect on improving the intestinal immunity of the mice with antibiotic-associated diarrhea. The results are shown in Table 6.

In conclusion, the Jingfang granules can effectively treat antibiotic-associated diarrhea.

TABLE 5 Effect of Jingfang granules on the number of intestinal flora in mice: (

n＝10)

TABLE 6 Effect of Jingfang granules on mouse serum DAO, D-lactic acid and intestinal mucosa SIgA content (II)

n＝10)

The experimental results show that the Jingfang granules can effectively treat viral myocarditis, reduce the serum concentrations of liver function markers ALT and AST and kidney function markers Cre and BUN, thereby effectively protecting liver and kidney functions, effectively inhibiting inflammatory reaction of acute respiratory distress syndrome, simultaneously effectively treating diarrhea, treating both symptoms and root causes, and having exact and remarkable treatment effect on new coronary sequelae.

Detailed Description

The invention is further illustrated by the following specific examples, which are not to be construed as limiting the invention in any way, as will be appreciated by those skilled in the art.

Example 1 preparation of Jingfang granules

C. b, preparing a 27% ethanol solution of the distilled water solution of the ligusticum wallichii and the fructus aurantii obtained in the step A as a solvent, and percolating the residues of the ligusticum wallichii and the fructus aurantii obtained in the step A and the poria cocos to obtain a percolate for later use;

D. decocting the residues of the schizonepeta, the divaricate saposhnikovia root, the incised notopterygium rhizome, the doubleteeth pubescent angilica root and the whiteflower hogfennel root obtained in the step A and the rest three medicines of the Chinese thorowax root, the platycodon root, the liquoric root and the like in water for 3 times, 2 hours for each time, combining decoction liquids, filtering, and concentrating the filtrate to an extract with the relative density of 1.;

E. c, combining the percolated liquid obtained in the step C and the extract obtained in the step D, uniformly mixing, standing, filtering, and concentrating the filtrate to an extract with the relative density of 1.24 at 50-60 ℃;

F. and E, belt-type vacuum drying the extract obtained in the step E under the conditions of vacuum degree of-0.08 MPa to-0.10 MPa and drying temperature of 63 ℃, crushing the extract into fine powder, sieving the fine powder, adding the beta-cyclodextrin inclusion compound obtained in the step B, uniformly mixing the fine powder to obtain the Jingfang extract fine powder, adding the sucrose powder, the hydroxypropyl starch and the mannitol (weight ratio is 4: 2: 1) in the formula amount, uniformly mixing the mixture, preparing the mixture into granules, drying and finishing the granules to prepare 10kg of the extract.

EXAMPLE 2 preparation of Jingfang tablets

C. b, preparing a 24% ethanol solution of the distilled water solution of the ligusticum wallichii and the fructus aurantii obtained in the step A as a solvent, and percolating the residues of the ligusticum wallichii and the fructus aurantii obtained in the step A and the poria cocos to obtain a percolate for later use;

D. decocting the residues of the schizonepeta, the divaricate saposhnikovia root, the incised notopterygium rhizome, the doubleteeth pubescent angilica root and the whiteflower hogfennel root obtained in the step A and the rest three medicines of the Chinese thorowax root, the platycodon root, the liquoric root and the like in water for 2 times, wherein each time lasts for 1.5 hours, combining the decoctions, filtering the decoction, and concentrating the filtrate to an extract with the relative;

E. c, combining the percolated liquid obtained in the step C and the extract obtained in the step D, uniformly mixing, standing, filtering, and concentrating the filtrate to an extract with the relative density of 1.28 at 50-60 ℃;

F. and E, belt-type vacuum drying the extract obtained in the step E under the conditions of vacuum degree of-0.08 MPa to-0.10 MPa and drying temperature of 63 ℃, crushing the extract into fine powder, sieving the fine powder, adding the beta-cyclodextrin inclusion compound obtained in the step B, uniformly mixing the fine powder to obtain the Jingfang extract fine powder, adding the microcrystalline cellulose, the low-substituted hydroxypropyl cellulose and the mannitol (the weight ratio is 4: 3: 1) in a formula amount, uniformly mixing the mixture to prepare coarse granules, drying, crushing, sieving, granulating, performing low-temperature drying and finishing, adding 0.2% of magnesium stearate and 0.1% of talcum powder, uniformly mixing the mixture, and pressing the mixture into 10000 tablets.

Example 3 preparation of Jingfang microcapsules

C. b, preparing 29% ethanol solution of the water solution of the distilled rhizoma ligustici wallichii and the fructus aurantii obtained in the step A as a solvent, and percolating the residues of the rhizoma ligustici wallichii and the fructus aurantii obtained in the step A and the poria cocos to obtain percolate for later use;

E. c, combining the percolated liquid obtained in the step C and the extract obtained in the step D, uniformly mixing, standing, filtering, and concentrating the filtrate to an extract with the relative density of 1.27 at 50-60 ℃;

F. adding the extract obtained in the step E into the cyclodextrin inclusion compound obtained in the step B, uniformly mixing, preparing into granules, carrying out belt type vacuum drying under the conditions that the vacuum degree is-0.08 MPa to-0.10 MPa and the drying temperature is 55 ℃, and crushing to obtain fine powder of the Jingfang extract for later use;

G. weighing fine powder of the Jingfang extract in the step F and sodium complexing protein according to the formula: maltodextrin 5:3 mixture as capsule wall material, octadecanol: titanium dioxide 2: 1 is antiadherent, polyethylene glycol: citric acid 4: adding the capsule wall material, the anti-sticking agent and the plasticizer into purified water, heating and stirring at 52 ℃ to dissolve the capsule wall material, preparing a capsule wall material solution with the mass fraction of 33.5%, cooling to room temperature, adding the fine powder of the Jingfang extract under the stirring state, adding 1.17 percent of sucrose fatty acid ester of the total amount of the preparation formula: homogenizing and emulsifying soybean phospholipid 8: 5 composite emulsifier to obtain emulsion;

H. and G, carrying out spray drying on the emulsion in the step G under the conditions of the air inlet temperature of 166 ℃, the spray pressure of 0.40MPa and the feeding speed of 22.5ml/min, collecting the microcapsules, and cooling to obtain the microcapsule.

Example 4 preparation of Jingfang capsules

D. decocting the residues of the schizonepeta, the divaricate saposhnikovia root, the incised notopterygium rhizome, the doubleteeth pubescent angilica root and the whiteflower hogfennel root obtained in the step A and the rest three medicines of the Chinese thorowax root, the platycodon root, the liquoric root and the like in water for 3 times, wherein each time lasts for 1.5 hours, combining the decoctions, filtering the decoction, and concentrating the filtrate to an extract with the relative;

F. and E, belt-type vacuum drying the extract obtained in the step E under the conditions of vacuum degree of-0.08 MPa to-0.10 MPa and drying temperature of 63 ℃, crushing the extract into fine powder, sieving the fine powder, adding the beta-cyclodextrin inclusion compound obtained in the step B, uniformly mixing the fine powder to obtain the Jingfang extract fine powder, adding the starch and the microcrystalline cellulose (the weight ratio is 6: 1) in a formula amount, uniformly mixing, granulating, drying, grading, filling, polishing in a polishing machine, and removing damaged capsules to obtain the Jingfang extract.

Example 5 preparation of Jingfang granules

F. and E, belt-type vacuum drying the extract obtained in the step E under the conditions of vacuum degree of-0.08 MPa to-0.10 MPa and drying temperature of 63 ℃, crushing the extract into fine powder, sieving the fine powder, adding the beta-cyclodextrin inclusion compound obtained in the step B, and uniformly mixing the fine powder to obtain the Jingfang extract fine powder, wherein the weight ratio of the extract to the sucrose powder in the formula is as follows: hydroxypropyl starch: mannitol 3: 1: 0.5 excipient, mixing, granulating, drying, and grading.

EXAMPLE 6 preparation of Jingfang capsules

C. b, preparing the water solution of the distilled hemlock parsley and the bitter orange obtained in the step A into 26 percent ethanol solution as a solvent, and percolating the dregs of the hemlock parsley and the bitter orange obtained in the step A and the tuckahoe to obtain percolate for later use;

D. decocting the residues of the schizonepeta, the divaricate saposhnikovia root, the incised notopterygium rhizome, the doubleteeth pubescent angilica root and the whiteflower hogfennel root obtained in the step A and the rest three medicines of the Chinese thorowax root, the platycodon root, the liquoric root and the like in water for 2 times, 2 hours for each time, combining decoction liquids, filtering, and concentrating the filtrate to an extract with the relative density of 1.;

E. c, combining the percolated liquid obtained in the step C and the extract obtained in the step D, uniformly mixing, standing, filtering, and concentrating the filtrate to an extract with the relative density of 1.23 at 50-60 ℃;

F. and E, belt-type vacuum drying the extract obtained in the step E under the conditions of vacuum degree of-0.08 MPa to-0.10 MPa and drying temperature of 63 ℃, crushing the extract into fine powder, sieving the fine powder, adding the beta-cyclodextrin inclusion compound obtained in the step B, uniformly mixing the fine powder to obtain the Jingfang extract fine powder, adding the starch, the superfine silica gel powder and the low-substituted hydroxypropyl cellulose (weight ratio is 5: 2: 2) in the formula amount, uniformly mixing, granulating, drying, grading, filling, polishing in a polishing machine, and removing damaged capsules to obtain the Jingfang extract.

Example 7 preparation of Jingfang capsules

C. b, preparing a 30% ethanol solution of the distilled water solution of the ligusticum wallichii and the fructus aurantii obtained in the step A as a solvent, and percolating the residues of the ligusticum wallichii and the fructus aurantii obtained in the step A and the poria cocos to obtain a percolate for later use;

F. and E, belt-type vacuum drying the extract obtained in the step E under the conditions of vacuum degree of-0.08 MPa to-0.10 MPa and drying temperature of 63 ℃, crushing the extract into fine powder, sieving the fine powder, adding the beta-cyclodextrin inclusion compound obtained in the step B, uniformly mixing the fine powder to obtain the Jingfang extract fine powder, adding the starch and the superfine silica gel powder (weight ratio is 4: 1) in a formula amount, uniformly mixing, granulating, drying, grading, filling, polishing in a polishing machine, and removing damaged capsules to obtain the Jingfang extract.

EXAMPLE 8 preparation of Jingfang tablets

C. b, preparing a 22% ethanol solution of the water solution of the distilled rhizoma ligustici wallichii and the fructus aurantii obtained in the step A as a solvent, and percolating the residues of the rhizoma ligustici wallichii and the fructus aurantii obtained in the step A and the poria cocos to obtain a percolate for later use;

E. c, combining the percolated liquid obtained in the step C and the extract obtained in the step D, uniformly mixing, standing, filtering, and concentrating the filtrate to an extract with the relative density of 1.22 at 50-60 ℃;

F. and E, belt-type vacuum drying the extract obtained in the step E under the conditions of vacuum degree of-0.08 MPa to-0.10 MPa and drying temperature of 63 ℃, crushing the extract into fine powder, sieving the fine powder, adding the beta-cyclodextrin inclusion compound obtained in the step B, uniformly mixing the fine powder to obtain the Jingfang extract fine powder, adding the starch, the dextrin and the sucrose (the weight ratio is 3: 2: 1) in a formula amount, uniformly mixing the mixture to prepare coarse granules, drying, crushing, sieving, granulating, drying at low temperature, finishing granules, adding 0.3% of magnesium stearate, uniformly mixing the granules and pressing the granules into 10000 tablets to obtain the Jingfang extract.

Example 9 preparation of Jingfang granules

C. b, preparing the water solution of the distilled hemlock parsley and the bitter orange obtained in the step A into 20 percent ethanol solution as a solvent, and percolating the dregs of the hemlock parsley and the bitter orange obtained in the step A and the tuckahoe to obtain percolate for later use;

F. and E, belt-type vacuum drying the extract obtained in the step E under the conditions of vacuum degree of-0.08 MPa to-0.10 MPa and drying temperature of 63 ℃, crushing the extract into fine powder, sieving the fine powder, adding the beta-cyclodextrin inclusion compound obtained in the step B, uniformly mixing the fine powder to obtain the Jingfang extract fine powder, adding the sucrose powder and the dextrin (weight ratio of 5: 2) in the formula amount, uniformly mixing the mixture to prepare granules, drying the granules, and preparing 10kg of granules to obtain the Jingfang extract.

Claims

1. Application of JING FANG ZHI JI in preparing medicine for treating new coronary sequelae is provided.

2. The use according to claim 1, wherein the Jingfang preparation is an oral pharmaceutical preparation, preferably the oral pharmaceutical preparation is one of granules, tablets, capsules and microcapsules.

3. The use of claim 2, wherein the oral pharmaceutical formulation is prepared from the following Chinese medicinal components:

preferably, the oral pharmaceutical preparation is prepared from the following traditional Chinese medicine components:

4. the use according to claim 2 or 3, wherein the process for the preparation of the oral pharmaceutical formulation comprises the following steps:

5. The use according to claim 4, wherein the preparation of the granules comprises the following steps:

6. Use according to claim 4, characterized in that the microcapsules are prepared by a process comprising the following steps:

7. The method of claim 6, wherein the belt vacuum drying conditions in step F are vacuum degree of-0.08 MPa-0.10 MPa, and drying temperature is 56 ℃.

8. The method of claim 6, wherein step G the capsule material is sodium complexing: maltodextrin 3:2, anti-tack agent octadecanol: titanium dioxide 3: 1, the plasticizer is polyethylene glycol: citric acid ═ 3: 1.

9. the method according to claim 6, wherein the emulsifier of step C is a sucrose fatty acid ester: the soybean lecithin is 8: 5 of the composite emulsifier, and the dosage of the composite emulsifier is 1.18 percent of the total amount of the preparation formula according to the mass fraction.