CN110585195B

CN110585195B - Anti-influenza virus medicine

Info

Publication number: CN110585195B
Application number: CN201910768161.8A
Authority: CN
Inventors: 王雪; 王林林; 赵军; 史玉柱; 司丽君; 刘燕; 马雪萍; 姚华
Original assignee: XINJIANG INSTITUTE OF MATERIA MEDICA
Current assignee: XINJIANG INSTITUTE OF MATERIA MEDICA
Priority date: 2019-08-20
Filing date: 2019-08-20
Publication date: 2022-09-09
Anticipated expiration: 2039-08-20
Also published as: CN110585195A

Abstract

The invention discloses an anti-influenza virus medicament, the active component of which is esculetin, the anti-influenza virus medicament is a tablet or a capsule, wherein the esculetin is esculetin extracted from viola tianschanica or violaceae plants or esculetin artificially synthesized. The invention firstly provides that the esculetin has definite anti-influenza virus effect, can obviously inhibit cytopathia caused by influenza virus infection, and provides an important idea for the esculetin in the aspect of treating influenza.

Description

Anti-influenza virus medicine

Technical Field

The invention relates to an anti-influenza virus medicament, and belongs to the field of medicines.

Background

Influenza is considered to be the most harmful infectious disease so far, and because the pathogen influenza virus has strong pathogenicity and is easy to mutate, the most effective measure for preventing influenza is difficult to inject influenza vaccine to play a role in time. Seasonal influenza epidemics which occur all over the world every year seriously affect people's life and work, even endanger life, and cause heavy economic burden to society. In recent years, the emergence of new highly pathogenic influenza viruses such as H5N1 and the like has made human beings face greater threat, and "human avian influenza", "pandemic influenza" and "seasonal influenza" have become public problems of high concern in today's society.

For a long time, anti-influenza drug research mainly focuses on targets of components closely related to replication and transmission of influenza virus, such as Hemagglutinin (HA), Neuraminidase (NA), Matrix protein2 (M2), influenza virus RNA polymerase and the like, and approved drugs on the market mainly include M2 protein channel blockers amantadine (amantadine) and rimantadine (rimantadine), NA inhibitors zanamivir (zanamivir) and oseltamivir (osehamimivi) and the like. The other is a nucleoside antiviral drug, the most commonly used at present is Ribavirin (Ribavirin), which has broad-spectrum strong efficacy and is widely used for treating influenza clinically. Although medical institutions in many countries currently use the medicines as reserve medicines in the influenza pandemic period, clinical studies show that amantadine and rimantadine can rapidly generate drug resistance and cross drug resistance in vitro and in vivo, the drug-resistant strains of zanavir and oseltamivir have been clinically isolated, the clinical curative effect is not ideal when the influenza symptoms are severe, and in addition, ribavirin can not only cause various general adverse reactions in clinical use, but also possibly cause severe adverse reactions such as hemolytic anemia and hypotension.

Once influenza viruses infect the body, the body rapidly initiates immune mechanisms. The immune reaction causes a large amount of immune cells and inflammatory factors to proliferate and gather in the lung, so that the inflammatory chain reactions such as cell apoptosis, capillary permeability increase, leucocyte exudation, pulmonary edema, airway blockage and the like are caused, the lung function is damaged, even the human or animal is finally killed, and the excessive host immune reaction is suggested to be one of the main reasons for mediating pathological damage.

In recent years, researches show that over-expression of influenza virus double-stranded RNA (dsRNA), HA, NP or M1 protein can be recognized by host cell retinoic acid and Toll-like receptor 3, so that the defense function of the host autoimmune system is stimulated, particularly the secretion of antiviral cytokine interferon alpha/beta (IFN alpha/beta) is caused, and signal channels such as NF-kappa B, Raf/MEK/ERK, IRF-3, PI3K-Akt and the like can be activated. Transcription of influenza virus is known to occur in the nucleus of infected cells, viral genomic RNA forms ribonucleoprotein bodies (RNPs) with nucleoproteins, and Raf/MEK/ERK signaling pathways are essential for RNP export out of the nucleus and viral proliferation.

NF-kB is an important signal molecule in a TLR 7-mediated MyD 88-dependent signal pathway, after the TLR7 signal pathway is activated, an NF-kB inhibitor IkB is degraded, the inhibition effect on the NF-kB is relieved, and free NF-kB translocates into nucleus and participates in the transcriptional regulation of various inflammatory factor genes by combining with DNA. Many studies have confirmed that NF- κ B expression is enhanced after influenza virus infection, accompanied by massive secretion of various inflammatory cytokines such as IL-6, IL-1, IL-8, TNF-a, etc. and release of the adhesion factor ICAM-1, thereby causing excessive release of inflammatory factors from the body to cause lung inflammatory injury. It is now clear that there is a positive correlation between NF-. kappa.B activation and the expression of various cytokines involved in the inflammatory response in the lung (e.g., TNF-a) and lung injury caused by influenza virus. Activation of NF-kB is taken as a sign of proliferation of influenza virus in host cells, and plays an important role in a signal transduction pathway of a body against influenza virus infection. It was found that influenza virus Hemagglutinin (HA) also activates NF-. kappa.B binding to DNA and the transcription process. NF- κ B not only indirectly participates in the exacerbation of viral pneumonia by mediating the release of proinflammatory factors, etc., but also determines the susceptibility of cells to influenza virus.

It follows that host factors also play an important role in the pathogenesis of influenza. It has been reported that drugs having both direct antiviral activity and immunomodulatory activity are effective in protecting A/H5N1 infected mice than simple antiviral drugs. In the pathogenic process of influenza virus infection, if immune regulation and anti-inflammatory drugs can be applied while antiviral drugs are administered, the abnormality of the immune system can be corrected, the progress of pneumonia can be intervened, and the lung damage caused by over-immunity can be reduced to the maximum extent, so that the symptoms caused by virus infection can be remarkably alleviated, and the death of human or animals can be reduced.

The traditional Chinese medicine has the characteristics of multiple components and multiple links, not only has direct antiviral effect, but also can regulate the complex process of immunopathological injury caused by viruses, and has unique advantages in the treatment of influenza. In reviewing the experimental research of the traditional Chinese medicine for preventing and treating influenza since the nineties of the last century, the mechanism exploration of the traditional Chinese medicine for promoting the antiviral immunity of the organism becomes a subject of general attention. Wherein, more and more researches discuss the intervention effect of the traditional Chinese medicine on inflammatory cytokines which are key links in the influenza immune regulation network, thereby disclosing the mechanism of the traditional Chinese medicine on the immune regulation effect of influenza virus infected organisms to a certain extent. In the aspect of influence on immune mechanisms, the enhancement effect of the medicine on the immune functions is not only emphasized, and more researches pay attention to the effect of the traditional Chinese medicine on resisting immune inflammatory injury, namely the inhibition of proinflammatory cytokines and the improvement of the anti-inflammatory cytokines are paid attention.

Esculetin, also called aesculetin or esculetin, is a coumarin compound with high content in violet, and the subject group has completed the separation and purification of esculetin from violet, and the chemical formula structure is as follows:

according to previous research reports, esculetin has pharmacological actions of resisting bacteria, relieving cough, eliminating phlegm, relieving asthma, resisting tumor, etc. However, the anti-influenza virus effect of esculetin and the application of esculetin in the preparation of anti-influenza virus drugs are not reported at home and abroad, and related documents are not retrieved.

Disclosure of Invention

In view of the above, the invention provides an anti-influenza virus medicament, which specifically adopts the following technical scheme:

an anti-influenza virus medicine contains esculetin as active ingredient.

Preferably, the anti-influenza virus medicament is a tablet or capsule;

the tablet or capsule contains esculetin with acceptable dose for human body, wherein esculetin content per tablet is 10-100 mg, and esculetin content per capsule is 10-100 mg.

The esculetin has better direct anti-influenza virus activity, can reduce the generation and development of inflammatory reaction in virus infection by inhibiting the activation of NF-kappa B, MAPK signal path, and has indirect antiviral effect by immunoregulation, so the esculetin has good prospect in preparing anti-influenza virus drugs.

The invention provides for the first time that esculetin has definite anti-influenza virus effect, can obviously inhibit cytopathic effect caused by influenza virus infection, and has half inhibitory concentration IC50 of influenza virus A/H1N 1/Jingfang/262/95 (A262/95), A/H3N 2/Guangdong fang/243/72 (A243/72), A/H3N 2/Jifang 15/90 (A15/90) and B/Jifang/13/97 (B97/13) as follows: 18.3, 17.3, 18.9 and 22.8, which shows that the esculetin has the activity of resisting influenza virus.

In addition, the esculetin can obviously inhibit LPS (low-temperature lipoprotein) to induce the release of proinflammatory factors TNF-alpha, IL-6 and inflammatory mediator NO in RAW264.7 cells, reduce the expression of iNOS (nitric oxide synthase), translocate p-p65, p-p38 and p-ERK1/2 proteins from cytoplasm to nucleus and reduce I kappa B alpha protein degradation, so the esculetin can reduce inflammatory reaction caused by virus infection by inhibiting the activation of an NF-kappa B, MAPK signal channel; esculetin can also significantly inhibit ConA from inducing T lymphocyte proliferation, inhibit T lymphocyte from secreting TNF-a, IL-6 and IL-2, and significantly inhibit LPS-induced B lymphocyte proliferation, which indicates that esculetin may play indirect antiviral role through immunoregulation.

Further, the aescin is obtained by extracting from Viola tianschanica or Violaceae plant or artificially synthesized aescin.

Furthermore, the anti-influenza virus drug also comprises pharmaceutically acceptable pharmaceutical excipients, wherein the pharmaceutical excipients are any one or a mixture of a plurality of cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, binders, fillers, disintegrants, lubricants, preservatives and antioxidants, such as microcrystalline cellulose, sodium carboxymethyl starch and magnesium stearate.

Drawings

FIG. 1 is a graph showing the effect of escin on the activity of RAW264.7 cells in example 2 of the present invention;

FIG. 2 is a graph showing the effect of escin on the secretion of TNF- α upon acute inflammation induced by LPS in RAW264.7 in example 2 of the present invention;

FIG. 3 is a graph showing the effect of escin on the secretion of IL-6 when RAW264.7 is induced by LPS in example 2 of the present invention;

FIG. 4 is a graph showing the results of the effects of esculetin on the expression level of iNOS protein and the production of NO when RAW264.7 is induced by LPS in example 2 of the present invention;

FIG. 5 is a graph showing the effect of escin on the expression of p-p65 protein when RAW264.730min was induced by LPS in example 2 of the present invention;

FIG. 6 is a graph showing the effect of esculetin on the expression of I.kappa.B.alpha.protein when RAW264.715min was induced by LPS in example 2 of the present invention;

FIG. 7 is a graph showing the effect of escin on the expression of p-p38 protein when RAW264.745min was induced by LPS in example 2 of the present invention;

FIG. 8 is a graph showing the effect of escin on the expression of p-ERK1/2 protein when RAW264.745min was induced by LPS in example 2 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Example 1

Esculetin has inhibitory effect on influenza virus (A/H1N 1/Jingfang/262/95, A/H3N 2/Jifang 15/90, A/H3N 2/Guangdong/243/72, B/Jifang/13/97)

The experimental principle is as follows: MDCK (dog kidney) cells are taken as virus hosts, and the inhibition effect of the samples on virus cytopathic degree (CPE) is determined.

Experimental materials: the sample escin was extracted from Viola tianschanica by the institute of Sinkiang pharmaceutical research, lot number 20180926. Influenza A virus strains (A/H1N 1/Jingfang/262/95, A/H3N 2/Jifang 15/90, A/H3N 2/Yuejang/243/72) and influenza B virus strains (B/Jifang/13/97) are subcultured in allantoic cavities of chicken embryos, and virus liquid is collected and stored at-80 ℃ for later use. Positive control drug Ribavirin (RBV) tablet with specification of 100 mg/tablet, and 0.15 g/d and 3 times/d for adult once, namely 0.45 g.d ^-1 Chongqing Kerui pharmaceutical Co., Ltd, national drug Standard H20073882, batch number 247001.

The experimental steps are as follows: MDCK cells were seeded in 96-well plates at a cell density of 2X 10 ⁴ And (4) sucking and removing the culture solution after the culture solution grows into a single layer, and arranging a cell blank control group, a virus control group, a positive control medicament ribavirin group and a esculetin group, wherein each medicament is provided with 5 (the concentration is diluted by 4 times under TC0 and TC 0) different concentrations, and each concentration is provided with 4 multiple wells. Each group was inoculated with 100TCID in addition to the cell control group ₅₀ The virus solution (100 μ L) was put at 35 ℃ and 5% CO ₂ Adsorbing for 1 hr in incubator, removing virus solution, washing cell surface with maintenance solution for 2 times, adding medicinal liquid with different concentrations into administration group, adding maintenance solution into cell control group and virus control group, 100 μ L/well, placing culture plate at 35 deg.C and 5% CO ₂ And (5) carrying out conditioned culture, observing the cytopathic condition every day, and recording the experimental result when the cytopathic condition of the virus control group reaches 4 grades. Calculation of half inhibitory concentration IC according to Reed-Muench method ₅₀ Selecting index SI ═ TC ₅₀ /IC ₅₀ . The results are shown in tables 1-2.

TABLE 1MDCK cell TC ₀ And TC ₅₀ Measurement of (2)

Test agent	Esculetin	Ribavirin
			TC ₀ (μg·mL ^-1 )	3.12	312.5
TC ₅₀ (μg·mL ^-1 )	36.6	1233.2

Note: TC (tungsten carbide) ₅₀ : half toxic concentration of drug; TC (tungsten carbide) ₀ : the maximum nontoxic concentration of the medicine.

TABLE 2 Effect of esculetin on Virus cytopathic CPE

Note: IC (integrated circuit) ₅₀ : the median inhibitory concentration of the drug on the virus; and (3) SI: selecting an index, SI ═ TC ₅₀ /IC ₅₀ 。

As can be seen from tables 1-2, esculetin has better inhibitory activity against influenza virus A/H1N 1/Jingfang/262/95, A/H3N 2/Jifang 15/90, A/H3N 2/Guangdong/243/72 and B/Jifang/13/97 under the experimental conditions, the result of the positive control drug is consistent with the previous result in the laboratory, the experimental system is established, and the experimental result is credible.

Example 2

Studies on anti-inflammatory action and mechanism of esculetin

The experimental principle is as follows: inflammatory response is a common pathophysiological process involved in the development and progression of various diseases. Inflammatory responses are a complex defense response, involving a variety of cytokines, such as TNF- α, IL-1, IL-6, IFN- γ, NO, and the like. During the immune reaction, macrophages play an important role, and a plurality of cytokines are derived from activated macrophages, wherein NO, TNF-alpha and IL-6 are indexes for evaluating sensitivity of inflammatory reaction, and NF-kappa B, MAPK signal path is an important inflammatory signal path. The experiment is to simulate the inflammation generating process in vitro, a Lipopolysaccharide (LPS) is adopted to induce a mouse abdominal cavity macrophage (RAW264.7) model, a Griess reagent method is adopted to detect the inhibition effect of esculetin (QYNZ) on cell secretion NO, an ELISA method is adopted to detect the inhibition effect of an extract on TNF-alpha and IL-6 secreted by cells, and a cell immunofluorescence method is adopted to evaluate the anti-inflammatory action mechanism of the NF-kappa B, MAPK signal path.

Experimental materials: a sample of esculetin (QYNZ), extracted from Viola tianshanensis by the institute for pharmaceutical research, Xinjiang, under code number 20180926. RAW264.7 cells (mouse peritoneal macrophages) were purchased from the cell center of the basic medical college of the beijing cooperative medical college. Lipopolysaccharide (LPS Escherichia Coli 055: B5), Sigma, USA, Cat. No.: l2880; CellTiter

AQueous One Solution Cell promotion Assay, Promega corporation, USA, Cat #: g3581; griess kit, bi yuntian, cat # cargo: s0021; mouse TNF alpha ELISA Ready-SET-GO, cat #: 88-7324-88, Mouse IL-6 ELISA Ready-SET-Go, cat #: 88-7064-88, Thermo Fischer Scientific; TritonX-100 is available from Thermo Fischer Scientific, Cat #: 8511; BSA purchased from Sigma, usa, cat #: a1933; p-p44/42(Erk1/2) (Thr202/Tyr204) antibody, cat #: 4370S, p-p38 MAPK (Thr180/Tyr182) antibody, cat #: 9211S, CST corporation, USA; p-NF- κ B p65 (Ser536) antibody, cat #: 3033S, I κ B α (E130) antibody, cat no: ab32518, abcam, usa; hoechst 33342 nuclear dye, japan east dong renk chemical company, cat #: h342; fluorescent secondary antibodies Alexa Fluor 546 gat anti-mouse IgG or Alexa Fluor 546 gat anti-rabbitIgG, Thermo Fischer Scientific, cat #: a11035 or a 11030; indomethacin, Sigma, usa, cat #: I7378.

the experimental steps are as follows: RAW264.7 cells at 37 ℃ in 5% CO ₂ Culturing in culture medium containing 10% fetal calf serum 1640 under saturated humidity environment, passaging when cells grow logarithmically, and adjusting cell concentration to 6 × 10 ⁵ Inoculating 100 μ L of the suspension to 96-well culture plate at 37 deg.C with 5% CO ₂ And incubating in a saturated humidity environment, and randomly dividing the cells into 7 groups when the cells grow to be 90 percent: the drug solutions were added to the blank control group, the model group, the compound QYNZ-treated group (final concentrations of 0.16. mu.M, 0.8. mu.M, 4. mu.M, 20. mu.M) and the indomethacin-treated group (10. mu.M) at the respective prescribed doses, LPS was added to each well at a final concentration of 1. mu.g/mL except for the blank control group, the final volume was 100. mu.L, and the cells were incubated in a 37 ℃ 5% CO2 incubator for 24 hours and then subjected to the correlation detection.

Taking cell supernatant, and respectively detecting the levels of TNF-alpha and IL-6 according to the specification of an ELISA kit;

② adopting Griess reagent to detect the concentration of NO in cell supernatant.

And detecting the influence of QYNZ on the expression level of iNOS.

After 24h incubation, adding 4% paraformaldehyde according to 100 mu L/hole, fixing at room temperature for 20min, discarding and washing with PBS; adding 0.25% Triton X-100 punching solution into 100 μ L/hole, acting at room temperature for 15min, and washing with PBS; adding 100 mu L/hole of 3% BSA for blocking for 45min, removing blocking solution, adding Anti-iNOS antibody, incubating overnight at 4 ℃, washing with PBS for the next day, adding goat Anti-rabbit fluorescent secondary antibody containing Hochest 33342, incubating for 2h at room temperature in the dark, washing with PBS, and detecting and analyzing in a Cellomics Arrayscan high content cell analysis system.

And fourthly, the action mechanism of QYNZ on NF-kB pathway and MAPK signal pathway. Compound QYNZ (0.16. mu.M, 0.8. mu.M, 4. mu.M, 20. mu.M) and indomethacin (10. mu.M) were added in advance for incubation for 4h, and 10. mu.L/well PBS was added in parallel to other groups; adding 1 mu g/mL LPS into cells, incubating for 15min to detect the degradation of I kappa B alpha, incubating for 30min to detect the nuclear expression of p-p65, and incubating for 45min to detect the nuclear expression of p-p38 and p-ERK 1/2.

After the cell treatment is finished, adding 4% paraformaldehyde into 100 mu L/hole, fixing at room temperature for 20min, and washing with PBS; adding 0.25% Triton X-100 punching solution into the mixture according to the concentration of 100 μ L/hole, reacting at room temperature for 15min, and washing with PBS; adding 100 mu L/hole of 3% BSA for blocking for 45min, removing blocking liquid, respectively adding an anti-I kappa B alpha antibody, an anti-p-p65 antibody, an anti-p-p38 antibody and an anti-p-ERK1/2 antibody, and incubating overnight at 4 ℃; washing with PBS for the next day, adding fluorescent secondary antibody of goat anti-rabbit (or anti-mouse) containing Hochestt 33342, incubating at room temperature in dark for 2h, and detecting and analyzing in Cellomics Arrayscan high content cell analysis system.

The results are shown in FIGS. 1-8:

the influence of esculetin on the activity of RAW264.7 cells is shown in figure 1, the influence of esculetin on the secretion of TNF-alpha when RAW264.7 is induced by LPS is shown in figure 2, the influence of esculetin on the secretion of IL-6 when RAW264.7 is induced by LPS is shown in figure 3, and in figures 1-3, compared with a blank control group, the LPS model group has a # P < 0.001; p <0.05, P <0.01, P <0.001 in the administration group compared to the LPS model group;

the effects of esculetin on the expression level of iNOS protein and the production of NO when RAW264.7 is induced by LPS are shown in FIG. 4(a) and FIG. 4(b), respectively, in which the LPS model group is compared with the blank control group, and # P is less than 0.001; administration group vs LPS model group P <0.05,. P <0.01,. P < 0.001;

the effect of esculetin on the expression of P-P65 protein when RAW264.730min was induced by LPS is shown in FIG. 5, in which the LPS model group is compared with the blank control group, and # P is less than 0.001; p <0.05, P <0.01, P <0.001 in the administration group compared to the LPS model group;

the influence of esculetin on expression of I kappa B alpha protein when RAW264.715min is induced by LPS is shown in FIG. 6, in which the LPS model group is compared with the blank control group, and # P is less than 0.001; p <0.05, P <0.01, P <0.001 in the administration group compared to the LPS model group;

the influence of esculetin on the expression of P-P38 protein when RAW264.745min is induced by LPS is shown in FIG. 7, wherein the # P of LPS model group is less than 0.001; p <0.05, P <0.01, P <0.001 in the administration group compared to the LPS model group;

the influence of esculetin on the expression of P-ERK1/2 protein when RAW264.745min is induced by LPS is shown in FIG. 8, wherein compared with the blank control group, the LPS model group has # P < 0.001; p <0.05, P <0.01, P <0.001 in the administration group compared to the LPS model group;

as shown in FIGS. 1-8, esculetin QYNZ can obviously inhibit LPS from inducing the release of TNF-alpha, IL-6 and NO in RAW264.7 cells, slow down the expression of iNOS and the translocation of p-p65, p-p38 and p-ERK1/2 proteins from cytoplasm to nucleus and reduce the degradation of IkappaB alpha protein, therefore, QYNZ can reduce the occurrence of inflammatory reaction by inhibiting the activation of NF-kappa B, MAPK signal path.

Example 3

Effect of escin on proliferation of T, B lymphocytes

Experimental materials: the sample escin, extracted from Viola tianshanensis by Xinjiang institute of medicine phytochemistry, lot number 20180926. Canavalin A (ConA), Sigma; LPS, Escherichia coli 0111: B4, Sigma; NH (NH) ₄ Cl, GIBCO 1640 medium, Gibco fetal bovine serum, MTS, and viola tianschanica esculetin 10mg dissolved in 200. mu.L DMSO to obtain a mother liquor with a concentration of 50 mg/mL. Mother liquor is diluted 2 times to a series of concentration gradients by incomplete 1640, and the series of concentrations are 100, 50, 25, 12.5, 6.25, 3.12 and 1.56 mu g/mL.

The experimental steps are as follows: preparation of spleen lymphocyte suspension: the 200 mesh nylon net is placed in a small culture dish, and a proper amount of incomplete 1640 culture medium is added from a screen. Killing 3 mice, sterilizing with 75% alcohol, placing spleen on a screen, grinding, sieving with 200 mesh sieve, sucking spleen cell suspension, centrifuging at 1500r/min, discarding supernatant, and adding NH ₄ CL is shaken gently, supernatant is discarded after 1500r/min centrifugation, incomplete 1640 culture solution is added to resuspend cells, cells are collected after 1500r/min centrifugation, complete 1640 culture solution is added to suspend, and the density of spleen cells is adjusted to be 200 multiplied by 10 ⁴ mL, seeded in 96-well plates, 100 μ L/well.

Effect on proliferation of T, B lymphocytes: t lymphocyte proliferation assay: setting a cell control group, a Con A control group and a esculetin administration group with different concentrations. B lymphocyte proliferation assay: setting a cell control group, an LPS control group and an esculetin administration group with different concentrations. Adding 10 μ L of medicinal liquid with different concentrations into each administration group, and adding into each wellAdding 160 mu g/mL ConA or 100 mu g/mL LPS in turn, finally complementing a 200 mu L system by using complete 1640 culture solution, placing the cells in a 5% CO2 incubator at 37 ℃ for culturing for 48h, and collecting cell supernatant for detecting the contents of cell factors TNF-alpha, IL-2 and IL-6. In addition, 10. mu.L of MTS was added to each well of the cell control group, Con A or LPS control group and each administration group, and 5% CO was added at 37 ℃ ₂ Incubate for 2h, detect OD490 with M2 microplate reader, i.e.: the effect of the drug on T lymphocyte proliferation induced by ConA and B lymphocyte proliferation induced by LPS was examined and the results are shown in tables 3-5.

TABLE 3 Effect of Tianshan Viola esculenta Hippolide on ConA-induced proliferation of mouse T lymphocytes

Note: compared with the blank control group, the composition of the composition, ^## P<0.01; compared with the group induced by ConA, ^＊＊ P<0.01。

TABLE 4 Effect of Viola odorata esculetin on LPS-induced proliferation of mouse B lymphocytes

Note: compared with the blank control group, the preparation method has the advantages that, ^## P<0.01; compared with the LPS-induced group, ^＊ P<0.05， ^＊＊ P<0.01。

TABLE 5 Effect of Dolicholide on ConA-induced secretion of cytokines from mouse T lymphocytes

as shown in the tables 3-5, esculetin can obviously inhibit ConA from inducing T lymphocyte proliferation and T lymphocyte from secreting TNF-a, IL-6 and IL-2, and also has obvious inhibition effect on LPS-induced B lymphocyte proliferation, which indicates that esculetin has certain immunoregulation effect.

Claims

1. The application of esculetin in the preparation of anti-influenza virus drugs is characterized in that the active ingredient of the anti-influenza virus drugs is esculetin; the influenza virus is A/H1N 1/Jingfang/262/95, A/H3N 2/Guangdong Fang/243/72, A/H3N 2/Jifang 15/90 and B/Jifang/13/97.

2. The use of claim 1, wherein the anti-influenza virus agent is a tablet or capsule.

3. The use as claimed in claim 1 or 2, wherein the escin is escin extracted from viola tianschanica or violaceae or artificially synthesized escin.

4. The use according to claim 1 or 2, wherein the anti-influenza virus medicament further comprises pharmaceutically acceptable pharmaceutical excipients, and the pharmaceutical excipients are any one or a mixture of several of cosolvent, emulsifier, solubilizer, osmotic pressure regulator, binder, filler, disintegrant, lubricant, preservative and antioxidant.