CN113288896A - Application of sophoridine in preparation of anti-herpes virus medicine - Google Patents
Application of sophoridine in preparation of anti-herpes virus medicine Download PDFInfo
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- A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
- A61P31/22—Antivirals for DNA viruses for herpes viruses
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Abstract
The invention provides application of sophoridine in preparation of a medicament for resisting herpes viruses, and belongs to the field of pharmacy. The experiment result shows that the sophoridine can effectively inhibit the formation of HSV-1 virus plaques and obviously inhibit the generation of progeny viruses; the sophoridine can also obviously inhibit gB and gD protein expression levels of HSV-1 virus, and obviously inhibit UL27 and US6 gene expression levels. The sophoridine has obvious inhibition effect on the growth of type 1 herpes simplex virus, and can be used for preparing medicine for resisting type 1 herpes simplex virus and medicine for treating type 1 herpes simplex virus infectious diseases. In addition, the sophoridine has low toxicity to normal cells, and has wide application prospect in preparing anti-herpes virus medicines and medicines for preventing and treating herpes virus infection diseases.
Description
Technical Field
The invention belongs to the field of pharmacy, and particularly relates to application of sophoridine in preparation of a medicament for resisting herpes viruses.
Background
Herpes Simplex Virus (HSV), a typical representative of herpes viruses, is known as herpes simplex due to the development of vesicular dermatitis in the acute phase of infection. Herpes simplex virus can cause various diseases in human, such as gingivitis, keratoconjunctivitis, encephalitis, genital system infection, neonatal infection, and the like. Based on the difference of the antigenicity of herpes simplex virus, the virus is classified into type 1 (HSV-1) and type 2 (HSV-2). HSV-1 mainly causes diseases of the waist, such as vesicular stomatitis, herpetic keratitis, herpetic encephalitis and the like, and the incidence rate caused by virus infection accounts for about 5 to about 5 percent of all encephalitis
20 percent, and the fatality rate is as high as 70 percent; pregnancy infection is not only harmful to the mother, but also can cause intrauterine infection to cause abortion, premature birth and dead fetus. HSV-2 is frequently associated with neonatal and genital infections.
Due to the lack of an effective viral vaccine, drug therapy has become the primary route to treat HSV infection. The drugs mainly used in clinic at present comprise nucleoside analogue antiviral drugs or acyclovir analogues and acyclovir analogues. Among them, nucleoside analogue antiviral drugs are mainly suitable for primary and recurrent infections of virus replication, and are difficult to be effective against latent infections. Early nucleoside analogs included iododeoxyuridine, vidarabine, and trifluorothymidine, but they had the disadvantage of low selectivity and greater toxicity to normal cells. Acyclovir (Acyclovir, abbreviated as ACV) is a typical Acyclovir analog that can reduce toxicity to normal cells to some extent, but for Acyclovir drugs, when Thymidine Kinase (TK) or DNA polymerase of HSV mutates, the virus can develop resistance, and the development of resistant strains increases the difficulty of treating herpes simplex virus-infected diseases. Therefore, the search for highly effective and low-toxic anti-herpes simplex virus drugs has become a subject of attention in the medical field.
Sophoridine (SRI) is monomer alkaloid extracted from Sophora alopecuroides L of Sophora of Leguminosae, is one of main active ingredients of Sophora alopecuroides L, Sophora flavescens and Sophora davidii L.with molecular formula of C15H24N2And O. The research shows that the sophoridine has various pharmacological activities of resisting tumor, resisting inflammation, resisting arrhythmia, relieving pain, etc. The application No. 200610041709.1 discloses the use of Sophora alopecuroides total alkaloid in preparing medicine for treating oral herpes simplex, wherein the Sophora alopecuroides total alkaloid is used as active ingredient and comprises more than 20 compounds such as matrine, oxymatrine, sophocarpine, oxysophocarpine, sophoridine, etc. However, in the drug for treating herpes simplex in the oral cavity disclosed in this application, since the active ingredient, sophora alopecuroide total alkaloid, is complex in composition, the problems of unstable active ingredient content and activity intensity tend to occur. Therefore, the development of a new anti-herpes simplex virus medicament with high efficiency, low toxicity and simple active ingredients has important significance for treating herpes simplex virus infection diseases.
Disclosure of Invention
The invention aims to provide the application of sophoridine, pharmaceutically acceptable salts thereof, stereoisomers thereof, crystals thereof or derivatives thereof in preparing anti-herpes virus medicaments.
The invention provides application of sophoridine, pharmaceutically acceptable salts, stereoisomers, crystals or derivatives thereof in preparation of anti-herpes virus medicines.
The invention also provides the application of the sophoridine, pharmaceutically acceptable salts, stereoisomers, crystals or derivatives thereof in preparing medicaments for preventing and/or treating herpes virus infection diseases.
Further, the herpes virus is a herpes simplex virus.
Further, the herpes simplex virus is herpes simplex virus type 1 or herpes simplex virus type 2.
Further, the herpes simplex virus is a herpes simplex virus type 1.
Further, the medicament is capable of inhibiting herpes virus proliferation; and/or the medicament is capable of inhibiting the formation of herpes viral plaques.
Further, the medicament is capable of inhibiting herpes virus gB and gD protein expression; and/or, the medicament is capable of inhibiting herpes virus UL27 and US6 gene expression.
Further, the herpesvirus infection diseases are herpetic stomatitis, keratoconjunctivitis, herpetic encephalitis, genital system infection, and neonatal infection.
Furthermore, the medicament is a preparation prepared by taking sophoridine, pharmaceutically acceptable salts thereof, stereoisomers thereof, crystals thereof or derivatives thereof as an active ingredient and adding pharmaceutically commonly used auxiliary ingredients.
The invention also provides an anti-herpes virus medicament which is a preparation prepared by taking sophoridine, pharmaceutically acceptable salts, stereoisomers, crystals or derivatives thereof as active ingredients and adding pharmaceutically commonly used auxiliary ingredients.
The experiment result shows that the sophoridine can effectively inhibit the formation of HSV-1 virus plaques and obviously inhibit the generation of progeny viruses; the sophoridine can also obviously inhibit gB and gD protein expression levels of HSV-1 virus, and obviously inhibit UL27 and US6 gene expression levels. The sophoridine has obvious inhibition effect on the growth of type 1 herpes simplex virus, and can be used for preparing medicine for resisting type 1 herpes simplex virus and medicine for treating type 1 herpes simplex virus infectious diseases.
In addition, the sophoridine has low toxicity to normal cells, and has wide application prospect in preparing anti-herpes virus medicines and medicines for preventing and treating herpes virus infection diseases.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 shows the result of the cytotoxicity test of sophoridine.
FIG. 2 shows the result of the test of inhibition of HSV-1 plaque formation by sophoridine.
FIG. 3 shows the results of the inhibition test of sophoridine against the virulence of progeny viruses; p < 0.001 compared to HSV-1 group.
FIG. 4 is a graph of the effect of sophoridine on the expression levels of the viral HSV-1gB and HSV-1gD proteins in Vero cells (A and B) and Hela cells (C and D); p < 0.05, P < 0.01, P < 0.001, compared to HSV-1.
FIG. 5 shows the effect of sophoridine on the expression levels of late HSV-1 virus genes UL27 and US6 mRNA in Hela cells; p < 0.05, P < 0.01, P < 0.001, compared to HSV-1.
Detailed Description
The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.
First, experimental material
Herpes simplex virus type 1 (HSV-1) F strain, purchased from institute of virology, college of medicine, Wuhan university, was passaged through Vero cells and then cryopreserved at-80 ℃ for later use.
Vero cells, human cervical carcinoma cells (Hela) cells, purchased from ATCC company, usa, and stored in liquid nitrogen for later use.
Sophoridine: purchased from oddment biotechnology limited, lot number: MUST-19113001, purity: not less than 96 percent.
Positive drug: acyclovir, batch No. 1411201, manufactured by Sunjiang pharmaceutical company, Hubei.
Second, the preparation of the medicine
Dissolving Sophoridine (SRI) with PBS buffer solution to obtain 20mg/ml mother solution, filtering with 0.22um filter for sterilization, storing in refrigerator at-20 deg.C, and diluting with maintaining solution to desired concentration before use.
Positive control Acyclovir (AVC) was prepared as a 10mg/ml stock solution with PBS, filtered through a 0.22um filter to sterilize, stored at-20 ℃ and diluted to 0.25mg/ml with a maintenance solution before use.
Third, statistical method
The original recording is recorded simultaneously by a paper writing and a computer system. Metering data adoption
Data obtained from each group are compared with the model group, and are in accordance with normal distribution, SPSS 19.0 One-way ANOVA (One-WayANOVA) or independent sample T test is adopted, and non-compliant persons are analyzed by a non-parametric test rank-sum method (P < 0.05, the samples are considered to have significant difference between the groups).
Example 1: plaque experiment detection of inhibition effect of medicine on virus plaque
1. Experimental methods
And (3) virus titer determination: taking Vero cells in logarithmic growth phase, digesting with pancreatin, centrifuging, counting according to 1.3 × 105Inoculating to 96-well plate at density of 100 μ l/well, placing at 37 deg.C and 5% CO2Culturing in an incubator. After the cells grow into a monolayer of cells, the supernatant is discarded, and the virus stock solution is diluted to 10 times by a 10-fold dilution method by using a maintenance solution-1To 10-9Series of gradients, 6 replicate wells per concentration, 100. mu.l of virus dilution per well seeded on Vero cells at 37 ℃ in 5% CO2Adsorbing in incubator for 2 hr, discarding supernatant, adding 100 μ l of maintenance solution per well, and culturingSetting a normal cell control group. Cytopathic effect (CPE) was observed and recorded under an inverted microscope every day, the results were recorded after three days of observation, and wells with cytopathic effect of 50% or more were recorded as cytopathic wells. Calculating half tissue infection (TCID) of virus according to Reed-Muench method50)。
Plaque experiment detection of the inhibition of the drug on viral plaque: taking Vero cells in logarithmic growth phase, digesting with pancreatin, centrifuging, counting according to 1.5 × 105Inoculating to 12-well plate at a density of 1 ml/well, standing at 37 deg.C and 5% CO2Culturing in an incubator. After the monolayer had formed, the supernatant was discarded and the amount of the supernatant was 100TCID50HSV-1 virus fluid infected cells, 37 ℃, 5% CO2Adsorbing the supernatant in an incubator for 2h, removing the supernatant, washing with PBS, mixing 2 times of high, medium and low concentrations (0.8, 0.4 and 0.2mg/ml) of sophoridine with 2% of methylcellulose covering fluid at a ratio of 1:1, inoculating to cells at a volume of 1ml per well, and setting cell control and virus control. And after 72h, removing the covering liquid containing methylcellulose, fixing the covering liquid at room temperature for 20min by using 4% paraformaldehyde, adding 0.5% crystal violet for dyeing for 30min after removing the paraformaldehyde, slowly washing the covering liquid with tap water, and counting the plaques after drying in the air. Plaque inhibition rate (viral control plaque-drug group plaque)/viral control plaque × 100%. The experiment was repeated 3 times, with virus controls and normal cell controls.
2. Results of the experiment
The Vero cells infected with HSV-1 are treated by sophoridine with different concentrations, the antiviral effect of the Vero cells is detected by a plaque experiment, and the result (figure 2) shows that sophoridine has an inhibition effect on the formation of HSV-1 plaques, and the inhibition effect is enhanced along with the increase of the concentration of sophoridine.
Example 2: plaque experiment method for detecting influence of drug on virulence of progeny virus
1. Experimental methods
Inoculating Vero cells on a 12-hole plate at a proper density, adding HSV-1 to infect the cells after the cells are fused the next day, culturing at 37 ℃ for 2h in 5% CO2, removing supernatant, adding sophoridine with different concentrations to treat the cells, and culturing in an incubator for 24 h. After 24h of culture, 1ml of maintenance liquid is added into each hole of the supernatant, the 12-hole plate is transferred to the temperature of minus 80 ℃ and 37 ℃ for repeated freeze thawing for 3 times, so that virus particles are released, and the supernatant is collected and placed at minus 80 ℃ for later use. The progeny virus titers were determined using a plaque assay.
2. Results of the experiment
The result of the plaque experiment is shown in fig. 3, and it can be seen that sophoridine has a significant inhibitory effect (P < 0.001) on the generation of progeny virus of HSV-1, and is dose-dependent.
Example 3: western blotting experiment for detecting influence of drug on gB and gD expression of virus protein
1. Experimental methods
Vero cells or Hela cells were seeded in 6-well plates and grown as a monolayer with 100TCID50Infecting cells with HSV-1 virus liquid, adsorbing with a 5% CO2 incubator at 37 ℃ for 2h, removing supernatant, washing with PBS, adding gradient diluted sophoridine solutions with different concentrations, and culturing in a 5% CO2 incubator at 37 ℃ for 24 h. After 24h, the supernatant was discarded and washed 2 times with 3min each time by adding pre-cooled PBS. 120. mu.L of RIPA lysate (containing 1% protease inhibitor and 1% phosphatase inhibitor) was added and lysed on ice for 30min, during which time the lysate was gently blown with a pipette to bring the lysate into intimate contact with the cells. After sufficient lysis, the cells were centrifuged at 12000g at 4 ℃ for 15 min. And (4) taking the supernatant, and determining the total protein content by using a BCA protein quantitative kit, wherein the total protein of all samples is adjusted to be in an equal concentration. According to the protein sample: adding 5 XLoading Buffer into the protein sample at a ratio of 5 Xloading Buffer 4:1, mixing well, centrifuging briefly, heating in boiling water (not less than 95 deg.C) for 5min to denature protein, cooling rapidly to obtain protein sample, packaging, and storing at-20 deg.C. And then detecting the expression levels of HSV-1 virus proteins gB and gD by using a western blotting method.
2. Results of the experiment
The results (fig. 4) show that sophoridine can significantly reduce the expression of HSV-1 viral proteins HSV-1gB and HSV-1gD levels (P < 0.001), that the inhibition appears dose-dependent, and that the inhibitory effect appears consistent in Vero (fig. 4A and 4B) and Hela (fig. 4C and 4D) cells.
Example 4: RT-PCR experiment for detecting influence of drug on viral gene US6 and UL27 mRNA expression
1. Experimental methods
The density of Hela cells was adjusted to 3X 105one/mL, seeded in 6-well plates, 2mL per well. Virus control group, sophoridine group (0.1, 0.2, 0.4mg/mL), and acyclovir control group (0.25 mg/mL). After 2h, the supernatant was discarded and HSV-1 was added, 2ml per well. After 2h, removing the supernatant, adding 2ml of maintenance liquid containing corresponding drugs or no drugs, and extracting total RNA of the cells after 3h, 9h and 16h according to the requirements of the kit. Then detecting the total RNA concentration and purity of the cells, carrying out reverse transcription on the RNA to synthesize cDNA, and finally carrying out real-time fluorescence quantitative polymerase chain reaction according to requirements to detect the influence of the medicament on viral genes US6 and UL 27.
2. Results of the experiment
The results are shown in fig. 5, and compared with the virus group, the sophoridine can obviously inhibit the expression of the virus genes US6 and UL27 after 3h, 9h and 16h (P is less than 0.001); the inhibition effect of different concentrations of drugs on the viral genes is detected within 16h, and the result shows that the sophoridine in each concentration group in the experiment has the effect of obviously inhibiting the expression of the viral genes (P is less than 0.001).
Example 5: cell toxicity assay for sophoridine
1. Experimental methods
Taking Vero and Hela cells in logarithmic growth phase, digesting with pancreatin, centrifuging, counting, and respectively preparing to density of 1.3 × 105One/ml and 1.8X 105The cell suspension/ml was inoculated into a 96-well plate at 100. mu.l/well, and the plate was incubated at 37 ℃ with 5% CO2Culturing in an incubator. After the cells grow into a monolayer, the supernatant is discarded, sophoridine is diluted to 8 concentrations of 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05, 0.025 and 0.0125mg/ml by using a maintenance solution, 100 mu l of liquid medicine containing different concentrations is added into each well, 6 multiple wells are formed in each group, and a normal cell control group is arranged at the same time. Standing at 37 deg.C for 5% CO2Culturing in an incubator, and observing the morphological change of the cells every day under a microscope. Culturing for 72h and 48h respectively, adding 100 μ l 0.5mg/ml MTT into each well, culturing for 3h, removing supernatant, adding 100 μ l DMSO, shaking in dark for 10min, and loading onto enzyme labeling instrument 490Measuring OD at nm, and calculating cell survival rate and half Toxic Concentration (TC)50). The experiment was repeated 3 times with normal cells as blank.
2. Results of the experiment
Treating Vero and Hela cells with sophoridine of different concentrations, observing cell survival rate, and drawing growth curve with drug concentration as abscissa and cell survival rate as ordinate, the result is shown in FIG. 1. As can be seen from FIG. 1, sophoridine is less toxic to Vero and Hela cells, and even at a high concentration of 0.8mg/ml, the survival rates of the cells were as high as 88.6% and 95.1%, respectively.
The experimental results show that the sophoridine can effectively inhibit the formation of HSV-1 virus plaques and remarkably inhibit the generation of progeny viruses; in addition, the sophoridine can also obviously inhibit gB and gD protein expression levels of HSV-1 virus, and can obviously inhibit UL27 and US6 gene expression levels. The sophoridine has obvious inhibition effect on the growth of type 1 herpes simplex virus, and can be used for preparing medicine for resisting type 1 herpes simplex virus and medicine for treating type 1 herpes simplex virus infection diseases.
In conclusion, the invention provides the application of sophoridine in preparing anti-herpes virus medicaments. The experiment result shows that the sophoridine can effectively inhibit the formation of HSV-1 virus plaques and obviously inhibit the generation of progeny viruses; the sophoridine can also obviously inhibit gB and gD protein expression levels of HSV-1 virus, and obviously inhibit UL27 and US6 gene expression levels. The sophoridine has obvious inhibition effect on the growth of type 1 herpes simplex virus, and can be used for preparing medicine for resisting type 1 herpes simplex virus and medicine for treating type 1 herpes simplex virus infectious diseases. In addition, the sophoridine has low toxicity to normal cells, and has wide application prospect in preparing anti-herpes virus medicines and medicines for preventing and treating herpes virus infection diseases.
Claims (10)
1. The application of sophoridine, pharmaceutically acceptable salts thereof, stereoisomers thereof, crystals thereof or derivatives thereof in preparing anti-herpes virus medicaments.
2. The sophoridine, pharmaceutically acceptable salts thereof, stereoisomers thereof, crystals thereof or derivatives thereof, and the application thereof in preparing medicines for preventing and/or treating herpes virus infection diseases.
3. Use according to claim 1 or 2, characterized in that: the herpes virus is herpes simplex virus.
4. Use according to claim 3, characterized in that: the herpes simplex virus is herpes simplex virus type 1 or herpes simplex virus type 2.
5. Use according to claim 4, characterized in that: the herpes simplex virus is type 1 herpes simplex virus.
6. Use according to any one of claims 1 to 5, characterized in that: the medicament is capable of inhibiting herpes virus proliferation; and/or the medicament is capable of inhibiting the formation of herpes viral plaques.
7. Use according to any one of claims 1 to 5, characterized in that: the medicament can inhibit the expression of herpesvirus gB and gD proteins; and/or, the medicament is capable of inhibiting herpes virus UL27 and US6 gene expression.
8. Use according to claim 2, characterized in that: the herpesvirus infection diseases include herpetic stomatitis, keratoconjunctivitis, herpetic encephalitis, genital system infection, and neonatal infection.
9. Use according to any one of claims 1 to 5, characterized in that: the medicament is a preparation prepared by taking sophoridine, pharmaceutically acceptable salt, stereoisomer, crystal or derivative thereof as an active ingredient and adding pharmaceutically commonly used auxiliary ingredients.
10. An anti-herpesvirus medicament characterized by: the sophoridine preparation is prepared with sophoridine, its pharmaceutically acceptable salt, its stereoisomer, its crystal or its derivative as component and pharmaceutically acceptable supplementary components.
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