Application of radix tetrastigme extract in preparation of medicine for resisting Crimean-Congo hemorrhagic fever virus

Abstract

The invention discloses application of a radix tetrastigme extract in preparation of a medicine for resisting Crimean-Congo hemorrhagic fever virus, and belongs to the technical field of medicine. The anti-CCHFV effect of the radix tetrastigme extract is evaluated on the cellular level by utilizing a CCHFV virus-like particle system capable of simulating virus endocytosis and transcriptional activity, and the result shows that the radix tetrastigme extract has remarkable antiviral activity, is dose-dependent, and has a good application prospect. The antiviral activity mechanism of the radix tetrastigme extract is related to virus cell entry and virus transcription stages. The radix tetrastigme extract provided by the invention shows the activity of inhibiting the CCHFV, and has a wide prospect of being developed into a medicine for treating human CCHFV infection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of Tetrastigma hemsleyanum extract in preparation of a drug for resisting Crimean-Congo hemorrhagic fever virus. BACKGROUND

[0002] Crimean-Congo hemorrhagic fever virus (CCHFV) belongs to the Nairovirus genus of the Bunyaviridae family, and is a highly pathogenic virus with a single-stranded negative-sense RNA genome. According to the high pathogenicity and population epidemic risk of CCHFV, the World Health Organization (WHO) defines it as a Class 4 virus. The virus is widely prevalent in Eastern Europe, the Middle East, Asia and Africa, and is mainly transmitted through tick bites, blood contact and tissue contact. After infection, it can cause acute fever, hemorrhagic tendency and other clinical symptoms. Severe patients will develop shock and multiple organ failure, with a mortality rate of 10% to 40%, posing a serious threat to human health and seriously challenging global public health security.

[0003] At present, there is no approved vaccine or specific treatment drug for CCHFV infection, and only symptomatic supportive treatment can be taken in the clinic. Existing broad-spectrum antiviral drugs such as ribavirin have problems such as unclear efficacy and significant toxicity, and cannot meet the clinical treatment needs. Due to the high pathogenicity of CCHFV, related research needs to be carried out in a biosafety level 4 (BSL-4) laboratory, which greatly hinders the development process of therapeutic drugs, making the treatment of CCHFV infection a major problem in the global public health field that needs to be solved urgently.

[0004] Tetrastigma hemsleyanum, also known as Jinxialiaohuolu, is a perennial plant of the grape family, and is one of the high-value medicinal species in China. It has been included in the list of "New Zhejiang Eight Flavors" of native medicinal materials, and is naturally distributed in many provinces such as Zhejiang, Fujian and Jiangxi. Traditional Chinese medicine theory believes that Tetrastigma hemsleyanum has the effects of clearing heat and detoxifying, dispelling wind and reducing phlegm, and promoting blood circulation and relieving pain, and is often used to relieve heat and toxic accumulation, rheumatic pain and other symptoms. Modern pharmacological studies have further confirmed that the active ingredients of Tetrastigma hemsleyanum exhibit significant biological activity in antiviral, immunomodulatory and antitumor aspects, highlighting its good prospects for drug development. Existing technologies have disclosed the application of Tetrastigma hemsleyanum extract in resisting common viruses such as influenza virus, herpes simplex virus (HSV) and respiratory syncytial virus (RSV), and the n-butanol and ethyl acetate extract fractions of its ethanol extract have shown significant antiviral effects.

[0005] However, the structural features of viruses have high specificity with infection mechanisms, and there are essential differences in the pathogenic mechanisms of viruses of different families. As a single-stranded negative-sense RNA virus, the entry and transcription process of CCHFV is completely different from viruses such as RSV and HSV-1. The existing technology only discloses the inhibitory effect of Radix Smilacis Glabrae on common viruses, does not mention the activity of Radix Smilacis Glabrae extract on viruses of the Bunyaviridae family, and does not disclose the therapeutic effect and application scheme of Radix Smilacis Glabrae extract on CCHFV. In view of the clinical blank of CCHFV treatment drugs and the research basis of the antiviral properties of Radix Smilacis Glabrae, the development of the application of Radix Smilacis Glabrae extract in anti-CCHFV drugs has important theoretical value and practical significance. SUMMARY

[0006] In view of the defects that CCHFV infection lacks specific treatment drugs and the application range of Radix Smilacis Glabrae extract is limited in the prior art, the purpose of the present application is to provide a new use of Radix Smilacis Glabrae extract, that is, the application of Radix Smilacis Glabrae extract in the preparation of a drug for treating CCHFV infection, and to fill the research and development blank of CCHFV treatment drugs.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: The application of Radix Smilacis Glabrae extract in the preparation of an anti-Crimean-Congo hemorrhagic fever virus drug.

[0008] In the preferred scheme, the Radix Smilacis Glabrae extract is prepared by the following method: Radix Smilacis Glabrae powder is added to an extraction solvent for ultrasonic extraction, and after the ultrasonic extraction is completed, the mixture is centrifuged, and the supernatant is taken, thereby obtaining a solution containing Radix Smilacis Glabrae extract.

[0009] In the preferred scheme, the extraction solvent is selected from deionized water, ethanol or dimethyl sulfoxide (DMSO).

[0010] In the preferred scheme, the mass ratio of Radix Smilacis Glabrae powder to extraction solvent is 1:3-10.

[0011] In the preferred scheme, the centrifugation speed is 7500-8500 rpm, and the centrifugation time is 1-5 min.

[0012] In the preferred scheme, the ultrasonic extraction frequency is 35-45 KHz, and the time is 0.5-1.5 h.

[0013] In the preferred scheme, the anti-Crimean-Congo hemorrhagic fever virus drug uses Radix Smilacis Glabrae extract as the only active ingredient.

[0014] In the preferred scheme, the preparation form of the anti-Crimean-Congo hemorrhagic fever virus drug is an oral preparation, an injection preparation or a topical preparation.

[0015] In a preferred embodiment, the oral preparation is selected from tablets, capsules, granules, oral liquids or powders, and the injectable preparation is selected from sterile powders or injection solutions for injection.

[0016] In a preferred embodiment, the anti-Crimea-Congo hemorrhagic fever virus drug comprises an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is a *Trifolium repens* extract, which accounts for 5%-50% of the drug by mass.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention is the first to demonstrate that the extract of *Trifolium repens* has significant inhibitory activity against CCHFV. Validated using the tc-VLP system (transcriptionally active virus-like particle system), its inhibition rate against CCHFV at non-toxic concentrations is dose-dependent, and it can act on the virus's entry into cells and transcriptional stages. This provides a novel treatment option for CCHFV infection and solves the problem of the lack of specific drugs for this virus in existing technologies.

[0018] 2. The extract of *Trifolium repens* has the advantage of being a natural source. Existing studies have confirmed that it has no liver or kidney toxicity with long-term use. Compared with traditional antiviral drugs such as ribavirin, it has fewer side effects and is better tolerated by patients, making it suitable for the treatment of patients of different ages.

[0019] 3. This invention expands the antiviral application of *Trifolium repens* extract from common viruses such as RSV and HSV to highly pathogenic level 4 virus CCHFV, breaking through its traditional medicinal scope. At the same time, the tc-VLP system reduces the dependence of the research process on BSL-4 laboratories, providing a scalable technical model for subsequent drug development. Attached Figure Description

[0020] Figure 1 This is a graph showing the cytotoxicity data of *Trifolium repens* extract on BSR-T7 / 5 cells.

[0021] Figure 2 A graph showing the data on the anti-CCHFV virus activity of *Trifolium repens* extract.

[0022] Figure 3 This is a graph showing the data on the inhibition of CCHFV entry into cells by the extract of *Trifolium repens*.

[0023] Figure 4 A graph showing the data on the inhibition of CCHFV transcriptional activity by *Trifolium repens* extract. Detailed Implementation

[0024] The technical solution of the present invention will be further described and illustrated below through examples. All raw materials used in the examples are commercially available or prepared using conventional methods.

[0025] Example 1 Preparation of *Trifolium repens* extract: Weigh 10 mg of *Trifolium repens* powder (purchased from Anhui Yaozhiyuan Traditional Chinese Medicine Pieces Co., Ltd.) and place it in a 1.5 mL EP tube; add deionized water at a material-to-liquid ratio of 1:4 to dissolve the *Trifolium repens* powder and shake to mix well; place the mixture in an ultrasonic oscillator and sonicate at a frequency of 40 kHz for 1 h; centrifuge the EP tube at 8000 rpm for 3 min, and take the supernatant as the *Trifolium repens* extract solution, which is stored in a -80 ℃ refrigerator for later use.

[0026] Example 2 Preparation of *Trifolium repens* extract: Weigh 10 mg of *Trifolium repens* powder (purchased from Anhui Yaozhiyuan Traditional Chinese Medicine Pieces Co., Ltd.) and place it in a 1.5 mL EP tube; add ethanol at a material-to-liquid ratio of 1:4 to dissolve the *Trifolium repens* powder and shake to mix well; place the mixture in an ultrasonic oscillator and sonicate at a frequency of 40 kHz for 1 h; centrifuge the EP tube at 8000 rpm for 3 min, and take the supernatant as the *Trifolium repens* extract solution, which is stored in a -80 ℃ refrigerator for later use.

[0027] Example 3 Preparation of *Trifolium repens* extract: Weigh 10 mg of *Trifolium repens* powder (purchased from Anhui Yaozhiyuan Traditional Chinese Medicine Pieces Co., Ltd.) and place it in a 1.5 mL EP tube; add DMSO to dissolve the *Trifolium repens* powder at a material-to-liquid ratio of 1:4, and shake to mix well; place the mixture in an ultrasonic oscillator and sonicate at a frequency of 40 kHz for 1 h; centrifuge the EP tube at 8000 rpm for 3 min, and take the supernatant as the *Trifolium repens* extract solution, and store it in a -80 ℃ refrigerator for later use.

[0028] Example 4 The cytotoxicity verification of the *Trifolium repens* extract includes the following steps: P1. 24 hours in advance, BSR-T7 / 5 cells were prepared at a rate of 2×10⁻⁶. 4 One cell was seeded in a 96-well cell culture plate; P2. The *Trifolium repens* extract solutions from Examples 1-3 were serially diluted with DMEM cell culture medium (GIBCO) containing 10% FBS (GIBCO). The highest concentration was 1%, and the dilution was 2-fold. Four gradients were set up, with three replicates for each group. Different concentrations of *Trifolium repens* extract solutions were added to the cells in the 96-well cell culture plate of step P1. P3. The cells treated in step P2 were placed in a 37 ℃, 5% CO2 incubator and cultured for 48 h (Trifolium repens extract experimental group). Cell viability was detected according to the CCK-8 (Cell Counting Kit-8, Sigma-Aldrich) instructions. 10 µL of the detection reagent was added to each well and incubated at 37 ℃ for 1 h. The OD450 value was read using a multi-functional microplate reader (EnSight, PerkinElmer) and cell viability was further calculated. P4. Data from each group were normalized using the blank control group without *Trifolium repens* extract as the standard group. Cell viability = *Trifolium repens* extract experimental group / standard group × 100%. Graphpad software was used for plotting.

[0029] The results are as follows Figure 1 As shown in the figure, the aqueous extract of *Trifolium repens* corresponds to the extract solution prepared in Example 1, the ethanol extract of *Trifolium repens* corresponds to the extract solution prepared in Example 2, and the DMSO extract of *Trifolium repens* corresponds to the extract solution prepared in Example 3. At the tested concentrations, the extract solutions of *Trifolium repens* from Examples 1 to 3 exhibited weak or no cytotoxicity to BSR-T7 / 5 cells, with a half-maximal toxicity concentration (CC50) greater than or close to 1%.

[0030] Example 5 Study on the antiviral activity of Trifoliate Orange Extract 1. Preparation of CCHFV tc-VLPs particles includes the following steps: S1. 24 hours in advance, BSR-T7 / 5 cells (the BSR-T7 / 5 cell line from golden hamster kidney tissue, purchased from Wuhan Pronosei Life Science Technology Co., Ltd.) in the logarithmic growth phase were injected at a rate of 1.5 × 10⁻⁶. 5 The cells / mL were seeded into 6-well plates; In S2 and 6-well plates, when the cell confluence was approximately 80%, plasmid transfection was performed according to the instructions of Lipofectamine 2000 (Thermo Fisher Scientific). 30 µL of transfection reagent was used, and the total amount of plasmid was 5 µg (the mass ratio of each plasmid was Pcaggs-T7: Pcaggs-L: Pcaggs-GPC: Pcaggs-NP: PSMART-nLuciferase = 4:2:10:4:1). S3. The transfected cells were placed in a 5% CO2 incubator at 37°C and cultured for 48 h. S4. Collect the cell supernatant virus solution, centrifuge at 2000 g for 10 min at 4℃ to remove the cell precipitate, aliquot the supernatant to obtain CCHFV tc-VLP, and store at -80℃ for later use.

[0031] 2. Verification of the antiviral activity of *Trifolium repens* extract, including the following steps: (1) 24 hours in advance, BSR-T7 / 5 cells were prepared at a rate of 2×10⁵ 5 One cell was seeded in a 96-well cell culture plate and the cells were allowed to adhere to the plate for later use. (2) Remove CCHFV tc-VLP from the -80 ℃ freezer and thaw it on ice; infect cells with tc-VLPs virus with MOI=0.1; (3) Add the *Trifolium repens* extract solution from Examples 1-3 respectively. The *Trifolium repens* extract solution is serially diluted with cell culture medium. The drug concentration starts from the highest 1% and is diluted 2 times. Four gradients are set up, and three replicates are made for each group. (4) After adding the extract of Trifoliate orange, the cells were cultured in a 37 ℃, 5% CO2 incubator for 48 h (Trifoliate orange extract experimental group). (5) Detect luciferase activity in cells according to the instructions. Mix 50× substrate and buffer at a ratio of 1:50. Prepare fresh and use immediately. Wash cells twice with 1×PBS (Gibco). After washing, add 100 uL of luciferase substrate nluc detection solution (Luciferase Reporter Gene Assaykit, Sigma-Aldrich) to the detection well and incubate at room temperature for about 10 min to allow the cells to lyse fully. (6) After cell lysis, mix the lysis buffer, transfer the liquid to a white opaque microplate, and use a microplate reader to measure the chemiluminescence at 560 nm.

[0032] When calculating the inhibition rate of *Trifolium repens* extract against the virus, the data of each group were normalized using the control group of virus infection without *Trifolium repens* extract as the standard group. The inhibition rate of *Trifolium repens* extract against the virus = 100% - (Trifolium repens extract experimental group - cell control group) / (virus infection control group - cell control group) × 100%, and the graph was plotted using Graphpad software.

[0033] The results are as follows Figure 2As shown in the figure, the aqueous extract of *Trifolium repens* corresponds to the extract solution prepared in Example 1, the ethanol extract of *Trifolium repens* corresponds to the extract solution prepared in Example 2, and the DMSO extract of *Trifolium repens* corresponds to the extract solution prepared in Example 3. The extracts of *Trifolium repens* in Examples 1 to 3 all showed the ability to inhibit the virus at non-toxic concentrations, and exhibited gradient-dependent inhibitory activity.

[0034] 3. Detection of the effect of *Trifolium repens* extract on the virus entry stage, including the following steps: (1) 24 h in advance, BSR-T7 / 5 cells were prepared at a rate of 2×10 4 One cell was seeded in a 96-well cell culture plate and the cells were allowed to adhere to the plate for later use. (2) Take CCHFV tc-VLP out of the -80 °C freezer and thaw it on ice; prepare the virus infection solution using cell culture medium and add 50 μL of virus solution to each well; (3) At the same time, diluents of the *Trifolium repens* extract solutions in Examples 1 to 3 were prepared respectively. The concentration of *Trifolium repens* extract in the diluent of *Trifolium repens* extract in Example 1 was 1%, the concentration of *Trifolium repens* extract in the diluent of *Trifolium repens* extract in Example 2 was 1%, and the concentration of *Trifolium repens* extract in the diluent of *Trifolium repens* extract in Example 3 was 0.5%. 50 μL of extract diluent was added to each well. (4) After treating the cells at 37 °C for 1 h, wash the cells twice with 1×PBS, add 100 μL of DMEM containing 10% FBS, and place in 5% CO2 solution. 2， Incubate at 37 ℃ for 2 days; (5) Detect luciferase activity in cells according to the instructions. Mix 50× substrate and buffer at a ratio of 1:50. Prepare fresh and use immediately. Wash cells twice with 1×PBS. After washing, add 100 uL of luciferase substrate nluc detection solution (Luciferase Luciferase Detection Kit, Luciferase Reporter Gene Assay kit, Sigma-Aldrich) to the detection well and incubate at room temperature for about 10 min to allow the cells to fully lyse. (6) After cell lysis, mix the lysis buffer, transfer the liquid to a white opaque microplate, and use a microplate reader to measure the chemiluminescence at 560 nm.

[0035] When calculating the inhibition rate of the Trifoliate Orange Extract against the virus, the data of each group were normalized with the virus-infected control group as the standard group. The antiviral inhibition rate of the extract = 100% - (Trifoliate Orange Extract experimental group - cell control group) / (virus-infected control group - cell control group) × 100%, and the graph was plotted using Graphpad software.

[0036] The results are as follows Figure 3 As shown in the figure, the aqueous extract of *Trifolium repens* corresponds to the extract solution prepared in Example 1, the ethanol extract of *Trifolium repens* corresponds to the extract solution prepared in Example 2, and the DMSO extract of *Trifolium repens* corresponds to the extract solution prepared in Example 3. At a non-cytotoxic concentration, the extracts of *Trifolium repens* in Examples 1-3 exhibited activity in inhibiting viral entry.

[0037] 4. Detection of the effect of *Trifolium repens* extract on the viral transcription stage, including the following steps: (1) 24 h in advance, BSR-T7 / 5 cells were prepared at a rate of 2×10 4 One cell was seeded in a 96-well cell culture plate and the cells were allowed to adhere to the plate for later use. (2) Take CCHFV tc-VLP out of the -80 ℃ freezer and place it on ice to thaw; prepare the virus infection solution using cell culture medium and add 100 μL of virus solution to each well; (3) The cells were placed at 37 °C for 1 h to allow the virus to fully infect them; (4) Wash the cells twice with 1×PBS, add 100 μL of culture medium containing the extract, and add 100 μL of the dilution buffer of the *Trifolium repens* extract solution in Examples 1-3 to each well. The concentration of *Trifolium repens* extract in the dilution buffer of *Trifolium repens* extract in Example 1 is 1%, the concentration of *Trifolium repens* extract in the dilution buffer of *Trifolium repens* extract in Example 2 is 1%, and the concentration of *Trifolium repens* extract in the dilution buffer of *Trifolium repens* extract in Example 3 is 0.5%. (5) Incubate in a 5% CO2, 37 ℃ incubator for 2 days; (6) Detect luciferase activity in cells according to the instructions. Mix 50× substrate and buffer at a ratio of 1:50. Prepare fresh and use immediately. Wash cells twice with 1×PBS. After washing, add 100 uL of luciferase substrate nluc detection solution (Luciferase Reporter Gene Assay kit, Sigma-Aldrich) to the detection well and incubate at room temperature for about 10 min to allow the cells to lyse fully. (6) After cell lysis, mix the lysis buffer, transfer the liquid to a white opaque microplate, and use a microplate reader to measure the chemiluminescence at 560 nm.

[0038] When calculating the inhibition rate of the Trifoliate Orange Extract against the virus, the data of each group were normalized with the virus-infected control group as the standard group. The antiviral inhibition rate of the extract = 100% - (Trifoliate Orange Extract experimental group - cell control group) / (virus-infected control group - cell control group) × 100%, and the graph was plotted using Graphpad software.

[0039] The results are as follows Figure 4 As shown in the figure, the aqueous extract of *Trifolium repens* corresponds to the extract solution prepared in Example 1, the ethanol extract of *Trifolium repens* corresponds to the extract solution prepared in Example 2, and the DMSO extract of *Trifolium repens* corresponds to the extract solution prepared in Example 3. At a non-cytotoxic concentration, the extracts of *Trifolium repens* in Examples 1-3 showed certain inhibitory activity against viral transcription.

[0040] It should be understood that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Application of Trifoliate Orchid Extract in the Preparation of Anti-Crimea-Congo Hemorrhagic Fever Virus Drugs.

2. The application according to claim 1, characterized in that, The *Trifolium repens* extract is prepared by the following method: *Trifolium repens* powder is added to an extraction solvent for ultrasonic extraction. After ultrasonic extraction, the mixture is centrifuged and the supernatant is taken to obtain a solution containing *Trifolium repens* extract.

3. The application according to claim 2, characterized in that, The extraction solvent is selected from deionized water, ethanol, or dimethyl sulfoxide.

4. The application according to claim 2, characterized in that, The mass ratio of the *Trifolium repens* powder to the extraction solvent is 1:3-10.

5. The application according to claim 2, characterized in that, In the preferred embodiment, the centrifugation speed is 7500-8500 rpm and the centrifugation time is 1-5 min.

6. The application according to claim 2, characterized in that, The ultrasonic extraction frequency is 35-45 kHz, and the duration is 0.5-1.5 h.

7. The application according to claim 1, characterized in that, The aforementioned drug for treating Crimean-Congo hemorrhagic fever virus uses *Trifolium repens* extract as its sole active ingredient.

8. The application according to claim 1, characterized in that, The aforementioned anti-Crimea-Congo hemorrhagic fever virus drug is available in oral, injectable, or topical formulations.

9. The application according to claim 8, characterized in that, The oral preparations are selected from tablets, capsules, granules, oral liquids or powders, and the injectable preparations are selected from sterile powders or injection solutions for injection.

10. The application according to claim 1, characterized in that, The aforementioned anti-Crimea-Congo hemorrhagic fever virus drug comprises an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient is *Trifolium repens* extract, which accounts for 5%-50% of the drug by mass.

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