CN108542940B

CN108542940B - Application of traditional Chinese medicine composition in preparation of medicine for preventing and/or treating hand-foot-and-mouth disease

Info

Publication number: CN108542940B
Application number: CN201810796232.0A
Authority: CN
Inventors: 于春江
Original assignee: Jilin Yizheng Pharmaceutical Group Co ltd
Current assignee: Yu Mingxi
Priority date: 2018-07-19
Filing date: 2018-07-19
Publication date: 2021-06-15
Anticipated expiration: 2038-07-19
Also published as: CN108542940A

Abstract

The invention discloses application of a traditional Chinese medicine composition in preparation of a medicine for preventing and/or treating hand-foot-and-mouth diseases. The traditional Chinese medicine composition is prepared from the following raw materials in parts by mass: 4-36 parts of radix bupleuri, 2-18 parts of radix scutellariae, 4-36 parts of radix isatidis, 2-18 parts of radix puerariae, 2-20 parts of honeysuckle, 1-9 parts of cornu bubali, 2-22 parts of fructus forsythiae and 1-9 parts of rheum officinale. The preparation method comprises the following steps: distilling bupleuri radix, flos Lonicerae and fructus forsythiae to extract volatile oil, and collecting the distilled water solution; decocting cornu Bubali with water, mixing with the residue of bupleuri radix and the rest four materials, collecting decoction, filtering, mixing the filtrate with the above aqueous solution, concentrating, cooling, adding ethanol to ethanol content of 50-80%, stirring, standing, collecting supernatant, concentrating to appropriate amount, and mixing with the volatile oil.

Description

Application of traditional Chinese medicine composition in preparation of medicine for preventing and/or treating hand-foot-and-mouth disease

Technical Field

The invention relates to application of a traditional Chinese medicine composition in preparation of a medicine for preventing and/or treating hand-foot-and-mouth diseases.

Background

Hand-foot-and-mouth disease is a common infectious disease caused by enterovirus infection. The disease mostly occurs in children under 5 years old, herpes or ulcer is caused at the extremities and oral cavity of children patients in the early stage of disease, and children patients are cured within 1-2 weeks. Meningitis, aseptic encephalitis, brainstem encephalitis, encephalomyelitis, pulmonary edema, circulatory disturbance and the like can appear in a few children patients after about 1-5 days of disease occurrence, the disease condition of few cases is critical, death can be caused, and sequelae can be left in survival cases. The disease has the main clinical symptoms of herpes of hand, foot and oral mucosa or ulcer after ulceration.

The hand-foot-and-mouth disease is an infectious disease caused by viruses of small RNA virus family and enterovirus genus and has great threat to the health of infants and public health safety. Since 2000 years, the large-scale outbreak of the hand-foot-and-mouth disease of children appears many times in China and Asian regions, and the large threat to the life health of tens of thousands of children is caused. Hand-foot-and-mouth disease can be caused by enteroviruses such as coxsackievirus a4, a5, a9, a10, B2, B5 and enterovirus 71, but is mainly caused by enterovirus 71 (EV71) and coxsackievirus 16 (cox. a 16). At present, the strategy of symptomatic treatment is mainly adopted clinically, and the propaglobin and antiviral drugs, such as acyclovir, ganciclovir, interferon and the like are used for preventing the disinfection by a pasteurization solution; there is no specific drug that targets viruses directly. However, most of the medicines are characterized by treatment, and the death rate of the children with serious diseases is still high after the medicines are used. With the continuous and deep understanding of traditional Chinese medicine, the treatment advantages of traditional Chinese medicine on the disease are continuously and obviously shown, and the disease is mainly considered to belong to the categories of 'damp poison', 'warm disease' and 'epidemic disease' by traditional Chinese medicine, so that the development of a traditional Chinese medicine preparation for treating the hand-foot-and-mouth disease has important practical significance according to the traditional Chinese medicine rules of 'damp poison', 'warm disease' and 'epidemic disease'.

Disclosure of Invention

The invention aims to provide a new medicinal application of a traditional Chinese medicine composition.

The new application of the pharmaceutical composition is the application of the pharmaceutical composition in preparing medicines for preventing and/or treating hand-foot-and-mouth diseases.

The hand-foot-and-mouth disease is caused by at least one of the following viruses: enterovirus type 71, coxsackievirus type A16, coxsackievirus type A4, coxsackievirus type A5, coxsackievirus type A9, coxsackievirus type A10, coxsackievirus type B2 and coxsackievirus type B5.

The traditional Chinese medicine composition is prepared from the following raw materials in parts by mass: 4-36 parts of radix bupleuri, 2-18 parts of radix scutellariae, 4-36 parts of radix isatidis, 2-18 parts of radix puerariae, 2-20 parts of honeysuckle, 1-9 parts of cornu bubali, 2-22 parts of fructus forsythiae and 1-9 parts of rheum officinale.

The traditional Chinese medicine composition can be further prepared from the following raw materials: 10-30 parts of radix bupleuri, 5-15 parts of radix scutellariae, 10-30 parts of radix isatidis, 5-15 parts of radix puerariae, 5-17 parts of honeysuckle, 2-8 parts of buffalo horn, 6-18 parts of fructus forsythiae and 2-8 parts of rheum officinale.

The traditional Chinese medicine composition is further prepared from the following raw materials: 16-24 parts of radix bupleuri, 8-12 parts of radix scutellariae, 16-24 parts of radix isatidis, 8-12 parts of radix puerariae, 9-14 parts of honeysuckle, 4-6 parts of cornu bubali, 9-14 parts of fructus forsythiae and 4-6 parts of rheum officinale.

The most preferred raw materials consist of: 20 parts of radix bupleuri, 10 parts of radix scutellariae, 20 parts of radix isatidis, 10 parts of radix puerariae, 11 parts of honeysuckle, 5 parts of cornu bubali, 12 parts of fructus forsythiae and 5 parts of rheum officinale.

The raw material medicines for preparing the pharmaceutical composition are prepared according to the mass parts, and can be increased or decreased according to the corresponding proportion during production, for example, the mass production can be in kilogram or ton units, the small-scale production can also be in gram units, the weight can be increased or decreased, but the mass proportion of the raw material medicines among the components is unchanged.

The traditional Chinese medicine composition is prepared by the following steps:

1) mixing bupleuri radix, flos Lonicerae and fructus forsythiae, extracting volatile oil by distillation, and collecting volatile oil, distilled water solution and residue;

2) adding water into buffalo horn, decocting for 2-4 hours, decocting the buffalo horn dregs and decoction with the dregs and the rest four medicines of scutellaria baicalensis, isatis roots, kudzuvine roots and rheum officinale in the step 1), and collecting decoction;

3) mixing the distilled water solution obtained in the step 1) and the decoction obtained in the step 2), concentrating into clear paste, cooling, adding ethanol into the clear paste to ensure that the volume content of the ethanol reaches 50-80%, stirring uniformly, standing, taking supernatant, recovering ethanol, concentrating to a proper amount, and mixing with the volatile oil obtained in the step 1) to obtain the traditional Chinese medicine composition.

The Chinese medicinal liquid can be administered directly or mixed with appropriate amount of correctant and/or purified water to make into mixture or oral liquid.

Wherein, in the distillation method (steam distillation method) in the step 1), the added water amount is usually 5-10 times of the total mass of the bupleurum, the honeysuckle and the forsythia, the mixture is soaked for 20-60 minutes after the water is added, and then the distillation is carried out again.

In the separate decoction in the step 2), the added water amount is 8-12 times of the weight of the buffalo horn (the common condition is that 10 times of water is added and the decoction is carried out for 3 hours). The common decoction can be carried out for 1-3 times, the time for each decoction is 1-3 hours, and the water amount added for each decoction is 5-8 times of the total mass of the eight medicinal materials.

The clear paste in the step 3) is clear paste with the relative density of 1.20-1.25 measured at 85 ℃. The standing time may be 24 to 72 hours.

To further obtain the drug in a solid state, the method further comprises: concentrating the traditional Chinese medicine composition into thick paste, drying, crushing, and sieving by a sieve of 80-120 meshes to obtain traditional Chinese medicine composition powder.

The medicinal materials used in the invention are all cleaned medicinal materials.

The medicine for preventing and/or treating the hand-foot-and-mouth disease, which is prepared by taking the traditional Chinese medicine composition as an active ingredient, also belongs to the protection scope of the invention. The medicinal liquid and powder prepared by the above process can be made into any pharmaceutically acceptable dosage forms, such as mixture, oral liquid, syrup, granule, capsule, tablet, powder, pill, dripping pill, etc., directly or by adding adjuvants. The medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field.

Taking the preparation mixture as an example, the Chinese medicinal composition liquid medicine prepared by the method is added with a proper amount of flavoring agent, the pH value is adjusted to 4.5-7.0, and water is added to dilute the mixture to 1000ml (the content is 0.233-2.092g crude drug/ml), and the mixture is uniformly mixed, stood, filtered, filled and sterilized to obtain the Chinese medicinal composition liquid medicine. The application and dosage are as follows: is administered orally. 2.5-5 ml for one year within one year old, 5-10 ml for one year from one year old to three years old, 10-15 ml for one year from three years old to seven years old, 15-20 ml for one year from seven years old to twelve years old, and 3-4 times a day.

The traditional Chinese medicine composition provided by the invention has small toxic and side effects, and can effectively inhibit cytopathic effect caused by hand-foot-and-mouth disease viruses, inhibit virus replication, reduce virus load, reduce or eliminate death of infected mice, prolong survival rate and the like. The traditional Chinese medicine composition makes up the blank and the defect of the existing medicine in the aspect of treating or preventing the hand-foot-and-mouth disease, and has wide application prospect.

Detailed Description

The method of the present invention is illustrated by the following specific examples, but the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included within the scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The bupleurum, the scutellaria, the isatis root, the kudzuvine root, the honeysuckle, the buffalo horn, the forsythia and the rhubarb used in the following embodiments all accord with the relevant regulations under the terms of each medicinal material in the part of Chinese pharmacopoeia (2015 edition). Before feeding, the material objects are identified to be consistent with the names of the medicinal materials, and the quality is in accordance with the standard.

First, preparation examples

Example 1 preparation of a mixture (oral liquid) of Chinese medicinal composition

Adding 3225ml of water into 250g of radix bupleuri, 137.5g of honeysuckle and 150g of fructus forsythiae, soaking for 30 minutes, heating and distilling for 2 hours to extract volatile oil, and collecting the distilled water solution in another container. Decocting 62.5g of cornu Bubali with 625ml of water for 3 hours, decocting the mixture with the residues of the three medicines such as radix bupleuri and the rest of scutellaria baicalensis (125g), isatis root (250g), kudzuvine root (125g) and rhubarb (62.5g) with 6975ml of water twice, 1 hour each time, mixing the decoctions, filtering, mixing the filtrate with the aqueous solution, concentrating to a relative density of 1.20-1.25 (85 ℃), cooling, adding ethanol to make the volume content of the ethanol reach 65%, stirring, standing for 48 hours, taking the supernatant, recovering the ethanol, concentrating to a proper amount, mixing with the volatile oil, adding a proper amount of sucrose, steviosin and potassium sorbate, adjusting the pH value to 7.0, adding water to 1000ml, mixing uniformly, standing, filtering, filling and sterilizing to obtain the traditional Chinese medicine.

The characteristics are as follows: the product is a reddish brown clear liquid; fragrant, sweet and slightly bitter.

Example 2 preparation of a mixture (oral liquid) of the Chinese medicinal composition

Adding 2925ml of water into 200g of radix bupleuri, 112.5g of honeysuckle and 175g of fructus forsythiae, soaking for 20 minutes, heating and distilling for 2 hours to extract volatile oil, and collecting the distilled water solution in another container. Adding 600ml of water into 50g of water buffalo horn, decocting for 3 hours, then adding 8700ml of water into the residues of the three medicines such as the radix bupleuri and the like and the rest of scutellaria baicalensis (150g), isatis root (200g), kudzuvine root (150g) and rhubarb (50g), decocting for two times, 3 hours each time, combining the decoctions, filtering, combining the filtrate with the aqueous solution, concentrating until the relative density is 1.20-1.25 (85 ℃), cooling, adding ethanol until the volume content of the ethanol reaches 50%, stirring uniformly, standing for 72 hours, taking the supernatant, recovering the ethanol, concentrating to a proper amount, combining with the volatile oil, adding a proper amount of sucrose, steviosin and potassium sorbate, adjusting the pH value to 6.0, adding water into 1000ml, mixing uniformly, filtering, filling and sterilizing to obtain the traditional Chinese medicine.

Example 3 preparation of a mixture (oral liquid) of the Chinese medicinal composition

Adding 300g of radix bupleuri, 175g of honeysuckle and 112.5g of fructus forsythiae into 3525ml of water, soaking for 30 minutes, heating and distilling for 2 hours to extract volatile oil, and collecting the distilled water solution in another container. Adding 750ml of water into 75g of water buffalo horn, decocting for 3 hours, then adding 6187.5ml of water into the medicine residues of three ingredients such as the radix bupleuri and the rest of scutellaria baicalensis (100g), isatis root (300g), kudzuvine root (100g) and rhubarb (75g), decocting for 1 hour, filtering, mixing the filtrate with the aqueous solution, concentrating until the relative density is 1.20-1.25 (85 ℃), cooling, adding ethanol until the volume content of the ethanol reaches 80%, stirring uniformly, standing for 24 hours, taking the supernatant, recovering the ethanol, concentrating to an appropriate amount, mixing with the volatile oil, adding an appropriate amount of sucrose, steviosin and potassium sorbate, adjusting the pH value to 6.5, adding water to 1000ml, mixing uniformly, standing, filtering, filling and sterilizing to obtain the traditional Chinese medicine. The characteristics are as follows: the product is a reddish brown clear liquid; fragrant, sweet and slightly bitter.

Example 4 preparation of a mixture (oral liquid) of the Chinese medicinal composition

Adding 412.5ml water into 125g radix bupleuri, 212.5g flos Lonicerae and 75g fructus forsythiae, soaking for 60 min, heating and distilling for 2 hr to obtain volatile oil, and collecting distilled water solution in another container. Decocting 100g of cornu Bubali with 1000ml of water for 3 hours, decocting the mixture with dregs of the three medicines such as radix bupleuri and the rest of scutellaria baicalensis (62.5g), isatis root (375g), kudzuvine root (62.5g) and rhubarb (100g) with 6675ml of water twice, each time for 1 hour, mixing decoctions, filtering, mixing the filtrate with the aqueous solution, concentrating to a relative density of 1.20-1.25 (85 ℃), cooling, adding ethanol to make the volume content of the ethanol reach 65%, stirring, standing for 48 hours, taking supernatant, recovering ethanol, concentrating to a proper amount, mixing with volatile oil, adding a proper amount of sucrose, steviosin and potassium sorbate, adjusting the pH value to 5.0, adding water to 1000ml, mixing uniformly, standing, filtering, filling, and sterilizing to obtain the traditional Chinese medicine.

Example 5 preparation of a mixture (oral liquid) of the Chinese medicinal composition

Adding 375g bupleuri radix, 62.5g flos Lonicerae, 225g fructus forsythiae into 3975ml water, soaking for 30 min, heating and distilling for 2 hr to obtain volatile oil, and collecting distilled water solution in another container. Adding 250ml of water into 25g of water buffalo horn, decocting for 3 hours, then adding 7275ml of water into the dregs of the three medicines such as the bupleurum and the rest of the four medicines such as the scutellaria baicalensis (187.5g), the isatis root (125g), the kudzuvine root (187.5g) and the rhubarb (25g), decocting twice, each time for 2 hours, merging decoction, filtering, merging filtrate with the aqueous solution, concentrating until the relative density reaches 1.20-1.25 (85 ℃), cooling, adding ethanol until the volume content of the ethanol reaches 75%, stirring uniformly, standing for 48 hours, taking supernatant, recovering the ethanol, concentrating to a proper amount, merging with volatile oil, adding a proper amount of sucrose, steviosin and potassium sorbate, adjusting the pH value to 7.0, adding water to 1000ml, mixing uniformly, standing, filtering, filling and sterilizing to obtain the traditional Chinese medicine.

Example 6 preparation of syrup of Chinese medicinal composition

Adding 2175ml water into 50g bupleuri radix, 37.5g flos Lonicerae, and 275g fructus forsythiae, soaking for 30 min, heating and distilling for 2 hr to obtain volatile oil, and collecting the distilled water solution in another container. Adding 1125ml of water into 112.5g of water of cornu bubali, decocting for 3 hours, then adding 5925ml of water into the dregs of the three medicines such as the radix bupleuri and the rest of scutellaria baicalensis (225g), isatis root (50g), kudzuvine root (225g) and rhubarb (12.5g), decocting twice, 1 hour each time, combining the decoctions, filtering, combining the filtrate with the aqueous solution, concentrating to the relative density of 1.20-1.25 (85 ℃), cooling, adding ethanol until the volume content of the ethanol reaches 65%, stirring uniformly, standing for 48 hours, taking supernatant, recovering the ethanol, adding a proper amount of water into the concentrated solution, 600g of sucrose and 3g of sodium benzoate, boiling for dissolving, filtering, cooling, adding the volatile oil, stirring uniformly, adding a proper amount of sucrose, adding water for adjusting the total amount to 1000ml, and stirring uniformly to obtain the traditional Chinese medicine.

Example 7 preparation of granules of Chinese medicinal composition

Adding 450g of radix bupleuri, 250g of honeysuckle and 25g of fructus forsythiae into 3625ml of water, soaking for 30 minutes, heating and distilling for 2 hours to extract volatile oil, and collecting the distilled water solution in another container. Decocting 12.5g of cornu bubali with 125ml of water for 3 hours, then decocting the mixture with dregs of three medicines such as radix bupleuri and the rest of scutellaria baicalensis (25g), isatis root (450g), kudzuvine root (25g) and rhubarb (112.5g) with 8100ml of water for two times, 1 hour each time, merging decoction, filtering, merging filtrate with the aqueous solution, concentrating the mixture until the relative density reaches 1.20-1.25 (85 ℃), cooling, adding ethanol until the volume content of the ethanol reaches 65%, uniformly stirring, standing for 24 hours, taking supernatant, recovering the ethanol, concentrating the supernatant into clear paste with the relative density of 1.20-1.35 (80 ℃), drying, crushing, adding a proper amount of sucrose and dextrin, uniformly mixing to prepare granules, drying, spraying the volatile oil, and uniformly mixing to obtain 1000g of granules.

Example 8 preparation of a tablet of a Chinese medicinal composition

Adding 250g of radix bupleuri, 137.5g of honeysuckle and 150g of fructus forsythiae into 5375ml of water, soaking for 30 minutes, heating and distilling for 2 hours to extract volatile oil, and collecting the distilled water solution in another container. Decocting 62.5g of cornu bubali with 625ml of water for 3 hours, then decocting the mixture with dregs of the three medicines such as the radix bupleuri and the rest of scutellaria baicalensis (125g), isatis root (250g), kudzuvine root (125g) and rhubarb (62.5g) with 6975ml of water for two times, 1 hour each time, merging decoction, filtering, merging filtrate with the aqueous solution, concentrating the mixture until the relative density reaches 1.20-1.25 (85 ℃), cooling, adding ethanol until the volume content of the ethanol reaches 65%, uniformly stirring, standing for 48 hours, taking supernatant, recovering the ethanol, concentrating the mixture into thick paste with the relative density of 1.15-1.35, drying and crushing to obtain medicine powder (passing through a 100-mesh sieve), adsorbing the volatile oil by using starch, uniformly mixing the medicine powder, granulating, adding magnesium stearate, uniformly mixing, tabletting, and coating sugar or film to obtain the tablet.

Second, embodiment of the drug action

Example 9 examination of the in vitro inhibitory effect of the traditional Chinese medicine composition on EV71 and CoxA16 viruses

1. Experimental Material

1.1 test drugs and reagents:

the mixture of the Chinese medicinal composition prepared in example 1 of the present invention was filtered through a 0.22 μm microfiltration membrane and stored at 4 ℃.

The positive control drug Ribavirin granule is purchased from Sichuan Baili pharmaceutical industry, Inc., dissolved in culture medium, filtered by 0.22 μm microporous membrane, and stored at 4 deg.C.

Cell culture medium: DEME culture solution is added with 10% fetal bovine serum.

1.2 cells:

vero cells were purchased from the chinese academy of sciences type culture collection committee cell bank.

1.3 Virus:

EV71 and Cox A16 were purchased from American Type Culture Collection (ATCC)

2. Experimental methods

2.1 cytotoxicity assay

Diluting the drug mother liquor by using a culture solution in a ratio of 1: 2-1: 256, adding the diluted drug mother liquor into a Vero cell culture plate grown into a single layer, wherein each hole is 100 mu L, each diluted drug solution is provided with 4 multiple holes, and a normal cell control is set. The plates were incubated at 37 ℃ with 5% CO₂Culturing in incubator, observing cytopathic Condition (CPE) under inverted microscope every day, determining the lowest dilution (maximum nontoxic concentration) of cell without obvious lesion, and calculating 50% cytotoxic concentration (TC50) and maximum nontoxic concentration (TC0) according to Reed-Muench method.

2.2 in vitro antiviral efficacy test (CPE method)

Collecting culture plate with grown monolayer cells, pouring off culture medium, washing cell surface with cell maintenance solution for 3 times, inoculating enterovirus solutions with different dilutions, placing each strain at 37 deg.C and 5% CO for 3 times₂Adsorbing for 1h in the incubator, adding 100 mu L/hole of the wind-dispelling and detoxifying particles with corresponding dilution, setting normal cell control and virus control, and taking ribavirin as positive control. Placing at 37 ℃ and 5% CO₂Culturing in incubator, observing cytopathic condition under inverted microscope every dayTest results were recorded when the viral control group cytopathic effect reached 4.

Cytopathic effects were judged by grade 6 criteria and 50% Inhibitory Concentration (IC) was calculated according to the Reed-Muench method₅₀). Therapeutic Index (TI) ═ TC₅₀/IC₅₀。

-: the cells grow normally and no disease is generated; + -: cytopathic effects are less than 10% of the entire monolayer; 1: cytopathic effects account for less than about 25% of the entire monolayer of cells; 2: cytopathic effects comprise less than about 50% of the total monolayer of cells; 3: cytopathic effects account for less than about 75% of the total monolayer of cells; 4: cytopathic effects account for more than about 75% of the total monolayer of cells. 3. Results of the experiment

The traditional Chinese medicine composition mixture prepared in the embodiment 1 has obvious inhibition effect on Vero cytopathic effect caused by enterovirus EV71 strain and Cox A16 strain in vitro. See table 1.

TABLE 1 in vitro anti-enterovirus Effect of the drugs

Example 10 therapeutic protection Effect of the Chinese medicinal composition of the invention on the infection of BALB/c suckling mice with virus of hand foot and mouth disease

1. Experimental Material

1.1 test drugs:

the traditional Chinese medicine composition mixture prepared in the embodiment 1 of the invention is filtered by a 0.22 mu m microfiltration membrane, stored in a penicillin bottle and placed in a refrigerator at minus 40 ℃ for standby; in vivo experiments, the clinical dosage (0.64g/kg) of a 3-year-old child is reduced into high, medium and low dosages of a mouse, which are respectively 18.92, 9.46 and 4.73g/kg (equivalent to 2, 1 and 0.5 times of equivalent dosage)), and the high, medium and low dosages are administrated by intragastric administration to 20mL/kg, and are prepared into 50g/L mother liquor by high-purity water before in vitro experiments, and the mother liquor is filtered and sterilized for later use, and is diluted according to the required times.

The positive control drug Ribavirin granules are purchased from Sichuan Baili pharmaceutical industry Limited liability company, the equivalent dose of a mouse is 1.26g/kg according to the clinical dosage (0.12g/kg) of a 3-year-old child in an in vivo experiment, and the administration is carried out according to intragastric administration of 20 mL/kg; the high-purity water is used for preparing 50g/L mother liquor before in vitro test, and dilution is carried out in a multiple ratio during test.

1.2 Virus:

EV71 and Cox A16 were purchased from American Type Culture Collection (ATCC)

1.3 animals:

BALB/c suckling mice, 3-5 days old, 8 mice per litter, provided by Beijing Wittingle laboratory animal technology, Inc.

2. Experimental methods

The BALB/c suckling mice are randomly divided into a normal control group, a virus control group, a ribavirin control group, a high-dose group, a medium-dose group and a low-dose group of the traditional Chinese medicine composition according to litter, each group is provided with 4 mice, each administration group is provided with distilled water according to the dose of 20mL/kg ig for 1 time/d and 3 days continuously, and the normal control group and the virus control group are provided with distilled water; on the 4 th day of administration, except for a normal control group, other groups of ip hand-foot-and-mouth disease virus stock solutions are infected by 0.4 mL/mouse, 75% ethanol is sprayed on a suckling mouse to wrap padding after each administration or infection, the suckling mouse is placed back into a cage to be fed together with a mother mouse, the survival state of the suckling mouse is observed every day after infection, the survival state is observed for 14d in total, and the severity of infection is calculated according to the following standard scores, namely 0 point: health care; 1 minute: arch, vertical hair (observed after hair growth), emaciation, decreased activity, etc.; and 2, dividing: the hind limb strength is weakened; and 3, dividing: unilateral hind limb paralysis or paralysis; and 4, dividing: bilateral hind limb paralysis or paralysis; and 5, dividing: dying or dying.

Mortality and life extension rates were calculated for each group and statistically analyzed using SPSS software.

Mortality rate is the number of deaths/total number of animals x 100%;

life extension rate ═ 100% (test group survival days-virus control group survival days)/virus control group survival days) ×

3. Results of the experiment

In the observed 14d of the model group, the animals die or are in an endangered state, the death rate is 100 percent, and the infection degree integral and the death rate of the model group are obviously different from those of the normal group; after ribavirin therapeutic administration, animals have the symptoms of emaciation, reduced activity, weakened hind limb strength, unilateral hind limb paralysis or bilateral hind limb paralysis, 1 animal dies, and the infection degree integral and the death rate of the animals have significant differences compared with a model group; after the traditional Chinese medicine composition is used for therapeutic administration, the mortality rates of high, medium and low doses are respectively 0, 12.5 percent and 25 percent, the life prolonging rates are respectively 76.15 percent, 50.58 percent and 40.22 percent, the infection degrees of 3 dose groups of the traditional Chinese medicine composition provided by the invention are obviously reduced, and the infection degrees and the mortality rates of the high dose groups have significant differences compared with those of a model group.

TABLE 2 protective Effect of drugs on the degree of infection of Enterovirus EV71 infected BALB/c suckling mice (n ═ 8)

Note: compared with the model group, a) P is less than 0.05, and b) P is less than 0.01.

TABLE 3 protective Effect of drugs on Enterovirus EV71 infected BALB/c suckling mice (n ═ 8)

Example 11 clinical efficacy observation of the Chinese medicinal composition for treating hand-foot-and-mouth disease

1. Diagnostic criteria

The patients with hand-foot-and-mouth disease are diagnosed by using clinical technical guidelines for treating hand-foot-and-mouth disease in traditional Chinese medicine (2012 edition) as a diagnosis standard. The clinical manifestations are as follows: the oral mucosa has disperse herpes, rice grain size and obvious pain; herpes of rice grain size appears in palms, soles or buttocks, inflammatory halos appear around the herpes, and liquid in the herpes is less; some children with fever are usually about 38 ℃ and some children with nausea and vomiting are usually caused.

2. Standard of therapeutic effect

The clinical cure is as follows: the general symptoms disappear, the rash is healed, and various biochemical indexes in the laboratory are recovered to be normal;

the effect is shown: the symptoms of the whole body are improved, the rash is not completely healed, and various biochemical indexes in a laboratory are improved;

the method has the following advantages: the symptoms of the whole body are improved, the rash is not completely healed, and various biochemical indexes in a laboratory are basically unchanged;

and (4) invalidation: the symptoms of the whole body are not improved or aggravated, and various biochemical indexes in a laboratory are obviously abnormal.

3. Method of administration

(1) Sample group: the traditional Chinese medicine composition mixture prepared in the embodiment 1 of the invention has the following application and dosage: orally administered 10ml at a time, 3 times a day, for 6 days;

(2) control group: the ribavirin oral liquid is taken for 3 times a day by 5ml each time for 6 days continuously according to the specification, symptomatic treatment is strengthened, oral care is well done, and cephalosporin medicines are given to combined infected persons for antibacterial treatment.

4. Application results

a. Clinical manifestations of children in different groups after treatment are compared

The defervescence time after treatment, the resolution time of the hand-foot herpes, the healing time of the oral ulcer and the average hospitalization time of each group of children patients are observed and recorded, and the t test is carried out in parallel, and the results are shown in table 4.

TABLE 4 comparison of clinical manifestations of children patients after treatment (day, x + -s)

Note: p < 0.05, P < 0.01 compared to control.

b comparison of clinical effects of the treated infants

The clinical effect of each group of children patients after treatment was recorded, and the effective and effective cases were added to calculate the total effective case, see table 5.

TABLE 5 comparison of clinical effects of the treated infants

Group of	Example number (example)	Show effect	Is effective	Invalidation	Total effective rate
						Sample set	30	18(60％)	10(33％)	2(7％)	93％
Control group	30	12(40％)	12(40％)	6(20％)	80％

As can be seen from table 5, the total effective rate of the Chinese medicinal composition mixture prepared in example 1 of the present invention is significantly better than that of the control group.

As can be seen from the data in tables 4 and 5, the Chinese medicinal composition prepared in example 1 of the present invention has a better therapeutic effect on the hand-foot-mouth disease than ribavirin.

Claims

1. An application of a Chinese medicinal composition as sole active ingredient in preparing medicine for preventing and/or treating hand-foot-and-mouth disease;

the hand-foot-and-mouth disease is caused by at least one of the following viruses: enterovirus type 71, coxsackievirus type a 16;

the traditional Chinese medicine composition is prepared from the following raw materials in parts by mass: 20 parts of radix bupleuri, 10 parts of radix scutellariae, 20 parts of radix isatidis, 10 parts of radix puerariae, 11 parts of honeysuckle, 5 parts of cornu bubali, 12 parts of fructus forsythiae and 5 parts of rheum officinale;

1) mixing the radix bupleuri, the honeysuckle and the fructus forsythiae, extracting volatile oil by a distillation method, and collecting the volatile oil, the distilled water solution and dregs of a decoction;

2) adding water into buffalo horn, decocting for 2-4 hours, decocting the buffalo horn dregs and decoction with the dregs obtained in the step 1) and the rest four medicines of scutellaria baicalensis, isatis roots, kudzuvine roots and rheum officinale, and collecting decoction;

3) mixing the distilled water solution obtained in the step 1) and the decoction obtained in the step 2), concentrating into clear paste, cooling, adding ethanol into the clear paste to ensure that the volume content of the ethanol reaches 50-80%, stirring, standing, taking supernatant, recovering ethanol, concentrating to a proper amount, and mixing with the volatile oil obtained in the step 1) to obtain the traditional Chinese medicine composition;

in the distillation method in the step 1), the added water amount is 5-10 times of the total mass of the bupleurum, the honeysuckle and the forsythia;

in the step 2), during the separate decoction, the added water amount is 8-12 times of the weight of the buffalo horn;

the common decoction is carried out for 1 to 3 times, the time of each decoction is 1 to 3 hours, and the water amount added in each decoction is 5 to 8 times of the total mass of the eight medicinal materials;

the clear paste in the step 3) is clear paste with the relative density of 1.20-1.25 measured at 85 ℃; the standing time is 24-72 hours.

2. Use according to claim 1, characterized in that: the method further comprises the steps of: concentrating the medicinal composition into thick paste, drying, pulverizing, and sieving with 80-120 mesh sieve to obtain Chinese medicinal composition powder.

3. Use according to claim 1, characterized in that: the dosage form of the traditional Chinese medicine composition is an oral dosage form.

4. Use according to claim 3, characterized in that: the oral dosage form is any one of the following: mixture, oral liquid, syrup, granule, capsule, tablet, powder, pill and dripping pill.