CN117717590A

CN117717590A - Pharmaceutical composition for preventing and treating novel coronavirus, and preparation method and application thereof

Info

Publication number: CN117717590A
Application number: CN202311401601.9A
Authority: CN
Inventors: 王智森; 刘水娟; 陈君; 李赛
Original assignee: Shijiazhuang Zangnuo Pharmaceutical Co ltd
Current assignee: Shijiazhuang Zangnuo Pharmaceutical Co ltd
Priority date: 2023-10-26
Filing date: 2023-10-26
Publication date: 2024-03-19

Abstract

The invention provides a pharmaceutical composition for preventing and treating novel coronaviruses, a preparation method and application thereof. The pharmaceutical composition is prepared from the following components in parts by weight: 224-250 parts of astragalus membranaceus, 56-70 parts of rhizoma anemarrhenae, 168-200 parts of rhodiola rosea, 168-200 parts of ageratum, 112-150 parts of Tibetan schizonepeta, 224-250 parts of roasted loquat leaves, 168-200 parts of fructus forsythiae, 56-70 parts of Tibetan saussurea and 56-70 parts of amur corktree bark. The traditional Chinese and Tibetan medicine combination prescription can inhibit the replication of viruses, prevent cytopathy caused by the viruses, regulate the immune function of organisms, improve the pulmonary circulation, relieve pain, resist inflammation and the like in clinic, and the viruses are difficult to generate drug resistance. The inhibition effect of the prescription on the novel coronavirus HCoV-OC43 is determined through in-vivo and in-vitro inhibition tests, and a test basis is provided for the clinical application of the traditional Chinese and Tibetan medicine combined with clinical proved recipe for resisting the novel coronavirus.

Description

Pharmaceutical composition for preventing and treating novel coronavirus, and preparation method and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a pharmaceutical composition for preventing and treating novel coronaviruses, a preparation method and application thereof.

Background

Coronavirus is a virus that can infect a variety of hosts, including humans, causing acute respiratory, digestive tract infections. Current research on anti-coronavirus drugs, especially anti-SARS-CoV-2 drugs, is mainly focused on S protein and enzyme inhibitors. The research and development of new drugs require higher economic and time investment, and the anti-coronavirus drugs are screened from the clinical drugs which are obtained to be applied, namely, the strategy of 'old drugs for new use' can effectively reduce the research and development cost of the drugs.

The traditional Chinese and Tibetan medicine combined prescription can clinically inhibit the replication of viruses, prevent cytopathy caused by the viruses, regulate the immune function of organisms, improve the pulmonary circulation, relieve pain, resist inflammation and the like, and the viruses are difficult to generate drug resistance.

Disclosure of Invention

The invention aims to provide a pharmaceutical composition for preventing and treating novel coronaviruses, and a preparation method and application thereof.

To achieve the object of the present invention, in a first aspect, the present invention provides a pharmaceutical composition for preventing and treating a novel coronavirus, which is prepared from the following components in parts by weight: 224-250 parts of astragalus membranaceus, 56-70 parts of rhizoma anemarrhenae, 168-200 parts of rhodiola rosea, 168-200 parts of ageratum, 112-150 parts of Tibetan schizonepeta, 224-250 parts of roasted loquat leaves, 168-200 parts of fructus forsythiae, 56-70 parts of Tibetan saussurea and 56-70 parts of amur corktree bark.

Preferably, the pharmaceutical composition is prepared from the following components in parts by weight: 224 parts of astragalus, 56 parts of rhizoma anemarrhenae, 168 parts of rhodiola rosea, 168 parts of ageratum, 112 parts of Tibetan schizonepeta, 224 parts of roasted loquat leaves, 168 parts of fructus forsythiae, 56 parts of Tibetan saussurea and 56 parts of amur corktree bark.

Further, the pharmaceutical composition also comprises auxiliary materials, and the bulk drugs and the auxiliary materials are mixed according to the weight ratio of 2:1.

Wherein the auxiliary material is at least one selected from dextrin, starch, lactose, maltose, mannitol, mannose and the like.

Preferably, the auxiliary materials are prepared by mixing dextrin, lactose and mannitol according to the weight ratio of 5:3:2.

In a second aspect, the present invention provides a method for preparing the pharmaceutical composition comprising the steps of:

(1) Weighing the raw materials according to a certain proportion, mixing, heating and reflux-extracting with 60% ethanol for 2 times, adding 10 times of 60% ethanol (namely 10 times of the total weight of the medicinal materials) into the mixture each time for 2.5 hours, combining and filtering the extracting solutions, concentrating the extracting solutions to a relative density of 1.20-1.30 g/mL (60 ℃), and vacuum-drying the thick paste until the water content is less than 2.5%, thereby obtaining extract powder I;

(2) Adding 8 times of water into the residue, extracting for 2 hours at 80 ℃, filtering, concentrating to a relative density of 1.20-1.30 g/mL (60 ℃), and vacuum drying the thick paste until the water content is less than 2.5%, thus obtaining extract powder II;

(3) Mixing the extract powder I and the extract powder II, and micronizing;

(4) Mixing the extract powder with adjuvants (dextrin, lactose and mannitol at weight ratio of 5:3:2) at weight ratio of 2:1, spray granulating with 90% ethanol, drying, and packaging to obtain "HONGLIANQINGFEI granule".

Further, the extract powder in the step (3) is crushed by adopting an ultrafine crushing technology, and the granularity is controlled to be 300 meshes.

The technical scheme for preparing the pharmaceutical composition for preventing and treating the novel coronavirus is shown in fig. 1.

In a third aspect, the present invention provides a formulation for the treatment of novel coronavirus infections, the active ingredient of which is said pharmaceutical composition.

Preferably, the formulation is in the form of a granular formulation.

In a fourth aspect, the invention provides the use of the pharmaceutical composition in the manufacture of a novel therapeutic agent for coronavirus infection.

The medicament reduces the generation of the final product MDA of lipid peroxidation by alleviating lipid peroxidation damage of an organism, improves SOD activity and enhances the antioxidant capacity of the organism, thereby slowing down the lung injury effect caused by novel coronaviruses.

The prescription of the 'Honglian lung-heat-clearing granule' consists of astragalus, rhizoma anemarrhenae, rhodiola rosea, ageratum, tibetan herba schizonepetae, roasted loquat leaf, fructus forsythiae, tibetan saussurea involucrata, phellodendron bark and the like, and aims at clearing away the heat and toxic materials, clearing lung and dissipating heat and clearing the exterior and interior. The Chinese medicinal composition is used for preventing influenza, pneumonia and novel coronavirus infection of people suffering from symptoms such as heat toxin attack on the lung, wind heat attack on the lung, and the like, and has the following symptoms: cough, excessive phlegm, nasal obstruction, watery nasal discharge, dry throat, pharyngalgia, fever, aversion to cold, muscular soreness, reddish tongue, yellow or yellow greasy coating, etc. are also indicated for infantile lung heat. Has the functions of broad antivirus, bacteriostasis and improving the immunity of the organism.

In view of the above, the formulation "Honglian Qingfei granule" is a formulation for clearing away heat and toxic materials and clearing lung-heat.

Huang Qigan in the recipe, the drugs for invigorating qi and controlling essence are as follows: astragalus root "five are used: the deficiency is compensated, the primordial qi is also benefited, the spleen and stomach are also strengthened, and the spleen and stomach are also strengthened; according to Ke Miaozeng, the fire of Mingmen is yang in water, the body of water is static and the flow of qi is not in return, the power of qi and the use of fire are …, the "qi" of Korotkoff's ' power of qi ' can be expanded to mean kidney qi, astragalus root can both benefit qi to control essence and assist the flow of water to return, and decoction of concentrated herbal medicine is called astragalus root, which is a medicine with three good functions of upper, middle, lower, inner and outer aspects.

Rhizoma anemarrhenae, bitter, sweet and cold in nature, nourish yin and dispel heat. For this, one helps astragalus root, zhang Xichun, if it is used for tonifying qi, it is terrible that it has heat-free, and it is also constantly supplemented with rhizoma anemarrhenae. Furthermore, water vapor is possible. It is known that Zhi mu can cover yin, clear heat and quench thirst in the book of this Jing Shu Zhi Shi (the syndrome of dredging meridians), but it is very rare for old people to use it and the reason is unknown after learning.

Huang Bai is bitter and cold in nature, and clears heat and detoxifies, and it is used to supplement water with Huang Bai in the cloud of De Ji Ben Cao, so it can clear yin fire from the lower part to the upper part, and fire clear water can be coagulated and not supplemented. "Yuan Zhi and Huang Bai in Yi Zhi Yi (medicine transformation of meaning)" are used together, not being the effect of reducing pathogenic fire and helping water ", but also being the effect of invigorating qi of Huang mao, and" Yuan Bai Liu Shen and Yao Fu Yao … in Huang Qi Tang in Yi Zhi Yi (medical Start Source) "are added to make the strength of both knees surrender.

Rhizoma anemarrhenae and cortex Phellodendri are ministerial drugs in the recipe; rhodiola rosea, wrinkled giant hyssop, tibetan schizonepeta herb and roasted loquat leaf are used as adjuvant drugs; fructus forsythiae and herba Saussureae Involueratae are used as guiding drugs. The whole formula has the effects of clearing away the heat and toxic materials, clearing away the lung-heat and dissipating the heat, and clearing the exterior and the interior. Can be used for preventing influenza, pneumonia and coronavirus infection.

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

the invention develops an innovative pharmaceutical preparation-HONGLIANQINGFEI granule which has simple preparation, obvious curative effect, small side effect and better effect on preventing and treating novel coronavirus according to the traditional Chinese medicine and Tibetan medicine pharmacological discussion experience.

In order to increase bioavailability of the medicinal components in vivo, the extract powder is crushed by superfine grinding technology, and the granularity is controlled to 300 meshes. The preparation process adopts a fluidized bed spray granulation method for one-step granulation, the production process is clean, the production period is shortened, the yield is improved, the energy is saved, and the production efficiency is high.

The inhibition of novel coronavirus OC43 (HCoV-OC 43) is determined by in vivo and in vitro inhibition tests of the formulation. Further researches on the action mechanism of the traditional Chinese medicine and Tibetan medicine and provides a test basis for the clinical application of combining clinical proved recipe with the novel coronavirus resistance.

The in vitro inhibition test results show that: half-Toxicity Concentration (TC) of the combination prescription for Tibetan medicine on MDCK cells ₅₀ ) 1458. Mu.g/mL. The effect of the combination of the traditional Chinese and Tibetan medicine on HCoV-OC43 coronavirus infection shows that the combination can inhibit the replication and proliferation effects of the absorbed viruses, and the average half-Effective Concentration (EC) of the three different drug concentrations and the action modes of the viruses ₅₀ ) 212.2, 210.6 and 240.3 μg/mL, respectively, and Therapeutic Indices (TI) of 7.5, 7.6 and 6.6, respectively.

The in vivo inhibition test results show that: the virus control group has serious lesions, such as pulmonary capillary congestion, most pulmonary alveolar structure destruction, partial pulmonary solid variable region fusion, alveolar septal thickening, alveolar space and inflammatory cell infiltration in interstitium. After the drug treatment, the health of mice is obviously improved compared with the virus control group in each drug group.

The research result of the action mechanism shows that: 1) Effects of the combination of the traditional Chinese and Tibetan medicine formulations on immune organ index and lymphocyte proliferation in infected mice: the thymus index and spleen index were significantly increased in the traditional chinese and Tibetan medicine combination formula (P < 0.05) compared to the virus control group, and the lymphocyte a value was significantly increased in the traditional chinese and Tibetan medicine combination formula (T, B) compared to the virus control group (P < 0.05). 2) Effects of the Tibetan zone binding formulation on cytokine expression in infected mice: compared with the virus control group, the IL-6 in the traditional Chinese and Tibetan medicine combined prescription is significantly reduced (P < 0.05), and the IL-10 is significantly increased (P < 0.05); the TNF-alpha in the middle and low dosage groups is extremely obviously reduced (P < 0.01) and the TNF-gamma is extremely obviously increased (P < 0.01) in the combination prescription of the traditional Chinese medicine and the Tibetan medicine; the high dose group of traditional Chinese and Tibetan medicine combined prescription has obviously raised TNF-gamma (P < 0.05). 3) Influence of Tibetan medicine combination prescription on antioxidant capacity of infected mice: compared with the virus control group, the SOD of the traditional Chinese and Tibetan medicine combined prescription and the SOD of the low-dose group are obviously increased (P < 0.01), the SOD of the traditional Chinese and Tibetan medicine combined prescription and the SOD of the high-dose group are obviously increased (P < 0.05), and the MDA of each medicine group is obviously reduced (P < 0.01).

In summary, in vivo and in vitro experiments show that the Tibetan medicine combination prescription has remarkable inhibition effect on novel coronavirus infection. The traditional Chinese and Tibetan medicine combined prescription has a certain antiviral function in the aspects of regulating the immune function and cytokine secretion of infected mice, relieving peroxidation stress injury caused by coronaviruses and the like.

Drawings

FIG. 1 is a technical route diagram for preparing the Honglian lung-heat clearing granule of the invention. Wherein the dashed box represents a hundred thousand grade denuded zone.

FIG. 2 shows the effect of the Tibetan medicine combination formula preparation on the pathological morphology of the lung tissue of a BALB/c mouse infected with coronavirus HCoV-OC43 in the preferred embodiment of the invention. A: blank control group, B: virus control group, C: adefovir group, D: dosage group in the formulation of the combination of traditional Chinese and Tibetan medicine, E: high dose group of traditional Chinese and Tibetan medicine combination formula preparation, F: low dose group of the combination formulation of traditional chinese and Tibetan medicine, scale bar = 100 μm.

FIG. 3 shows morphological changes of epidemic virus infection of cells according to the preferred embodiment of the present invention. A: blank control cells, B: coronaviruses infect MIJCK cells.

Fig. 4 is a schematic diagram showing the toxic effects of the Tibetan medicine combination formula on MDCK cells in the preferred embodiment of the invention. A: control cells, B: the Tibetan medicine combination formula preparation (1250 mug/mL) treats group cells, C: the Tibetan medicine combination formula preparation (2500 mug/mL) treats the group cells.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.

Example 1 pharmaceutical composition for preventing and treating novel coronavirus and process for preparing the same

The present embodiment provides a pharmaceutical composition for preventing and treating a novel coronavirus.

A. Dosage form selection and prescription determination

The recipe is a novel recipe for patients with coronavirus infection, which hurts the stomach, does not take capsules, tablets and the like directly, is not easy to take dosage forms which are easy to damage the spleen and the stomach, and is also not easy to take sugar-containing medicament for patients with diabetes. Therefore, the product can be better taken by kidney patients, brings good news to the majority of diabetes patients, and selects sugar-free granular preparation.

Prescription: 224g of astragalus, 56g of rhizoma anemarrhenae, 168g of rhodiola rosea, 168g of ageratum, 112g of Tibetan schizonepeta, 224g of roasted loquat leaf, 168g of fructus forsythiae, 56g of Tibetan saussurea, 56g of amur corktree bark and a proper amount of auxiliary materials.

B. Selection of a process route

The astragalus mainly contains astragalus polysaccharide, astragalus saponin, amino acid and various trace elements, and has the functions of invigorating qi, supporting yang, strengthening exterior, arresting sweating, inducing diuresis and relieving edema. Cortex Phellodendri mainly contains berberine, which is an isokaline alkaloid, and has antiinflammatory and analgesic effects. Pharmacological studies show that the berberine can reduce the infiltration of mononuclear macrophages in lung tissues, especially M1 type mononuclear macrophages, through inhibiting the activation of NF- κB signaling pathway, and inhibit inflammatory reaction, thereby reducing chronic kidney injury of mice induced by fat-free diet, and reducing the micro-autoprotein excretion of urine of type II diabetics in 24 hours, and the effects of the berberine are possibly related to the blood glucose reduction, lipid regulation, insulin resistance improvement and the like of the berberine. The rhizoma anemarrhenae polysaccharide, saponin and aglycone contained in rhizoma anemarrhenae have pharmacological activities of resisting bacteria and inflammation, lowering blood pressure, lowering blood sugar and blood lipid, etc., so as to protect damaged tissues. The effective components such as the saponin in the Tibetan herba schizonepetae and the ageratum can be used for resisting bacteria, diminishing inflammation, activating blood and protecting kidney tissue cells. The rhodiola rosea contains various chemical components such as flavonoid compounds, can regulate nonspecific immunity, can strengthen body immunity, and can improve disease resistance of human body. Therefore, the extraction process of the product aims at making the following processes with the aim of extracting the active ingredients to the maximum extent:

The astragalus root, the rhizoma anemarrhenae and other medicinal materials are firstly extracted by alcohol for 2 times and then extracted by water, firstly, the effective components such as astragaloside IV, timosaponin, mangiferin, berberine, ferulic acid and the like can be extracted with high efficiency, and secondly, the combined extraction effect of the astragalus root on the rhizoma anemarrhenae and the cortex phellodendri can be exerted. The preparation process uses dextrin as filler, lactose and mannitol as filler and corrective, and the granule is prepared.

C. Selection of process conditions

1. Identification and pretreatment of medicinal materials

Radix astragali, rhizoma anemarrhenae, radix Rhodiolae, herba Agastaches, herba Schizonepetae, folium Eriobotryae Preparata, fructus forsythiae, herba Saussureae Involueratae, and cortex Phellodendri according to the related specification of one part of the pharmacopoeia of the people's republic of China 2015. All the nine medicinal materials are medicinal decoction pieces, pretreatment is not needed before extraction, and the medicinal decoction pieces are directly added.

2. Measurement of index component

2.1 method for measuring astragaloside IV content

And (3) spectrum inclusion condition and system adaptability test, namely, octadecylsilane chemically bonded silica is used as a filler, acetonitrile-water (36:64, volume ratio) is used as a mobile phase, an evaporative light scattering detector detects the mixture, and the theoretical plate number is not lower than 3000 according to astragaloside IV peak.

Preparation of a control solution: taking appropriate amount of astragaloside IV reference substance, precisely weighing, and adding methanol to obtain solution containing 0.4mg per 1 mL.

Preparation of test solution: taking about 2g of powder, precisely weighing, adding 20mL of water, slightly heating to dissolve, shaking and extracting with water saturated n-butanol for 4 times, 30mL each time, combining n-butanol solutions, fully washing with ammonia test solution for 2 times, 30mL each time, discarding ammonia solution, evaporating n-butanol solution to dryness, dissolving residues with methanol, quantitatively transferring to a 10mL measuring flask, adding methanol to scale, shaking, filtering, and taking the subsequent filtrate as a sample solution.

Assay: precisely sucking 5 mu L of reference substance solution and 10 mu L of test substance solution, respectively, injecting 10-20 mu L of test substance solution into a liquid chromatograph, measuring, and calculating by using an external standard two-point logarithmic equation to obtain the final product.

2.2 method for measuring berberine content

Octadecylsilane chemically bonded silica is used as a filler for chromatographic conditions and system adaptability tests: mobile phase: acetonitrile-0.1% phosphoric acid [ (0.1 g of sodium dodecyl sulfonate per 100 mL) 50:50] as phase A, acetonitrile as phase B, A-B (79:21); the detection wavelength is 265nm; column temperature 40' C; the flow rate is 1.0mL/min; theoretical plate number: calculated according to berberine hydrochloride peak not less than 2800.

Preparation of a control solution: taking a proper amount of berberis hydrochloride Fan Jian reference substance, precisely weighing, adding 70% methanol solution containing 0.2% hydrochloric acid to prepare a solution containing 16 mug per 1mL, and shaking uniformly to obtain the final product.

Preparation of test solution: taking 0.6g of powder, precisely weighing, placing into a 100mL measuring flask, adding mobile phase to scale, shaking, filtering, precisely sucking 3mL of continuous filtrate, placing into a 10mL measuring flask, adding mobile phase to scale, shaking, filtering, and collecting continuous filtrate.

Assay: respectively precisely sucking 10 μl of each of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.

2.3 timosaponin B II

Chromatographic conditions and system suitability test: octyl silane bonded silica gel is used as a filler; acetonitrile-water (25:75) as mobile phase; the evaporative light scattering detector detects. The theoretical plate number is not less than 10000 calculated according to timosaponin B II peak.

Preparation of a control solution: taking a proper amount of timosaponin B II reference substance, precisely weighing, adding 30% acetone to prepare a solution containing 0.50mg per 1 mL.

Preparing a test sample solution: about 1g of powder is taken, precisely weighed, placed in a conical flask with a plug, precisely added with 25mL of 30% acetone, weighed, subjected to ultrasonic treatment (power 400W, frequency 40 kHz) for 30 minutes, taken out, cooled, weighed again, complemented with 30% acetone to the lost weight, and shaken well. Filtering, and collecting the subsequent filtrate.

Assay: precisely sucking 5-10 μl of reference solution and sample solution, respectively, and injecting into liquid chromatograph, measuring, and calculating by external standard two-point logarithmic equation.

2.4 polysaccharide content determination method

The preparation of the test solution comprises precisely measuring lmL of mixed solution No. 1-9 with a constant volume of 100mL, respectively placing into 10mL centrifuge tubes, adding 6mL of ethanol, mixing, centrifuging at 4000rpm for 5min, and discarding supernatant. Adding ethanol to the precipitate for 6mL washing, centrifuging, removing supernatant, adding water to the precipitate, quantitatively transferring to a 25mL measuring flask, dissolving, filtering, and taking the subsequent filtrate as a sample solution of polysaccharide.

Glucose standard solution preparation: precisely weighing 10 tons of D (+) -anhydrous glucose reference substance dried at 105 ℃ for 6 hours, placing in a 100m1 measuring flask, adding water to the scale, and shaking uniformly to obtain the D (+) -anhydrous glucose reference substance.

Preparing a 5% phenol solution: 100g of trypan is taken, 0.1g of aluminum sheet and 0.05g of sodium bicarbonate are added, distillation is carried out, 182 ℃ fractions are collected, 5g of the obtained product is weighed, 100mL of water is added for dissolution, and the obtained product is placed in a brown bottle and put in a refrigerator for standby.

Standard curve: precisely measuring 0.2,0.4,0.6,0.8 and 1.0mL of D (+) -anhydrous glucose reference substance (0.l mg/mL), placing into a dry scale test tube, respectively adding water to 2mL, adding 5% phenol to 2mL, adding 7mL of concentrated sulfuric acid, fully shaking, standing at room temperature for 25 minutes, accompanying with blank, and measuring absorbance at 490 nm. And drawing a standard curve and calculating a regression equation.

Measuring according to ultraviolet spectrophotometry (appendix VA of 2010 edition of Chinese pharmacopoeia), precisely sucking 0.4mL of the prepared polysaccharide sample solution, respectively adding water to 2mL, adding 5% phenol to 2mL, rapidly adding 7mL of concentrated sulfuric acid, fully shaking, standing at room temperature for 25 minutes, and measuring absorbance at a wavelength of 490 nm. And calculating the polysaccharide content in 9 samples by adopting a regression equation under a standard curve term.

D. Preliminary stability study

In order to ensure the effectiveness and safety of clinical medication, the law of the change of the preparation along with time under the influence of temperature and humidity is inspected, a basis is provided for production and packaging, and long-term test and accelerated test inspection are performed.

1. Long-term test: three batches of samples were subjected to stability studies under the conditions of packaging to be marketed, and placed at a temperature of 25 ℃ + -2 ℃ and a relative humidity of 60% + -5%. And (5) examining the change conditions of the properties, the identification, the inspection, the content, the microorganism limit and the like of the sample in the placing process. The initial measurement results were taken as 0 month test data, and each index was measured once a year after sampling at the end of 3 rd, 6 th, 9 th, 12 th, 18 th and 24 th months of the test period. The stability study was currently performed for 15 months, after which the long-term stability test of the existing batch samples was continued until the recommended expiration date.

2. Acceleration test: three batches of samples were subjected to stability studies under the conditions of packaging to be marketed, and placed at a temperature of 40 ℃ + -2 ℃ and a relative humidity of 75% + -5%. Stability studies were performed for 6 months. And (5) examining the change conditions of the properties, the identification, the inspection, the content, the microorganism limit and the like of the sample in the placing process. The initial measurement results were used as 0 month test data, and each index was measured by sampling once at the end of 1 st, 2 nd, 3 rd and 6 th months of the test period.

The results show that the stability of the product is good in comparison with 0 month in the investigation period, regardless of the long-term test or the acceleration test. According to the result of the accelerated stability test, the validity period of the product is predicted to be about two years.

E. Pharmacological and toxicological studies

1. Pharmacodynamic test

SPF female BALB/c mice of 6-8 weeks old, 300, 18-22 g, purchased from Experimental animal research center, hubei province, license number SCXK E2020-0018.BALB/c mice were fed back into the barrier system, kept at 50-70% humidity, fed adaptively for 5 days, and started to enter the formal test.

Experimental grouping:

90 female BALB/C mice were cut for adaptive feeding, weighed and randomly divided into 6 groups of 15, 5/cage. The 6 groups are air-self control group, virus control group and Ruidexivir control group respectively, and the combination formula of the traditional Chinese medicine and the Tibetan medicine is high, medium and low dose groups. The grouping and stomach filling dosage experiment method comprises the following steps:

After the traditional Chinese and Tibetan medicine combination preparation is continuously infused with stomach for 2 days, the blank control group is dripped with 50 mu L of PBS solution per unit, and the concentration of coronavirus challenge experiments of other groups is 10LD ₅₀ 50. Mu.L. Starting to irrigate the stomach after 4 hours from virus infection, and then continuously abusing for 5 days, wherein the blank control group and the model control group are irrigated with physiological saline which is equal to the traditional Chinese and Tibetan medicine combined preparation in volume, and the time and the mode of the lavage are the same as those of the traditional Chinese and Tibetan medicine combined preparation; the daily gastric lavage dose of the Ruidexiwei group is 100g/kg BW, and the time and the mode of the gastric lavage are the same as those of the Tibetan medicine combination formula preparation. The stomach was irrigated 1 time/d, 0.2mL/10gBW. The results show that the results of the traditional Chinese and Tibetan medicine combination formula for the change of lung index of mice infected with coronavirus HCoV-OC43 after 5d show that: the lung index of the virus control group was extremely significantly elevated compared to the blank group (p<0.01). Compared with the virus control group, the lung index of the dosage group in the Redewei group and the traditional Chinese and Tibetan medicine combination formula preparation is extremely obviously reduced (p<0.01 The lung index of the low-dose group of the traditional Chinese and Tibetan medicine combination formula preparation and the high-dose group of the traditional Chinese and Tibetan medicine combination formula preparation is obviously reduced (p is less than 0.05); hemagglutination titers in lung tissue after coronavirus HCoV-OC43 infection of mice were determined using the Hemagglutination Assay (HA), reflecting the replication of the coronavirus. After 5d of medicines with different doses are continuously infused into the stomach, compared with a virus control group, the pulmonary tissue hemagglutination titer of the mice in the medicine group is extremely obviously reduced (p is less than 0.01), which indicates that each group of medicines has different degrees of effect of inhibiting coronavirus, the blood coagulation titer of the Ruidexiwei group is the lowest, and the effect of inhibiting coronavirus is the best. Traditional Chinese and Tibetan medicine combination preparation dose of each pharmaceutical composition blood coagulation titer The group is lower than the high-dose group and the low-dose group, which indicates that the anti-coronavirus group effect of the dose group in the traditional Chinese and Tibetan medicine combination formula preparation is better in the three dose groups, and the pathological morphology results of the traditional Chinese and Tibetan medicine combination formula preparation on mice infected with coronavirus HCoV-OC43 after 5d show that: the HE staining observes the lung tissue structure of the normal group mice, and the terminal bronchiole part extends to the periphery to form a respiratory bronchiole, and then branches into alveolar ducts (acinus) and alveoli, so that the bronchiole structure at each stage is clear, and inflammatory cells which infiltrate in the alveolar tissue are basically not exuded. Alveolar wall capillaries are packed with red blood cells. The peribronchial spaces of each stage are pulmonary arteries, pulmonary veins, lymphoid tissue and loose connective tissue (a in fig. 2). Most of the lung tissues of the virus control group had damaged alveolar structure, some of the lung tissue solid variable regions fused, the alveoli thickened, the alveolar space and interstitial endoinflammatory cells diffused, the lesions were heavier, the capillaries were engorged, the alveoli destroyed, and inflammatory cell infiltration (B in fig. 2). The adefovir dipivoxil group was infused for 7 days continuously, which significantly improved the inflammatory lesion degree of the lung, significantly reduced or resolved inflammatory cell infiltration, and approached the normal lung tissue structure (C in FIG. 2). After the traditional Chinese and Tibetan medicine combined preparation is continuously infused for 7 days in different dosage groups, the lesions of lung tissues are improved to different degrees, and the inflammatory cells of the lung tissues are obviously reduced in about 1/2 of the dosage groups and congestion bleeding is not seen in the slice photographs (D in figure). The effect after low dose treatment was about 1/3 of the lung tissue inflammatory cells were significantly reduced, the remaining lung tissue inflammatory cells were reduced (F in fig. 2). The high dose group showed about 1/3 of the lung tissue inflammatory cells significantly decreased, and the inhibition was less pronounced than the medium dose group (E in FIG. 2).

The effect of the Tibetan formulation on the lung index and the lung index inhibition rate of novel coronavirus infected BALB/c mice is shown in Table 1.

Table 1 Effect of Tibetan formulations on the lung index and the inhibition rate of the lung index in novel coronavirus infected BALB/C mice

Injecting; the AAP of the virus control group is less than 0.01 compared with the blank control group; each drug group had a P < 0.01P < 0.05 compared to the virus control group

The result shows that the effect test of the Tibetan-to-Chinese-medicinal preparation on pneumonia caused by coronavirus HCoV-OC43 infection of BALB/c mice proves that after the Tibetan-to-Chinese-medicinal preparation is used for treating the infected mice, the average lung index and the average hemagglutination titer are reduced, and the pathological morphology of lung tissues is obviously improved. The Tibetan medicine combination formula preparation is shown to alleviate the lung infection caused by coronavirus by inhibiting the proliferation of coronavirus in the lung.

Effects of the Tibetan medicine combination formula preparation on the hemagglutination titer of coronavirus HCoV-OC43 in lung tissue of infected mice: hemagglutination titers in lung tissue after coronavirus HCoV-OC43 infection of mice were determined using the Hemagglutination Assay (HA), reflecting the replication of the coronavirus. After 5d of medicines with different doses are continuously infused into the stomach, compared with a virus control group, the pulmonary tissue hemagglutination titer of the mice in the medicine group is extremely obviously reduced (p is less than 0.01), which indicates that each group of medicines has different degrees of effect of inhibiting coronavirus, the blood coagulation titer of the Ruidexiwei group is the lowest, and the effect of inhibiting coronavirus is the best. The middle dose group of the blood coagulation titer of each medicine group of the traditional Chinese and Tibetan medicine combined preparation is lower than that of the high dose group and the low dose group, which indicates that the anti-coronavirus group effect of the dose group of the traditional Chinese and Tibetan medicine combined preparation is better in the three dose groups. The results are shown in Table 2.

Table 2 Effect of Tibetan medicine combination formula preparation on hemagglutination titres in coronavirus infected BALB/C mouse lung tissue

Note that: p < 0.01 for each drug group compared to the virus control group

2. Drug toxicity test

2.1 acute toxicity test

Experimental animals and groupings: the BALB/c mice of 40 females are taken, adaptively fed for 5 days, weighed and randomly divided into 4 groups, and 10 groups are divided into a blank control group and a high, medium and low (1.5 g/kgBW,0.75g/kgBW and 0.375 g/kgBW) dosage group of the traditional Chinese and Tibetan medicine combination formula preparation, and the high, medium and low dosage concentrations are respectively 75mg/ml,37.5mg/ml and 18.8mg/ml. BALB/c mice were given different concentrations of the traditional Chinese and Tibetan medicine combination formula preparation by gavage for a fixed time every day, 0.2ml/10gBW, 7d by continuous gavage, equal volumes of deionized water by stomach nourishment in the control group, 14d by daily observation of clinical manifestations, weighing every week, no water forbidden for 12-16 hours before the end of the test, blood collection for blood biochemistry (ALT, AST, BUN and CRE) and blood routine detection. The results are shown in Table 3.

During the experimental results, the weight gain and mental status of mice and SD rats were normal, and none of the mice died.

The Tibetan medicine combination formula in Table 3 changes the weight of each group

Note that: p > 0.05 compared with the blank group

The preparation of the combination formula of traditional Chinese and Tibetan medicine is prepared by continuously lavaging different doses of preparation 7d of mice, and after 14d of observation, blood is taken for biochemical detection. Compared with the blank control group, the blood biochemical indexes of the traditional Chinese and Tibetan medicine combination formula preparation are not obviously different (p is more than 0.05). The results are shown in Table 4.

Blood biochemical index changes of each group of Tibetan medicine combination formula preparation in Table 4

Note that: p > 0.05 compared with the blank group

The Tibetan medicine combination formula preparation is prepared by continuously lavaging different doses of preparations 7d of mice, and blood is collected for routine blood detection after 14d observation. Compared with the blank control group, the blood routine index of each group of the traditional Chinese and Tibetan medicine combination formula preparation has no obvious difference (p is more than 0.05). The results are shown in Table 5.

Blood routine index changes for each group of Tibetan medicine combination formula preparation in Table 5

Note that: p > 0.05 compared with the blank group

Conclusion: the dosage of the mice and SD rats is 3.54g/Kg/d, the dosage of the Honglian lung-heat clearing granule is 0.3g/Kg/d, and the dosage of the mice for acute toxicity test is 118 times of clinical medication silty according to the weight, which indicates that the acute toxicity of the drug is low and the drug is clinical safe.

3. Long-term toxicity test

80 SD rats (male and female halves) with the weight of about 220g are tested;

experimental grouping: the high dose group, the medium dose group, the low dose group and the control group are 10 female SD rats and 10 female SD rats;

Experimental samples: the high dose group, the medium dose group and the low dose group are respectively filled with red-link lung-heat clearing granule solutions with different concentrations, and the contrast group is filled with physiological saline;

the experimental method comprises the following steps: preparing 1-3 of medicinal liquid with active ingredient concentration of 0.08g/mL, 0.04g/mL and 0.01g/mL respectively, and respectively feeding the medicinal liquid into a high-dose group, a medium-dose group and a low-dose group according to the dosage of 0.2mL/20g.Bw for 1-3,2 times/day, and continuously feeding for 60 days, wherein the dosage of the medicinal liquid from the dose group, the medium-dose group and the low-dose group is 1.6g/Kg/d, 0.8g/Kg/d and 0.4g/Kg/d respectively; the control group was fed with the same amount of physiological saline in the same manner. The animals were observed for growth and general behavior during dosing. The next day of the last administration, 6 animals (male and female half) are sacrificed in each group, and viscera coefficient measurement, urine biochemical examination, blood biochemical and pathological histology examination are carried out; the remaining 4 animals were stopped and observed for two weeks to see how reversible the toxic response was and the possible delayed toxicity, and the results are shown in tables 6-8.

The test results show that the basic signs of body weight, body temperature, hair and the like of rats in each group are normal during the administration and withdrawal observation period, the mental state is good, abnormal behaviors do not occur, and no mice die during the test period; no delayed toxicity occurred during withdrawal recovery. The urine biochemical examination and the blood biochemical examination of each dosage group are in a normal range, no obvious difference exists, and the organ coefficient measurement and the pathological tissue examination of each group are not obvious abnormal.

Influence of Tibetan medicine combination formula preparation on physiological index of long-term toxicity test of rats in Table 6

Table 7 Effect of Tibetan medicine combination formula preparation on long-term toxicity test urine protein of rats (mg/24 h)

Table 8 shows the results of biochemical tests of long-term toxicity blood of Tibetan medicine combination formula preparation on rats

The long-term toxicity test research proves that in the long-term toxicity test, the traditional Chinese and Tibetan medicine combination preparation has no delayed toxicity and no obvious toxic or side effect on kidney and cardiac muscle, and the clinical dosage of the traditional Chinese and Tibetan medicine combination preparation is very safe.

Example 2 in vitro efficacy test of Tibetan medicine in combination with clinical prescription for inhibiting HCoV-OC43 coronavirus infection

1.1 materials

1.1.1 medicaments

The clinical prescription is provided by Shijia Tibetan noro pharmaceutical industry Co., ltd, and the test drug is brown formula particles prepared according to the compound preparation dosage of example 1;

control drug adefovir: purchased from Target Mol company, U.S.;

thiazole blue (MTT) and dimethyl sulfoxide mock (DMSO) were purchased from sigma company; DEME medium, membrane protease and fetal bovine serum were purchased from Hyclone company, usa.

1.1.2 cells and viruses

Coronavirus HCoV-OC43 was cryopreserved by an affiliated Hospital of the university of science and technology, china;

Canine kidney epithelial cells (MDCK) were maintained by the institute of combined chinese and western medicine, university of china and sciences, and passaged 1 time for 2-3 d.

1.2 method

1.2.1 pharmaceutical formulation

1.2.1.1A Tibetan medicinal preparation

1g of the Tibetan medicine combination formula preparation brown particles are weighed by an analytical balance and placed in a 100mL beaker, then 20mL of deionized water at 100 ℃ is added into the beaker, after rapid and uniform stirring, 80mL of hot water is added, stirring is continued until the mixture is fully dissolved, cooling and filtering sterilization are carried out, and then a preparation solution with the concentration of 10000 mug/mL is obtained, and is used as a mother solution to be stored at 4 ℃ and diluted to the required concentration.

1.2.1.2 Rede West formulation

Weighing 0.1g of Ruidexi Wei Yu mL of PE pipe by an analytical balance, adding 80mL of water for injection, uniformly mixing and dissolving to obtain 1250 mu g/mL of Ruidexi-vir sterile solution, and then diluting to the required concentration to obtain the product.

1.2.2MTT method for determining half-Toxicity Concentration (TC) of Tibetan medicine combination formula preparation on MDCK cells ₅₀ )

MDCK cells were inoculated into 96-well plates with 200. Mu.L of cells in each well, 2X 10 cells ⁴ Individual wells/well, 37 ℃,5% co ₂ Culturing for 24h, and growing the cells into a monolayer. The supernatant was aspirated, and 10000, 5000, 2500, 1250, 625, 312.5, 156.25, 78.125, 39.062. Mu.g/mL of each of the Tibetan medicine combination formula preparations was added, and cell controls (cells+medium) were set in each of 4 wells. Let Ruidexivir be the positive control drug with concentration gradients of 312.5, 156.25, 78.125 and 39.062 mug/mL, 4 holes per concentration, and end the test after 72h observation Testing, adding MTT for 4h, absorbing supernatant, adding DMSO for dissolving for 0.5h, measuring OD570nm absorption value by using an enzyme-linked detector, performing Probit regression analysis on data by using statistical software SPSS13.0, and calculating TC of the medicine ₅₀ 。

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1.2.3 Cytopathic (CPE) method to determine the half-size infection (TCID) of viruses on MDCK cells ₅₀ )

Coronavirus HCoV-OC43 was passaged on MDCK cells and cytopathic observed, and after apparent lesions were found and stabilized for 3 passages, TCID of the virus was determined ₅₀ MDCK cells were seeded in two 96-well plates at 200. Mu.L each containing 2X 10 cells ⁴ Culturing at 37deg.C for 24 hr to form single layer, diluting a virus solution with culture solution 10 times for 10 times, respectively, for each 96-well plate ^-1 ～10 ^-10 10 total concentrations were added to the first 10 rows of cell wells, 8 wells per concentration, and the last two rows were used as cell control wells, with only cell maintenance fluid, 37℃and 5% CO ₂ Culturing in an incubator. Cytopathy was observed daily with an inverted microscope. Cytopathic extent was recorded and the test was ended when the virus control wells were 80-90% CPE. Reed-Muench method for calculating TCID of coronavirus ₅₀ 。

1.2.4 in vitro efficacy test of Tibetan medicine combination formula preparation for inhibiting coronavirus HCoV-OC43 infection

1.2.4.1 inhibition of replication and proliferation of coronavirus HCoV-OC43 after adsorption by Tibetan medicine combination formula preparation

MDCK cells were seeded in two 96-well plates at 200. Mu.L each containing 2X 10 cells ⁴ Individual wells/well, 37 ℃,5% co ₂ Culturing for 24h, and growing the cells into a monolayer. Two 96-well plates were each loaded with 100. Mu.L (100 TCID ₅₀ ) After adsorption of the coronavirus in lh, the supernatant was discarded, and then 1250, 625, 312.5, 156.25, 78.125, 39.062. Mu.g/mL of the Tibetan medicine combination formula preparation was added, 100. Mu.L per well, 4 wells per concentration, respectively. Simultaneously, virus control (cytochalasin+culture solution), cell control (cell+culture solution) and preparation control of the combination formula of traditional Chinese medicine and Tibetan medicine (1250 mug/mL preparation)+ cells + culture medium), and adefovir as a positive control drug, with concentration gradients of 312.5, 156.25, 78.125, 39.062 μg/mL, 4 wells per concentration. Cell morphology changes were observed daily, cytopathic effect was recorded when virus control wells were diseased, and the test was ended when virus control wells CPE were 80% -90%. MTT staining method for cell activity. The test was repeated 3 times each. The data were subjected to Probit regression analysis using statistical software SPSS13.0, and the median Effective Concentration (EC) of the drug was calculated separately ₅₀ ). Using Therapeutic Index (TI) as evaluation index for evaluating replication and proliferation effect of traditional Chinese and Tibetan medicine combination preparation after adsorbing coronavirus, TI=TC ₅₀ /EC ₅₀ . The method for calculating the virus inhibition rate comprises the following steps:

1.2.4.2 prevention of coronavirus HCoV-OC43 infection by Tibetan medicine combination formula preparation

MDCK cells were seeded in two 96-well plates at 200. Mu.L each containing 2X 10 cells ⁴ Individual wells/well, 37 ℃,5% co ₂ Culturing for 24h, removing supernatant, and adding 1250, 625, 312.5, 156.25, 78.125, 39.062 μg/mL of Tibetan medicine combination formula preparation into two 96-well plates respectively, wherein each well is 100 μl, and each concentration is 4 wells. After 24h of pre-interaction with the cells, the supernatant was discarded, washed 2 times with PBS, and 100TCID was added separately ₅₀ Is 100uL of virus liquid. After adsorption of lh, the supernatant was discarded, and 100 μl of cell maintenance solution containing 2% fbs was added per well. The test was performed in a normal cell control group and a positive drug group and a virus control group. Cell morphology changes were observed daily, cytopathic effect was recorded, and when the virus control wells CPE were 80% -90%, the assay was ended and the cell activity was determined by MTT staining. The test was repeated 3 times each. The data were subjected to Probit regression analysis using statistical software SPSS13.0 to calculate drug EC, respectively ₅₀ . Calculate ti=tc ₅₀ /EC ₅₀ . The prevention effect of the Tibetan medicine combination formula preparation on coronaviruses is observed.

1.2.4.3 direct inactivation of Tibetan medicine combination formula infection

MDCK cells were seeded in two 96-well plates at 200. Mu.L each containing 2X 10 cells ⁴ Individual wells/well, 37 ℃,5% co ₂ Culturing for 24h, and growing the cells into a monolayer. Discarding supernatant, and respectively mixing 2 times of medicine with equal volume of 200TCID ₅₀ Coronavirus was mixed well in sterile EP tubes, 37℃at 5% CO ₂ After 1h of incubator action, the mixed solution was inoculated to the cell plates, respectively. Normal cell control group, positive drug and virus control were established. Adsorbing for 1 hr, removing supernatant, adding cell maintenance solution, 37deg.C, 5% CO ₂ The incubator continues to cultivate. Cell morphology changes were observed daily, cytopathic effect was recorded, and when the virus control wells CPE were 80% -90%, the assay was ended and the cell activity was determined by MTT staining. The test was repeated 3 times each. The data were subjected to Probit regression analysis using statistical software SPSS13.0 to calculate drug EC, respectively ₅₀ . Calculate ti=tc ₅₀ /EC ₅₀ . Direct inactivation of coronavirus HCoV-OC43 by Tibetan medicine combination formula preparation was observed.

1.3 experimental results

1.3.1 TCID of coronavirus HCoV-OC43 against MDCK cells ₅₀

Lesions were observed after infection of cells with coronavirus HCoV-OC 43: cells become trapped, vacuoles appear, the cell refraction is enhanced, cells are expanded, collapsed, star-shaped aggregated into clusters, fall off, nuclei are solidified and disintegrated, and finally the whole cells are broken into fragments, while MDCK cells in control wells are in the form of shuttle-shaped or polygonal epithelial cells, grow well, are closely arranged, and the culture solution is transparent and clear (fig. 3). TCID of coronavirus HCoV-OC43 ₅₀ Is 1v ^-4.12 /100μL。

1.3.2 half-maximal toxic concentration of coronavirus HCoV-OC43 on MDCK cells (TCID) ₅₀ ) Is (are) determined by

In the experiment, the concentration of the traditional Chinese and Tibetan medicine combination formula preparation is more than or equal to 2500 mug/mL, the cell proliferation is slow, the refractive index is changed, swelling and rounding are carried out, part of cells are reduced in wall attaching capability, broken and shed, intracellular particles are increased, the concentration of the traditional Chinese and Tibetan medicine combination formula preparation with very low cell survival rate is less than or equal to 1250 mug/mL, the difference between the cell survival rate and the cell growth of a control group is small after 72 hours, and the final survival rate of the cells is more than 75 percent. When the concentration of the adefovir dipivoxil is 312.5 mug/mL, the cell survival rate is more than or equal to 80%, and the maximum nontoxic concentration of the adefovir is more than 312.5 mug/mL. SPSS13.0 data were subjected to a inhibition regression analysis with a drug half toxicity concentration TCID50 of 1458. Mu.g/mL on MDCK cells. The effect of the formulation of the combination of traditional Chinese and Tibetan medicine on the cytotoxicity is shown in Table 9 and FIG. 4.

Table 9 results of drug toxicity to MDCK

1.3.3 in vitro therapeutic results of Tibetan medicine combination formula preparation for inhibiting coronavirus HCoV-OC43 infection

Results of inhibition of replication and proliferation of coronavirus HCoV-OC43 by the combination of traditional Chinese and Tibetan medicine preparation

In the safety range of the medicine, the inhibition rate of the medicine against coronavirus is enhanced along with the increase of the medicine concentration, and the medicine has a certain dose-effect relationship, and both the Tibetan medicine combination formula preparation and the Ruidexivir have obvious effect of inhibiting the replication and proliferation after the adsorption of the coronavirus.

When the virus infected cells without the drug show obvious CPE, the cell survival number and the growth state of the drug holes at each concentration are different, but the cells are obviously more than those of the virus control holes, and when the concentration of the Tibetan medicine combination formula preparation is higher than 312.5 mug/mL and the concentration of the Ruidexi Wei Gaoyu 78.12.12 mug/mL, more than 50% of the cells survive in the visual field. Half of the effective concentrations of 3 test replicates for the combination of traditional Chinese and Tibetan medicine formulation were 197.4 μg/mL, 206.7 μg/mL and 232.3 μg/mL, with a Therapeutic Index (TI) of 7.5; the half-effective concentrations of 3 trials of adefovir were 62.3 μg/mL, 71.3 μg/mL and 76.6 μg/mL with a Therapeutic Index (TI) of > 4.5. The results are shown in tables 10 and 11.

Inhibition of proliferation after adsorption of coronavirus HCOV-OC43 by Tibetan medicine combination formula in Table 10

Therapeutic index of Tibetan medicine combination formula preparation on replication and proliferation after adsorption of coronavirus HCOV-OC43 in Table 11

Results of the preventive action of the Combined preparation of the traditional Chinese and Tibetan medicine on the infection of coronavirus HCoV-OC43

In the safety range of the medicine, the OD value is increased along with the increase of the medicine concentration, and the inhibition rate of coronavirus is increased, so that the traditional Chinese and Tibetan medicine combination preparation and the Ruidexivir have obvious effect of preventing the coronavirus.

When the virus infected cells without the drug show obvious CPE, the cell growth of the drug holes with different concentrations is complete, but the cell growth is obviously more than that of the virus control holes, and when the concentration of the Tibetan medicine combination formula preparation is higher than 312.5 mug/mL and the concentration of the Ruidexi Wei Gaoyu 78.12.12 mug/mL, more than half of cells survive in the visual field. Half of the effective concentrations of 3 test replicates for the combination of traditional Chinese and Tibetan medicine formulation were 209.5 μg/mL, 186.8 μg/mL, and 235.5 μg/mL, with a Therapeutic Index (TI) of 7.6; half-effective concentrations of 3 trials of adefovir were 78.9 μg/mL, 73.3 μg/mL and 62.6 μg/mL with a Therapeutic Index (TI) of > 4.4. The results are shown in Table 12 and Table 13.

Prevention effect of Tibetan medicine combination formula preparation in Table 12 on coronavirus HCOV-OC43 infection/>

Therapeutic index of the combined Tibetan medicine formulations in Table 13 for the prevention and treatment of coronavirus HCOV-OC43 infection

Direct inactivation results of the Combined preparation of the traditional Chinese and Tibetan medicine on coronavirus HCoV-OC43 infection

Within the safe range of drug concentration, the inhibition rate of the traditional Chinese and Tibetan medicine combination formula preparation and the Ruidexivir to the coronavirus HCoV-OC43 is enhanced along with the increase of the concentration of the traditional Chinese and Tibetan medicine combination formula preparation. When obvious CPE appears in the virus control hole after the medicine is given, the cell growth of each concentration medicine group shows different states and numbers, but the cell growth is obviously more than that of the virus control hole, and when the concentration of the Tibetan medicine combination formula preparation is higher than 312.5 mug/mL and the concentration of the Ruidexi Wei Gaoyu 78.12.12 mug/mL, more than 50% of cells survive. The half-effective concentration of the traditional Chinese and Tibetan medicine combination formula preparation for 3 test replicates is 257.5 mug/mL, 225.8 mug/mL and 237.7 mug/mL, the Therapeutic Index (TI) is 6.6, the half-effective concentration of the Ruidexivir for 3 test replicates is 63.4ug/mL, 71.4 mug/mL and 74.5 mug/mL, and the Therapeutic Index (TI) is > 4.5. The results are shown in tables 14 and 15.

Direct inactivation of coronavirus HCOV-OC43 infection by Tibetan medicine combination preparation in Table 14 />

Therapeutic index of Tibetan medicine combination formula preparation in Table 15 on coronavirus HCOV-OC43 infection

1.4 statistical analysis

The CPE method and the MTT method are combined with microscopic observation, and the traditional Chinese and Tibetan medicine combination formula preparation has obvious effects of preventing the adsorption of the coronavirus HCoV-OC43 and directly killing the coronavirus on the replication and proliferation effect of the coronavirus HCoV-OC43 after the adsorption.

EXAMPLE 3 in vivo efficacy test of Tibetan medicine combination formula preparation for inhibiting coronavirus HCoV-OC43 infected mice

The in-vivo animal test reflects the antiviral effect of the organism on the whole level, and is closer to the antiviral state of the human body than the in-vitro test. The in vivo test is performed on the basis of the in vitro test, and the in vitro test result is further verified. Coronavirus infection is an acute infection, and mainly invades the body from the respiratory tract, replicates and proliferates in cells of the respiratory tract, causes cell degeneration necrosis and shedding, further affects bronchi, bronchioles, alveoli and surrounding tissues of bronchi, and seriously causes the pulmonary alveoli wall to engorge water feet, exudates and fibrin to increase, and neutrophil infiltration and mononuclear cell infiltration, etc. The lung is used as a target organ of coronavirus, and whether the drug can inhibit the replication of coronavirus in the target organ is one of indexes for measuring antiviral efficacy. After a model is successfully built by coronavirus HCoV-OC43 infected mice, the protection effect of the traditional Chinese and Tibetan medicine combination preparation on death of the coronavirus infected mice and the intervention effect of the traditional Chinese and Tibetan medicine combination preparation on lung injury caused by the coronavirus infected mice are observed, so that the in vivo inhibition effect of the traditional Chinese and Tibetan medicine combination preparation on the coronavirus infected mice is verified.

1.1 materials

1.1.1 animals

SPF female BALB/c mice, 300, 18-22 g, purchased from Hubei province laboratory animal research center under license number SCXKE2020-0018, 6-8 weeks old. BALB/c mice are fed into a barrier system after being retrieved, the humidity is 50-70%, the mice are fed for 5 days in an adaptive mode, and the mice start to enter a formal experiment.

1.1.2 viruses and drugs

The coronavirus HCoV-OC43 is stored in the same medical college affiliated with the same medical college of university of science and technology in China, is multiplied by chicken embryo for 2 rounds, filtered and stored at 4 ℃ for standby, and then LD is measured ₅₀ . All viruses are preserved at-80' deg.C for standby.

The clinical prescription is provided by Shijia Tibetan noro pharmaceutical industry stock limited company, and the test drug is brown formula particles prepared by Shijia Tibetan noro pharmaceutical industry stock limited company according to compound preparation dosage;

control drug adefovir: purchased from Target Mol company, U.S.A.

1.1.3 chick embryo

SPF chick embryo purchased from Beijing Mei Liya vitamin laboratory animal technology Co., ltd., license number SCXK (Beijing) 2020-0017.

1.1.4 reagents

Hydrated chloric acid, formic acid, ethanol, xylene, eosin, hematoxylin, hydrochloric acid were purchased from beijing chemical reagent company.

1% chicken red blood cell suspension: collecting at least 3 SPF-grade adult cock lower wing veins to collect blood 1mL, adding 1mL sodium citrate for anticoagulation, washing with 5-10 times of physiological saline for several times, removing plasma and leucocyte layers until the supernatant is cool and transparent, preparing erythrocyte into suspension with volume fraction of Sichuan by using physiological saline, and storing at 4 ℃ for standby.

Reagent used for preparing HE stained tissue section:

(1) 0.5-1% eosin alcohol solution:

weighing 0.5-1 g of eosin, adding a small amount of distilled water for dissolution, and then dripping glacial acetic acid until the mixture is pasty. Filter paper was used for filtration, and the residue was baked in an oven and then dissolved in 100ml of 95% alcohol.

(2) Hematoxylin dye liquor formula:

6g of hematoxylin is dissolved in 100mL of absolute ethanol, 150g of aluminum potassium sulfate is dissolved in 2000mL of distilled water, 900mL of glycerin is poured into the solution after the dissolution and mixed together, and finally 1.2g of sodium iodate and 120mL of glacial acetic acid are added.

(3) 1% hydrochloric acid alcohol differentiation solution: 1 ml of concentrated hydrochloric acid is added into 99 ml of 70% alcohol.

1.2 method

1.2.1 test of acute toxicity of Tibetan medicine combination preparation for repeated administration

The BALB Deng Xiaoshu of 40 females is taken, adaptively fed for 5 days, weighed and randomly divided into 4 groups, and 10 groups are divided into a blank group and a high, medium and low (1.5 g/kgBW,0.75g/kgBW and 0.375 g/kgBW) dosage group of the traditional Chinese and Tibetan medicine combination formula, and the high, medium and low dosage concentrations are respectively 75mg/ml,37.5mg/ml and 18.8mg/ml. BALB/c mice were given different concentrations of the traditional Chinese and Tibetan medicine combination formula preparation by gavage for a fixed time every day, 0.2ml/10gBW, 7d by continuous gavage, equal volumes of deionized water by gavage of the control group, 14d by daily observation of clinical manifestations, weighing every week, no water forbidden for 12-16 hours before the end of the test, blood collection for blood biochemical CALT, AST, BUN and CRE) and blood routine tests.

1.2.2. 2 half Lethal Dose (LD) of coronavirus HCoV-OC43 ₅₀ ) Is (are) determined by

Sequentially diluting the collected chick embryo allantoic fluid by 10 times to 10 times ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 Concentration of virus liquid. Taking 70 female BALB/c mice, adaptively feeding for 5 days, weighing, and randomly dividing into 7 groups of 10, blank control group and 10 ^-1 ～10 ^-6 Group, virus control group BALB/c mice are anesthetized by 5% chloral hydrate of 0.05ml/10g, and then are respectively infected with 50 mu L of HCoV-OC43 virus liquid with different dilutions by nasal drops, and blank control group is infected with 50 mu L of PBS by nasal drops; daily observation of clinical response, recording of death time, statistics of death number (number of death not counted in 24 h), observation of 14d, calculation of half-number of deaths (LD) of the strain using Reed-Muench method formula ₅₀ )。

1.2.3 test of protection of Tibetan medicine combination formula preparation against death of coronavirus HCoV-OC43 infected BALB/c mice

1.2.3.1 test animals grouping

90 female BALB/c mice were randomly divided into 6 groups of 15, 5/cage after weighing for adaptive feeding. The 6 groups are blank control group, virus control group and Ruidexivir control group respectively, and the combination formula of the traditional Chinese medicine and the Tibetan medicine is high, medium and low dose groups. The grouping and lavage doses are shown in Table 16.

Table 16 grouping of laboratory animals

1.2.3.2 administration to test animals

After the traditional Chinese and Tibetan medicine combination preparation is continuously infused with stomach for 2 days, the air-to-air ratio control group is dripped with 50 mu L/piece of PBS solution, and the concentration of coronavirus challenge experiments of other groups is 10LD ₅₀ 50. Mu.L. And starting to perfuse the stomach after 4 hours from virus infection, and then continuously perfusing the stomach for 5 days, wherein the blank control group and the model control group are perfused with physiological saline which is the same volume as the traditional Chinese and Tibetan medicine combined preparation, the time and mode of the stomach perfusing are the same as the traditional Chinese and Tibetan medicine combined preparation, the daily stomach perfusing dose of the Ruidexiwei group is 100g/kg BW, and the time and mode of the stomach perfusing are the same as the traditional Chinese and Tibetan medicine combined preparation. The stomach was irrigated 1 time/d, 0.2mL/10gBW.

1.2.3.3 observations index

Continuous observation of 14d from the day of infection, mainly observed the onset symptoms of BALB/c mice, recorded the time to death and the number of deaths, calculated mortality, mortality protection and mean survival time.

Mortality protection (%) = (mortality of virus control group mortality test group mortality)/mortality of virus control group×100%

Life extension (%) = (average survival days of test group-average survival days of virus control group)/average survival days of virus control group×100%

1.2.4 experiments of the Effect of Tibetan Property combination formula preparation on pneumonia caused by coronavirus HCoV-OC43 infection in BALB/c mice

1.2.4.1 test animals group: and 1.2.3.

1.2.4.2 methods of dosing test animals: and 1.2.3.

1.2.4.3 observations index

Lung index:

after 5d of virus infection, after 12 hours of no water retention after fasting, weighing, killing after taking blood, opening the chest cavity, picking up the whole lung, washing cleanly with normal saline, sucking water with filter paper, weighing by an electronic balance, and calculating lung index and lung index inhibition rate according to the following formula;

lung index= (mouse lung weight/mouse body weight) ×100%

Lung index inhibition = (average lung index of virus control group average lung index test group average lung index)/average lung index of virus control group x 100%.

Viral hemagglutination potency of lung tissue homogenate suspension:

6 lungs of each group are randomly taken and placed in a homogenizer, physiological saline [ lung weight (g)/physiological saline (mL) =1/9 ] is added, the mixture is ground in an ice bath to prepare homogenate, and supernatant is taken after centrifugation at 4 ℃ for a micro-hemagglutination test. The method comprises the following steps:

(1) 25 μLPBS was added to each well of 96-well V-type microplatelet l Kong.

(2) 25. Mu.L of the lung tissue homogenate to be examined was added to the first well of each row and mixed.

(3) 25 mu L of lung tissue homogenate is sucked from the 1 st hole by a multi-channel mascot and is put into the 2 nd hole, and the lung tissue homogenate is blown uniformly without generating bubbles. After mixing, 25. Mu.L of the mixture was aspirated, and the mixture was added to the 3 rd well, diluted to 11 wells in the order of multiple ratio, and 25. Mu.L of the mixture was aspirated after mixing, and the 12 th row was used as a control well.

(4) Then 1% chicken erythrocytes were added to each well (the erythrocyte suspension was thoroughly shaken before addition).

(5) Mixing by light shaking, standing at 20-25 ℃ for 40min, and observing the result (or standing at 4 ℃ for 60 min).

(6) The results determined that (well 12) erythrocytes should be precipitated in the bottom of the well in a pronounced button-like manner. The plate was tilted to observe whether or not the red blood cells were present as teardrop droplets, and the highest dilution factor for complete clotting represents 1 hemagglutination unit.

1.2.4.4 data processing and statistical analysis

The SPSS13.0 software is adopted to count the obtained data, the metering data is processed by single factor analysis of variance or T test, and the test result is usedThe representation is: the counting data adopts χ ² And (5) checking.

1.3 experimental results

1.3.1 results of the test for acute toxicity of continuous administration of Tibetan Properties

Tibetan medicine combination formula preparation group BALB/c mice in 1.3.1.1 the Tibetan medicine combination formula preparation high dose group, the medium dose group and the low dose group mice in general clinical manifestations have normal diet, active and active, sensitive response and good mental state, and have no difference compared with the mice in the air-free control group.

1.3.1.2 Effect of Tibetan medicine combination formula preparation on body weight of BALB/c mice

Formulation of the combination of traditional Chinese and Tibetan medicine each group of mice was continuously perfused with different doses of formulation 7d, and after 14d observation, the mice were weighed weekly. Compared with the blank control group, the weight difference of each group of the traditional Chinese and Tibetan medicine combination formula preparation is not obvious (p is more than 0.05). The results are shown in Table 17.

The Tibetan medicine combination formula in Table 17 changes the weight of each group

Note that: p > 0.05 compared with the blank group

1.3.1.3 Effect of Tibetan medicine combination formula preparation on Biochemical indicators of blood of BALB/c mice

The preparation of the combination formula of traditional Chinese and Tibetan medicine is prepared by continuously lavaging different doses of preparation 7d of mice, and after 14d of observation, blood is taken for biochemical detection. Compared with the blank control group, the blood biochemical indexes of the traditional Chinese and Tibetan medicine combination formula preparation are not obviously different (p is more than 0.05). The results are shown in Table 18.

Blood biochemical index changes of Tibetan medicine combination formula preparation in Table 18/>

Note that: p > 0.05 compared with the blank group

Influence of Tibetan medicine combination formula preparation in 1.3.1.4 on routine index of BALB/c mouse blood

The Tibetan medicine combination formula preparation is prepared by continuously lavaging different doses of preparations 7d of mice, and blood is collected for routine blood detection after 14d observation. Compared with the blank control group, the blood routine index of each group of the traditional Chinese and Tibetan medicine combination formula preparation has no obvious difference (p is more than 0.05). The results are shown in Table 19.

Blood routine index changes for each group of Tibetan medicine combination formula preparation in Table 19

Note that: p > 0.05 compared with the blank group

1.3.2 LD of coronavirus HCoV-OC43 ₅₀ Is measured according to the measurement results of (2)

The BALB/c mice in the blank group have agile actions, normal diet, uniform respiration, good mental condition and glossy fur. 10 ^-1 Group, 10 ^-2 Group, 10 ^-3 Group, in 3d morning after challenge, was found that BALB/c mice were anorexia, matted with Mao Song, accumulated with cold tremors, aversion to motion, shortness of breath, abdominal respiration, wasting, etc. Continuous observation, death occurred in the 5d BALB/c mice, 5 to 9d 10 ^-1 All mice in group BALB/c died, 10 ^-2 All mice in group BALB/c died, 10 ^-3 Group BALB/c mice died 8. From 10d no more deaths occurred, and the status of diet and activity of BALB/c mice also began to improve. 10 ^-4 Group, 10 ^-5 Group, 10 ^-6 The mice in the group and the control group are agile in action, glossy in hair, active in BALB/c, normal in diet and increased in weight, and 14d is observed in the experiment, the weights and death numbers of the mice in each group are counted, the results are shown in tables 20 and 21, and the LD50 of the virus is calculated to be 10 ^-3.375 /50μL。

TABLE 20 variation of body weight of coronavirus-HCOV-OC 43 challenge BALB/c mice/>

Table 21 half Lethal Dose (LD) of coronavirus HCOV-OC43 ₅₀ ) Calculation of (2)

1.3.3 results of protective effects of Tibetan Property formulations on death of coronavirus HCoV-OC43 infected BALB/c mice

In the 14d observation period, the mice in the blank control group do not die, the death rate is 0, the death rate of the mice in the virus control group is 86.6%, the death rate of the mice in the Ruidexiwei group is 26.6%, P is less than 0.01 after being tested by X2, the mice in the dosage group in the Tibetan medicine combination formula preparation have obvious death protection effect, and the death rate of the mice in the dosage group is 40%, and P is less than 0.01 after being tested by X2. The other groups were not statistically significant (P > 0.05) compared to the virus control group.

The survival time of the mice in the virus control group is 9.0d, and the average survival time of the mice is extremely obviously reduced (P < 0.01) compared with that of the blank control group. Compared with the virus control group, the average survival time of mice in each dose group of the traditional Chinese and Tibetan medicine combination formula preparation and the Ruidexivir control group is obviously prolonged, the average survival time of mice in the Ruidexivir group is 12.5d, the average survival time of mice in the dose group of the traditional Chinese and Tibetan medicine combination formula preparation is 12.0d, and the average survival time of the mice in the two groups is obviously prolonged (P is less than 0.01) compared with the virus control group.

The other groups were not statistically significant (P > 0.05) compared to the virus control group. The results are shown in Table 22.

Protection of coronavirus infection BALB/C by Tibetan medicine combination proved recipe preparation in Table 22/>

Note that: virus control groupCompared with the blank control group and the virus control group, ^** p＜0.01 ^* p < 0.05: each drug group was compared with the blank group, ^ΔΔ P＜0.01

1.3.4 effects of Tibetan medicine combination formula preparation on pneumonia caused by coronavirus HCoV-OC43 infected mice

1.3.4.1 Effect of Tibetan medicine combination formula preparation on the lung index of coronavirus HCoV-OC43 infected mice

The result of the change of the lung index of the traditional Chinese and Tibetan medicine combination formula preparation on the mice infected by the coronavirus HCoV-OC43 for 5d shows that the lung index of the virus control group is extremely obviously higher (p is less than 0.01) compared with that of the blank control group. Compared with a virus control group, the lung index of a dosage group in the Ruidexivir group and the traditional Chinese and Tibetan medicine combination formula preparation is extremely obviously reduced (p is smaller than v.01), and the lung index of a low dosage group in the traditional Chinese and Tibetan medicine combination formula preparation and a high dosage group in the traditional Chinese and Tibetan medicine combination formula preparation is obviously reduced (p is smaller than 0.05). The results are shown in Table 1.

1.3.4.2 Effect of Tibetan medicine combination formula preparation on hemagglutination titres of coronavirus HCoV-OC43 in pulmonary tissue of infected mice

Hemagglutination titers in lung tissue after coronavirus HCoV-OC43 infection of mice were determined using the Hemagglutination Assay (HA), reflecting the replication of the coronavirus. After 5d of medicines with different doses are continuously infused into the stomach, compared with a virus control group, the pulmonary tissue hemagglutination titer of the mice in the medicine group is extremely obviously reduced (p is less than 0.01), which indicates that each group of medicines has different degrees of effect of inhibiting coronavirus, the blood coagulation titer of the Ruidexiwei group is the lowest, and the effect of inhibiting coronavirus is the best. The middle dose group of the blood coagulation titer of each medicine group of the traditional Chinese and Tibetan medicine combined preparation is lower than that of the high dose group and the low dose group, which indicates that the anti-coronavirus group effect of the dose group of the traditional Chinese and Tibetan medicine combined preparation is better in the three dose groups. The results are shown in Table 2.

Effects of Tibetan medicine combination formula preparation on lung tissue pathology of coronavirus HCoV-OC43 infected BALB/c mice in 1.3.4.3

The results of the pathomorphology of the traditional Tibetan medicine combination formula preparation after 5d infection of mice with coronavirus HCoV-OC43 show that the lung tissue structure of normal mice is observed by HE staining, the terminal bronchiole part stretches peripherally to form respiratory bronchioles, and then branches into alveolar ducts (acinus and alveoli, the structures of all levels of bronchi are clear, and inflammatory cells which are basically not infiltrated in the alveolar tissues are exuded, the capillary vessels of the alveoli wall are filled with erythrocytes, and the surrounding matrix of all levels of bronchi is provided with pulmonary arteries, pulmonary veins, lymphatic tissues and loose connective tissues (figure 2).

1.4 statistical analysis

The protection effect test of the traditional Chinese and Tibetan medicine combination preparation on the death of the BALB/c mice infected by the coronavirus HCoV-OC43 proves that after the mice infected by the traditional Chinese and Tibetan medicine combination preparation are treated, the survival time is prolonged, the death rate is reduced, the death protection rate is improved, the protection effect on the mice which are lethal infected by the coronavirus is obvious, and the medium-dose group is superior to the high-dose group and the low-dose group.

The effect test of the traditional Chinese and Tibetan medicine combination preparation on pneumonia caused by coronavirus HCoV-OC43 infection of BALB/c mice proves that after the traditional Chinese and Tibetan medicine combination preparation is used for treating the infected mice, the average lung index and average blood coagulation titer are reduced, and the pathological morphology of lung tissues is obviously improved. The Tibetan medicine combination preparation is proved to reduce the lung inflammation caused by coronavirus by inhibiting the proliferation of coronavirus in the lung.

EXAMPLE 5 study of the mechanism of action of Tibetan medicine in combination with clinical prescriptions in inhibiting coronavirus HCoV-OC43 infection

After the coronavirus invades the body, the virus replicates in the host cell and then enters the blood, forming viremia. After the virus acts on the body, the immune cells of the body generate immune responses, and a large amount of small molecular protein cytokines with biological activity are generated. These cytokines regulate cell growth, differentiation and effects by binding to the corresponding receptors, regulating functions such as innate and adaptive immunity and repair of tissue damage. I.e. coronaviruses act on the lung of the target organ, causing immune cell interactions with a significant increase in free radicals, resulting in damage to normal tissue cells. Thus, maintaining a dynamic balance between oxygen radicals and antioxidants is extremely important to maintain the normal structure and function of tissue cells. The test is based on the in vivo inhibition effect of the traditional Chinese and Tibetan medicine combined clinical prescription preparation, and the action mechanism of the traditional Chinese and Tibetan medicine combined clinical prescription preparation for resisting coronavirus infection is studied by measuring immune organ index and immune cell proliferation capacity after the traditional Chinese and Tibetan medicine combined clinical prescription preparation intervenes in BALB/c mice infected by coronavirus HCoV-OC 43.

1.1 materials

Test animals: SPF-class female BALB/c mice, 90, 18-22g, purchased from laboratory animal research center, hubei province under license number SCXKE2020-0018, were used 6-8 weeks. BALB/c mice were fed in barrier system after being retrieved, humidity was 50-70%, and were adapted for 5d. A formal test is started.

1.2 test methods

1.2.1 test animals grouping and method: the animals were grouped and tested as in example 4.

1.2.2 sampling as a method

After BALB/c mice were anesthetized 5d after challenge, and after dissection and weighing, 6 sterile blood samples of 0.5mL were randomly selected for each group, and serum was collected and isolated from the remaining mice of each group. After taking blood, the mice are sacrificed, the thoracic cavity and the abdominal cavity are opened to respectively pick up the lung, the thymus and the spleen, after the blood is washed by normal saline, the surface moisture is sucked by using a water absorbing paper, and then the weights of the lung, the thymus and the spleen are respectively weighed by dividing by an electronic day.

1.2.3 observations index

1.2.3.1 Effect of coronavirus HCoV-DC43 on immune organ index and immune cell proliferation Capacity in BALB/c mice

Effect of the Tibetan medicine combination formula preparation on spleen index and thymus index of coronavirus HCoV-OC43 infected BALB/c mice:

after a BALB/c mouse is infected by coronavirus HCoV-OC43 for 5d, after no water forbidden for 12h, weighing, after taking blood and killing, opening the chest and the abdominal cavity to respectively find out thymus and spleen, washing the blood with normal saline, sucking surface water with absorbent paper, and then equally weighing the weights of the thymus and the spleen respectively by using an electronic day, and respectively calculating thymus index and spleen index according to the following formula:

Spleen index= (mouse spleen weight/mouse body weight) ×100%

Thymus index= (mouse thymus weight/mouse body weight) ×100%

Effect of the Tibetan medicine combination formula preparation on T lymphocyte proliferation in coronavirus HCoV-OC43 infected BALB/c mice:

after 5d infection of BALB/c mice with coronavirus HCoV-OC43, 6 mice were randomly selected, 0.5mL of peripheral blood was aseptically taken from each group, placed in a 2mL EP tube just wetted with 50 units heparin, lmL PBS was added and mixed well, and the mixed blood sample was gently added to the liquid surface of 2mL lymphocyte separation liquid. Centrifuging at 2000rpm for 20min, carefully sucking white foggy cells between the separating liquid and PBS into a new test tube, namely peripheral blood mononuclear cells. The pellet was resuspended in 1mL of 640 complete nutrient solution (eventually containing 10% calf serum, 5. Mu.g/mL conc. Protein) and 100. Mu.l per well, 4 duplicate wells per group, and ConA-free control wells, and ConA control wells were provided, and to prevent edge effects, 100. Mu.l of cell-free 1640 complete nutrient solution was added to the wells surrounding the cell plates. Cell plates were incubated at 37℃with 5% CO ₂ After 2d of incubator, 5mg/mL of 20 mu LMTT is added, the culture is continued for 4 hours, the mixture is taken out, placed on an ultra-clean workbench, 150 mu l of DMSO is added for shaking bath for 10 minutes, and the mixture is read by an enzyme-labeling instrument. The proliferation capacity of T lymphocytes is represented by a value of each sample.

Effect of the Tibetan medicine combination formula preparation on B lymphocyte proliferation in coronavirus HCoV-OC43 infected BALB/c mice:

after 5d infection of BALB/c mice with coronavirus HCoV-OC43, 0.5mL of sterile peripheral blood from 6 mice each group was placed in a 50 unit heparin-just-wetted 2mL EP tube, 1mL PBS was added and mixed well, and the blood samples were gently added to the surface of 2mL lymphocyte separation solution. Centrifugation at 2000rpm for 20min, carefully aspirate white tertiary cells between the isolate and PBS into a new tube, i.e., peripheral blood mononuclear cells. The cells were resuspended in 1640 complete nutrient solution and stained with trypan blue and the number of viable cells therein was visualized. Adjusted to 2X 10 ⁶ The mixture was centrifuged at lmL at room temperature at 2000rpm/15min to obtain a precipitate. The pellet was then suspended with lmL 640 complete nutrient solution (eventually 10% calf serum, 10. Mu.g/mL LPS) and 100. Mu.L per well, 4 duplicate wells per group, and LPS-free control wells, and LPS control wells were provided, and to prevent edge effects, 100. Mu.L of cell-free 1640 complete nutrient solution was added to the wells around the cell plate. Cell plates were incubated at 37℃with 5% CO ₂ After 2d in the incubator, 5mg/mL of 20. Mu. LMTT was added. After the culture is continued for 4 hours, the mixture is taken out and placed on an ultra-clean workbench, 150 mu L of DMSO is added for shaking and dissolving for 10 minutes, and the mixture is read by an enzyme-labeled instrument. The proliferation capacity of B lymphocytes is represented by a value of each sample.

1.2.3.2 Effect of Tibetan medicine combination formula preparation on cytokine levels in lung tissue of coronavirus HCoV-OC43 infected BALB/c mice

After BALB/c mice are infected by coronavirus HCoV-OC43 for 5d, the mice are fasted and not forbidden for 12h, and then are sacrificed, the chest is opened and the whole lung is taken out, the mice are washed clean by normal saline, and the filter paper is used for sucking water, the electronic balance is named after it, the normal saline (lung weight (g)/normal saline (ml) =1/9) is cooled in the lung straight homogenizer, and the homogenate is prepared by grinding in ice bath, and the supernatant is prepared for detecting the cytokine after centrifugation at 4 ℃. The IL-6, IL-10, TNF-a and IFN gamma cytokines are all measured by a double antibody sandwich method, and the operation is strictly carried out according to the requirements of the kit instruction.

1.2.3.3 Effect of Tibetan medicine combination formula preparation on SOD and MDA in serum of coronavirus HCoV-OC43 infected BALB/c mice

After 5d infection of mice with coronavirus HCoV-OC43, serum was isolated after taking blood after a 12h fast without water withdrawal for measuring superoxide dismutase (SOD) activity and malonic acid (MDA) content.

1.2.4 data processing and statistical analysis

The SPSS13.0 software package is adopted to count the obtained data, the variance alignment test is carried out on the test data, the variance alignment person is processed by adopting single-factor variance analysis or T test, the data variance is irregular, the non-parameter test is adopted, and the test result is expressed by (x+/-SD).

1.3 experimental results

1.3.1 effects of Tibetan medicine combination formula preparation on the immune organ index and the immunocyte proliferation Capacity of coronavirus HCoV-OC 43-infected BALB/c mice

Effects of Tibetan medicine combination formula preparation in 1.3.1.1 on spleen index and thymus index of coronavirus HCoV-OC43 infected BALB/c mice

After infection of BALB/c mice with coronavirus HCoV-OC43, the spleen index and thymus index were significantly reduced (p < 0.01) in the virus control compared to the blank. After the traditional Chinese and Tibetan medicine combination preparation and the Ruidexivir are adopted for treatment, compared with a virus control group, the thymus index of a low-dose group in the traditional Chinese and Tibetan medicine combination preparation is remarkably increased (p < 0.01), and the spleen index of a low-dose group in the traditional Chinese and Tibetan medicine combination preparation is remarkably increased (p < 0.05); other groups had increased spleen index and thymus index, but were not statistically significant (p < 0.05); the spleen index and thymus index were not statistically significant (p > 0.05) in the combination of the traditional Chinese and Tibetan medicine in the group of high, medium and low doses, and the results are shown in Table 23.

Effects of Tibetan medicine combination formula preparation on spleen index and chest leap index of coronavirus infected BALB/c mice in Table 23

Note that: the virus control group is less than 0.01 in comparison with the blank control group, and each drug group is less than 0.01 in comparison with the virus control group in the x p < 0.05.

1.3.1.2 effects of Tibetan Property formulations on proliferation of T, B lymphocytes in coronavirus HCoV-OC43 infected BALB/c mice

After mice are infected with coronavirus HCoV-OC43, the virus control T, B lymphocyte A has a very significant value (p < 0.01) compared with the normal blank; after the drug treatment, compared with a virus control group, the T, B lymphocyte A value of a dosage group in the traditional Chinese and Tibetan medicine combination formula preparation is remarkably increased (p is less than 0.05), and the other T, B lymphocyte A values are increased to different degrees but have no statistical significance (p is less than 0.05); the results of the comparison of T, B lymphocyte A values between the high, medium and low dose groups of the combination formula for both traditional and Tibetan medicine (p < 0.05) are shown in Table 24.

Effect of Tibetan medicine combination formula preparation on proliferation of T, B lymphocytes in coronavirus infected mice in table 24

Note that: the virus control group was p < 0.01 compared with the blank group: p < 0.05 for each drug group compared to control group

1.3.2 Effect of Tibetan medicine combination formula preparation on cytokine levels in lung tissue of coronavirus HCoV-DC43 infected BALB/c mice

1.3.2.1 Effect of Tibetan medicine combination formula preparation on the content of TNF-a and IL-6 in the lung tissue of coronavirus HCoV-QC43 infected BALB/c mice

After 5d of coronavirus infection of mice, the virus control group had significantly elevated TNF-alpha and IL-6 (p < 0.01) compared to the blank group; compared with a virus control group, the dose group in the traditional Chinese and Tibetan medicine combination formula preparation and the low dose group of the traditional Chinese and Tibetan medicine combination formula preparation have extremely obviously reduced TNF-alpha (p < 0.01), the dose group in the traditional Chinese and Tibetan medicine combination formula preparation has obviously reduced IL-6 (p < 0.05), other groups have different degrees of reduction but have no statistical significance (p < 0.05), and the high, middle and low dose groups of the traditional Chinese and Tibetan medicine combination formula preparation have no statistical significance (p < 0.05) when comparing TNF-alpha and IL-6 in pairs. The comparison results are shown in Table 25.

Effect of Tibetan medicine preparation on TNF-alpha and IL-6 content in coronavirus infected BALB/c mouse lung tissue in Table 25

Note that: the virus control group is compared with the blank control group, and p is less than 0.01; p < 0.05 for each group compared to the control group

1.3.2.2. Effect of the Tibetan medicine combination formula preparation on the IL-10 content in the lung tissue of coronavirus HCoV-OC 43-infected BALB/c mice

The virus control group showed very significant decrease in IL-10 (p < 0.01) compared with the blank control group, the combination formula of the traditional Chinese medicine showed significant increase in IL-10 (p < 0.05) in the dosage group after the stomach-nourishing administration compared with the virus control group, and the other groups showed different increases, but no statistical significance (p < 0.05), and the combination formula of the traditional Chinese medicine showed no statistical significance (p < 0.05) in the high, medium and low dosage groups compared with TL-10.

Effect of Tibetan medicine combination formula preparation on IL-10 content in coronavirus infected BALB/c mouse lung tissue in table 26

/>

Note that: the virus control group is compared with the blank control group, and p is less than 0.01; each drug group was compared to the control group with p < 0.05.

1.3.2.3 effects of Tibetan Property formulation on IFN-y content in lung tissue of coronavirus HCoV-OC43 infected BALB/c mice

Compared with the blank control group, the IFN-gamma of the virus control group is extremely obviously reduced (p is less than 0.01), the IFN-gamma of the traditional Chinese and Tibetan medicine combination formula preparation and the IFN-gamma of the low-dose group are extremely obviously increased (p is less than 0.01), the IFN-gamma of the traditional Chinese and Tibetan medicine combination formula preparation and the IFN-gamma of the high-dose group (p is less than 0.05), and the IFN-gamma of the traditional Chinese and Tibetan medicine combination formula preparation in pairs is not statistically significant (p is less than 0.05), and the results are shown in Table 27.

Effect of Tibetan medicine combination formula preparation on IFN-y content in lung tissue of New coronavirus infected BALB/c mice in Table 27

Note that: the virus control group is p < 0.01 compared with the blank control group, and each drug group is p < 0.01 and p < 0.05 compared with the virus control group.

1.3.3 results of the influence of Tibetan Property formulations on SOD and MDA in serum of coronavirus HCoV-OC43 infected BALB/c mice

Compared with a blank control group, the SOD activity of a virus control group is extremely obviously reduced (p is less than 0.01), the SOD activity of a medium-Tibetan medicine combination formula preparation and a low-dose group is extremely obviously improved (p is less than 0.01), the SOD activity of a high-dose group of a medium-Tibetan medicine combination formula preparation is obviously improved (p is less than 0.05), the SOD activity of a medium-Tibetan medicine combination formula preparation and the SOD activity of a low-dose group of a medium-Tibetan medicine combination formula preparation are extremely higher than the SOD activity of a high-dose group of a medium-Tibetan medicine combination formula preparation (p is less than 0.01), the MDA content of a virus control group is extremely obviously improved (p is less than 0.01), the MDA content of a medium-Tibetan medicine combination formula preparation is extremely obviously reduced (p is less than 0.01), the MAD content of a medium-Tibetan medicine combination formula preparation is not statistically significant (p is less than 0.05) in a pairwise comparison between the high-Tibetan medicine combination formula preparation, the medium-Tibetan medicine combination formula preparation and the low-dose group, and the comparison result is shown in table 28.

Influence of Tibetan medicine combination prescription on SOD and MDA in coronavirus BALB/c mouse serum in Table 28/>

Note that: (1) the virus control group is compared with the blank control group, p is less than 0.01, and p is less than 0.05.

1.4 statistical analysis

The influence test of the traditional Chinese and Tibetan medicine combination formula preparation on the immune organ index and the immune cell proliferation capability of the coronavirus infected mice proves that the dosage group in the traditional Chinese and Tibetan medicine combination formula preparation can reduce the spleen and thymus of the immune organ, and through the gonocyte proliferation test, the T cell immunity and the B lymphocyte humoral immunity functions of the influenza infected mice are improved, the immunity of the organism is improved, and the damage of the influenza virus to the organism is improved.

The influence test of the traditional Chinese and Tibetan medicine combination formula preparation on the lung tissue cell poverty of mice infected by coronavirus proves that the traditional Chinese and Tibetan medicine combination formula preparation can effectively reduce pro-inflammatory poverty TNF-alpha and IL-6, improve anti-inflammatory factors IL-10 and improve the level of antiviral factors IFN-gamma, and shows that the traditional Chinese and Tibetan medicine combination formula preparation can play the antiviral effect by regulating the secretion of organism cytokines.

The influence test of the traditional Chinese and Tibetan medicine combination formula preparation on the antioxidant capacity of mice infected by coronaviruses proves that the traditional Chinese and Tibetan medicine combination formula preparation can reduce the lipid peroxidation damage of organisms, reduce the MDA generation of the final product of lipid peroxidation, improve the activity of SOD and enhance the antioxidant capacity of organisms, thereby slowing down the lung injury effect caused by coronaviruses to a certain extent.

Conclusion:

the in vitro inhibition test results show that: half-Toxicity Concentration (TC) of the combination prescription for Tibetan medicine on MDCK cells ₅₀ ) The effect of the Tibetan medicine combination prescription on HCoV-OC43 coronavirus infection in 1458 mug/mL shows that the effect of inhibiting replication and proliferation after virus adsorption is achieved, and the average half-Effective Concentration (EC) of three different drug concentrations and the mode of action of the virus ₅₀ ) 212.2, 210.6 and 240.3 μg/mL, respectively, and Therapeutic Indices (TI) of 7.5, 7.6 and 6.6, respectively.

The in vivo inhibition test results show that: the average survival time of mice in a protective effect virus control group for the death of coronavirus infected mice by the combination prescription of the traditional Chinese medicine and the Tibetan medicine is 9.0d, the average survival time of mice in a dosage group in the combination prescription of the traditional Chinese medicine and the Tibetan medicine is 12.0d, and the difference is very obvious compared with the virus control group (P < 0.01); the mortality rate of the virus control group is 86.6%, the mortality rate of the traditional Chinese and Tibetan medicine combined prescription dosage group is 26.6%, and the difference between the two is very obvious (P < 0.01) through the X2 test. The traditional Chinese medicine and Tibetan medicine combined prescription has the effect of inhibiting lung inflammation of infected mice: the average lung index of the traditional Chinese and Tibetan medicine combined prescription is extremely obviously reduced (P < 0.01) compared with a virus control group, the average lung index of the traditional Chinese and Tibetan medicine combined prescription is obviously reduced (P < 0.05), and the average hemagglutination titer of the test group is extremely obviously reduced (P < 0.01) compared with the virus control group; the virus control group has serious lesions, such as pulmonary capillary congestion, most pulmonary alveolar structure destruction, partial pulmonary solid variable region fusion, alveolar septal thickening, alveolar space and inflammatory cell infiltration in interstitium. After the drug treatment, the health of mice is obviously improved compared with the virus control group in each drug group.

The results of the study on the action mechanism show that (the thymus index and spleen index of the traditional Chinese and Tibetan medicine combination prescription are obviously increased (P < 0.05) in the traditional Chinese and Tibetan medicine combination prescription, the lymphocyte A value of the traditional Chinese and Tibetan medicine combination prescription is obviously increased (P < 0.05) in the traditional Chinese and Tibetan medicine combination prescription, the IL-6 of the traditional Chinese and Tibetan medicine combination prescription is obviously reduced (P < 0.05), the IL-10 of the traditional Chinese and Tibetan medicine combination prescription is obviously increased (P < 0.05), the TNF-a of the traditional Chinese and Tibetan medicine combination prescription is extremely obviously reduced (P < 0.01), the TNF-gamma of the traditional Chinese and Tibetan medicine combination prescription is extremely obviously increased (P < 0.01), the SOD of the traditional Chinese and Tibetan medicine combination prescription is extremely obviously increased (P < 0.01) in the traditional Chinese and Tibetan medicine combination prescription, the SOD of the traditional Chinese and Tibetan medicine combination prescription is obviously increased (P < 0.05), and the MDA of each medicine combination prescription is extremely reduced (P < 0.01).

In conclusion, in vivo and in vitro experiments show that the Tibetan medicine combination prescription has remarkable inhibition effect on novel coronavirus infection. The traditional Chinese and Tibetan medicine combined prescription has a certain antiviral function in the aspects of regulating the immune function and cytokine secretion of infected mice, relieving peroxidation stress injury caused by coronaviruses and the like.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. The pharmaceutical composition for preventing and treating the novel coronavirus is characterized by comprising the following components in parts by weight: 224-250 parts of astragalus membranaceus, 56-70 parts of rhizoma anemarrhenae, 168-200 parts of rhodiola rosea, 168-200 parts of ageratum, 112-150 parts of Tibetan schizonepeta, 224-250 parts of roasted loquat leaves, 168-200 parts of fructus forsythiae, 56-70 parts of Tibetan saussurea and 56-70 parts of amur corktree bark.

2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is prepared from the following components in parts by weight: 224 parts of astragalus, 56 parts of rhizoma anemarrhenae, 168 parts of rhodiola rosea, 168 parts of ageratum, 112 parts of Tibetan schizonepeta, 224 parts of roasted loquat leaves, 168 parts of fructus forsythiae, 56 parts of Tibetan saussurea and 56 parts of amur corktree bark.

3. The pharmaceutical composition according to claim 1 or 2, further comprising an adjuvant, wherein the drug substance and the adjuvant are mixed in a weight ratio of 2:1;

wherein the auxiliary material is at least one selected from dextrin, starch, lactose, maltose, mannitol and mannose.

4. A pharmaceutical composition according to claim 3, wherein the adjuvant is mixed with dextrin, lactose and mannitol in a weight ratio of 5:3:2.

5. The method of preparing a pharmaceutical composition of claim 4, comprising the steps of:

(1) Weighing the raw materials according to a proportion, mixing, heating and reflux-extracting with 60% ethanol for 2 times, adding 10 times of 60% ethanol for 2.5 hours each time, mixing the extracting solutions, filtering, concentrating to a relative density of 1.20-1.30 g/mL, and vacuum drying the thick paste until the water content is less than 2.5%, thus obtaining extract powder I;

(2) Adding 8 times of water into the dregs, extracting for 2 hours at 80 ℃, filtering, concentrating to a relative density of 1.20-1.30 g/mL, and drying the thick paste in vacuum until the water content is less than 2.5%, thus obtaining extract powder II;

(3) Mixing the extract powder I and the extract powder II, and micronizing;

(4) Mixing the extract powder with adjuvants according to the weight ratio of 2:1, granulating with 90% ethanol by spraying, drying, and packaging.

6. The method according to claim 5, wherein the extract powder in step (3) is pulverized by ultra-fine pulverizing technique, and the particle size is controlled to 300 mesh.

7. A formulation for use in the treatment of novel coronavirus infections, characterized in that the active ingredient thereof is a pharmaceutical composition according to any one of claims 1-4.

8. The formulation of claim 7, wherein the formulation is in the form of a granular formulation.

9. Use of a pharmaceutical composition according to any one of claims 1-4 for the preparation of a novel therapeutic agent for coronavirus infection.

10. The use according to claim 9, wherein the medicament reduces the effects of lung injury caused by novel coronaviruses by reducing lipid peroxidation damage in the body, reducing MDA formation, which is the end product of lipid peroxidation, increasing SOD activity, and enhancing antioxidant capacity in the body.