CN111166737B

CN111166737B - Application of mangiferin

Info

Publication number: CN111166737B
Application number: CN201910999332.8A
Authority: CN
Inventors: 秦枫; 朱善元; 郭长明; 刘云; 吴双; 吴植; 王安平; 王永娟; 封琦; 洪伟鸣
Original assignee: Jiangsu Agri Animal Husbandry Vocational College
Current assignee: Jiangsu Agri Animal Husbandry Vocational College
Priority date: 2019-10-21
Filing date: 2019-10-21
Publication date: 2022-04-01
Anticipated expiration: 2039-10-21
Also published as: CN111166737A

Abstract

The invention relates to the technical field of traditional Chinese medicines and discloses a new application of mangiferin. The invention utilizes Western-Blot, qRT-PCR, apoptosis and other technologies to prove through experiments of various layers that mangiferin can obviously inhibit PRRSV infection and replication, has obvious PRRSV virus resistance, is expected to become a novel medicament for preventing and treating porcine reproductive and respiratory syndrome, and has good application prospect in the prevention and treatment of porcine reproductive and respiratory syndrome.

Description

Application of mangiferin

Technical Field

The invention relates to the technical field of traditional Chinese medicines, in particular to application of mangiferin.

Background

Porcine Reproductive and Respiratory Syndrome (PRRS), commonly known as Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), is an acute, contact, highly contagious disease caused by the Porcine Reproductive and Respiratory Syndrome virus. The primary infected subjects of PRRSV are pregnant sows, which present a reproductive barrier, and piglets, which, in addition to severe respiratory symptoms and persistent infections, also cause growth retardation and neurological symptoms. The disease was first reported in 1987 in the united states, followed by successive reports in germany, the netherlands, canada, etc., and then rapidly spread to asia and other regions, with enormous losses to the swine industry worldwide.

Scholars at home and abroad do a lot of work on the research of PRRS prevention and control, and certain progress is made on the aspects of vaccine prevention and western medicine prevention and control. Currently, the PRRSV vaccines on the market mainly comprise inactivated vaccines and attenuated vaccines, but both vaccines have serious efficacy and safety problems. The first vaccine used for clinical treatment of PRRSV was the inactivated vaccine. The inactivated vaccine has safety, does not cause virus transmission and epidemic diseases, and has the advantages of convenient storage and transportation. However, the protective effect of inactivated vaccines is not ideal. After the inactivated vaccine is used, only humoral immunity can be generated, so that secondary immunity is needed to play a certain protection role, the time from immunization to immune protection of the inactivated vaccine is long, the generation amount of neutralizing antibodies in the period is possibly little, or the neutralizing antibodies are not generated, so that the condition of ineffective immunity occurs, and the inactivated vaccine cannot effectively control the attack and the spread of PRRSV. In contrast, the attenuated vaccine can be induced to generate neutralizing antibody rapidly, and has the advantages of long immunization time, strong resistance and the like. However, attenuated vaccines present the risk of virus spread and may be virulent back during the course of immunization.

Because the PRRSV is strictly intracellular parasitic and needs to be copied depending on various functions of cells, western medicines are difficult to exert the efficacy. The antiviral western medicines ribavirin, zanamivir and amantadine are mainly used, but the western medicines are forbidden clinically in veterinarians. The traditional Chinese medicine has unique superiority in preventing and controlling virus diseases. The Chinese medicine antiviral can not only directly play an antiviral role in the aspects of preventing the adsorption, penetration, replication and the like of viruses, but also regulate the immune function of an organism, participate in the immune reaction of the organism and play immune monitoring and antiviral roles.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of mangiferin and/or a mangiferin-containing plant extract in the preparation of a medicament for treating and/or preventing porcine reproductive and respiratory syndrome;

the invention also aims to provide the application of mangiferin and/or a plant extract containing mangiferin components in preparing a medicament for resisting porcine reproductive and respiratory syndrome virus;

another object of the present invention is to provide a Chinese medicinal composition for treating porcine reproductive and respiratory syndrome virus, comprising mangiferin and/or a plant extract containing mangiferin components.

Mangiferin is a carbon glycoside of tetrahydroxy bisphenone. The mango leaves have the activity of resisting herpes simplex virus (HSV-1 and HSV-2), can inhibit the replication of infectious HSV-2 in cells infected by wounds, and the active component of the anti-herpes virus is mangiferin. Chen H et al reported that mangiferin is an effective ingredient in limiting mouse infection with influenza virus. Mangiferin was found to inhibit human herpes virus type 1 (HSV-1), mouse encephalomyocarditis virus (EMCV) and dengue virus type 2 (DENV-2) by Brando GC et al. The danga and the like report that mangiferin has good effect of inhibiting Duck Hepatitis B Virus (DHBV) infection, and the phenomenon of rebound is not easy to occur after the medicine is stopped. The research shows that scholars at home and abroad make certain research on the resistance of mangiferin to human and animal viruses, but relevant reports on the PRRSV resistant effect of the mangiferin are not available.

The mangiferin is researched and found to have a remarkable antiviral effect on porcine reproductive and respiratory syndrome virus for the first time, and specifically, the mangiferin is prepared, the antiviral effect on PRRSV is analyzed through different action modes and different action times of the mangiferin on PRRSV, ribavirin is used as a positive control drug, Western-Blot, qRT-PCR, apoptosis and other technologies are utilized, experiments on various layers prove that the mangiferin can remarkably inhibit the infection and the replication of PRRSV, has a remarkable effect on resisting the PRRSV virus, is expected to become a novel drug for preventing and treating the porcine reproductive and respiratory syndrome, and has a good application prospect in the prevention and treatment aspect of the porcine reproductive and respiratory syndrome.

Therefore, the invention provides the application of mangiferin or a plant extract containing mangiferin components in preparing medicaments for treating and/or preventing porcine reproductive and respiratory syndrome virus and medicaments for resisting the porcine reproductive and respiratory syndrome virus; the mangiferin is present in fruits, leaves and barks of mangoes (Mangifera indica L.) in the family of Anacardiaceae, rhizomes and aerial parts of Anemarrhena asphodeloides Bge in the family of Liliaceae, flowers and leaves of Belamcanda chinensis DC in the family of Iridaceae, so the mangiferin-containing plant provided by the invention mainly refers to the above plant extract, and the mangiferin-containing extract can be obtained by the existing extraction methods in the field, such as water decoction, organic solvent extraction and the like.

More specifically, the application provided by the invention is the application of mangiferin or a plant extract containing mangiferin components in the preparation of a medicament for treating and/or preventing porcine reproductive and respiratory syndrome virus and a medicament for resisting porcine reproductive and respiratory syndrome virus; or the application of the mixture of mangiferin and plant extract containing mangiferin component in preparing medicine for treating and/or preventing porcine reproductive and respiratory syndrome virus; or the application of the mangiferin and/or the plant extract containing the mangiferin and other traditional Chinese medicine components without rejection in the preparation of medicines for treating and/or preventing porcine reproductive and respiratory syndrome virus.

According to the application, the invention provides a traditional Chinese medicine composition for resisting porcine reproductive and respiratory syndrome virus or treating porcine reproductive and respiratory syndrome, which comprises mangiferin and/or a mangiferin-containing plant extract. Preferably, the weight ratio of the mangiferin and/or the mangiferin-containing plant extract in the composition is 60-99.9995%; more preferably, the weight ratio of the mangiferin and/or the mangiferin-containing plant extract in the composition is 80-99.9995%; more preferably, the weight ratio of the mangiferin and/or the mangiferin-containing plant extract in the composition is 95-99.9995%.

In the specific implementation mode of the invention, the mango leaves are separated and purified through organic solvent extraction, chromatographic column purification and the like, and the purity of the mango leaves is 99.9995 percent calculated by an area normalization method; the molecular structure is determined by nuclear magnetic resonance. The content determination and molecular structure determination maps are shown in FIG. 1 and FIG. 2, respectively.

According to the technical scheme, experiments on various layers prove that mangiferin can obviously inhibit PRRSV infection and replication by using Western-Blot, qRT-PCR, apoptosis and other technologies, has an obvious PRRSV virus resistance effect, is expected to become a novel medicament for preventing and treating porcine reproductive and respiratory syndrome, and has a good application prospect in the prevention and treatment aspect of the porcine reproductive and respiratory syndrome.

Drawings

FIG. 1 is an HPLC chromatogram of mangiferin;

FIG. 2 is a nuclear magnetic resonance spectrum of mangiferin;

FIG. 3 shows the pathological changes of cells under the blocking, inhibiting and direct inactivation effects of mangiferin on PRRSV;

FIG. 4 is a diagram showing the identification of recombinant plasmids;

FIG. 5 shows that mangiferin blocks PRRSV, and qRT-PCR detects N gene expression level after 24h, 48h and 72 h;

FIG. 6 shows that mangiferin inhibits PRRSV, and after 24h, 48h and 72h, qRT-PCR detects the expression level of N gene;

FIG. 7 shows that mangiferin has direct inactivation effect on PRRSV, and after 24h, 48h and 72h, qRT-PCR detects N gene expression level;

FIG. 8 shows that the mangiferin blocks PRRSV, and Western Blot detects the expression level of N protein after 24h, 48h and 72 h; wherein 1 is mangiferin group; 2 is positive control ribavirin group; 3 is PRRSV control group; 4 is cell control group; graph a shows the effect of different treatment groups on PRRSV N protein expression, graph B shows the effect of different treatment groups on the PRRSV N protein gray value ratio, and the ordinate represents the gray value ratio of the target protein to the responsive internal reference;

FIG. 9 shows that the mangiferin inhibits PRRSV, and Western Blot detects the expression level of N protein after 24h, 48h and 72 h; wherein 1 is mangiferin group; 2 is positive control ribavirin group; 3 is PRRSV control group; 4 is cell control group; graph a shows the effect of different treatment groups on PRRSV N protein expression, graph B shows the effect of different treatment groups on the PRRSV N protein gray value ratio, and the ordinate represents the gray value ratio of the target protein to the responsive internal reference;

FIG. 10 shows that the mangiferin has direct inactivation effect on PRRSV, and Western Blot detects the N protein expression level after 24h, 48h and 72 h; wherein 1 is mangiferin group; 2 is positive control ribavirin group; 3 is PRRSV control group; 4 is cell control group; graph a shows the effect of different treatment groups on PRRSV N protein expression, graph B shows the effect of different treatment groups on the PRRSV N protein gray value ratio, and the ordinate represents the gray value ratio of the target protein to the responsive internal reference;

FIG. 11 shows the apoptosis of cells detected by flow cytometry after 24h, 48h and 72h of culture of uninfected PRRSV and infected PRRSV, respectively; wherein the lower left quadrant represents live cells, the lower right quadrant represents early apoptotic cells, the upper right quadrant represents late apoptotic or dead cells, and the upper left quadrant represents dead cells; LL is the living cell rate, LR is the early apoptosis rate, UR is the late apoptosis or death rate, and UL is the cell death rate; n1, N2 and N3 respectively represent cell control groups of 24h, 48h and 72h, and D1, D2 and D3 respectively represent virus control groups of 24h, 48h and 72 h;

FIG. 12 shows that mangiferin blocks PRRSV, and after 24h, 48h and 72h, the influence of the mangiferin on apoptosis is detected by a flow cytometer; wherein LL is the living cell rate, LR is the early apoptosis rate of the cell, UR is the late apoptosis or death rate of the cell, and UL is the death rate of the mechanical damage of the cell; a1, A2 and A3 respectively represent mangiferin groups of 24h, 48h and 72h, B1, B2 and B3 respectively represent positive control ribavirin drug groups of 24h, 48h and 72 h;

FIG. 13 shows that mangiferin inhibits PRRSV, and after 24h, 48h and 72h, the influence of the mangiferin on apoptosis is detected by a flow cytometer; wherein LL is the living cell rate, LR is the early apoptosis rate of the cell, UR is the late apoptosis or death rate of the cell, and UL is the death rate of the mechanical damage of the cell; a1, A2 and A3 respectively represent mangiferin groups of 24h, 48h and 72h, B1, B2 and B3 respectively represent positive control ribavirin drug groups of 24h, 48h and 72 h;

FIG. 14 shows that mangiferin has direct inactivation effect on PRRSV, and after 24h, 48h and 72h, the influence of mangiferin on apoptosis is detected by a flow cytometer; wherein LL is the living cell rate, LR is the early apoptosis rate of the cell, UR is the late apoptosis or death rate of the cell, and UL is the death rate of the mechanical damage of the cell; a1, A2 and A3 respectively represent mangiferin groups of 24h, 48h and 72h, B1, B2 and B3 respectively represent positive control ribavirin drug groups of 24h, 48h and 72 h;

FIG. 15 is monitoring of changes in body temperature of groups of piglets after infection with virus;

FIG. 16 is the clinical symptom scores of groups of piglets after infection with virus;

FIG. 17 is a lung lesion observation of various groups of piglets after infection with virus; the upper graph is a lung visual image, and the lower graph is a pathological section image;

FIG. 18 is a dynamic monitoring of serum viremia for groups of piglets after infection with virus;

FIG. 19 is RT-PCR detection of tissue and organ viral loads of piglets in each group after infection with virus;

FIG. 20 is PRRSV antibody detection of various groups of test piglets after infection with virus;

in each of the above figures, P <0.05, P < 0.01.

Detailed Description

The invention discloses application of mangiferin, and can be realized by appropriately improving process parameters by referring to the content in the text by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the techniques of the invention can be implemented and practiced with modification, or with appropriate modification, and combination of parts and features without departing from the spirit and scope of the invention.

Statistical analysis of the following examples of the invention: all experiments were repeated at least 3 times independently, and single-factor analysis of variance was performed using software SPSS17.0, all data expressed as Mean ± SD, Student's test statistical analysis. And analyzing the obtained grey value of the protein band by using Image J, and dividing the grey value of the target band by the corresponding grey value of the beta-actin band to obtain a ratio which is a relative expression value. All histograms were plotted using graph Prism 5.

The invention is further illustrated by the following examples.

Example 1: cytotoxicity of mangiferin and ribavirin

Cell maintenance solution (DMEM with 2% FBS) containing 0.1% DMSO (non-toxic to cells at this concentration) was used to dissolve mangiferin and dilute 2 fold. Cells grown to a monolayer in cell flasks were digested with complete cell culture medium (DMEM containing 10% FBS) and diluted to about 2X 10⁵Perml, add to 96-well plate, 100. mu.L cell suspension per well. After the cells grow to a monolayer, the old culture solution is discarded, the diluted liquid medicine is added into the 1 st to 10 th rows in sequence from high concentration to low concentration, each concentration is repeated for 4 holes, each hole is 100 mu L, and the cell maintenance solution containing 0.1% DMSO is added into the 11 th to 12 th rows to serve as cell control. Culturing for 72h, discarding the medicinal liquid, adding MTT solution, culturing for 4h, discarding the MTT solution, adding DMSO to dissolve the crystal completely, and measuring OD_490nmThe value is obtained. The calculation formula is as follows:

growth inhibition (%) (cell control OD)_490nmValue-drug group OD_490nmValue)/cell control OD_490nmThe value is multiplied by 100%

When the inhibition rate is 0, the drug has no toxic effect on cells at that concentration, and the maximum safe concentration of the drug is determined, and the results are shown in table 1.

TABLE 1 inhibition ratio (%) -145 cells by different drug concentrations

Note: the initial concentration of mangiferin is 50 mug/mL and the initial concentration of ribavirin is 1 mg/mL.

As shown in Table 1, when the mangiferin concentration is 0.2. mu.g/mL or less, and the ribavirin concentration is 15.63. mu.g/mL or less, the mangiferin composition has no toxicity to Marc-145 cells. Therefore, the initial concentration of mangiferin and ribavirin used in the subsequent experiments was 0.2. mu.g/mL and 15.63. mu.g/mL.

Example 2: viral Titer (TCID)₅₀) Measurement of

Sequentially diluting virus solution to 10 times by using DMEM culture solution without FBS according to 10 times ratio^-1～10^-88 concentration gradients, seeded on 96 well cell culture plates at 100. mu.L/well. 8 wells were inoculated at each dilution, and a virus control group and a cell control group were simultaneously established and placed in 5% CO₂Culturing in an incubator at 37 ℃ for 2h, discarding virus solution, adding cell maintenance solution, observing for 5-7 days, recording cytopathic condition, and calculating TCID by Reed-Muench method₅₀The calculation formula is as follows:

LgTCID₅₀log dilution of virus above 50% lesion rate + (above 50% lesion rate-50%)/(above 50% lesion rate-below 50% lesion rate) x log of dilution multiple

The results of the virus titer determination are shown in Table 2, and the TCID of PRRSV on Marc-145 cells is calculated according to the formula₅₀Value of 10^-4.60.1mL, i.e., 0.1mL of virus was inoculated at a concentration of 10^-4.6The virus liquid can cause 50% of cells to be diseased.

TABLE 2 PRRSV TCID₅₀Measurement results

Example 3: MTT method for detecting effect of mangiferin on resisting PRRSV in vitro

3 action modes of blocking, inhibiting and directly inactivating are adopted to detect the effect of mangiferin on resisting PRRSV in vitro. The 1 st mode is that the medicine is added firstly and then the virus is added (blocked), so as to observe whether the medicine can be combined with the cell membrane surface receptor to prevent the adsorption and entry of the virus; the 2 nd mode of action is that the virus is added firstly and then the medicine is added (inhibited), aiming at observing whether the medicine can enter the cell to act on the virus in the cell and enter the cell to inhibit the biosynthesis and the replication of the virus; the 3 rd mode of action is that the medicine and the virus are added after a period of time of action (direct inactivation), so as to observe whether the medicine has direct inactivation effect on the virus or not, and directly inhibit and kill the virus outside host cells before infection. The 3 action modes are respectively matched with the design of clinical prevention, treatment and disinfection tests, and clinically, the 1 st action mode is the prevention action of the medicament on the viral diseases, and the last two action modes are the treatment action of the medicament on the viral diseases.

(1) Blocking effect of mangiferin on PRRSV

After Marc-145 cells grow to a single layer in a 96-well plate, discarding old culture solution, adding a drug solution with the maximum safe mass concentration as the initial concentration, diluting by 2 times and 4 concentration gradients with 100 mu L of each well, culturing for 4h, discarding liquid medicine, adding 100TCID₅₀And culturing PRRSV solution in each well for 2h in 100 mu L, discarding virus solution, and adding DMEM growth maintenance solution containing 2% FBS. And calculating the inhibition rate of the medicament on the virus through a formula.

The results are shown in table 3, the maximum inhibition rates of mangiferin and ribavirin on PRRSV at the maximum safe concentrations are 91.7% and 63.3%, respectively, and the inhibition rate of mangiferin on ribavirin is significantly higher than that of ribavirin (P <0.01), which indicates that mangiferin has a strong blocking effect on PRRSV.

TABLE 3 protection of Marc-145 cells by mangiferin in blocking mode%

Note: the above experimental results were all subjected to three independent experiments, with four replicates established for each experiment. C1, C2, C3, C4 represent 4 different concentrations diluted 2-fold from the maximum safe concentration of the drug, all data are expressed as Mean ± standard deviation (Mean ± SD). Indicates that the significance of the difference between the drug group and the ribavirin control group in each column is P <0.05 and P <0.01 (enhancement effect), respectively, as follows.

(2) Inhibitory effect of mangiferin on PRSV

After Marc-145 cells grow to a single layer in a 96-well plate, old culture solution is discarded, 100TCID50PRRSV solution is added into each well, 100 mu L of each well is cultured for 2 hours, virus solution is discarded, drug solution with the maximum safe mass concentration as the initial concentration is added, 100 mu L of each well is diluted by 2 times for 4 concentration gradients, after 4 hours of culture, drug solution is discarded, and cell growth maintenance solution is added. And calculating the inhibition rate of the medicament on the virus through a formula.

The results are shown in table 4, the maximum inhibition rates of mangiferin and ribavirin on PRRSV are 71.3% and 68.9%, respectively, and there is no significant difference, which indicates that mangiferin and ribavirin both have the effect of inhibiting PRRSV in vitro, and the effects are similar.

TABLE 4 protection of Marc-145 cells by mangiferin in the inhibition mode%

(3) Direct inactivation effect of mangiferin on PRRSV

After Marc-145 cells grew to a monolayer in a 96-well plate, the old culture solution was discarded, and 50. mu.L of the Chinese medicinal extract solution and 50. mu.L of the virus solution (the maximum concentration of the liquid drug was the maximum safe mass concentration and the final concentration of the virus solution was 100 TCID) were added simultaneously₅₀) After culturing for 4h, the liquid medicine is discarded, and a DMEM growth maintenance liquid containing 2% FBS is added. And calculating the inhibition rate of the medicament on the virus through a formula.

The results are shown in table 5, the maximum inhibition rates of mangiferin and ribavirin on PRRSV are 65.7% and 65.5%, respectively, and there is no significant difference, which indicates that mangiferin and ribavirin both have the effect of directly inactivating PRRSV and have similar effects.

TABLE 5 protection of Marc-145 cells by mangiferin under direct inactivation mode%

(4) Observation of cytopathic effects of 3 modes of action

The cell morphology of 3 modes of action of mangiferin and ribavirin on PRRSV is shown in fig. 3. As can be seen from the figure, the cells in the virus control group mostly have the phenomena of shrinkage, aggregation and shedding, while the cells in the cell control group keep a monolayer intact. The positive control drug ribavirin group has obvious cytopathic effect under 3 action modes of PRRSV, and the cells have the phenomena of aggregation and rounding and fall off from a small part of cells. The mangiferin drug group has better protective effect on cells under the blocking effect, the cells have no obvious pathological changes and basically keep a single layer intact, and the cells have obvious pathological changes under the other 2 action modes, and the pathological change degree is similar to that of the ribavirin group which is a positive control drug.

Example 4: influence of mangiferin on N gene, N protein expression and apoptosis under PRRSV action

1. Influence of mangiferin on N gene expression under PRRSV action

1.1 primer design

PRRSV N gene primers were designed using the software Primer Premier 5.0, see table 6, with reference to the NCBI PubMed gene bank.

TABLE 6 primer sequences and PCR product sizes

1.2 preparation of standards

Extracting RNA from the virus liquid, carrying out reverse transcription on the extracted RNA, carrying out PCR amplification by using a specific primer aiming at the N gene code, connecting a pMD-18T vector, transforming DH5 alpha competent cells, extracting a plasmid by using the kit, and measuring the concentration. And carrying out PCR amplification on the plasmid, detecting the size of the amplified product fragment by agarose gel electrophoresis, determining the concentration of the N gene recombinant plasmid, and calculating the template amount of the standard substance.

Sequencing results show that the homology of the recombinant plasmid gene sequence and the corresponding sequence of the NCBI database reaches 99.6 percent. The recombinant plasmid was amplified by ordinary PCR, and the amplified product was electrophoresed to see a clean band, which was identical to the expected band size, as shown in FIG. 4. The recombinant plasmid OD260/OD280 value was 1.89, and the copy number of the recombinant plasmid was calculated to be 2.94X 10 based on the conversion formula of the concentration and the copy number⁹copies/μL。

1.3 recombinant plasmid concentration determination and Standard Curve establishment

A standard curve was prepared. A standard substance (10-1 to 10-6) diluted by 10 times of EASY Dilution is used as a template, an ABI 7300 fluorescence quantitative PCR instrument is used for amplification, and the reaction system is shown in the following table 7:

TABLE 7 fluorescent quantitative PCR reaction System

Performing fluorescent quantitative PCR reaction by a two-step method: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing and extension at 60 ℃ for 31s, 40 cycles, ending extension, and collecting fluorescence signals; and finally, preparing a melting curve in the process of 60-90 ℃.

1.4 Effect of Mangiferin on N Gene expression under PRRSV action

Discarding old culture solution from a six-hole plate of the Marc-145 cells growing to a single layer, and adopting a mode of adding the liquid medicine first and then adding the virus solution, adding the virus solution first and then adding the liquid medicine and simultaneously adding the virus solution and the liquid medicine to ensure that the concentration of the liquid medicine is the maximum safe concentration and the final concentration of the virus solution is 100TCID₅₀，5％CO₂Incubations were continued for 24h, 48h, 72h at 37 ℃ respectively, with 3 replicates per time point. Meanwhile, a positive drug ribavirin control group (ribavirin and virus solution), a cell control group (only adding cell maintenance solution) and a virus control group (only adding virus solution) are arranged.

Extracting total RNA from cells according to the Kit protocol of TaKaRa MiniBEST Universal RNA Extraction Kit, reverse-transcribing the cDNA of each treatment group, and using RNase-free H₂And O, diluting the samples of each group to the loading concentration, and performing reaction according to the following reaction system: SYBR Green Premix Ex Taq II (2X) 10. mu.L, PCR Forward Primer (10. mu.M) 0.8. mu.L, PCR Reverse Primer (10. mu.M) 0.8. mu.L, ROX Reference Dye (50X) 0.4. mu.L, cDNA 2. mu.L, sterile water 6. mu.L, and the total amount is 20. mu.L, and the PRRSV N gene copy number in each group of samples was calculated with Reference to the Ct value of each group using a standard curve prepared from the same lot. The results are shown in tables 8-10 and in FIGS. 5-7;

from the blocking effect of mangiferin on PRRSV, compared with the PRRSV control group, at 48h, mangiferin significantly inhibited N gene synthesis (P < 0.01). At 24h, compared with PRRSV positive control, the drug ribavirin control group has extremely obvious inhibiting effect (P <0.01) on the synthesis of N gene, and within 24 h-72 h, ribavirin obviously inhibits the synthesis of N gene (P < 0.05). Compared with the positive control drug ribavirin, mangiferin remarkably inhibits the synthesis of N gene at 48h (P < 0.01).

TABLE 8 Effect of mangiferin on N Gene copy number under PRRSV blockade

Note: compared with the PRRSV control group,^*P<0.05，^**P<0.01; compared with Ribavirin (Ribavirin) of a positive control drug group,^##P<0.05，^##P<0.01; the same applies below.

From the inhibition effect of mangiferin on PRRSV, compared with the PRRSV control group, the mangiferin significantly inhibits the synthesis of N gene at 24h and 72h (P <0.01) and at 48h (P < 0.05). Compared with the PRRSV control group, the positive control medicament ribavirin has extremely obvious inhibiting effect (P is less than 0.01) on the synthesis of the N gene at 24h, has obvious inhibiting effect on the synthesis of the N gene at 48h, and has unobvious inhibiting effect on the synthesis of the N gene at 72 h. Compared with a positive control medicament ribavirin, the mangiferin has an insignificant effect of inhibiting the synthesis of the N gene within 24-72 h.

TABLE 9 Effect of mangiferin on the copy number of the N Gene under the inhibition of PRRSV

From the direct inactivation effect of mangiferin on PRRSV, compared with a PRRSV control group, the mangiferin and a positive control drug ribavirin have a remarkable inhibition effect (P <0.05) on the synthesis of an N gene at 72 hours, but compared with the positive control drug ribavirin, the mangiferin has an insignificant inhibition effect on the synthesis of the N gene within 24-72 hours.

TABLE 10 Effect of mangiferin on the copy number of the N Gene under the direct inactivation of PRRSV

2. Influence of mangiferin on N protein expression under PRRSV action

Culturing cells according to the method in the step 1.4, removing the culture solution, completely sucking, adding 4 ℃ precooled PBS (PBS), slightly washing, removing, placing the plate on ice, adding 100 mu L of lysis solution containing PMSF (1mL of lysis solution containing 100 mu L of PMSF) into each cell, lysing for 30min on ice, and frequently shaking to fully lyse the cells; taking beta-actin as an internal reference to carry out western blot detection.

The results are shown in figures 8-10, where mangiferin and ribavirin both inhibited the expression of the N protein, which was consistent with the results of the gene level assay.

3. Effect of mangiferin on apoptosis under PRRSV action

Culturing cells according to the method in the 1.4, collecting cell samples according to the instructions of the Annexin V-EGFP/PI double-staining apoptosis detection kit, and detecting the influence of mangiferin on apoptosis after the cells are infected with PRRSV by using a flow cytometer.

The apoptosis results of the virus control group and the cell control group within 24-72 h are shown in Table 11 and FIG. 11. The results show that the apoptosis rate of PRRV virus group is significantly higher than that of normal cell control group (P <0.01), indicating that PRRSV can induce apoptosis. The cells of the normal cell control group belong to natural apoptosis, the apoptosis rate at 24h is 1.80% + -0.02%, the apoptosis rate at 48h is 4.13% + -0.54%, the apoptosis rate at 72h is 7.11% + -2.04%, and the apoptosis rate of the normal control group is increased with time but has little difference. The apoptosis rate of the virus control group is obviously increased along with the increase of time, the peak is reached at 72h, the total apoptosis rate of the cells is 84.29% +/-0.77%, and the late apoptosis rate is obviously increased compared with 48 h.

TABLE 11 apoptosis assay results for Virus control and cell control groups

Note: compared with the cell control,^#P<0.05，^##P<0.01；

the influence of mangiferin blocking on apoptosis was detected by flow cytometry, the apoptosis rate results of each test group are shown in table 12 and fig. 12, and the obtained apoptosis rates were compared with the cell control group and the PRRSV control group. The result of the late apoptosis rate of the cells shows that the mangiferin treated group has no significant difference from a normal cell control group within 48-72 h, is significantly lower than a PRRSV control group (P <0.05) within 24-72 h, and is significantly lower than a positive control medicament ribavirin group (P <0.05) at 24h and 72 h. The positive control drug ribavirin group has no significant difference between the late apoptosis rate of the cells and the normal cell control group at 24-48 h, and the late apoptosis rate of the cells at 72h is significantly higher than that of the cell control group (P <0.05) but significantly lower than that of the PRRSV control group (P < 0.05). The result of the total apoptosis rate of the cells shows that the total apoptosis rate of the mangiferin treated group is remarkably higher than that of a cell control group (P <0.05) within 24-48 h, is remarkably higher than that of the cell control group (P <0.01) at 72h and is remarkably lower than that of a positive control medicament ribavirin group (P < 0.05). The positive control drug ribavirin group has the total apoptosis rate obviously higher than that of the cell control group (P <0.05) within 24-72 h and obviously lower than that of the PRRSV control group (P <0.01) within 48-72 h.

TABLE 12 Effect of mangiferin on apoptosis Rate under PRRSV blocking%

Note: compared with the PRRSV control group,^*P<0.05，^**P<0.01; compared with the cell control group,^#P<0.05，^##P<0.01; compared with the ribavirin control group,^aP<0.05，^aaP<0.01. the same applies below.

The influence of mangiferin inhibition on apoptosis was detected by flow cytometry, the apoptosis rate results of each test group are shown in table 13 and fig. 13, and the obtained apoptosis rates were compared with those of the cell control group and PRRSV control group. The result of the data of the late apoptosis rate of the cells shows that the late apoptosis rates of the mangiferin group and the ribavirin group as the positive control medicament have no significant difference with those of the normal cell control group within 24-72 h, and are significantly lower than that of the PRRSV control group (P <0.05), and the late apoptosis rate of the mangiferin group has no significant difference with those of the ribavirin medicament group. The results of the data of the total apoptosis rate show that the apoptosis rate of the mangiferin group is significantly higher than that of the cell control group (P <0.05), and is significantly lower than that of the PRRSV control group (P <0.01, P <0.05), and is significantly lower than that of the ribavirin group (P <0.05) at 48 h. The total apoptosis rate of the ribavirin group at 72h was significantly lower than that of the PRRSV control group (P < 0.01).

TABLE 13 Effect of mangiferin on apoptosis Rate under PRRSV inhibition%

The influence of mangiferin on apoptosis under direct inactivation effect is detected by using a flow cytometer, the apoptosis rate results of each test group are shown in table 14 and figure 14, and the obtained apoptosis rate is compared with a cell control group and a PRRSV control group. The data result of the late apoptosis rate of the cells shows that the late apoptosis rate of the mangiferin cells is not obviously different from that of a normal cell control group within 24-48 h, is obviously lower than that of a PRRSV control group (P <0.05) within 24h and 72h, and is obviously lower than that of a positive control medicament ribavirin group (P <0.05) within 72 h. The late apoptosis rate of cells of the positive control medicament ribavirin is remarkably higher than that of a cell control group (P <0.01) at 72 hours, but is remarkably lower than that of a PRRSV control group (P < 0.05). The result of the total apoptosis rate of the cells shows that the apoptosis rate of mangiferin is extremely lower than that of a cell control group (P <0.01) within 24-72 h, and is extremely lower than that of a PRRSV control group and a positive control medicament ribavirin group (P <0.01) within 72 h. The positive control drug ribavirin was significantly lower than the PRRSV control group at 72h (P < 0.05).

TABLE 14 Effect of mangiferin on apoptosis Rate in the direct PRRSV inactivation mode%

Example 5: in vivo assay

1. Test pig pathogen detection

Purchasing 12 large-growing ternary hybrid weaned pigs of 28-day old from a PRRS non-immune pig farm, feeding for 3-5 days by using a negative pressure isolator, and observing whether the pigs are abnormal or not; before experiment, the vena cava is used for blood sampling and serum separation, DNA/RNA is extracted by using a kit, a PRRSV specific primer (F: GGGGAATGGCCAGYCAGTCAA, R: GCCAGRGGAAAATGKGGCTTCTC) is synthesized, a virus genome stored in a laboratory is used as a positive control, the pathogen infection condition of a test pig is detected by using PCR/RT-PCR, and a PRRSV specific antibody is detected by using a Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) enzyme-linked immunosorbent assay kit.

The kit is used for detecting the PRRSV antibodies, and the piglets meet the test requirements.

2. Experimental protocol

Dividing 9 32-day-old weaned piglets into 4 groups, feeding 3 piglets in 4 negative pressure isolators respectively, injecting mangiferin liquid medicine into group 1, injecting ribavirin liquid medicine as positive control medicine into group 2, injecting no toxicity counteracting control group into group 3, and injecting no toxicity counteracting control group into group 4. After the 4 th day of administration, the piglets of the 1 st, 2 nd and 3 rd groups injected with intramuscular injection and inoculated with nasal drip SH1705 strain PRRSV (10)⁵TCID₅₀)。

3. Body temperature monitoring

After the experimental piglets are infected with the PRRSV, the rectal temperature is measured once every day at regular time, data are recorded, and a body temperature change trend graph is drawn. The results are shown in fig. 15, the body temperature of piglets in the challenge control group and the drug group was significantly increased at the start of day 4 of the virus infection, and the body temperature was decreased and gradually returned to normal at day 11. The body temperature of each group of piglets is observed, and the effect of mangiferin on resisting PRRSV in vivo cannot be seen.

4. Clinical symptom scoring

Clinical symptoms were observed 2 times daily and recorded, and scored according to the clinical symptom scoring criteria of table 15.

TABLE 15 clinical symptom score criteria

The results are shown in fig. 16, the piglets in the challenge control group began to have clinical symptoms (1-4 points) such as diarrhea and emaciation on day 1, then gradually became serious (4-10 points), had clinical symptoms such as rash, skin ulceration, shortness of breath and hair loss, and had symptom relief on day 17 and continued to day 22; the positive control drug ribavirin group piglets begin to have clinical symptoms such as diarrhea, rash and the like on the 1 st day and continue to the 22 nd day (2-3 min); the mangiferin group piglets show mild symptoms continuously on day 1 to day 19, then gradually recover to be normal (1-2 points), and the clinical symptom score on days 2-16 is obviously lower than that of the challenge control group (P < 0.05). The placebo pigs did not develop significant symptoms during the trial.

5. Sample collection and detection

On

days

1, 3, 5, 8, 11, 15, 18 and 22 after PRRSV infection, 5mL of serum isolate was collected from the anterior vena cava of each piglet, while 1g of feces collected from each group was resuspended in PBS; performing cesarean examination on the killed or experimental animal, collecting samples of heart, liver, spleen, lung, kidney, lymph node, etc., and freezing at-80 deg.C for later use. Collecting lung tissue blocks, and cutting into pieces of 1mm³The small blocks are fixed by 4% buffer solution at 4 ℃, fixed tissue samples are embedded by paraffin, and pathological histological changes of the small blocks are observed after conventional HE staining.

5.1 Lung dissection and pathological section Observation

The lung changes and pathological section observation results of each group of piglets are shown in figure 17; the lung of the piglets of the challenge control group is obviously bleeding and swelling, a large amount of necrotic foci can be seen on the surface, the lung of the piglets of the mangiferin group and the positive control drug ribavirin group has no obvious macroscopic change, and only a small amount of bleeding spots and necrotic foci are seen. The mangiferin group and the positive control drug ribavirin are shown to have the effect of resisting PRRSV in vivo.

Pathological section results show that interstitial pneumonia of lungs of the challenge control piglets is obvious, alveolar walls of the lungs are thickened, lymphocyte infiltration is achieved, a large amount of exudates appear in bronchi, pathological damage of a positive control group is reduced compared with that of the challenge control group, pathological damage of a mangiferin group is lighter than that of the positive control group, alveolar walls of the lungs are slightly thickened, the infiltration of lymphocytes and the exudates are reduced, the lungs of piglets of a blank control group are normal, no pathological damage is found, and the mangiferin and the ribavirin serving as a positive control medicament can reduce tissue damage and tissue lesions caused by PRRSV infection.

5.2 viral load detection

Viral RNA was extracted from each sample using TaKaRa MiniBEST viral RNA/DNA Extraction Kit, wherein 200. mu.L of serum was taken, 100mg of tissue and organ were taken, and the number of copies of viral genome in each sample was determined by the above-established fluorescent quantitative RT-PCR.

The viremia test result is shown in fig. 18, the PRRSV of the infected pig serum is detected to be positive on the 3 rd day, the RNA copy number of the challenge control group is increased to the maximum (4.6-5.1log10/mL) on the 8 th day, no piglet dies in the process, and the serum virus content of the piglet is reduced to 1.1-1.6log10/mL on the 22 th day; the positive control drug ribavirin group and mangiferin drug group piglets are also positive in PRRSV detection at 3 days after infection, RNA copy number is highest at 8 days, serum virus content is respectively 4.1-5.1log10/mL and 3.3-5.3log10/mL, and serum virus content is respectively reduced to 0.4-0.9log10/mL and 0.2-0.9log10/mL at 22 days.

The organ viral load detection results are shown in fig. 19, and the viral loads of heart, liver, spleen, lung, kidney and lymph node of the challenge control group are respectively 1.9-4.2, 1.2-1.9, 4.1-4.2, 4.1-4.7, 1.2-2.1 and 4.4-4.6log10/0.1 g; the positive control drug ribavirin groups are respectively 0.9-2.0, 0.5-1.6, 4.1-4.7, 1.4-2.3, 1.4-1.7 and 3.5-4.9log10/0.1g, wherein the viral load of the lung is obviously lower than that of the challenge control group (P < 0.05); the mangiferin drug groups are respectively 1.0-2.2, 1.2-1.4, 3.7-4.9, 3.3-4.2, 0.7-1.8 and 1.5-1.7log10/0.1g, wherein the viral load of lymph nodes is remarkably lower than that of a challenge control group (P <0.01), and the viral load of lung is remarkably higher than that of a positive control drug ribavirin group (P < 0.05). The results show that the positive control drugs ribavirin and mangiferin both have the effect of inhibiting PRRSV in vivo.

5.3PRRSV antibody detection

After infecting PRRSV SH1705, the serum of each group of piglets is periodically collected, and the PRRSV antibody level is detected according to the specification of a Jianglai Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) enzyme-linked immunosorbent assay kit.

The results are shown in fig. 20, on day 1 after challenge, antibody detection of the challenge control group, the positive control medicament ribavirin group and the mangiferin medicament group is positive, then antibody levels of the challenge control group and the mangiferin group are rapidly increased, the antibody levels reach the highest values on day 8, and on days 3-8, the antibody levels of the mangiferin group are significantly higher than those of the challenge control group, which indicates that the mangiferin can stimulate organism immunity and promote antibody generation, so that the antibody levels are rapidly increased. The mangiferin drug group antibody level gradually decreased at days 9-15, and tended to level off at days 15-22, indicating that mangiferin might play a direct virucidal role during this period. Similarly, the level of the positive control drug ribavirin antibody is obviously lower than that of the challenge control group at days 1-15, which indicates that ribavirin does not have the function of regulating immunity but plays the role of directly killing viruses. The detection results of the PRRSV antibodies of the piglets of the blank control group in the test period are negative.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. Application of mangiferin in preparing medicine for treating and/or preventing porcine reproductive and respiratory syndrome is provided.

2. Application of mangiferin in preparing medicine for resisting porcine reproductive and respiratory syndrome virus is provided.

3. The application of a mangiferin-containing plant extract in preparing a medicament for treating and/or preventing porcine reproductive and respiratory syndrome is disclosed, wherein the mangiferin-containing plant extract is a mango extract, a rhizoma anemarrhenae extract or a blackberry lily extract.

4. The application of a mangiferin-containing plant extract in preparing a medicament for resisting porcine reproductive and respiratory syndrome virus is disclosed, wherein the mangiferin-containing plant extract is a mango extract, a rhizoma anemarrhenae extract or a blackberry lily extract.

5. Application of mangiferin and a mangiferin-containing plant extract in preparation of medicines for treating and/or preventing porcine reproductive and respiratory syndrome, wherein the mangiferin-containing plant extract is a mango extract, a rhizoma anemarrhenae extract or a blackberry lily extract.

6. Application of mangiferin and a mangiferin-containing plant extract in preparation of a medicament for resisting porcine reproductive and respiratory syndrome virus, wherein the mangiferin-containing plant extract is a mango extract, a rhizoma anemarrhenae extract or a blackberry lily extract.

7. The use according to any one of claims 3 to 6, wherein the extract is an aqueous extract or an alcoholic extract.

8. The use of claim 2, 4 or 6, wherein the porcine reproductive and respiratory syndrome virus is direct porcine reproductive and respiratory syndrome virus.