CN116637158A - Anti-influenza virus traditional Chinese medicine composition and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and in particular relates to a traditional Chinese medicine composition for resisting influenza virus, which is characterized in that: the proportion of each component is as follows: 15 parts of dyers woad leaf, 9 parts of mulberry leaf, 12 parts of reed rhizome, 9 parts of baical skullcap root, 30 parts of gypsum, 6 parts of chrysanthemum, 6 parts of bitter apricot seed, 9 parts of Ningpo Yam rhizome and 4 parts of liquoric root. The Qing Sang Fang disclosed by the invention has the effects of clearing heat and detoxicating, relieving exterior syndrome and ventilating the lung, and has the effects of strengthening body resistance (improving immunity). The qing Sang Fang provided by the invention not only has a better curative effect in clinic, but also takes up a great advantage in price, and obtains higher acceptance of parents of children patients. According to the invention, the influence of the green Sang Fang on the hemagglutination titer of the influenza virus in the chick embryo and the treatment effect of infected mice are researched by two experimental examples, so that a theoretical basis is provided for resisting the influenza virus in the traditional Chinese medicine; experiments show that the green Sang Fang has an inhibiting effect on the hemagglutination titer of the influenza virus, can improve the symptoms, the weight, the lung index, the pathological manifestations and the scores of mice, and proves the reliability of the formula.

Description

Anti-influenza virus traditional Chinese medicine composition and preparation method thereof

Technical Field

The invention belongs to the technical field of anti-influenza traditional Chinese medicines, and in particular relates to an anti-influenza traditional Chinese medicine composition and a preparation method thereof.

Background

Influenza (influenza for short) is a respiratory disease caused by influenza virus. Symptoms such as hyperpyrexia, muscular soreness, weakness and the like are main clinical manifestations, and some children may have atypical gastrointestinal symptoms such as vomiting, diarrhea and the like.

The traditional Chinese medicine corresponding to influenza is named as "time-consuming cold", exogenous pestilence toxin epidemic pathogenic factors and deficiency of vital qi are two main causes of disease, the disease location is usually in lung defensive qi, but spleen and stomach are easily involved, the heart is invading by serious people, and the total pathogenesis is epidemic toxin exogenous pathogenic factors and attack lung defensive qi.

Oseltamivir is the western medicine with the best treatment and prevention effects in clinic at present, and is suitable for influenza A (A) and influenza B (B). However, it is required to take the medicine within 48 hours of symptoms of infection to obtain the best curative effect; from 2008, reports of drug resistance of oseltamivir are gradually increased, and the highest drug resistance rate of some countries can reach 67.3%.

The children influenza virus pneumonia still has higher fatality rate. The traditional Chinese medicine has unique advantages in preventing and treating infectious diseases such as influenza and the like, and accumulates rich theoretical and clinical experience for thousands of years.

In recent years, the research of traditional Chinese medicine becomes a popular direction for resisting influenza virus, and more clinical experience reports are provided, and the invention patent is also published. However, there are still certain limitations in the published anti-influenza patents, including: the traditional Chinese medicine has the advantages of multiple medicine tastes, heavy bitter taste and poor compliance of children; the influenza syndrome type applicable to the medicine is not obvious: some drugs still contain components of the bib , affect safety, etc. For example, "a traditional Chinese medicine mixture for treating viral influenza and a preparation method thereof," application number (201410223497.3): 60 parts of gypsum, 150 parts of cordate houttuynia, 150 parts of platycodon grandiflorum, 60 parts of bitter apricot seed, 120 parts of chrysanthemum, 150 parts of mulberry leaf, 60 parts of schizonepeta, 90 parts of peppermint, 150 parts of reed rhizome, 150 parts of weeping forsythia, 90 parts of perilla leaf, 120 parts of rhizoma anemarrhenae, 150 parts of honeysuckle, 90 parts of baical skullcap root, 120 parts of elsholtzia, 150 parts of kudzuvine root, 90 parts of liuyisan, 150 parts of dyers woad leaf, 120 parts of lithospermum, 150 parts of isatis root and 60 parts of liquorice, wherein 22 types (including six yisan) of the total weight of the traditional Chinese medicine composition are obtained, and only clinical case statistics data are lacked; meanwhile, children are not mainly used as treatment objects.

Through animal experiments, the anti-influenza curative effect of the traditional Chinese medicine can be better verified, related action mechanisms are discussed, and more reliable support is provided for clinical popularization and use.

The domestic influenza is summarized and summarized into 7 types of syndromes by combining with the latest diagnosis and treatment scheme and expert consensus guidance of the country, and the corresponding treatment methods, ancient prescription and provenance and symptoms are shown in table 1.

The green Sang Fang of the invention is added and subtracted on the basis of Maxingshi Gantn Tang Mulberry leaf-chrysanthemum drink according to clinical experience. Is mainly suitable for wind-heat invading exterior syndrome, and the syndrome: high fever, slight aversion to cold, debilitation, pharyngalgia, headache, body pain, conjunctival congestion, cough and asthma and the like, and has good effects.

Disclosure of Invention

Aiming at the technical problems, the invention provides an anti-influenza virus traditional Chinese medicine composition and a preparation method thereof. Influenza is usually caused by epidemic toxin invading the body and struggling with the healthy qi and pathogenic factors, which causes fever, weakness, headache and body pain, etc., and toxic heat is paced in the throat to cause reddish swelling and pain. The lung is prone to feel exogenous pathogenic factors and has weakened dispersing function, and the descending and incorporating forces are not good enough, so that the lung qi is reversed and the cough and asthma are caused. Therefore, qing Sang Fang takes clearing heat and detoxicating, relieving exterior syndrome and ventilating lung as a treatment method, and has the effects of strengthening body resistance (improving immunity), and is suitable for wind-heat invading exterior syndrome caused by influenza virus infection. The green Sang Fang of the invention has the characteristics of few medicinal herbs, bitter Xin Weidan, easy taking by children, good clinical antipyretic and cough relieving effects and the like. Experimental example researches show that the green Sang Fang has antipyretic effect on experimental rats, influence on the hemagglutination titer of influenza viruses in chick embryos and treatment effect on infected mice, and provides theoretical basis for anti-influenza viruses of traditional Chinese medicines; experiments show that the green Sang Fang has an inhibiting effect on the hemagglutination titer of influenza viruses, and can improve the symptoms, the weight, the lung index, the pathological manifestations and the scores of mice, thereby proving the reliability of the influenza virus resistance of the formula. The infant who is clinically determined to be diagnosed with influenza virus infection has good treatment effect by using the green Sang Fang.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

a Chinese medicinal composition for resisting influenza virus comprises folium Isatidis, folium Mori, rhizoma Phragmitis, scutellariae radix, gypsum Fibrosum, flos Chrysanthemi, semen Armeniacae amarum, ningpo Yam rhizome and Glycyrrhrizae radix.

Preferably, the proportions of the components are: 15 parts of dyers woad leaf, 9 parts of mulberry leaf, 12 parts of reed rhizome, 9 parts of baical skullcap root, 30 parts of gypsum, 6 parts of chrysanthemum, 6 parts of bitter apricot seed, 9 parts of Ningpo Yam rhizome and 4 parts of liquoric root.

A preparation method of an anti-influenza virus traditional Chinese medicine composition comprises the following steps:

step 1: decocting 3 agents, namely 300g of gypsum with 300ml of pure water with strong fire for 15-30min;

step 2: 150 parts of dyers woad leaf, 90 parts of mulberry leaf, 120 parts of reed rhizome, 90 parts of baical skullcap root, 60 parts of chrysanthemum, 60 parts of bitter apricot seed, 90 parts of Ningpo Yam rhizome and 40 parts of liquorice are added into the medicine in the step 1, and 1500ml of pure water is added for soaking for 1 hour;

step 3: heating and decocting all the medicines in the step 2 with slow fire to 300mL, filtering the decoction with 4 layers of sterile gauze, collecting the filtered decoction, adding 1500mL of pure water into the residual filter residue, heating and decocting with slow fire to 300mL again, repeating the filtering and decocting operation for 5 times, finally combining the 5 times of filtrates, and concentrating the medicines to 6g/mL;

step 4: sterilizing the medicine prepared in the step 3 at high temperature and high pressure for 30min, and diluting with pure water sterilized by filtration to form 6 concentrations of 6g/ml, 3g/ml, 1.5g/ml, 0.75g/ml, 0.375g/ml and 0.1875 g/ml.

The beneficial effects are that:

the Qing Sang Fang disclosed by the invention has the effects of clearing heat and detoxicating, relieving exterior syndrome and ventilating the lung, and has the effects of strengthening body resistance (improving immunity). The qing Sang Fang provided by the invention not only has a better curative effect in clinic, but also takes up a great advantage in price, and obtains higher acceptance of parents of children patients.

According to the invention, the influence of the green Sang Fang on the hemagglutination titer of the influenza virus in the chick embryo and the treatment effect of infected mice are researched by two experimental examples, so that a theoretical basis is provided for resisting the influenza virus in the traditional Chinese medicine; experiments show that the green Sang Fang has an inhibiting effect on the hemagglutination titer of the influenza virus, can improve the symptoms, the weight, the lung index, the pathological manifestations and the scores of mice, and proves the reliability of the formula.

Drawings

The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention.

In the drawings:

FIG. 1 is a 24h histogram of the mean viral titer of each group of example 1;

FIG. 2 is a histogram of the mean value of the viral titers of each group for 48h in example 1;

FIG. 3 is a graph of the redistribution of the various groups: a: a base weight; b: successful molding; c: day 1; d: day 2; e: day 3; f: day 4; g: day 5; h: day 6; day 7;

Fig. 4 shows lung tissue pathology in mice of each group: a: blank group; b, model group; c: a high concentration group; d: medium concentration group; e: a low concentration group; f: an OS group;

fig. 5 is a group pathology score histogram.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Modern pharmacological research of Qing Sang Fangzu

Folium Isatidis (a leaf of Isatis tinctoria) is known to have effects of clearing heat, cooling blood and removing toxic substances in traditional medicine, and researchers find that it can effectively inhibit in vivo influenza virus proliferation, clear heat, detumescence and enhance human immune system function ^] . The mulberry leaf contains a large amount of beta-sitosterol, and the active ingredient can prevent mice from infecting deadly influenza A and improve immune disorder, and can inhibit inflammatory reaction in cells infected by influenza A virus through signals such as NF-kappa B, MAPK and the like.

The rhizoma Phragmitis extract not only has inhibiting effect on Staphylococcus aureus, but also can inhibit herpesvirus replication without affecting cell physiological activity. Reed rhizome also reduces pain response from inflammation, which may be associated with anti-inflammatory and antioxidant activity. Bai Huang belongs to a heat-clearing herb, which is dedicated to purging fire of upper energizer.

Baicalin and baicalein in baikal skullcap root belong to flavonoids, and the anti-influenza virus mechanism is related to inhibiting the activity of host epithelial cell sialidase, fusion of influenza virus and cell membrane and budding process of influenza virus, and apoptosis enzyme, inflammatory factor and oxidoreductase are interfered by flavonoid substances in baikal skullcap root to reduce influenza A lung injury. The influenza virus usually causes high fever of organism, and researches show that the gypsum has compact structure, small pore diameter, anti-inflammatory effect, and can reduce the body temperature of the fever rats, and the fever mechanism can inhibit NF-kB signal path and reduce the PGE as a fever medium ₂ Related to the following.

Chlorogenic acid in flos Chrysanthemi can promote production of interferon and tumor necrosis factor in human lymphocyte and peripheral leukocyte, and inhibit proliferation of influenza virus.

Bitter almonds can significantly reduce the immune injury of the lung and intestinal tracts of influenza mice, and the action mechanism is possibly related to inhibiting the release of TNF-alpha and IL-6 in the serum of the influenza mice and down regulating the expression level of TLR7 protein.

Ningpo Yam rhizome contains a naturally occurring steroid saponin which not only can block virus infection in the initial stage, but also can reduce the sensitivity of host cells to virus infection and inhibit airway inflammation indexes (such as IL-6 and IL-1 beta) of asthmatic mice. Triterpenes present in licorice can prevent fusion of influenza virus with host epithelial cell membrane in early stage, so that virus replication activity after infection of cells is inhibited.

Based on the pharmacological mechanism and clinical treatment experience, an anti-influenza virus traditional Chinese medicine composition is developed and comprises dyers woad leaf, mulberry leaf, reed rhizome, baical skullcap root, gypsum, chrysanthemum, bitter apricot seed, ningpo Yam rhizome and liquoric root.

The proportion of each component is as follows: 15 parts of dyers woad leaf, 9 parts of mulberry leaf, 12 parts of reed rhizome, 9 parts of baical skullcap root, 30 parts of gypsum, 6 parts of chrysanthemum, 6 parts of bitter apricot seed, 9 parts of Ningpo Yam rhizome and 4 parts of liquoric root.

A preparation method of an anti-influenza virus traditional Chinese medicine composition comprises the following steps:

step 1: decocting 3 agents, namely 300g of gypsum with 300ml of pure water with strong fire for 15-30min;

step 2: 150 parts of dyers woad leaf, 90 parts of mulberry leaf, 120 parts of reed rhizome, 90 parts of baical skullcap root, 60 parts of chrysanthemum, 60 parts of bitter apricot seed, 90 parts of Ningpo Yam rhizome and 40 parts of liquorice are added into the medicine in the step 1, and 1500ml of pure water is added for soaking for 1 hour;

step 3: heating and decocting all the medicines in the step 2 with slow fire to 300mL, filtering the decoction with 4 layers of sterile gauze, collecting the filtered decoction, adding 1500mL of pure water into the residual filter residue, heating and decocting with slow fire to 300mL again, repeating the filtering and decocting operation for 5 times, finally combining the 5 times of filtrates, and concentrating the medicines to 6g/mL;

step 4: sterilizing the medicine prepared in the step 3 at high temperature and high pressure for 30min, and diluting with pure water sterilized by filtration to form 6 concentrations of 6g/ml, 3g/ml, 1.5g/ml, 0.75g/ml, 0.375g/ml and 0.1875 g/ml.

Furthermore, the traditional Chinese medicine composition can be prepared into water aqua, oral liquid, granules and the like.

Example 1: experiment of anti-H1N 1 influenza A virus in green Sang Fang chicken embryo

1 materials and instruments

1.1 viral strains

The murine lung adapted strain is the strain A H1N1 virus NYMCX-179A (titre of viral fluid hemagglutination 1:640) and is supplied by the laboratory animal center of Jiangsu Dike Biotechnology Co., ltd. All influenza virus tests were performed in the biosafety laboratory class II (ABSL-2).

1.2 therapeutic agents

(1) Green Sang Fang

The prescription is composed of 9 traditional Chinese medicines (15 g of dyers woad leaf, 9g of mulberry leaf, 12g of reed rhizome, 9g of baical skullcap root, 30g of gypsum (decocted first), 6g of chrysanthemum, 6g of bitter apricot seed, 9g of Ningpo Yam rhizome and 4g of liquoric root), 100g in total, and is purchased in a second affiliated hospital of Shanxi traditional Chinese medicine university, and is identified as a genuine product by the university of medicine university, and the medicine dosage accords with 2020 edition of Chinese pharmacopoeia.

(2) Control drug

Oseltamivir capsules (manufacturer and trade name are Changjiang pharmaceutical industry Co., ltd., and kewei, product lot number: H20065415, respectively).

1.3 Experimental chick embryo

SPF grade fertilized chick embryo without pathological change, 9-11 days old, provided by Hangzhou Xiaoshan Wei farm.

1.4 chicken erythrocyte suspension

Fresh cock blood is taken from healthy cock bred in laboratory animal center of Jiangsu Dike Biotechnology Co., ltd, after being anticoagulated by adding Aldrich solution, the mixture is centrifuged for 3 times by using 0.9% NaCl solution, the time is controlled to be about 10 minutes, the upper serum and the leucocyte layer of the centrifuge tube are fully sucked to leave chicken erythrocytes, 49.5ml of 0.9% sterile normal saline solution is sucked by using a micropipette, 0.5ml of chicken erythrocytes are added to prepare 1% sterile erythrocyte suspension according to a proportion, and the preservation condition is 4 ℃.

1.5 instruments

The experimental field and equipment are all provided by Jiangsu Dike Biotechnology Co., ltd.

Name of the name	Company (Corp)	Model number
			Biological safety cabinet	Jiangsu purifying plant	BBS-SDC
Refrigerator with a heat exchanger	Qingdao sea	HYC-1378
			Centrifugal machine	Hunan instruments laboratory instruments Co.Ltd	TDZ5-WS
Pressure steam sterilizing pot	LICHEN	DGS-280B
			Water bath kettle	Rui-stationary instrument and equipment factory	DK-S11
Water purifier	Shanghai Lei Ci	UPW-H2-15
			Chick embryo incubator	Plain hatching equipment factory	ZX-88
96-well plate	Biyundian biotechnology Co.Ltd	FPT0166
			Liquid transfer device	EPPENDORF	Q37533

2. Method of

2.1 pharmaceutical preparation

(1) Decocting green Sang Fang

300g (3 doses) of green Sang Fangzhong gypsum is firstly decocted with 300mL of pure water with strong fire for 15-30min, then 1500mL of pure water is added and the rest of the medicines are soaked for about 1 hour, all the medicines are heated and decocted again with slow fire to 300mL, the medicine juice is collected after being filtered by 4 layers of sterile gauze, 1500mL of pure water is added into the residual filter residue and heated and decocted again with slow fire to 300mL, the filtering and the decoction operation are repeated for 5 times, and finally the 5 times of filtrate are combined and the medicines are concentrated to 6g/mL. In order to avoid bacteria in the medicine polluting chick embryo, the medicine with the concentration is sterilized at high temperature and high pressure for 30min, and then diluted by pure water which is filtered and sterilized to form 6 concentrations of 6g/ml, 3g/ml, 1.5g/ml, 0.75g/ml, 0.375g/ml, 0.1875g/ml and the like, and each diluted concentration is used for the experiment of the lowest lethal concentration of the medicine on chick embryo. The remaining 3g/ml concentration of the medicine is used as an antiviral medicine in chicken embryo bodies for standby.

(2) Oseltamivir

300mg OS is added into 10ml pure water to prepare 30mg/ml sterile suspension, and the sterile suspension is diluted into 15mg/ml, 7.5mg/ml, 3.75mg/m, l.88mg/ml and 0.94mg/ml, and the 6 concentrations are used as the lowest lethal concentration experiment of chick embryo for standby. 225mgOS is added into 100ml of pure water to prepare 2.25mg/ml of medicine solution which is used as an antiviral medicine in chicken embryo body for standby.

2.2 influenza Virus chick embryo amplification

Taking 9-11 days old chick embryo, using a marker pen to mark the boundary between the air chamber and the embryo after the chick embryo is photographed in a darkroom, sterilizing eggshells at the air chamber part by alcohol, and opening a small opening with the length of about 2mm at the position about 1mm on the line and avoiding a conical eggbeater for blood vessels, thus obtaining the injection point. The virus stock (titer 1:640) was removed from-80 ℃, the centrifuge tube was rinsed with natural running water to thaw the virus, and 0.2ml of virus solution was injected per chick embryo, 5 total chick embryos, and the syringe was required to penetrate the allantoic membrane and avoid puncturing chick embryo blood vessels. And (3) after melting paraffin in an alcohol burner flame, plugging small holes in eggshells of the chick embryos, adjusting the temperature of an incubator to 33-35 ℃, incubating the chick embryos for 48 hours, harvesting allantoic fluid, observing survival conditions of the chick embryos inoculated for 24 hours and 48 hours, and removing dead chick embryos caused by improper operation in 24 hours. The chick embryos are placed in a refrigerator at-20 ℃ for 1-2 hours before harvest, and care should be taken not to excessively long to avoid freezing allantoic fluid into a solid. The eggshells are disinfected again before the allantoic fluid is harvested, eggshells at the top of the air chamber are knocked out by using a pair of sterilizing scissors, the allantoic membrane is broken by using the needle point of a disposable sterile syringe, the needle point of the syringe avoids blood vessels to extract the allantoic fluid after the chick embryos are inclined, and 5-10ml of allantoic fluid is extracted from each chick embryo.

2.3 detection of the hemagglutination titre of Virus fluid

Extracting 5ul of the harvested virus allantoic fluid, adding the first hole at the left side on a 96-well plate, adding 45ul of physiological saline, and repeatedly and uniformly blowing by a pipetting gun to obtain 1:10 virus liquid, adding 25ul of physiological saline into each row of the remaining 11 holes, then sucking 25ul of influenza virus liquid 1:10 from the 1 st hole, adding the 2 nd hole, sequentially diluting the solution to the 11 th hole by 2 times, discarding 25ul of virus diluent sucked by the 11 th hole, and only keeping 25ul of physiological saline in the 12 th hole as a blank control; after 1% of fresh chicken red blood cells are blown and sucked uniformly, the chicken red blood cells are sequentially added into each plate hole, 25 ul/hole is vibrated for 15 seconds, then the chicken red blood cells are kept stand for 30 seconds, the result is observed, the virus titer is calculated according to relevant standards, and allantoic fluid with higher titer is selected and placed at 4 ℃ for standby.

2.4 experiments with lowest lethal concentration of drug

The aim of the experiment of the lowest lethal concentration of the drug is to ensure the safe and reliable concentration of the drug used for resisting influenza virus in the chick embryo. After wet sterilization of 6g/ml of green Sang Fanggao under high pressure and 2-fold serial dilution, 6 gradient drugs (6 g/ml-0.1875 g/ml) were obtained. Each gradient was inoculated with 5 chick embryos, 0.2ml each. After filtration sterilization at 30mg/ml OS, 2-fold serial dilutions (30 mg/ml-2.5 mg/ml) were made for a total of 6 gradients, inoculated virus amounts and chick embryo numbers were identical to those of green Sang Fang. Blank 5 chick embryos were not subjected to any treatment. The total number of the 13 groups is 65 chick embryos, the incubation condition of the incubator is 34+/-1 ℃, the survival condition is observed in 24h, the death caused by improper operation is eliminated, the survival condition is observed in 72h, and the lowest concentration of chick embryo death is regarded as the lowest lethal concentration.

2.5 research on anti-influenza Virus efficacy of Qing Sang Fang in chick embryo

The SPF fertilized chick embryos without lesions were selected and randomly divided into 14 groups of green Sang Fang, oseltamivir (OS) 6, virus positive, blank, 12 chick embryos each, 9-11 days old. Because there is no embryo correlation coefficient in the human and animal dose conversion, the highest concentration used in this study of embryos is consistent with the highest concentration used in subsequent mice experiments. Sterilizing green Sang Fang (3 g/ml) in biosafety cabinet according to 2 ^-1 -2 ^-6 Dilution of 6 gradients (Green Sang Fang 2 ^-1 Consistent with the high dose of mice, i.e., 1500 mg/ml), 3ml of each gradient was taken and added with an equal amount of virus stock (titer 1:640), incubated in a 37℃water bath for 1 hour, and inoculated into chick embryos at 0.2ml per each gradient.OS (2.25 mg/ml) was first pressed 2 ^-1 -2 ^-6 Dilution of 6 gradients (OS 2 ^-1 At the same mouse dose, i.e., 1.125 mg/ml), the remaining treatments were performed on the same green Sang Fang group. The positive group was taken from 3ml of PBS solution and added with an equal amount of virus stock, and the blank group was not treated at all before incubation and inoculation. Virus hemagglutination titers in each group of chicken embryo were measured at 24h and 48h post inoculation.

2.6 statistical analysis

ˉ

Statistical software is graphpadrism, data of measurement data are represented by X+ -S, single-factor analysis of variance is used, multiple comparisons are selected, average value of each column is compared with average value of each other column, and P < 0.05 is statistically significant.

3 results

3.1 virus hemagglutination titre after chick embryo expansion

Observations and recordings on 96-well plates, where positive indicates a "#, ++, +,", negative is indicated as "-". "+" indicates that a small lump with unsmooth edges is formed in the plate hole, and small agglomerations are gathered around the small lump; "++" is only formed into a ring, there is no agglutination in the ring, and there are small agglomerations around it; tidy and tidy degree of uniformity of degree better than "++", but not as much as "#", the uniform coverage area in the plate holes is larger; "#" represents binding of influenza virus hemagglutinin to the red blood cell mucin receptor, with red blood cells evenly spread across the plate wells; the red blood cells in the negative "-" plate holes form a red cluster with smooth edges. Table 1 shows that most of the hemagglutination titers of the influenza viruses tested after replication and amplification can be maintained at 1:640, extracting the allantoic fluid of the chick embryo as virus stock for later use.

TABLE 1 titers of influenza virus chick embryos after expansion

3.2 minimum lethal concentration of drug

The chick embryo death phenomenon starts to appear at the time of 6g/ml and 15mg/ml of green Sang Fang and OS respectively, the lowest lethal concentration of green Sang Fang is 6g/ml, and oseltamivir is 15mg/ml. So that the subsequent chick embryoMaximum concentration of the two drugs in the in vivo antiviral assay (Green Sang Fang 2 ^-1 And oseltamivir 2 ^-1 ) Are within a safe range.

Table 2 lowest lethal concentration of drug

3.3 antiviral results in green Sang Fang chick embryo

The positive group had higher virus titers at both 24h and 48h than the corresponding blank group (P < 0.05). In the aspect of drug treatment, compared with a virus positive group, the green Sang Fang and the OS can inhibit influenza viruses at 24 hours and 48 hours (P is less than 0.05). Green Sang Fang at 24h and 48h was 2 compared to OS group ^-1 、2 ^-2 、2 ^-3 The difference in concentration from the corresponding OS group was not statistically significant (P > 0.05), especially 2 ^-1 、2 ^-2 At concentration, both green Sang Fang and OS virus titers were 0; but green Sang Fang 2 ^-4 、2 ^-5 、2 ^-6 The virus titers at the concentrations were higher than the corresponding OS group at both 24h and 48h, with statistical differences (P < 0.05). From a time comparison, except for the foregoing cyan Sang Fang 2 ^-1 、2 ^-2 The infusion was 0 at both times, 48h green Sang Fang 2 ^-3 、2 ^-4 The mean value of the concentration virus titers is higher than the corresponding 24h, but the statistics show that the difference is not significant (P > 0.05); 48h green Sang Fang 2 ^-5 、2 ^-6 The virus titer of the concentration group is higher than the corresponding 24h, and the difference is statistically significant (P < 0.05). See table 3 and figures 1 and 2 for details.

TABLE 3 anti-influenza Effect in chick embryo with different concentrations of drug

(note: 0' represents negative reaction, positive to blank ratio: four represents P < 0.05; V represents P < 0.05; compared to positive group; compared to oseltamivir counterpart group): Representing P > 0.05, +.represents P < 0.05; comparison of groups corresponding to 24 h: wherein P is greater than 0.05, and T is less than 0.05. )

Conclusion 4

The influenza virus amplification culture method mainly comprises 3 steps: chicken embryo culture method, animal living body culture method, and animal cell culture method. The chick embryo culture method has the unique advantages of strong virus sensitivity, simple operation, sufficient source, low tissue differentiation degree and the like, and is widely applied to research and development and production of influenza vaccines. The living animals for culturing influenza virus mainly comprise mice, ferrets, chickens, pigeons, pigs, horses and the like, and the most sensitive animals in the living animals are ferrets, but the animals and the raising price are high, and the infection symptoms of the animals such as pigs are not obvious, so the price, the raising and the replicability determine that the mice are most commonly used in influenza experiments. The animal cell culture method mainly utilizes kidney cells of animals such as dogs, monkeys, cattle and the like to amplify influenza viruses, and dog kidney passage cells are tissue cells most commonly used in the cell culture method, and have the advantages of economy, convenience, less allergic reaction and the like, but also have the defects of poor sensitivity, low virus titer, inconvenient preservation and the like.

The simultaneous administration method is one of 3 main administration modes of the anti-influenza virus of the traditional Chinese medicine in the chick embryo. The simultaneous administration method comprises mixing the virus liquid and the Chinese medicinal materials with relevant concentration, incubating at 37deg.C for 1 hr, and injecting into chick embryo at a ratio of 0.2 ml/piece to observe the inhibiting effect of the medicine on influenza virus. The injection quantity of chick embryo needs to be noted, and operational death is avoided. The proliferation of influenza virus occurs 18-72h after the inoculation of the chick embryo, and the current time for detecting the hemagglutination titer of the influenza virus chick embryo is mainly 24h, 48h and 72h after infection.

Therefore, the test uses a simultaneous administration method in chick embryo culture, and the hemagglutination titer is detected 24h and 48h after infection. Experiments show that the hemagglutination titer of the positive group infected with the influenza virus is higher than that of the blank group, which indicates that the influenza virus is successfully replicated in the chick embryo model; cyan Sang Fang 2 ^-1 -2 ^-6 (1500 mg/ml-46.88 mg/ml) at 24h and 48h inhibits influenza virus, and overall virus titer is inversely related to concentration;cyan Sang Fang 2 ^-1 (1500mg/ml)、2 ^-2 (750 mg/ml) can completely inhibit influenza virus in chicken embryo to give blood coagulation titer of 0, and has effect similar to OS2 ^-1 、2 ^-2 The concentration groups are equivalent, which shows that the green Sang Fang and the OS can completely inhibit the influenza virus in chicken embryo bodies, and the minimum concentration of the completely inhibited virus is green Sang Fang 2 ^-2 (750 mg/ml) and OS2 ^-2 (562.5 ug/ml). Overall distribution, the titers of the cyan Sang Fang groups were not better than the corresponding OS group, in particular cyan Sang Fang 2 ^-4 (187.5mg/ml)2 ^-5 (93.75mg/ml)、2 ^-6 The virus titer of the (93.75 mg/ml) concentration is higher than that of the corresponding OS group at 24h and 48h, which shows that the anti-influenza effect of the OS still has great advantage, and further intensive research is needed in the aspect of anti-influenza curative effect of the traditional Chinese medicine. From time comparison, the 48h hemagglutination titer of the virus positive group is higher than that of the 24h, which indicates that the influenza virus is proliferated in chicken embryo along with the extension of time (within 48 h), so that the virus proliferation should be inhibited by timely administration after the infection of the influenza virus; low concentration of green Sang Fang (e.g. 2 ^-5 、2 ^-6 ) Titers at 48h were higher than 24h, indicating that low concentrations of drug did not completely inhibit the virus, and residual virus continued to proliferate over time suggesting that green Sang Fang anti-influenza virus would be best selected for use in higher dose groups within 24h (e.g., 2 ^-1 And 2 ^-2 A group).

The invention discloses a self-made prescription containing 9 traditional Chinese medicines, namely qing Sang Fang, which is obtained through clinical practice, and the syndrome of wind-heat invading exterior is the adaptation type of qing Sang Fang for treating influenza. The green Sang Fang not only has good curative effect in clinic, but also has obvious price advantage compared with oseltamivir, and the traditional Chinese medicine related to the green Sang Fang has huge market potential.

Example 2:

1 test materials

1.1 laboratory animals

108 female SPF-grade BALB/c mice were provided by Jiangsu Hua Xinnuo medical science and technology Co., ltd, and purchased at a weight of 18-20g, lot number: SCXK (su) 2020-009, randomly divided into 6 groups: blank group, model group, green Sang Fanggao, medium and low concentration group and Oseltamivir (OS) group, wherein 18 groups are fed separately, the environment temperature is set to 20-26 ℃, the humidity is automatically set to 40-70%, the complete sterile feed is eaten, and tap water is drunk and sterilized at high temperature and cooled.

1.2 strains

The strain A H1N1 virus NYMCX-179A (murine lung adapted strain) is provided by experimental animal center of Jiangsu Dike Biotechnology Co., ltd, and virus allantoic fluid with a titer of 1:640 is obtained after virus amplification by chick embryo experiment.

1.3 medicaments

(1) Green Sang Fang

As in example 1.

(2) Oseltamivir

As in example 1.

1.4 major equipment

The experimental field and equipment are all provided by Jiangsu Dike Biotechnology Co., ltd.

/>

1.5 major reagents

2 method

2.1 calculation of the dose administered to mice:

(1) Oseltamivir dose conversion

The dose of oseltamivir for adults is 75 mg/time and 2 times/day, so that the clinical dose of 60kg of adults is 2.5mg/kg. According to the conversion coefficient of the medical experiment animal science course, the equivalent dose of mice is 9.01x2.5mg/kg= 22.525 mg/kg=22.5 ug/g, and the daily dose of the mice with 20g is 450ug, and the mice are administrated once in the morning and evening, so that the drug content of each administration is 225ug, each mouse is administrated according to 0.2ml, and the concentration is 1.125mg/ml.

(2) Conversion of the amount of green Sang Fangji

The total weight of the green Sang Fang containing 9 traditional Chinese medicines is 100g, and the clinical dosage of 60kg adults is 1.667g/kg. The remaining calculation method was the same as oseltamivir, and the final clinical equivalent dose of the adult to mice was 750mg/ml, which was also considered as the concentration group in green Sang Fang, the high concentration group was 1500mg/ml, and the low concentration group was 375mg/ml.

2.2 decocting (compounding) the drugs

(1) Green Sang Fang

The 300g green Sang Fang decoction method is the same as that of the first part of chick embryo experiment, 5 times of filtering juice are combined and concentrated to 200mL, and the medicine concentration is 1500mg/mL (high dose group) at the moment, and then the medicine concentration is diluted to medium concentration (750 mg/mL) and low concentration (375 mg/mL) in sequence.

(2) Oseltamivir preparation

225mg of oseltamivir was dissolved in 200ml of pure water, and stirred using a magnetic stirrer to completely dissolve, to finally obtain oseltamivir solution having a concentration of 1.125 mg/ml.

2.3 nasal drop molding die

The blank group compartment is fed to avoid infection, drinking water and food normally without any treatment; the other 5 groups of mice are subjected to nasal drop molding for 2 days, the drop degree of the rhinovirus drops is 1:640, the grabbed mice are kept upright, the pipette tips are aligned to the left nasal cavity and the right nasal cavity of the mice, the nasal drop amount of each nasal cavity is 15 ul/time, 5-7 times a day, and the phenomenon that the nasal drop amount cannot be too large every time is noticed, so that the mice are prevented from choking is avoided. After nasal drip for 2 days, combined with the symptoms and pathological sections of the mice, the model can be clearly established to be successful and regarded as formal infection ^[128] 。

2.4 administration (Water) and recording of basic Condition

The treatment is carried out by continuously dosing according to the calculated gastric lavage concentration on days 1-5 of the infection of the green Sang Fangzu and OS groups, the dosing is carried out 2 times a day, the interval is 8-12 hours, each time is 0.2 ml/dose, and the water feeding time, frequency and quantity of the model group are equal to those of the drug treatment group; the blank group was normally fed. The basic status of mental state, diet, body weight, respiration, ocular and nasal secretion, etc. of each group of mice was observed and recorded.

2.5 sample acquisition and calculation of Lung index

Each group was 3 rd after infection, 5. 6 mice were randomly picked 7 days for carbon dioxide sacrifice ^[129] . The sacrificed mice are placed in an alcohol jar for pickling and sterilizing 1, the sterilized mice are placed in a newly opened sterile culture dish in an ultra-clean workbench, the chest cavity is slowly and gently sheared by scissors after high-temperature and high-pressure sterilization, lung tissues are required to be completely taken out under the precise matching of the scissors and forceps, the lung tissue weight is weighed and recorded, and the lung index (lung index = lung weight of mice/body weight of mice x 100%) is calculated. The left and right lungs of the mice were isolated and placed into the formaldehyde tube.

2.6 pathological sections, dehydration and wax dipping embedding. Slicing, dewaxing, staining and observing

The pathological changes are observed under a microscope and photographed by a computer. Pathology scoring is performed according to relevant pathology scoring criteria.

2.7 statistical analysis

Statistical analysis was performed as in example 1.

3 results

3.1 symptomatic manifestations of groups

Before modeling, the mental state, the food intake and the respiration of each group of mice are normal, and secretion does not exist at eyes and noses; compared with normal blank groups, the mice in each group after molding have poor mental state, diet, hair appearance, activity and the like, and abnormal secretion at eyes and noses is increased; after administration of green Sang Fang and oseltamivir, mice of each treatment group had improved mental, hair, activity, etc. aspects; the symptom improving effect of the mice in the green Sang Fanggao concentration group is better than that of the mice in the middle and low concentration groups, but the effect of the mice in the green Sang Fanggao concentration group can not be equivalent to that of oseltamivir by the 5 th day, and the symptoms of the mice in the middle and low concentration groups are consistent. The specific symptoms of each group are shown in Table 4.

Table 4 symptomatic manifestations of each group

3.2 weight changes

The basis body weights of the mice in each group were approximately equivalent before molding. Compared with blank groups, the weight of each group is obviously reduced (P is less than 0.05) after the molding is successful, and each group of mice does not die after the molding. Model group body weight fell to nadir on day 5, with a minimum average body weight of 15.33g, and slowly rising after that, but still lower than the blank group body weight (P < 0.05); at various time points after formal treatment, both green Sang Fang and oseltamium Wei Tichong were higher than model group (P < 0.05); on days 1 and 2, green Sang Fang had still a decreasing trend in body weight, but green Sang Fang had consistently higher body weight than model group (P < 0.05); the decrease trend was discontinued for each drug group on day 3 of treatment, and the mice body weight was gradually increased after that, especially on day 7, there was no significant difference between the body weight of the cyan Sang Fanggao concentration group and the OS group and the blank group (P > 0.05).

Table 5 weight distribution table for mice in each group

Comparison of base body weight: wherein P is > 0.05; compared to the blank:representing P > 0.05, +.represents P < 0.05; comparison with the corresponding set of time point models: delta represents P > 0.05, and V represents P < 0.05.

3.3 Lung index Change

After successful modeling, the lung index of the model group infected with influenza virus was higher than that of the blank group (P < 0.05). On day 3 of infection, the lung index was lower for the cyan Sang Fanggao concentration only group and the OS group than for the model group (P < 0.05). On day 5 of infection, green Sang Fanggao, medium and low concentrations all reduced lung index in infected mice, with differences statistically significant (P < 0.05). On day 7 of infection, treated green Sang Fanggao, medium concentration groups of mice had no significant difference in lung index (P > 0.05) from the blank group, particularly the green mulberry Fang Gao concentration group was the group with lung index reduced to the nearest normal level.

TABLE 3 results of mouse lung index at various time points

Blank comparison, +.; compared to the model set: where R represents P < 0.05 and delta represents P > 0.05.

3.4 pathological lesions of pulmonary tissue

Pathological changes in lung tissue

Blank group: the pulmonary tissue has clear reticular structure, the alveoli are not broken and have no hyperemia phenomenon, and no leukocyte infiltration exists; infection model group: the lung tissue is structurally changed, the alveoli are broken, the reticular structure disappears, the wall thickness of the alveoli is increased, congestion phenomenon exists in the alveoli, oblate concave red blood cells can be seen after enlargement, a large amount of leucocyte infiltration exists at the alveoli interval, and nuclei can be seen after enlargement; OS group: the structural damage is light, the alveoli are complete, the congestion is less, and the leukocyte infiltration is less; cyan Sang Fang: compared with the model group, the alveolar structure of the 3 concentration groups is clearer, the hyperemia phenomenon is less, and the inflammatory infiltration is lighter; group 3 lesion severity exhibited dose effects: high concentration group < medium concentration group < low concentration group; the high concentration group did not show pathology on day 3 and day 5 as compared to the OS group, the network structure was worse, congestion and leukocyte infiltration were more severe; in addition to the blank, the lesion improvement was more pronounced for each group over time, especially on day 7, with the high and low concentration groups restored to normal blank levels, but the medium and low concentration groups did not reach the OS group, as detailed in fig. 4.

3.5 pathology scoring

The lung tissue pathology scoring standard table of the influenza virus mice is prepared by referring to relevant experimental mouse pathology scoring standards at home and abroad. Specific scoring items and scores are shown in Table 4. In the experiment, the pathological scores of the model groups after successful modeling are higher than those of the blank groups (P is less than 0.05); compared with the model group, the green Sang Fanggao, medium and low concentration group and the OS group can reduce the lung tissue pathology scores on days 3, 5 and 7; on days 3 and 5, pathology scores were higher for the cyan Sang Fanggao, medium, low concentration group than for the OS group (P < 0.05); however, no significant difference (P > 0.05) was found between the group at the concentration of azure Sang Fanggao and the group OS on day 7, as shown in Table 5 and FIG. 3.

TABLE 4 pathological scoring criteria

TABLE 5 results of pathological scoring for each group of experiments

Comparison of model group with blank, +.representing P < 0.05, > 0.05 for four P, > 0.05, comparison with model group: wherein P is less than 0.05, and delta is more than 0.05; compared to the oseltamivir counterpart group: ● Represents P < 0.05,representing P > 0.05.

Discussion 4

4.1 symptomatic manifestations in mice

In influenza studies, mice are the most widely used animal model, and inbred mice such as BALB/C and C57BL/6 are used most frequently for the following reasons: (1) easy-to-operate genome, (2) rapid propagation and easy-to-handle, (3) easy-to-feed, (4) low cost, etc ^[134] . Influenza infected mice show clinical symptoms such as anorexia, poor mental state, reduced activity and the like, and the specific performance depends on the strain of the mice, the virus strain and the challenge dosage. Weight loss and survival in mice are also important indicators of influenza disease severity.

Influenza virus is an important cause of a large number of acute respiratory viral infections in the population, and many symptoms including high fever, aversion to cold, muscle pain and the like are generated after human infection, diarrhea is sometimes generated, and clinical symptoms of influenza are often more serious in infants and the elderly due to poor or weakened immune systems. Unlike humans, influenza virus infected mice often do not develop fever after macrophage increase, but rather exhibit clinical symptoms such as hypothermia, cyanosis and hemoptysis, which are not readily observed in mice. The particularity of the physiological structure of the mice makes it difficult to measure the self-sensation symptoms such as anal temperature, pharyngalgia, muscular soreness and the like by using a thermometer after the mice are infected with influenza viruses, and tongue pulse of the traditional Chinese medicine diagnosis and treatment cannot be observed and experienced on the mice, so that the study can only judge the infection condition by observing and recording the mental state, activity, weight, secretion and the like of the mice, and is similar to the symptom study of other influenza virus infected mice.

In the experiment, the blank group has normal diet and water inflow, and the front and rear symptoms have no obvious change; after the model group mice are successfully modeled, the symptoms of poor mental state, upright hair, unsmooth and smooth hair, tremble whole body, shrinkage to form clusters, reduced activity, wood chip pad, shortness of breath, reduced appetite and water intake, visible secretion at eyes and nose, frequent front limb face washing action and the like appear. The symptoms were also partially relieved after 5 days of water treatment in the model group, possibly associated with increased autoimmunity in mice. The mice of each treatment group of Qing Sang Fang are better in all aspects of spirit, hair, activity and the like, which shows that Qing Sang Fang can improve symptoms of infected mice, and the effect of a high concentration group is better than that of a medium concentration group and a low concentration group (the improvement effect of the symptoms of the two groups is consistent). However, the effect of the high concentration group can not be equivalent to the effect of oseltamivir until the 5 th day, which indicates that oseltamivir can still play an important role.

4.2 weight changes in mice

The study on the weight of the mice is widely applied to the fields of tumors, diabetes and the like, and the weight of the mice can be changed due to the infection of respiratory viruses. The mental state of mice is poor after the mice are infected with viruses, and the food and water intake are reduced to influence the weight, so that the weight of the mice infected with influenza viruses is generally reduced, and other influenza similar researches prove that the mice are changed. In this experiment, the base weights of the mice in each group were not significantly different before molding; the weight of each group was significantly lower than the base weight (P < 0.05) after 2 days of nasal drip, except for the blank group, indicating successful molding. Model group body weight fell to nadir on day 5, with a minimum average body weight of 15.33g, and slowly increased later, possibly associated with a better before improvement in mental state and increased intake after autoimmune improvement; however, the model group body weight was always lower than the blank group, suggesting that the model group was recovering normal body weight as soon as possible, and still required drug intervention. Green Sang Fang each drug treatment group had a higher body weight than the model group (P < 0.05), suggesting that green Sang Fang could improve the body weight of the infected mice. Green Sang Fang treatment groups still showed a decreasing trend in body weight on days 1 and 2, but higher than model group (P < 0.05), indicating that green Sang Fang was able to reduce the extent of body weight loss in infected mice during the first two days of treatment. The concentration of the green Sang Fanggao can play a role similar to that of the OS on the 7 th day, and the weight of the infected mice can be recovered to be normal.

4.3 pulmonary index protection

The Lung index (Lung index) refers to the percentage of total mass of mice by the mass of Lung tissue taken intact from the mice body, and the severity of inflammatory lesions in Lung tissue is usually expressed by the magnitude of the Lung index. Inflammatory lesions may appear in lung tissue of influenza infected mice, and inflammatory exudates and pulmonary congestion edema increase the mass of mouse lung tissue. The more severe the symptoms of infection, the greater the mass of the lung tissue, and the corresponding lung index. The lung index can be used as an index of the protective effect of the drug on the lung tissue of the mice, and can objectively reflect the change degree of inflammatory lesions of the lung tissue. In the experiment, the lung index of the model group is higher than that of the blank group, and the success of model modeling of the mice in the model group is prompted; green Sang Fang can reduce the lung index of the infected mice, but the lung index reduction effect of all concentration groups can be guaranteed by the 5 th day, so that the treatment time is required to be at least 5 days; no obvious difference between the concentrations of green Sang Fanggao and the medium concentration on the 7 th day of infection and the blank group shows that the concentrations of green Sang Fanggao and the medium concentration can restore the lung index of the mice to be normal, wherein the concentration group of green Sang Fanggao is the group with the best curative effect of reducing the lung index.

4.4 pathological manifestations of pulmonary tissue

The reason for choosing formaldehyde as the fixative in this experiment is that formaldehyde is considered a classical solution in the fixation process, has a remarkable ability to penetrate tissue in a short time, ensures less tissue damage, and plays both a role as fixative and preservative, so it is called classical fixative. In the experiment, the lung tissue of the model group has obvious structural change, the alveoli of the mice are broken, the reticular structure disappears, the wall thickness of the alveoli is increased, the congestion phenomenon exists in the alveoli, inflammatory cell infiltration is consistent with similar research at home and abroad, and the authenticity of successful modeling is further proved.

Although the phenomena of network structure damage, alveolar wall thickening, congestion and the like also exist in the mice treated by the green Sang Fang and OS, compared with a model group, the pulmonary alveolus network structure integrity of the mice of the green Sang Fang and oseltamivir 4 drug groups is better, and the phenomena of congestion, leukocyte infiltration, cell nucleus number and the like are fewer, so that the green Sang Fang and oseltamivir have a certain protection effect on lung tissue injury of the mice infected with H1N1 influenza. The severity of lesions in the cyan Sang Fang dose group exhibited a dose effect: the higher the dose, the lighter the lung tissue lesions; the longer the treatment time of each group of green Sang Fang, the lighter the lung tissue lesions, which may be related to antiviral drugs such as dyers woad leaf, baical skullcap root and the like in the prescription, and also may relate to glycyrrhizin in liquorice (inhibiting influenza virus, improving immunity and alleviating immunopathogenic injury of organisms). The high concentration group did not show much pathology as the OS group on days 3 and 5, but the high concentration group and OS group had recovered to normal blank group levels on day 7, indicating that the green Sang Fanggao concentration group exhibited the best anti-influenza effect on day 7, and also suggesting that oseltamivir still had anti-influenza advantage.

4.5 pathology scoring

In this experiment, the scores were mainly based on 5 items of tracheal lumen exudates, substantial pneumonia, peripulmonary vascular infiltration, invasive lesions and qualitative in lung tissue of influenza virus infected mice. The model group pathology scores at 3 time points are higher than that of the blank group (P is less than 0.05), and are always at the highest position in the scores of each group, so that the mice are successfully modeled and the lung tissue injury is serious after being infected by the H1N1 influenza virus. The green Sang Fanggao, medium, low concentration and OS groups reduced lung tissue pathology scores on days 3, 5 and 7, and were statistically different (P < 0.05), indicating that green Sang Fang 3 concentration groups and OS groups both had protective effects on lung tissue in influenza virus infected mice. On days 3 and 5, the pathology scores of the cyan Sang Fang groups are higher than those of the OS group, which shows that the effect of early-stage pathology score reduction is inferior to that of the OS group, and the OS still plays a role in drug selection. The concentration group of cyan Sang Fanggao on day 7 was not statistically different from OS, indicating that the concentration group of cyan Sang Fanggao had a similar effect on OS in reducing pathology scores in infected mice.

Case examples

Case 1:

child, male, 10 years old, complaint: fever cough for more than 2 days, and the current medical history: fever and cough for more than 2 days, diarrhea, no herpes, no rash, no nausea and vomiting, no diarrhea, abdominal distension and other symptoms are generally found in the oral cavity. The appetite is still good, and the urination and defecation are normal. Checking: normal development, moderate nutrition, clear mind and good spirit. Bregma is soft, no yellow staining of skin and mucous membrane, bleeding points and rash, and superficial lymph node is not touching enlargement. The throat is free from congestion, the tonsils are free from swelling, abnormal secretions are not caused, and the oral cavity is free from blepharma. The chest has symmetrical and non-malformation, and the lung breathing is sound-absorbing and clear, and the lung breathing is not smelling and is dry and wet. No abnormality was seen in the abdomen. The limbs can move freely, no deformity exists, and the buttocks and hands do not see the skin. Physiological reflex exists, and pathological reflex is not led out.

Auxiliary inspection: detecting the alpha-influenza antigen, namely, detecting the alpha-influenza antigen positively; mycoplasma pneumoniae antibody IgM: positive.

Diagnosis: wind-heat syndrome, cold; acute upper respiratory tract infection.

The prescription of the traditional Chinese medicine:

5 doses of radix scutellariae 9g of gypsum, 9g of mulberry leaf, 12g of reed rhizome, 15g of dyers woad leaf, 6g of chrysanthemum, 6g of fried bitter apricot seed, 4g of Ningpo Yam rhizome and 4g of liquorice are taken together, one dose is taken every day, 200ml of decoction is taken orally: 100ml of the preparation is taken 2 times a day

The curative effect of the following treatment: the medicine is taken on day 3, and symptoms disappear on day 5.

Case 2:

child, woman, 13 years old, complaint: fever and headache are more than 1 day. The current medical history: fever for more than 1 day, headache, cough, expectoration, no herpes, no rash, no nausea, no vomiting, no diarrhea, no abdominal distention and other symptoms. The appetite is still good, and the urination and defecation are normal. Checking: normal development, moderate nutrition, clear mind and good spirit. Bregma is soft, no yellow staining of skin and mucous membrane, bleeding points and rash, and superficial lymph node is not touching enlargement. The throat is free from congestion, the tonsils are free from swelling, abnormal secretions are not caused, and the oral cavity is free from blepharma. The chest has symmetrical and non-malformation, and the lung breathing is sound-absorbing and clear, and the lung breathing is not smelling and is dry and wet. No abnormality was seen in the abdomen. The limbs can move freely, no deformity exists, and the buttocks and hands do not see the skin. Physiological reflex exists, and pathological reflex is not led out.

Auxiliary inspection: blood routine WBC 3.3X109/L NEUT 33.6NEUT#1.1CPR <10mg/L hCPR1.69mg/L, and influenza A antigen detection; mycoplasma pneumoniae antibody IgM: negative.

Diagnosis: wind-heat syndrome, cold; acute upper respiratory tract infection.

The prescription of the traditional Chinese medicine:

3 doses of radix scutellariae 9g of gypsum, 9g of mulberry leaf, 12g of reed rhizome, 15g of dyers woad leaf, 6g of chrysanthemum, 6g of fried bitter apricot seed, 4g of Ningpo Yam rhizome and 4g of liquorice are taken orally, wherein one dose is decocted with 200ml of water every day: 100ml each time 2 times daily.

The curative effect of the following treatment: symptoms are relieved on the 3 rd day of administration, and symptoms disappear on the 5 th day.

Case 3:

child, male, 3 years old, complaint: fever cough for 1 day, history of illness: in general, the oral cavity has no herpes, no rash, no nausea and vomiting, no diarrhea, no abdominal distention and other symptoms. The appetite is still good, and the urination and defecation are normal. Plain body is healthy. Checking: normal development, moderate nutrition, clear mind and good spirit. Bregma is soft, no yellow staining of skin and mucous membrane, bleeding points and rash are seen, and superficial lymph nodes are not touching enlargement. The throat is free from congestion, tonsils are free from swelling, abnormal secretion is not seen, and the oral cavity is free from herpes. The chest has symmetrical and non-malformation, and the lung breathing is sound-absorbing and clear, and the lung breathing is not smelling and is dry and wet. No abnormality was seen in the abdomen. The limbs can move freely without deformity, and the skin is not seen on the buttocks and hands. Physiological reflex exists, and pathological reflex is not led out. Auxiliary inspection: blood routine shows: n%71%, LYMPH%:17.7%, EOS%2%, PLT 356X 10 ⁹ L, PCT 0.3, SAA 16. And detecting the alpha-influenza antigen, namely positive. Mycoplasma pneumoniae antibody IgM: negative.

Diagnosis: wind-heat syndrome, cold; acute upper respiratory tract infection

The prescription of the traditional Chinese medicine:

6g of mulberry leaf, 6g of dyers woad leaf, 15g of gypsum and 5g of fried bitter apricot seed

Chrysanthemum flower 4g, baikal skullcap root 4g, ningpo Yam rhizome 4g, licorice root 3g, reed rhizome 6g

Total 5 doses, one dose per day, water decoction 200ml, oral: 100ml of the preparation is taken 2 times a day

The curative effect of the following treatment: after taking 3 doses, symptoms are relieved, symptoms disappear for one week, and the SAA <5 is obviously reduced before the outpatient inspection.

Case 4:

child, female, age 2, complaint: heating for 1 day. The current medical history: fever appears after catching cold before 1 day, the highest temperature is 38.7 ℃, cough, paroxysmal, phlegm, difficult expectoration, no chills and convulsion, nausea and vomiting, morbidity, excessive appetite and normal urination and defecation are caused. Checking: the mind is clear and the spirit is good. Smooth breathing, reddish throat, no swelling of tonsil, white and thick tongue coating, thick sound absorption of double lung breathing, no smell and dry and wet sound. No abnormality was seen in the examination of the heart, abdomen and nervous system.

Auxiliary inspection: blood convention: weak positive, MONO%15.2CPR <10mg/L hCPR1.22mg/L alpha-stream antigen detection; mycoplasma pneumoniae antibody IgM: negative.

Diagnosis: cold with excessive pathogenic factors (wind-heat syndrome), influenza.

The prescription of the traditional Chinese medicine:

radix scutellariae 6g gypsum 12g mulberry leaf 6g reed rhizome 4g dyers woad leaf 6g chrysanthemum 5g fried bitter apricot seed 3g Ningpo Yam rhizome 5g liquorice 3g total 5 dose, one dose per day, water decoction 200ml, oral administration: 100ml each time 2 times daily.

The curative effect of the following treatment: the symptoms are relieved on the 4 th day of administration, and the symptoms disappear on the 5 th day.

Case 5:

child, male, 5 years old, complaint: heating for 2 days for repeated diagnosis; the current medical history: : the infant has no obvious cause of fever before 2 days, the maximum body temperature is 38.4 ℃, no shivering or convulsion exists, and the body temperature is reduced to be normal after physical cooling; with pharyngalgia, headache, cough, expectoration, nasal obstruction, nasal discharge, nausea, emesis, abdominal distention and diarrhea, the family members can take the oral antiphlogistic antipyretic mixture (not detailed) by themselves, and still generate heat, thus the patient can visit the hospital further. The sick children have the symptoms of mental and appetite, normal urination and defecation. Checking: the health-care food has the advantages of clear mind, stable respiration, reddish throat, no tonsil swelling, white and thick tongue fur, thick sound absorption of double lung breathing, inaudible dry and wet sound. No abnormality was seen in the examination of the heart, abdomen and nervous system.

Auxiliary inspection: blood routine WBC 3.26X109/L LYMPH%17.7 CPR <28.3mg/LhCPR >10mg/L alpha-stream antigen detection; mycoplasma pneumoniae antibody IgM: negative.

Diagnosis: wind-heat syndrome, cold; acute upper respiratory tract infection

The prescription of the traditional Chinese medicine:

5 doses of radix scutellariae, gypsum 15g, mulberry leaf, reed rhizome, dyers woad leaf, chrysanthemum 6g, fried bitter apricot seed 4g, ningpo Yam rhizome 4g and liquorice 4g are taken together, one dose is taken every day, water decoction is carried out for 200ml, and oral administration is carried out: 100ml each time 2 times daily. The curative effect of the following treatment: six days later, the conventional CPR of the outpatient blood is obviously reduced by 18.3mg/L hCPR8.87mg/L compared with the prior method

The curative effect of the following treatment: the symptoms are obviously relieved after taking the medicine, and the symptoms disappear after 6 days.

The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (3)

1. An anti-influenza virus traditional Chinese medicine composition is characterized in that: comprises folium Isatidis, folium Mori, rhizoma Phragmitis, scutellariae radix, gypsum Fibrosum, flos Chrysanthemi, semen Armeniacae amarum, ningpo Yam rhizome and Glycyrrhrizae radix.

2. The anti-influenza virus traditional Chinese medicine composition according to claim 1, wherein the anti-influenza virus traditional Chinese medicine composition is characterized in that: the proportion of each component is as follows: 15 parts of dyers woad leaf, 9 parts of mulberry leaf, 12 parts of reed rhizome, 9 parts of baical skullcap root, 30 parts of gypsum, 6 parts of chrysanthemum, 6 parts of bitter apricot seed, 9 parts of Ningpo Yam rhizome and 4 parts of liquoric root.

3. A preparation method of an anti-influenza virus traditional Chinese medicine composition is characterized by comprising the following steps: comprising the following steps:

step 1: decocting 3 agents, namely 300g of gypsum with 300ml of pure water with strong fire for 15-30min;

step 2: 150 parts of dyers woad leaf, 90 parts of mulberry leaf, 120 parts of reed rhizome, 90 parts of baical skullcap root, 60 parts of chrysanthemum, 60 parts of bitter apricot seed, 90 parts of Ningpo Yam rhizome and 40 parts of liquorice are added into the medicine in the step 1, and 1500ml of pure water is added for soaking for 1 hour;

step 3: heating and decocting all the medicines in the step 2 with slow fire to 300mL, filtering the decoction with 4 layers of sterile gauze, collecting the filtered decoction, adding 1500mL of pure water into the residual filter residue, heating and decocting with slow fire to 300mL again, repeating the filtering and decocting operation for 5 times, finally combining the 5 times of filtrates, and concentrating the medicines to 6g/mL;

step 4: sterilizing the medicine prepared in the step 3 at high temperature and high pressure for 30min, and diluting with pure water sterilized by filtration to form 6 concentrations of 6g/ml, 3g/ml, 1.5g/ml, 0.75g/ml, 0.375g/ml and 0.1875 g/ml.