CN114099563A

CN114099563A - Traditional Chinese medicine composition for resisting respiratory syncytial virus and application thereof

Info

Publication number: CN114099563A
Application number: CN202111521796.1A
Authority: CN
Inventors: 王剑; 于健; 王京龙; 张立华; 杜健; 李伟; 田景振
Original assignee: Zaozhuang University
Current assignee: Zaozhuang University
Priority date: 2021-12-14
Filing date: 2021-12-14
Publication date: 2022-03-01

Abstract

The invention discloses a traditional Chinese medicine composition for resisting Respiratory Syncytial Virus (RSV) and application thereof, wherein the traditional Chinese medicine composition comprises the following components in parts by weight: 23-35 parts of baicalin, 1000 parts of honeysuckle polysaccharide, 10-30 parts of honeysuckle organic acid and 10-20 parts of honeysuckle flavone, and can be applied to preparation of respiratory syncytial virus resistant medicaments, including tablets, capsules or liquid preparations, wherein the concentration of each component in the liquid preparation is preferably 312 mu g/mL of baicalin, 12500 mu g/mL of honeysuckle polysaccharide, 113 mu g/mL of honeysuckle organic acid and 181 mu g/mL of honeysuckle flavone. The traditional Chinese medicine composition is prepared from four components of baicalin, honeysuckle polysaccharide, honeysuckle organic acid and honeysuckle flavone, can effectively inhibit the activity of respiratory syncytial virus, has the characteristics of low toxicity and safe use, provides a theoretical basis for clinically applying a traditional Chinese medicine compound to treating Respiratory Syncytial Virus (RSV) infectious diseases, provides a certain guiding significance for developing a medicament for resisting the Respiratory Syncytial Virus (RSV), and has an important reference value.

Description

Traditional Chinese medicine composition for resisting respiratory syncytial virus and application thereof

Technical Field

The invention belongs to the technical field of traditional Chinese medicine antivirus, and particularly relates to a traditional Chinese medicine composition for resisting respiratory syncytial virus and application thereof.

Background

Respiratory Syncytial Virus (RSV) belongs to the family Paramyxoviridae, and is an enveloped, non-segmented, single-stranded negative-strand RNA virus. RSV is the leading cause of lower respiratory tract disease in infants and young children, with pneumonia and bronchiolitis as the major clinical symptoms, while acute lower respiratory tract infection is the leading cause of death in children worldwide. Viral diseases seriously threaten the health of human beings, at present, the main obstacle to treating viral diseases is the lack of specific antiviral drugs, and vaccine drugs have hysteresis on epidemic viral diseases; antiviral synthetic drugs are very easy to generate drug resistance; and is liable to cause side effects. The compound honeysuckle and scutellaria baicalensis is a prescription for treating respiratory tract infection, but the research on RSV resistance of the compound honeysuckle and scutellaria baicalensis is rare, the chemical components of the traditional Chinese medicine are complex, particularly the antiviral effect of the compound traditional Chinese medicine can be the result of the comprehensive effect of various components,

traditional Chinese medicine has a long history in treating viral diseases, and antivirus is an advantageous field of traditional Chinese medicine. However, the chemical components of Chinese herbs are complex, and especially compound Chinese herbs have antiviral action probably as a result of the combined action of various components, or some component alone can act on the focus. Each traditional Chinese medicine usually contains a plurality of chemical components, but not all the chemical components can generate treatment effect, so the traditional Chinese medicines can be divided into effective components and ineffective components, and one traditional Chinese medicine usually contains a plurality of effective components which can generate synergistic action but also can generate restriction action and antagonism. In addition, the research on the cytotoxicity of the traditional Chinese medicine shows that certain components in the traditional Chinese medicine possibly generate side effects and seriously affect the dosage of the effective components. Therefore, the proportion and selection of each component in the compound can only depend on the experience of doctors, the optimal medicine compatibility and the proportion of each component cannot be scientifically and uniformly provided, and the scientific cognition on the effective substances and the action mechanism of the traditional Chinese medicine is lacked, so that the law of the traditional Chinese medicine prescription for exerting the curative effect is not sufficiently known, the stable exertion of the curative effect of the traditional Chinese medicine is influenced, and the improvement of the quality of the traditional Chinese medicine product is also hindered.

The uncontrollable quality of the traditional Chinese medicinal materials is always a big problem for related researchers. For example, the effective components of honeysuckle are organic acids, volatile oil, flavonoids, iridoids and triterpenoid saponins, but the content of each effective component in honeysuckle medicinal materials is unstable and is easily influenced by various internal and external factors, the effective components in honeysuckle can be influenced significantly by different production places and different collection times, for example, the difference of chlorogenic acid in honeysuckle of different species sources is obvious, and the species with the highest content and the species with the lowest content are different by dozens of times. In addition, the difference between the extraction method and the processing method of the honeysuckle also has important influence on the content of the effective components of the honeysuckle. Therefore, even for the same kind of medicinal material, it is very difficult to control the quality.

Therefore, it is of great significance to develop a Chinese medicinal composition comprising effective components of flos Lonicerae and Scutellariae radix, and to carry out quantitative compatibility.

Disclosure of Invention

The traditional Chinese medicine composition is prepared from four components of baicalin, honeysuckle polysaccharide, honeysuckle organic acid and honeysuckle flavone, can effectively inhibit the activity of the respiratory syncytial virus, has the characteristics of low toxicity and safe use, provides a theoretical basis for clinically treating Respiratory Syncytial Virus (RSV) infectious diseases by using a traditional Chinese medicine compound, provides a certain guiding significance for developing a medicament for resisting the Respiratory Syncytial Virus (RSV), and has an important reference value.

The invention is realized by the following technical scheme:

a traditional Chinese medicine composition for resisting respiratory syncytial virus comprises the following components in parts by weight: 23-35 parts of baicalin, 1300 parts of honeysuckle polysaccharide, 10-30 parts of honeysuckle organic acid and 10-20 parts of honeysuckle flavone.

Further, the traditional Chinese medicine composition for resisting the respiratory syncytial virus comprises the following components in parts by weight: 31.2 parts of baicalin, 1250 parts of honeysuckle polysaccharide, 11.3 parts of honeysuckle organic acid and 18.1 parts of honeysuckle flavone.

In the invention, the traditional Chinese medicine composition for resisting the respiratory syncytial virus is applied to the preparation of the medicine for resisting the respiratory syncytial virus.

Furthermore, the respiratory syncytial virus resistant medicine is a tablet, a capsule or a liquid preparation.

Further, the liquid preparation comprises the following components in concentration: 312 mu g/mL of baicalin, 12500 mu g/mL of honeysuckle polysaccharide, 113 mu g/mL of honeysuckle organic acid and 181 mu g/mL of honeysuckle flavone.

Advantageous effects

The traditional thinking of developing traditional Chinese medicines by clinical experience and decoction piece composition is broken through, the mechanism of treating diseases by the traditional Chinese medicines is favorably elucidated, and then the traditional Chinese medicine components with controllable quality are obtained.

Drawings

FIG. 1 is a microscopic picture of a virus control group with complete lesions in an antiviral experiment;

fig. 2 is a picture after HE staining of the model group, the normal group, and the administration group.

Detailed Description

The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.

In the following examples, the parts are parts by weight.

Example 1

A traditional Chinese medicine composition for resisting respiratory syncytial virus is composed of the following components in parts by weight: 31.2 parts of baicalin, 1250 parts of honeysuckle polysaccharide, 11.3 parts of honeysuckle organic acid and 18.1 parts of honeysuckle flavone.

Example 2

A traditional Chinese medicine composition for resisting respiratory syncytial virus is composed of the following components in parts by weight: 23.5 parts of baicalin, 1250 parts of honeysuckle polysaccharide, 29 parts of honeysuckle organic acid and 20 parts of honeysuckle flavone.

Example 3

A traditional Chinese medicine composition for resisting respiratory syncytial virus is composed of the following components in parts by weight: 29 parts of baicalin, 1000 parts of honeysuckle polysaccharide, 21 parts of honeysuckle organic acid and 18 parts of honeysuckle flavone.

Example 4

A traditional Chinese medicine composition for resisting respiratory syncytial virus is composed of the following components in parts by weight: 31.2 parts of baicalin, 1250 parts of honeysuckle polysaccharide, 29 parts of honeysuckle organic acid and 20 parts of honeysuckle flavone.

Comparative example 1

A traditional Chinese medicine composition for resisting respiratory syncytial virus is composed of the following components in parts by weight: 31.2 parts of baicalin and 1250 parts of honeysuckle polysaccharide.

Comparative example 2

A traditional Chinese medicine composition for resisting respiratory syncytial virus is composed of the following components in parts by weight: 1250 parts of honeysuckle polysaccharide, 11.3 parts of honeysuckle organic acid and 18.1 parts of honeysuckle flavone.

Comparative example 3

A traditional Chinese medicine composition for resisting respiratory syncytial virus is composed of the following components in parts by weight: 31.2 parts of baicalin, 11.3 parts of honeysuckle organic acid and 18.1 parts of honeysuckle flavone.

Comparative example 4

(1) And (3) honeysuckle extract: weighing a proper amount of honeysuckle decoction pieces, crushing, sieving with a 60-mesh sieve, placing in a round-bottom flask, respectively adding 10 times and 8 times of water, reflux-extracting for two times (2 h each time), combining filtrates, filtering, centrifuging at 3000 rpm for 15 min, taking supernatant, vacuum-filtering, concentrating under reduced pressure, and drying to obtain honeysuckle extract;

(2) weighing a proper amount of scutellaria baicalensis extract, respectively decocting with 10 times of water and 8 times of water for two times, each time for 1.5 hours, combining filtrates, filtering, centrifuging at 3000 rpm for 15 minutes, taking supernate, filtering, concentrating under reduced pressure to a proper amount, adjusting the pH of the concentrated solution to 1.8 by using 2 mol/L hydrochloric acid solution, preserving heat at 80 ℃ for 30 minutes, standing for 12 hours, filtering, adding a proper amount of water into precipitates, uniformly stirring, adjusting the pH to 7.0 by using 40% sodium hydroxide solution, adding an equal amount of 95% ethanol after dissolving, uniformly stirring, stirring to dissolve, filtering, adjusting the pH of the filtrate to 1.8 by using 2 mol/L hydrochloric acid solution, preserving heat at 60 ℃ for 30 minutes, standing for 12 hours, filtering, sequentially washing the precipitates to 7.0 by using a proper amount of water and ethanol with different concentrations, volatilizing the ethanol completely, vacuum drying, the content of baicalin is 90.13% by high performance liquid chromatography content measurement.

In vitro and in vivo antiviral research experiment

1 in vitro cell assay

1.1 preparation of monolayer cells in 96-well plates

Reviving Hep-2 (human laryngeal carcinoma epithelial cell) cells, passaging for 4 times until the cells grow well, digesting the cells in logarithmic phase, and adjusting the concentration to 1 × 10⁵each/mL, the mixture is blown, beaten and mixed evenly, 100 mu L of the mixture is inoculated on a 96-well plate, and the mixture is placed at 37 ℃ and 5 percent CO₂Culturing in a virus incubator for 24 h.

1.2 amplification of viruses

Inoculating 200 μ L RSV strain on the cell which has grown into a monolayer, adsorbing for 1 h, adding 5 mL of 2%1640 maintenance solution, standing at 37 deg.C and 5% CO₂Culturing in virus incubator, setting cell control, taking out when more than 90% of cells have pathological changes, repeatedly freezing and thawing for 3 times, blowing and centrifuging (1000 rpm, 10 min), and taking supernatantQuantitatively subpackaging the liquid in a freezing tube, sealing with a sealing film, and freezing at-80 deg.C for use.

1.3 viral virulence TCID₅₀Measurement of (2)

Continuously diluting the virus to be detected with 1640 maintenance solution at 10 times ratio to obtain 10^-1~10^-10Different concentrations are inoculated in a 96-well plate growing into a monolayer of cells, and the plate is longitudinally repeated for 4 wells, each well is 100 mu L, and a cell control and a blank control are arranged at the same time. 37 ℃ and 5% CO₂Culturing in virus incubator for 24 hr, adding MTT 10 μ L, 37 deg.C, 5% CO₂Discarding the staining solution after 3-4 hours in the incubator, adding DMSO100 μ L into each hole, measuring OD value with an enzyme-labeling instrument at 490nm wavelength after 15 min, and calculating half infection concentration (TCID) of the virus solution according to Reed-Muench formula₅₀). TCID of RSV (respiratory syncytial virus) calculated by applying Reed-Muench method₅₀Is 10^-4.94and/mL. The experimental time used 100 TCIDs₅₀As the concentration of the virus solution.

1.4 determination of cytotoxicity

The medicine is diluted by 1640 maintenance liquid according to a concentration gradient of 10 times of the maintenance liquid, and is sequentially inoculated in a 96-well plate which has been grown into a monolayer of cells, and each group is provided with 3 multiple wells, a blank control well and a cell control well. 37 ℃ and 5% CO₂Culturing in virus incubator for 24 hr, adding MTT 10 μ L, 37 deg.C, 5% CO₂3-4 hours in the incubator, removing the dye solution, adding DMSO (100 mu L), measuring OD (optical density) value at 490nm by using an enzyme-labeling instrument after 15 min, wherein the survival rate of more than 90% of cells is the maximum nontoxic concentration (TC) of the drug₀). In antiviral experiments, from the maximum nontoxic concentration (TC)₀) The dilution was started.

1.5 drug antiviral experiments

Respectively setting a drug group, a cell control group, a virus control group and a blank control group, and carrying out in-vitro antiviral drug solution, wherein the drug group is prepared by using 1640 maintenance solution to prepare corresponding concentrations (shown in the following table 1) of the traditional Chinese medicine composition for resisting respiratory syncytial virus in examples 1-4 and comparative examples 1-3, the honeysuckle extract in comparative example 4 and the scutellaria baicalensis extract (maximum nontoxic concentration), 3 wells are repeated at each concentration, 37 ℃ and 5% CO are carried out₂Culturing in incubator, observing cells under microscopeStopping culturing after more than 90% of cells in virus control group are diseased, recording antiviral state of medicine by CPE method, taking virus infected cell disease rate below 50% as effective hole, adding MTT 10 μ L, 37 deg.C, 5% CO₂After 4 hours in the incubator, the staining solution was discarded, 100. mu.L of DMSO was added, and after 15 minutes, the OD value was measured at a wavelength of 490nm with a microplate reader, and the virus inhibition ratio ER (%) was calculated.

ER (%) = (lesion well OD value-virus control OD value)/(cell control well OD value-virus control OD value) × 100%

The calculated virus inhibition rate ER (%) is shown in table 1 below, and the microscopic picture of the virus control group with complete lesions is shown in fig. 1.

As can be seen from fig. 1 and table 1, the traditional Chinese medicine composition for resisting respiratory syncytial virus in the application has a good virus inhibition effect, particularly when the concentrations of baicalin, honeysuckle polysaccharide, honeysuckle organic acid and honeysuckle flavonoid are respectively 312 μ g/mL, 12500 μ g/mL, 113 μ g/mL and 118 μ g/mL, the traditional Chinese medicine composition has an inhibition rate of 78.51% on virus, and has a relatively good anti-RSV activity.

Animal experiments

Diluting RSV virus to 100TCID in high sugar medium₅₀Except the normal group, the administration group (including the drug group and the positive drug group) and the model group are modeled. The mouse is first injected with 2% sodium pentobarbital (2 mL/kg) via abdominal cavity, the mouse is placed with abdomen upward and head upward, and the model group is dripped with 100TCID via pipette into nostril₅₀50 μ L, and put into a cage after all inhalation. The nasal drops are applied to the patient 1 time per day, 2 times in total, the normal group is applied to the high-sugar culture medium with the same volume in the same way, and except for the model group and the normal group, the drug group is applied 3 days in advance before the model is made.

The administration mode is intragastric administration, and the intragastric administration dosage is as follows: a drug group (baicalin: 31.2 mg/kg, flavone: 18.1 mg/kg, organic acid: 11.3 mg/kg, polysaccharide: 1250 mg/kg); the ribavirin group as the positive drug is converted into mouse dose (58.5 mg/kg) by clinical equivalent dose.

Mice in the model group, the normal group and the dosing group are sacrificed and taken on the 5 th day after the model building, 5 mice in each group are taken, diet is forbidden within 8 h before the sacrifice, and eyeballs are taken for standby after weighing. Whole mouse lung tissue was removed. After the lung tissue is washed with sterile normal saline for 2 times, surface water is sucked dry by sterile filter paper, the degree of lesion is observed and recorded, the lung is weighed, and the lung index is calculated. And the upper left lung lobe was placed in tissue fixative (4% paraformaldehyde) and the specimen was labeled for HE staining.

2.1 pulmonary index

Compared with the normal group, the lung index of the model group is obviously increased (P is less than 0.05), which indicates that the BALB/c mice have obvious lung inflammation after being infected with RSV. Compared with the model group, the lung indexes of the positive drug ribavirin group and the drug group are obviously reduced (P is less than 0.05), and the results are shown in table 2.

2.2HE staining

The pictures after HE staining are shown in figure 2. The normal group has uniform and consistent alveolar cavities, no inflammatory cell infiltration is found at the periphery, the alveolar walls have uniform thickness, no thickening and bleeding phenomena exist, and the alveolar cavities are clean; the alveolar wall of the model group is obviously thickened, a large amount of inflammatory cells infiltrate, mainly lymphocytes and neutrophils infiltrate, alveoli collapse, alveolar cavities become small or disappear, visible red blood cells and a small amount of serous fluid appear in the cavities, and interstitial pneumonia is obviously shown, so that the RSV infected mouse model is successfully established. Compared with the model group, the ribavirin group and the drug group have the advantages that the lung inflammation is obviously reduced, the alveolar wall is slightly thick, the infiltration of inflammatory cells is reduced, and the lung tissue injury is obviously improved.

Claims

1. The traditional Chinese medicine composition for resisting respiratory syncytial virus is characterized by comprising the following components in parts by weight: 23-35 parts of baicalin, 1300 parts of honeysuckle polysaccharide, 10-30 parts of honeysuckle organic acid and 10-20 parts of honeysuckle flavone.

2. The respiratory syncytial virus-resistant traditional Chinese medicine composition according to claim 1, which is characterized by comprising the following components in parts by weight: 31.2 parts of baicalin, 1250 parts of honeysuckle polysaccharide, 11.3 parts of honeysuckle organic acid and 18.1 parts of honeysuckle flavone.

3. Use of the respiratory syncytial virus-resistant traditional Chinese medicine composition of claim 1 or 2 in the preparation of a respiratory syncytial virus-resistant medicament.

4. The use according to claim 3, wherein the anti-respiratory syncytial virus medicament is in the form of a tablet, capsule or liquid formulation.

5. The use according to claim 4, wherein the liquid formulation has a concentration of each component: 312 mu g/mL of baicalin, 12500 mu g/mL of honeysuckle polysaccharide, 113 mu g/mL of honeysuckle organic acid and 181 mu g/mL of honeysuckle flavone.