CN110841024A - New use of fructus Tsaoko in preparing antiviral drugs - Google Patents

Abstract

The invention provides application of an tsaoko aqueous extract in preparing antiviral drugs. The research on the action mechanism discovers that the tsaoko amomum fruit water extract not only can play a role in the HSV-1 virus adsorption stage, but also can play a role in the virus replication stage, so that the tsaoko amomum fruit water extract has double functions of prevention and treatment in the aspect of the anti-HSV-1 effect, has good curative effect, high safety and wide application, and has comprehensive development and application prospects of new medicines for preventing and treating virus infection diseases such as HSV-1 and the like.

Description

New use of fructus Tsaoko in preparing antiviral drugs

Technical Field

The invention relates to a new application of tsaoko amomum fruit in preparing antiviral drugs.

Background

The virus is the smallest of pathogenic microorganisms, is propagated in a cell, has a core of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), has a shell of protein and does not have a cell structure, is parasitic in the host cell and is propagated and replicated depending on a host cell metabolic system, the virus replication process comprises the steps of recognizing and adsorbing ① virus to the surface of the host cell, ② penetrates into a susceptible cell through a host cell membrane, ③ is unshelled, ④ synthesizes early regulatory protein and nucleic acid polymerase, ⑤ virus genome (DNA or RNA) is replicated, ⑥ synthesizes later structural protein, ⑦ progeny virus is assembled, ⑧ susceptible cells release progeny virus, the process is a replication cycle, and the antiviral drug can play an antiviral role by targeting any step of virus replication.

Amomum tsaoko Crevost et Lemarie is a perennial herb of the genus Amomum in the family Zingiberaceae, the whole plant has pungent flavor, and is mainly distributed in Guangxi, Yunnan and Guizhou provinces in China at present. The dried and mature tsaoko fruit is an edible spice necessary in daily life, is a traditional Chinese medicinal material in China, has the functions of warming the middle-jiao, invigorating the stomach, promoting digestion and guiding qi downward, and is often used for relieving pain, resisting gastric ulcer and epilepsy, treating cold-dampness internal resistance, vomiting and diarrhea and the like. At present, the research on the tsaoko amomum fruits mainly comprises the aspects of extracting volatile oil, analyzing chemical components, separating active components of the tsaoko amomum fruits and the like.

At present, no therapeutic research report of the application of amomum tsao-ko to the anti-herpes simplex virus type 1 exists.

Disclosure of Invention

In order to solve the problems, the invention provides the application of tsaoko amomum fruit or tsaoko amomum fruit aqueous extract in preparing a medicament for resisting herpes simplex virus 1; the tsaoko water extract is an extract obtained by taking tsaoko as a raw material and adding water for extraction.

Furthermore, the extract in the medicine contains 1.6-63.7mg of crude drug per 1 ml.

Furthermore, the extract in the medicine contains 1.6-3.2mg crude drug per 1ml crude drug.

The invention also provides an anti-herpes simplex virus 1 medicament, which is a preparation prepared by using amomum tsao-ko as a raw material and adopting a water extraction method to extract, and adding pharmaceutically acceptable auxiliary materials or auxiliary components into the extract.

Further, the preparation is an oral preparation, an injection preparation or an external preparation.

Further, the oral preparation is an ointment, a pill, an oral liquid, a powder, a tablet, a granule or a capsule.

Further, the external preparation is a solution, lotion, liniment, ointment, plaster, paste or patch.

Further, the injection is a solution, a powder, a tablet.

Further, the extract in the medicine contains 1.6-63.7mg, preferably 1.6-3.2mg crude drug per 1ml crude drug.

The invention finally provides a process for the preparation of the aforementioned medicament, which comprises the steps of:

(1) weighing Chinese medicinal fructus Tsaoko, and grinding into powder;

(2) extracting fructus Tsaoko with water, and adding pharmaceutically-acceptable adjuvants or adjuvant ingredients into the extract.

The research on the action mechanism shows that the tsaoko amomum fruit aqueous extract can not only play a role in the HSV-1 virus adsorption stage, but also play a role in the virus replication stage, so that the tsaoko amomum fruit aqueous extract has double functions of prevention and treatment in the aspect of HSV-1 action, has good curative effect, high safety and wide application, and has comprehensive development and application prospects of new medicines for preventing and treating virus infection diseases such as HSV-1 and the like.

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 toxic Effect of aqueous extracts of Tsaoko on Vero cells

FIG. 2 shows the cytopathic effect (100X) of different concentrations of tsaoko amomum fruit aqueous extracts on HSV-1 infected cells (A: cell control; B: ACV positive control; C: virus control; D: inventive drug (0.4 mg/ml); E: inventive drug (0.8 mg/ml); F: inventive drug (1.6 mg/ml); G: inventive drug (3.2 mg/ml); H: inventive drug (6.4mg/ml))

FIG. 3 shows the antiviral effect of the aqueous extract of Amomum tsao-ko on HSV-1 (A: normal cell control, virus control and positive drug control B: Vero cells after HSV-1 infection with different concentrations of the inventive drug; green arrow indicates that cell nucleus is changed, chromatin is aggregated and nuclear envelope is discontinuous after HSV-1 virus infection; red arrow indicates virus particles and inclusion body formed)

FIG. 4 shows the adsorption effect (100X) of the aqueous extracts of Amomum tsao-ko on HSV-1 virus (A: cell control; B: ACV positive control; C: virus control; D: inventive drug (0.4mg/ml), E: inventive drug (0.8mg/ml), F: inventive drug (1.6mg/ml), G: inventive drug (3.2mg/ml), H: inventive drug (6.4mg/ml))

FIG. 5 shows that the water extracts of Amomum tsao-ko of different concentrations can inhibit the replication of HSV-1 virus (A: Western-blot experiment result; B: q-PCR detection result)

FIG. 6 shows that the tsaoko amomum fruit aqueous extract inhibits the expression of HSV-1 virus gene ICP34.5 (A: q-PCR detects the expression of ICP34.5 gene; B: electron microscope detects the formation of autophagic vacuoles (red arrow indicates autophagic vacuoles)

Detailed Description

Example 1

1 materials of the experiment

1.1 medicaments

The invention has the following advantages: the water extract of the tsaoko is used for extracting the Chinese medicinal tsaoko by a water extraction method.

Positive drug: acyclovir, batch No. 1411201, manufactured by Sunjiang pharmaceutical company, Hubei.

1.2 Virus strains

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.

1.3 cells

Vero cells, purchased from ATCC, usa, and stored in liquid nitrogen for later use.

1.4 Primary reagents

DMEM medium, lot number: SH30243.01, manufactured by Hyclone; trypsin, batch No.: j170024 manufactured by Hyclone; cell proliferation-toxicity test kit (CCK-8, batch No.: BS350A, manufactured by Biosharp, China); penicillin-streptomycin antibiotic, batch No.: j160019, manufactured by Hyclone corporation; newborn bovine serum, lot number: 130908, Zhejiang Hangzhou Biotechnology Ltd; reverse transcription kit, batch No.: 00612871, manufactured by Thermo Scientific;

green premix, batch number: 1712046, manufactured by Thermo Scientific, Inc.

1.5 Main Instrument

Microplate reader (Bio-Rad 680, USA); carbon dioxide incubator (Thermo 3111, usa), inverted microscope (nikon TS100-F, japan); an ultra-clean bench (SW-CJ-180, Suzhou Antai air technologies, Inc., China); transmission electron microscopy (Hitachi H600, Japan); fluorescent quantitative PCR instrument (analytikjena-qTOWER2.2, Germany); general PCR instrument (analytikjena-qTOWER2.2, Germany); a very small amount of nucleic acid protein analyzer (scandrop100, germany); centrifuge (SCILOGEX D3024R, USA).

2 method of experiment

2.1 pharmaceutical formulation

The invention is diluted to 100ml with sterile distilled water corresponding to 500mg crude drug, filtered and sterilized, stored in a refrigerator at 4 ℃ for standby, and diluted to 102.4, 51.2, 25.6, 12.8, 6.4, 3.2 and 1.6mg/ml with maintenance solution before use for cytotoxicity experiments and antiviral experiments. The positive control drug acyclovir is prepared into 10mg/ml mother liquor by PBS, filtered and sterilized, stored in a refrigerator at 4 ℃ for later use, and diluted to 0.25mg/ml by maintenance solution before use.

2.2 Virus titre assay

Taking Vero cells in logarithmic growth phase, digesting with pancreatin, and then adding 1 x 10⁵One/ml density was inoculated into 96-well plates, 100. mu.l per well, 5% CO₂The culture was carried out in an incubator at 37 ℃. After the cells grow into a monolayer, the virus stock solution is diluted to 10 times by using a cell maintenance solution by a 10-fold dilution method^-1To 10^-9Series of gradients, 6 replicate wells per concentration, 100. mu.l of virus dilution per well inoculated into Vero cells, 5% CO₂Adsorbing for 2h at 37 ℃ in an incubator, removing supernatant, adding 100 mu l of cell maintenance liquid, continuing culturing for 72h, observing and recording cytopathic effect (CPE) every day, observing and recording experimental results under an inverted microscope after cytopathic effect is stable, wherein the holes with CPE reaching 50% or more are cytopathic holes. Half of the infection amount of the virus was calculated by the Reech-Muench formula (TCID 50). The experiment was repeated 3 times, with normal cells as blank.

2.3 cytotoxicity of the inventive Agents on Vero cells

The cytotoxicity of the invention medicine to Vero cells is determined by adopting the same method for determining virus titer. The inventive drug was diluted in a cellular maintenance medium in multiple proportions to give a total of 7 concentration gradients of 102.4, 51.2, 25.6, 12.8, 6.4, 3.2 and 1.6 mg/ml. Inoculating Vero cells into a 96-well plate, abandoning the culture medium after the cells grow into a single layer, adding 100 mu l of diluent of the invention medicine into each well, setting 6 multiple wells for each concentration, and setting 5% CO₂Culturing at 37 deg.C in incubator, observing cell morphology change every day, adding 10 μ l CCK-8 solution into each well after 72 hr, culturing for 4 hr, measuring absorbance at 450nm with microplate reader, and calculating cell survival rate and half poisoning concentration of the medicine (TC 50). The experiment was repeated 3 times, with normal cells as blank.

2.4 anti-HSV-1 Activity of the inventive Agents

Median inhibitory concentration (IC50) determination: the cytopathic effect of the inventive drug on HSV-1 was determined by the same method as described above 2.2 (determination of viral titer). Inoculating Vero cells into a 96-well plate, discarding supernatant after the cells grow into a single layer, infecting the cells with 100 times of HSV-1 virus solution of TCID50, adsorbing at 37 ℃ for 2h, discarding the virus solution, respectively adding 100 mu l of invention medicine diluent with different concentrations into culture wells, setting 6 multiple wells for each concentration, and setting 5% CO₂Incubators were incubated at 37 ℃ for 72h and CPE was observed daily. When the CPE of the virus control group reaches "++++ - +++++" the experimental result is judged, the median inhibitory concentration (IC50) is analyzed according to the Reech-Muench method, and the Therapeutic Index (TI) of the HSV-1 treated by the invention is calculated, wherein the TI is TC50/IC 50. The experiment was repeated 3 times, with virus control, positive drug control and normal cell control.

Note: the nature of the lesions was determined according to the condition of the CPE, 0% for CPE, "-", 1% -25% for CPE, "+", 25% -50% for CPE, "+ + + + +", 50% -75% for CPE, "+ + + + + + + + +", and 75% and above.

Electron microscopy of cytopathic effects: the pretreatment method comprises measuring the half inhibition concentration (IC50), namely taking Vero cells in logarithmic growth phase, digesting with pancreatin, and configuring into 1 × 10⁵Inoculating 2ml of the mixture per ml, culturing, removing supernatant after the cells grow into a monolayer, infecting the cells with 100 times of HSV-1 virus of TCID50, adsorbing at 37 ℃ for 2h, removing virus liquid, adding 2ml of the invented medicine with 3 different concentrations (6.4, 3.2 and 1.6mg/ml) and 5% CO₂The cells were cultured in an incubator at 37 ℃ and observed daily for cytopathic conditions. Collecting cells when the CPE of the virus control group reaches "+++ - +++++", centrifuging for 15min at 1500-2000 r/min, removing supernatant, adding 500 mu l of 0.5% glutaraldehyde fixing solution, and standing for 10min at 4 ℃ for pre-fixing; centrifuging at 10000-13000 r/min for 15min, discarding the supernatant, adding 500 mu l of 3% glutaraldehyde for fixation, and inspecting by a projection electron microscope laboratory of the national institute of science and western university of Sichuan. And simultaneously setting virus control, positive drug control and normal cell control.

2.5 mechanism of action of the inventive drugs against HSV-1

The inventive medicine has the following adsorption effect on HSV-1 virus: taking Vero cells in logarithmic growth phase, preparing into 1 x 10 after trypsinization⁵Taking 2ml of the mixture per ml, inoculating the mixture into a 6-well plate, and waiting forAfter the cells grew into monolayers, precooling for 1h at 4 ℃, and mixing the mixture of 2 times of administration concentration and 200 times of TCID50 HSV-1: 1 mixing, inoculating to cells, standing at 4 deg.C for 2 hr, discarding supernatant, washing with precooled PBS for 2 times, adding 2ml maintenance solution, 5% CO₂The cells were incubated at 37 ℃ and the status of each group was observed microscopically when the virus control lesions reached "+ + + +". And simultaneously setting virus control, positive drug control and normal cell control.

The inventive drug can inhibit HSV-1 virus replication: taking Vero cells in logarithmic growth phase, preparing into 1 x 10 after trypsinization⁵Inoculating and culturing 2ml to 6cm culture dish, discarding supernatant after cell growth to form monolayer, infecting cells with 100 times of TCID50 HSV-1 virus, adsorbing at 37 deg.C for 2h, discarding virus solution, adding 2ml of 3 invented medicines with different concentrations (6.4, 3.2, 1.6mg/ml) and 5% CO₂Culturing in an incubator at 37 ℃, collecting cells for 18h, respectively extracting RNA and protein, detecting the transcription change of virus replication genes US6 and UL27 by fluorescent quantitative PCR, and detecting the expression change of proteins gB and gD by enzyme-linked immunoblotting (western-blot). And simultaneously setting virus control, positive drug control and normal cell control.

2.6 data statistics and analysis

Statistical analysis is carried out on the data by SPSS21.0 statistical software, wherein the comparison of the cell survival rate and the virus inhibition rate adopts t test, and the difference is that P <0.05 has statistical significance.

3 results and analysis

3.1 Virus Titers

The TCID50 of the HSV-1 virus is 10 according to the formula of Reech-Muench^-5.8I.e. a titer of 10 at 100. mu.l per well^-5.18The virus can cause obvious lesions on 50 percent Vero cells, and the concentration of the virus used in the experiment is 100 times of TCID 50.

3.2 cytotoxicity of the inventive Agents

Vero cells are treated by the invention drugs with different concentrations, the survival of the cells is observed, a growth curve is drawn by taking the drug concentration as the abscissa and the cell survival rate as the ordinate, and the result is shown in figure 1.

As is clear from FIG. 1, the inventive drug exhibited concentration-dependent inhibitory action on Vero cells, and the inhibitory action was enhanced with increasing concentration. An electron microscope observation result shows that the Vero normal cells are complete in shape, fusiform, compact and regular in arrangement and clear in cell membrane boundary; when the concentration of the inventive drug is 12.8mg/ml or less, the cell morphology has no obvious difference with a normal control group, and when the concentration reaches 25.6mg/ml, part of cells begin to appear the morphological changes such as rounding and shrinking, death and shedding, and the like, thereby reducing the number and slowing the proliferation. SPSS21.0 statistical analysis the inventive drug exhibited 63.7mg/ml TC50 for Vero cells.

3.3 inhibition of HSV-1 Virus proliferation by the inventive Agents

Vero cells infected with HSV-1 were treated with different concentrations of the invention drug and analyzed for changes in cytopathic effects, the results are shown in FIG. 2.

As can be seen from FIG. 2, HSV-1 can cause obvious cytopathy after infecting Vero cells, while the inventive drug can obviously inhibit cytopathy, and the inhibition effect is obviously concentration-dependent, i.e. the inhibition effect is obviously enhanced with the increase of the concentration of the inventive drug. When the inventive drug is 0.8mg/ml, the virus growth is obviously inhibited; when the concentration is 3.2mg/ml, the virus growth is completely inhibited, and Vero cells have no obvious lesion and no obvious difference from a blank control.

3.4 anti-HSV-1 Virus Effect of the inventive Agents

The results of observing the antiviral effect of the inventive drugs with different concentrations on HSV-1 by a transmission electron microscope are shown in figure 3

As can be seen from FIG. 3, the normal Vero nucleus is irregular, the chromatin in the nucleus is uniformly distributed, and the nuclear membrane is clear; the organelles such as mitochondria, rough endoplasmic reticulum, ribosome and the like can be seen in cytoplasm, and the structure is clear; the cell membrane is intact and the surface has a few microvilli. After HSV-1 infects Vero cells, cellular chromatin is gathered around the nuclear envelope, the nuclear envelope is discontinuous (shown by green arrows in figure 3A), obvious virus particles and inclusion bodies appear in cell membranes, cytoplasm and cell nucleus, and organelles such as mitochondria are destroyed to form cavities (shown by red arrows in figure 3A). The invention treats cells infected with HSV-1, and finds that the invention has obvious HSV-1 virus resistance and obvious concentration dependence, namely, the higher the concentration is, the stronger the virus inhibition effect is. The concrete expression is as follows: the invention medicine with low dose (1.6mg/ml) can lead Vero cell chromatin to be gathered to a certain degree, a small amount of virus particles can be seen under a high power microscope, the organelles are obviously reduced, and partial vacuoles (shown by a red arrow of 3B) are generated; the invention medicine (3.2mg/ml) with medium dosage can make Vero cell structure complete without virus particles; the high dose of the invention (6.4mg/ml) allowed the cellular structure to be intact and no viral particles were visible (FIG. 3B). The invention is proved to have similar HSV-1 virus resistance with the positive drug acyclovir under the action of high and medium dosage.

3.5 mechanism of action of the inventive drugs against HSV-1 virus

3.5.1 inhibition of HSV-1 Virus adsorption

Vero cells infected with HSV-1 were treated with different concentrations of the invention drug and analyzed for changes in cytopathic effects, the results are shown in FIG. 4.

As can be seen from FIG. 4, the inventive drug can significantly inhibit cytopathic effect, and the inhibitory effect is significantly concentration-dependent, i.e., the inhibitory effect is significantly enhanced with the increase of the drug concentration. In the test, the positive medicament has pathological changes, which shows that the medicament can inhibit HSV-1 virus adsorption, and the positive medicament has no inhibition effect on virus adsorption.

3.5.2 inhibition of replication of HSV-1 Virus

As can be seen from FIG. 5, with the increase of the concentration of the inventive drug, the expression of the late proteins gB and gD of HSV-1 virus replication is obviously reduced (FIG. 5A), and the transcription of the genes US6 and UL27 is obviously lower than that of the virus control (FIG. 5B), which indicates that the inventive drug has the function of inhibiting the late replication of HSV-1 virus.

3.5.3 the inventive drug inhibits the ICP34.5 expression of HSV-1 virus gene

The autophagy is closely related to the innate and adaptive immune responses of herpes virus infection, ICP34.5 is a virus late protein encoded by HSV genes, and can inhibit autophagy of cells through different ways to resist the antiviral effect of a host, and the experiment shows that the medicine can induce the formation of autophagy vesicles of the cells (shown by red arrows in figure 6B) by inhibiting the expression of the HSV-1 virus genes ICP34.5, so that the medicine has the antiviral effect.

In conclusion, the half poisoning concentration TC50 of the medicine for Vero cells is 63.7mg/ml, the half inhibition concentration IC50 of the medicine for HSV-1 is 1.6mg/ml, and the therapeutic index TI is 39.81; the research of action mechanism finds that the medicine can not only play a role in the HSV-1 virus adsorption stage, but also play a role in the virus replication stage, which indicates that the medicine has dual functions of prevention and treatment in the aspect of HSV-1 action, and the positive medicine acyclovir only has the function of inhibiting HSV-1 virus replication. Therefore, the medicine has good curative effect, high safety and wide application, and has comprehensive development and application prospect for new medicines for preventing and treating virus infection diseases such as HSV-1 and the like.

Claims (10)

1. Use of tsaoko amomum fruit or aqueous extracts of tsaoko amomum fruit for the preparation of a medicament for combating herpes simplex virus type 1; the tsaoko water extract is an extract obtained by taking tsaoko as a raw material and adding water for extraction.

2. Use according to claim 1, characterized in that: the medicine contains crude drug 1.6-63.7mg per 1 ml.

3. Use according to claim 2, characterized in that: the medicine contains crude drug 1.6-3.2mg per 1 ml.

4. An anti-herpes simplex virus type 1 medicament, characterized in that: the preparation is prepared by using tsaoko amomum fruits as raw materials, extracting by adopting a water extraction method, and adding pharmaceutically acceptable auxiliary materials or auxiliary components into the extract.

5. The medicament of claim 4, wherein: the preparation is oral preparation, injection or external preparation.

6. The medicament of claim 5, wherein: the oral preparation is paste, pill, oral liquid, powder, tablet, granule or capsule.

7. The medicament of claim 5, wherein: the external preparation is a solution, lotion, liniment, ointment, plaster, paste or patch.

8. The medicament of claim 5, wherein: the injection is solution, powder or tablet.

9. The medicament of claim 4, wherein: the medicine contains crude drug 1.6-63.7mg, preferably 1.6-3.2mg per 1 ml.

10. A process for the preparation of a medicament according to any one of claims 4 to 9, characterized in that: it comprises the following steps:

(1) weighing Chinese medicinal fructus Tsaoko, and grinding into powder;

(2) extracting fructus Tsaoko with water, and adding pharmaceutically-acceptable adjuvants or adjuvant ingredients into the extract.

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