Application of 7, 8-dihydroxyflavone in preparation of medicine for treating diseases caused by herpes virus infection

Abstract

The invention provides application of 7, 8-dihydroxyflavone in preparation of a medicine for treating diseases caused by herpes virus infection, and belongs to the technical field of biological medicine. The 7, 8-dihydroxy flavone can be used for remarkably inhibiting infection of herpes simplex viruses HSV-1 and HSV-2, and has broad-spectrum anti-HSV activity. According to the present invention, the HSV virus protein expression can be significantly inhibited by pretreating the virus with the 1, 7, 8-dihydroxyflavone and treating the cells after the virus adsorption, the virus plaque formation can be reduced, and the dose dependence can be provided; experiments show that the 7, 8-dihydroxyflavone plays the anti-herpes simplex virus activity mainly through targeting host protein fructose-diphosphate aldolase A. The 7, 8-dihydroxyflavone has the anti-herpes simplex virus activity. Mouse in-vivo experiments prove that the 7, 8-dihydroxyflavone can significantly improve the survival rate of HSV-1 mice, improve the weight reduction trend and reduce the virus titer of lung tissues; meanwhile, mouse genital organ lesions caused by HSV-2 infection can be remarkably relieved, and the weight of infected mice is improved. Therefore, the compound 7, 8-dihydroxyflavone is expected to be developed into a candidate drug for treating the herpes simplex virus.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the use of 7,8-dihydroxyflavone in the preparation of a drug for treating diseases caused by herpes virus infection. Background Art

[0002] Herpes Simplex Virus (HSV) is the most widely prevalent herpes virus globally. It belongs to the alpha subfamily of Herpesviridae and has two subtypes, HSV-1 and HSV-2. HSV-1 can cause herpes labialis, herpes keratitis, encephalitis, and even Alzheimer's disease, etc.; HSV-2 can cause genital herpes. Currently, the drugs for treating HSV clinically are mainly nucleoside analogs such as acyclovir and ganciclovir, nucleotide analogs such as cidofovir and adefovir dipivoxil, and pyrophosphate analogs such as foscarnet sodium. These three types of drugs all target viral DNA replication to exert anti-HSV activity, but these drugs have some common defects. Long-term use is prone to drug resistance, and there will also be side effects such as nephrotoxicity and low bioavailability.

[0003] The flavonoid compound 7,8-dihydroxyflavone is a dihydroxyflavone hydrate, which mainly exists in foods such as red wine, citrus, grains, tea, vegetables, fruits, and chocolate. Existing studies have shown that the compound 7,8-dihydroxyflavone is a specific agonist of the tyrosine kinase B (TrkB) receptor, which can activate the TrkB receptor and simulate the function of BDNF (brain-derived neurotrophic factor). In the research on nervous system-related diseases, it shows a protective effect on neurons, helping to reduce neuronal damage and death. The existing patents CN114916665A, CN 116549441 A, and CN108272787B have verified that the compound 7,8-dihydroxyflavone can enhance human memory and cognitive ability, improve cardiovascular health, prevent Alzheimer's disease, and promote neuronal growth and protection. In addition, the compound 7,8-dihydroxyflavone also has biological functions such as preventing retinal degenerative diseases (CN106265626A) and treating depression (CN102143744B). However, the anti-HSV effect of the compound 7,8-dihydroxyflavone has not been reported. Summary of the Invention

[0004] The object of the present invention is to provide the use of 7,8-dihydroxyflavone in the preparation of a drug for treating diseases caused by herpes virus infection. The present invention has experimentally proven that the natural product 7,8-dihydroxyflavone derived from plants has good anti-herpes simplex virus activity. The present invention has for the first time proven that the compound 7,8-dihydroxyflavone has anti-HSV-1 and HSV-2 virus activities and has low toxicity, and can be developed into a new drug for treating herpes simplex virus.

[0005] To achieve the above-mentioned invention objectives, the present invention is implemented by the following technical solutions: The present invention provides the use of 7,8-dihydroxyflavone in the preparation of a medicament for treating diseases caused by herpes virus infection.

[0006] Furthermore, the compound 7,8-dihydroxyflavone (7,8-Dihydroxyflavone, i.e., 7,8-DHF) is a natural flavonoid polyphenol compound with a molecular formula of C 15 H 10 O4, CAS No.: 38183-03-8, and its chemical structural formula is as follows: .

[0007] The present invention screened out that the compound 7,8-DHF has a significant anti-HSV virus effect through a CPE inhibition experiment.

[0008] Furthermore, the herpes virus includes herpes simplex virus HSV-1 and HSV-2.

[0009] Furthermore, the 7,8-DHF can significantly inhibit the in vitro proliferation of herpes simplex virus in infected cells and reduce plaque formation.

[0010] Furthermore, the 7,8-DHF significantly inhibits the infection of herpes simplex virus within the concentration range of 1.25 - 10 μM, inhibits the expression of key viral proteins, and shows a dose-dependent relationship.

[0011] Furthermore, the important viral proteins of HSV-1 are ICP0 and ICP27, and the important viral proteins of HSV-2 are gB and gD.

[0012] Furthermore, the 7,8-DHF exerts an inhibitory effect on the virus both during the pretreatment of herpes simplex virus and after virus adsorption.

[0013] Furthermore, the 7,8-DHF exerts an antiviral effect 6 - 9 h after virus adsorption and has a time cumulative effect.

[0014] Furthermore, the 7,8-DHF acts on the host protein ALDOA after adsorption and affects the anti-HSV virus activity.

[0015] Furthermore, the 7,8-DHF can improve the lung lesions in mice caused by HSV-1 infection, improve the survival status and weight loss trend of mice, and significantly reduce the viral load in the lung tissue of mice at the animal level.

[0016] Furthermore, 7,8-DHF can improve the genital tract lesions in mice caused by HSV-2 infection at the animal level, improve the survival status and the trend of weight loss in mice, and significantly reduce the amount of virus shed from the vagina of mice.

[0017] The present invention also provides a drug for preventing herpes simplex virus infection, and the drug contains the above-mentioned 7,8-DHF as an active ingredient and a pharmaceutically acceptable excipient.

[0018] Compared with the prior art, the advantages and positive effects of the present invention are as follows: The present invention selects the compound 7,8-DHF as the research object and reveals its inhibitory effect on herpes simplex virus at the in vivo and in vitro levels. At the cellular level, the compound 7,8-DHF has a significant inhibitory effect on the cytopathic effect induced by HSV, can inhibit the expression of key viral proteins in HSV-infected cells, and shows dose-dependence within a certain concentration range. The compound 7,8-DHF exerts its antiviral effect at two stages: pre-treating the virus and after virus adsorption; at the animal level, experiments show that the compound 7,8-DHF can improve the symptoms of mice infected with HSV-1 and HSV-2, reduce the weight loss, and improve the survival rate, thereby exerting an anti-HSV virus effect.

[0019] After reading the specific embodiments of the present invention in conjunction with the accompanying drawings, other features and advantages of the present invention will become clearer. Description of the Drawings

[0020] Figure 1 It is a result graph of the cytotoxicity detection of 7,8-DHF at different concentrations in Vero and the inhibition rate of anti-HSV cytopathic effect in Example 1.

[0021] Figure 2 It is a result graph of the inhibition of HSV virus plaque formation by 7,8-DHF at different concentrations in Example 1.

[0022] Figure 3 It is a result graph of the inhibition of the expression of key viral proteins in HSV-infected cells by 7,8-DHF in Example 1, and the key proteins are ICP0, gD, and gB.

[0023] Figure 4 It is a result graph of the inhibitory effect of 7,8-DHF on the expression of key proteins of HSV under different action modes in Example 2, and the key proteins are ICP0, ICP27, gB, and gD.

[0024] Figure 5 It is a result graph of the inhibitory effect of 7,8-DHF on the expression of key proteins at different action times after HSV-1 adsorption in Example 2, and the key proteins are the early viral proteins ICP0 and ICP27.

[0025] Figure 6It is the result diagram for verifying that 7,8-DHF may exert antiviral effects by targeting host protein ALDOA through a thermal drift experiment in Example 2.

[0026] Figure 7 It is the result diagram for the effect of 7,8-DHF on the body weight of mice infected with HSV-1 virus in Example 3.

[0027] Figure 8 It is the result diagram for the effect of 7,8-DHF on the survival rate of mice infected with HSV-1 virus in Example 3.

[0028] Figure 9 It is the result diagram for the virus titer in the lungs of mice infected with HSV-1 virus by 7,8-DHF in Example 3 Figure 10 It is the result diagram for 7,8-DHF to relieve genital tract inflammation in mice infected with HSV-2 virus in Example 3.

[0029] Figure 11 It is the result diagram for the effect of 7,8-DHF on the body weight of mice infected with HSV-2 virus in Example 3.

[0030] Figure 12 It is the result diagram for the vaginal virus titer of mice infected with HSV-2 virus by 7,8-DHF in Example 3. Detailed implementation manners

[0031] The technical solutions of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation manners, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0032] The present invention evaluated the inhibitory effect of 7,8-DHF on herpes simplex virus infection in vitro according to internationally recognized methods. The 7,8-DHF (CAS No.: 38183-03-8) used in the present invention can be a commercially available product.

[0033] Example 1: Experiment on the inhibitory effect of 7,8-DHF on HSV activity in vitro 1. Cytotoxicity experiment of 7,8-DHF Detect the cytotoxicity of 7,8-DHF in Vero cells. Seed Vero cells in a 96-well plate and place it in an incubator at 37 °C with 5% CO₂ for culture. After the cells grow into a monolayer, aspirate the original medium, and add 7,8-DHF diluted in gradients (final concentrations are 800 μM, 400 μM, 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM) into the 96-well plate. Set three replicates for each concentration, and set a blank control at the same time. After culturing the 96-well plate in the incubator at 37 °C with 5% CO₂ for 24 h, aspirate the medium, add 4% paraformaldehyde to fix at room temperature for 15 min, then aspirate, add 0.1% (w / v) crystal violet to stain at room temperature for 15 min, aspirate again, and rinse thoroughly with water. After drying, measure the A540nm value with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the concentration that reduces cell viability by 50% (CC50).

[0034] Cell survival rate = (A540nm value of the drug-treated group / A540nm value of the blank group) × 100%.

[0035] The experimental results are as Figure 1 shown. As the concentration of 7,8-DHF gradually increases, the cytotoxicity gradually becomes stronger. When the concentration is lower than 200 μM, the cell viability is above 60%. When the concentration is lower than 25 μM, the cell viability is above 80%. Thus, it can be seen that 7,8-DHF has certain cytotoxicity to Vero cells and shows a dose-dependent relationship.

[0036] 2. Determine the anti-HSV activity of 7,8-DHF in Vero cells using the CPE experiment Vero cells were inoculated into 96-well plates and cultured in an incubator at 37 °C with 5% CO₂. After the cells grew to confluence, the original culture medium was aspirated off. 7,8-DHF was diluted in gradients (final concentrations were 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0.78125 μM, 0.390625 μM), and the whole process of drug administration was carried out according to four drug administration methods (pretreating cells, pretreating virus, administering drugs during adsorption, and administering drugs after adsorption). Three replicates were set for each concentration, and a blank control group, a virus group, and a positive drug acyclovir (ACV) group were also set. Pretreating cells (Pre+Cell): 7,8-DHF was used to pretreat cells at 37 °C for 1 h, then aspirated off. HSV (MOI = 0.1) was adsorbed at 37 °C for 1 h, and then the maintenance medium was changed; Pretreating virus (Pre+Virus): 7,8-DHF and HSV (MOI = 0.1) were mixed and incubated at 37 °C for 1 h, then added to the 96-well plate from which the original medium had been aspirated off, adsorbed at 37 °C for 1 h, and then the maintenance medium was changed; Administering drugs during adsorption (Adsorption): 7,8-DHF and HSV (MOI = 0.1) were mixed and added to the 96-well plate and adsorbed at 37 °C for 1 h, then the maintenance medium was changed; Administering drugs after adsorption (Post-adsorption): HSV (MOI = 0.1) was adsorbed to cells at 37 °C for 1 h, and then the maintenance medium containing 7,8-DHF was changed. The cytopathic effect was observed. When the virus group was completely cytopathic, the original culture medium was aspirated off, 4% paraformaldehyde was added and fixed at room temperature for 15 min, then aspirated off, 0.1% (w / v) crystal violet was added and stained at room temperature for 15 min, then aspirated off, and rinsed thoroughly with water. After drying, the A540nm value was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the concentration that reduced the cytopathic effect by 50% (IC50) was calculated.

[0037] Cytopathic inhibition rate = (A540 nm value of the drug administration group / A540 nm value of the positive drug group - A540 nm value of the virus group) / (A540 nm value of the blank control group - A540 nm value of the virus group) × 100%.

[0038] The experimental results were as Figure 1 shown, demonstrating that 7,8-DHF has good anti-HSV-1 and anti-HSV-2 activities and shows a certain dose-dependence.

[0039] 3. It was verified by plaque assay that 7,8-DHF can inhibit the in vitro proliferation of HSV Vero cells were inoculated into 12-well plates and incubated in an incubator at 37 °C with 5% CO₂. After the cells grew to confluence, they were adsorbed with HSV-1 or HSV-2 (MOI = 0.1) at 37 °C for 1 h, and then 7,8-DHF (final concentrations of 10 μM, 5 μM, 2.5 μM, 1.25 μM) was added. Meanwhile, a blank control group and a virus group were set up. After 24 h, the supernatant was collected, diluted to 10 -2 times, adsorbed to cells at 37 °C for 1 h, and the unadsorbed virus was washed away with PBS. The prepared Overlay medium was added to the wells. After the medium solidified, the culture plates were inverted and placed in the incubator. During this period, the plaque growth was observed. After the plaque size was appropriate, 4% paraformaldehyde was added and the room temperature was maintained. The plaques were counted using Image J, and the data were processed using GraphPad Prism 8.0 to calculate the virus titer.

[0040] The plaque titer assay was used to investigate the effect of 7,8-DHF on the in vitro proliferation of HSV. The experimental results are as Figure 2 shown. After HSV adsorption, treatment with 7,8-DHF (final concentrations of 1.25 μM, 2.5 μM, 5 μM, 10 μM) was performed. Compared with the virus group, when the concentration of 7,8-DHF was 5 μM, most of the virus plaque formation could be inhibited. After HSV-1 adsorption, the virus titers treated with 7,8-DHF decreased by 0.25, 0.84, 1.73, and 5.37 log10 (PFU / mL) respectively. After HSV-2 adsorption, the virus titers treated with 7,8-DHF decreased by 0.34, 0.41, 1.6, and 3.91 log10 (PFU / mL) respectively. Therefore, the results of the plaque titer assay indicate that 7,8-DHF treatment significantly inhibits the proliferation of HSV in vitro.

[0041] 4. Determine the effect of 7,8-DHF on the expression of important HSV viral proteins by Western blot assay Different compound concentrations: Vero cells were seeded in 12-well plates and cultured in an incubator at 37 °C with 5% CO₂. After the cells grew to confluence, they were adsorbed with HSV-1 or HSV-2 (MOI = 0.1) at 37 °C for 1 h, and then 7,8-DHF (final concentrations of 10 μM, 5 μM, 2.5 μM, 1.25 μM) was added. At the same time, a blank control group and a virus group were set up. After 14 h, the culture medium was aspirated, and cell lysis buffer (800 μl Ripa lysis buffer + 200 μl 5× Loading buffer + 10 μl PMSF) was prepared. 70 μl of cell lysis buffer was added to each well, and the cells were lysed at 4 °C for 20 min. The lysate was collected and boiled at 100 °C for 20 min. After measuring the protein concentration extracted with a BCA kit, the proteins were separated by SDS-PAGE and transferred to an NC membrane. The NC membrane was blocked with 5% non-fat milk at 4 °C overnight, washed three times with 1×TBST for 10 min each time. Then, it was incubated with the diluted antibody at 4 °C overnight. After washing three times with 1×TBST, it was incubated with an AP-labeled secondary antibody (diluted 1:5000) at 37 °C for 2 h. After washing three times with 1×TBST again, it was developed with an alkaline phosphatase kit at room temperature. After taking pictures and saving them, the protein bands were quantitatively analyzed with Image J.

[0042] The experimental results are as Figure 3 shown. After HSV-1 adsorption and treatment with 7,8-DHF, the expression of ICP0 and gD decreased with increasing concentration. At 5 μM, the bands of ICP0 and gD were hardly detectable. After HSV-2 adsorption and treatment with 7,8-DHF, the expression of gB and gD decreased with increasing concentration. At 10 μM, the bands of gB and gD were very faint. This indicates that 7,8-DHF has a significant inhibitory effect on the expression of important viral proteins of HSV at concentrations of 1.25 - 10 μM.

[0043] Example 2: Mechanism of action of 7,8-DHF in inhibiting HSV virus 1. Evaluation of different modes of action of 7,8-DHF against HSV by Western blot Different modes of action: Vero cells were seeded in 12-well plates and cultured in an incubator at 37 °C with 5% CO₂. After the cells grew to confluence, 7,8-DHF (final concentration of 10 μM) was used to treat HSV in four modes of action (pretreatment of cells, pretreatment of virus, administration during adsorption, administration after adsorption). At the same time, a blank control group and a virus group were set up. After 14 h, the culture medium was aspirated, the cells were lysed, and the cell lysate was collected. Western blot experiments were performed according to the above method. After development, the bands were photographed and saved, and the protein bands were quantitatively analyzed with Image J.

[0044] The experimental results are asFigure 4 As shown, after HSV was treated by pre - treating the virus and administering the drug after adsorption, the protein expression level decreased significantly. The important viral proteins of HSV - 1 are ICP0 and ICP27, and the important viral proteins of HSV - 2 are gB and gD. After these two drug - administration methods, the protein bands became significantly lighter and even almost disappeared. This indicates that 7,8 - DHF exerts its antiviral effect at two stages: pre - treating the virus and administering the drug after adsorption.

[0045] 2. Results of exploring the action time of 7,8 - DHF after adsorption Different action times: Hela cells were seeded in 12 - well plates and incubated in an incubator at 37 °C and 5% CO₂. After the cells grew to confluence, they were adsorbed with HSV - 1 (MOI = 0.1) at 4 °C for 1 h. 7,8 - DHF (final concentration of 10 μM) was added at different time periods after adsorption (0 - 3 h, 3 - 6 h, 6 - 9 h, 9 - 12 h, 0 - 6 h, 0 - 12 h). At the same time, a blank control group and a virus group were set up. After 12 h, the total RNA of Hela cells was extracted using an RNA extraction kit according to the instructions. After detecting the concentration of the extracted RNA with Nanodrop, two - step RT - PCR was performed according to the instructions of the Evo M - MLV Reverse Transcription Kit and the SYBR® Green Premix Pro Taq HS qPCR Kit II.

[0046] Table 1 Primer sequences for RT - PCR

[0047] The experimental results are as Figure 5 shown. 7,8 - DHF mainly exerts its antiviral activity 6 - 9 h after the adsorption of HSV - 1. In the 0 - 12 h stage, the mRNA expression levels of ICP0 and ICP27 decreased significantly, and the inhibitory effect on the virus was remarkable, indicating that the compound has a cumulative effect. The longer the action time, the more significant the antiviral effect.

[0048] 3. Effect of 7,8 - DHF on the thermal stability of proteins Vero cells were inoculated into 6-well plates and cultured in an incubator at 37 °C with 5% CO₂. After the cells grew to confluence, they were adsorbed with HSV-1 for 1 h. After 24 h, 120 μL of NP-40 lysis buffer (added with 10× Cocktail protease inhibitor) was added to each well and lysed on ice for 20 min. The cells were scraped off with the tail of a pipette tip, and the cell lysate was collected. The cell lysate was centrifuged at 13,000 rpm at 4 °C for 10 min in a refrigerated centrifuge. The supernatant was collected into an EP tube and divided into two EP tubes, with 360 μL in each tube. 40 μL of 10× TNC buffer and 100 μM of compound 7,8-DHF were added to tube A, and 40 μL of 10× TNC buffer was added to tube B. The tubes were incubated with rotation at 4 °C overnight in a refrigerator. Then, tube A was divided into four tubes A1, A2, A3, and A4, and tube B was divided into four tubes B1, B2, B3, and B4. Tubes A1 and B1 were not treated. Tubes A2 and B2 were heated at 50 °C for 3 min in a PCR instrument. Tubes A3 and B3 were heated at 60 °C for 3 min in a PCR instrument. Tubes A4 and B4 were heated at 70 °C for 3 min in a PCR instrument. Equal amounts of supernatant were taken, 5× Loading Buffer was added, and the samples were boiled at 100 °C for 20 min. SDS-PAGE was used to detect differential bands, and mass spectrometry analysis was performed to identify possible proteins. Then, gradient thermal shift (54 - 68 °C) was carried out, and finally, Western blot experiments were used to verify whether the protein was the target.

[0049] 7,8-DHF exerts an antiviral effect during the post-adsorption administration stage. The thermal shift technique was used to explore the action target of 7,8-DHF, and observe which proteins may change the protein stability after binding with 7,8-DHF, and increase or decrease the thermal stability of the protein. The experimental results are as Figure 6 shown. Through SDS-PAGE experiments, it was found that when treated at 60 °C, a differential band appeared between 35 kd - 45 kd, and the addition of the compound made the protein thermal stability worse. With the help of protein mass spectrometry analysis and identification technology, the differential protein was identified as the host protein ALDOA. Then, secondary thermal shift (54 °C - 68 °C) was carried out in Hela cells. Western blot experiments showed that the thermal stability of protein ALDOA in the compound-added group decreased compared with the non-compound-added group. The results showed that the action target of 7,8-DHF against HSV was the host protein ALDOA.

[0050] Example 3: Effect of 7,8-DHF on treating HSV-infected mice 1. Clinical manifestations of mice after HSV-1 infection Female BALB / c mice, 3 - 4 weeks old, weighing 14 - 15 g, were divided into six groups: blank group, control group, positive drug (ACV) group, 7,8 - DHF low - dose (2.5 mg / kg) group, 7,8 - DHF medium - dose (5 mg / kg) group, and 7,8 - DHF high - dose (10 mg / kg) group, with 12 mice in each group. The bedding was changed daily, and sufficient feed and water were provided. After the mice adapted to the environment, they were inoculated with HSV - 1 by nasal drip. First, the mice were anesthetized intraperitoneally with 30% urethane. After the anesthesia took effect, the HSV - 1 virus suspension was diluted to the working concentration with sterile saline. 60 μL of the virus solution was slowly instilled into the nasal cavity using a pipette, and the control group was treated with an equal volume of sterile saline. The mice were kept in the supine position during the operation to ensure full absorption of the inoculum.

[0051] After the mice were infected with HSV - 1 and administered drugs once a day, the mice in the virus group began to show symptoms of illness. The mice showed obvious discomfort, reduced activity, a sluggish overall mental state, and a decrease in body weight. Some mice even showed abnormal symptoms such as incoordination and staggering gait, while the blank group and the drug - administered groups showed no obvious symptoms.

[0052] The changes in the body weight of the mice after infection with HSV - 1 are as Figure 7 shown. Compared with the blank group, the body weight of the mice in the virus group continued to decline and reached the lowest point on the 6th day, indicating that virus infection had a significant negative impact on the body weight of the mice; the body weight of the positive drug (acyclovir 10 mg / kg) group and the 7,8 - DHF - administered (10, 5, 2.5 mg / kg) groups decreased slightly and then gradually increased, and basically returned to the initial body weight after 14 days. The results showed that 7,8 - DHF could reduce the body weight loss caused by virus infection, and the high - dose treatment had the most significant effect.

[0053] The changes in the survival rate of the mice infected with HSV - 1 are as Figure 8As shown, the survival rate of the virome mice gradually decreased after virus infection, and the survival rate was 0% after the 13th day, showing the lethality of virus infection; the mice in the positive drug (ACV 10 mg / kg) group began to die on the 6th day, and the survival rate was about 70% on the 14th day; the mice in the high-dose (10 mg / kg) 7,8-DHF group began to die on the 5th day, and the survival rate was about 80% on the 14th day, higher than that of the positive drug (ACV 10 mg / kg) group; the mice in the medium-dose (5 mg / kg) 7,8-DHF group began to die on the 4th day, and the survival rate was about 70% on the 14th day, which was equivalent to that of the positive drug (ACV 10 mg / kg) group; the mice in the low-dose (2.5 mg / kg) 7,8-DHF group began to die on the 4th day, and the survival rate was about 45% on the 14th day. The results showed that low-dose 7,8-DHF failed to significantly improve the survival rate of virus-infected mice, and the therapeutic effect was average. While high-dose and medium-dose 7,8-DHF could significantly improve the survival rate of virus-infected mice, and the effect was equivalent to or even better than that of the positive drug, showing a good therapeutic effect.

[0054] 2. Anatomical method and tissue extraction of HSV-1-infected mice At 72 h after virus infection, 3 mice were randomly selected from each group for sample collection. The specific operation was as follows: First, the mice were sacrificed by cervical dislocation, and then the lung tissue was immediately dissected. The lung lobes were placed in a pre-cooled homogenization tube containing pre-placed homogenization beads and immediately stored on ice for tissue homogenate preparation.

[0055] 3. Detection of virus titer in the lung tissue of HSV-1-infected mice The lung tissue was weighed, and the corresponding volume of pre-cooled PBS was added at a ratio of 7 mL / g. The tissue was homogenized using a homogenizer for 3 times, 30 s each time. The homogenate was centrifuged at 4 °C and 12,000 rpm for 10 min, and the supernatant was carefully aspirated into a new centrifuge tube and immediately stored at -80 °C for later use. Vero cells were inoculated into 12-well plates in advance. After growing to a monolayer, a plaque assay was performed to calculate the virus titer in the mouse lung tissue.

[0056] The virus titer of the lung tissue of the mice was detected by plaque assay, and the experimental results are as Figure 9 shown. Compared with the virus group, the virus titers in the positive drug (ACV 10 mg / kg) group and the high- and medium-dose (5, 10 mg / kg) 7,8-DHF groups decreased significantly. The experimental results showed that high-dose and medium-dose 7,8-DHF could significantly reduce the viral load in the lung tissue of HSV-1-infected mice, further indicating that 7,8-DHF can play an anti-HSV-1 role in mice.

[0057] 4. Clinical manifestations of mice after HSV-2 infection In this invention, female BALB mice at 6 - 7 weeks of age, sexually mature, with a body weight of 18 - 19 g were selected and divided into six groups: blank group, control group, positive drug (ACV) group, 7,8 - DHF low - dose (2.5 mg / kg) group, 7,8 - DHF medium - dose (5 mg / kg) group, and 7,8 - DHF high - dose (10 mg / kg) group. There were 12 mice in each group. The bedding was changed daily, and sufficient feed and water were provided. Three days before modeling, each mouse was subcutaneously injected with 100 μl of 30 mg / ml medroxyprogesterone. On the day of modeling, the mice were anesthetized with 30% urethane according to their body weight. A cell brush was used to rub the vagina of the mice back and forth. After the vagina oozed blood and opened, 20 μl of compound gel and 20 μl of HSV - 2 (2×10 5 PFU / mL) virus solution were successively injected into the vagina. The mice were placed on their backs and left still for 5 min to allow full infection with the virus.

[0058] After the mice were infected with HSV - 2 and given drugs twice a day, morphological changes occurred in the vulva and surrounding tissues of the mice. In the virus group, the vulva of the mice showed redness, ulceration, hair loss, etc. The phenomenon was most severe on the 6th day after virus inoculation. In the drug - administered groups, the inflammatory conditions of the mice were alleviated to some extent. The experimental results are as Figure 10 shown. Compared with the mice in the virus group, the vulva of the mice in the low - dose drug - administered group showed redness, but no more severe phenomena such as ulceration and suppuration occurred. As the drug dose increased, the vulva state of the mice infected with HSV - 2 improved significantly, indicating that 7,8 - DHF also has good antiviral activity in vivo.

[0059] The weight change of the mice after infection with HSV - 2 is as Figure 11 shown. As time went by, the body weight of the mice in the blank group increased slowly, while the body weight of the mice in the virus group showed a downward trend and reached the lowest point on the 8th day. Compared with the virus group on the 8th day, the average body weight of the mice in the drug - administered groups and the positive - drug group was significantly higher than that of the mice in the virus group, indicating that 7,8 - DHF has a mitigating effect on the downward trend of body weight of the mice infected with HSV - 2 and has a certain therapeutic effect.

[0060] 5. Dissection method and tissue extraction On the 8th day after modeling, the vulva of the mice was photographed and recorded. Three mice were randomly selected from each group for dissection. After sacrifice, the vaginal tissues of the mice were extracted and stored in an EP tube containing formalin at 4 °C.

[0061] 6. Detection of virus titer in vaginal exfoliates of mice On the 3rd day after virus inoculation, prepare 200 μl of sterile PBS containing double antibiotics in an EP tube and place it on ice for standby. Use a pipette to aspirate 20 μl of PBS and inject it into the vagina of the mouse. After aspirating and blowing 20 times, aspirate it back into the original EP tube and mix well. Repeat the operation again, and keep all EP tubes on ice. Dilute the vaginal lavage fluid in a 10-fold concentration gradient and add it to the pre-cultured Vero cells in a 12-well plate. Adsorb at 37 °C for 1 h, aspirate and discard, add the pre-prepared Overlay medium, perform a plaque assay, count the number of plaques, and calculate the titer.

[0062] HSV-2 can damage the vaginal mucosa and skin and release the virus in vaginal secretions. Figure 12 It is the genital tract shedding virus titer of the genital herpes mice. Compared with the virus group, the amount of virus shed from the vagina in the positive drug (Acyclovir-10 mg / kg) group decreased by 0.72 Log10 (PFU / mL), while the amount of virus shed from the vagina in the high-dose (10 mg / kg) group of 7,8-DHF decreased most significantly, by 0.68 LogI0 (PFU / mL). The amount of virus shed from the vagina in the medium-dose (5 mg / kg) group of 7,8-DHF decreased by 0.41 LogI0 (PFU / mL), and the amount of virus shed from the vagina in the low-dose (2.5 mg / kg) group of 7,8-DHF decreased by 0.28 LogI0 (PFU / mL). This indicates that 7,8-DHF can significantly reduce the amount of virus shed from the vagina of genital herpes mice and has a good therapeutic effect on genital herpes.

[0063] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, for those of ordinary skill in the art, it is still possible to modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions required to be protected by the present invention.

Claims

1. Use of 7,8-dihydroxyflavone in the preparation of a drug for treating diseases caused by herpes virus infection.

2. The application according to claim 1, wherein The molecular formula of the 7,8-dihydroxyflavone is C 15 H 10 O4, and its chemical structural formula is as follows: 。 3. The application according to claim 2, wherein The herpes virus includes herpes simplex virus HSV-1 and HSV-2.

4. The application according to claim 2, wherein The 7,8-dihydroxyflavone can significantly inhibit the in vitro proliferation of herpes simplex virus in infected cells and reduce plaque formation.

5. The application according to claim 2, characterized in that, The 7,8-dihydroxyflavone significantly inhibits the infection of herpes simplex virus in the concentration range of 1.25 - 10 μM, inhibits the expression of key viral proteins, and shows a dose-dependent manner.

6. The application according to claim 2, wherein The 7,8-dihydroxyflavone exerts an inhibitory effect on the virus both during the pretreatment of herpes simplex virus and after virus adsorption.

7. The application according to claim 2, characterized in that, The 7,8-dihydroxyflavone exerts an antiviral effect 6 - 9 h after virus adsorption and has a time cumulative effect.

8. The application according to claim 2, characterized in that The 7,8-dihydroxyflavone acts on the host protein ALDOA after adsorption and affects the anti-HSV virus activity.

9. The application according to claim 2, wherein The 7,8-dihydroxyflavone can improve the pulmonary inflammation in mice caused by HSV-1 infection at the animal level, increase the survival rate of mice, improve the trend of weight loss, and significantly reduce the virus amount in the lung tissue of mice; The 7,8-dihydroxyflavone can improve the genital tract lesions in mice caused by HSV-2 infection at the animal level, improve the survival status and the trend of weight loss in mice, and significantly reduce the virus amount shed from the vagina of mice.

10. A drug for preventing herpes simplex virus infection, characterized in that, The drug contains the 7,8-dihydroxyflavone as an active ingredient and a pharmaceutically acceptable excipient.

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