Application of cucurbitacine compound in preparation of medicine for treating herpes virus infection

Abstract

The invention relates to the technical field of medicines, and discloses application of cucurbitacine compounds in preparation of a medicine for treating herpes virus infection. Particularly, the cucurbitacine I, the cucurbitacine IIA and the cucurbitacine B effectively inhibit HSV-1 infection in Vero cells while maintaining corresponding cell viability, and the cucurbitacine I, the cucurbitacine IIA and the cucurbitacine B have a wide effective dose window and better patent medicine potential. According to the invention, the effect of the cucurbitacine compound in prevention and treatment of herpes virus infection is found for the first time, and the cucurbitacine compound is expected to become a low-toxicity and high-efficiency medicine and is applied to the medicine for treating herpes virus infection.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to use of a cucurbitacin compound in preparing a drug for treating herpes virus infection. Background Art

[0002] Herpes viruses are a family of enveloped, double-stranded DNA viruses. Eight of these species have been found to infect humans and are referred to as human herpesviruses (HHVs). These include varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), Epstein-Barr virus (EBV), human cytomegalovirus (CMV), human herpesvirus type 6 (HHV-6), human herpesvirus type 7 (HHV-7), and Kaposi's sarcoma herpesvirus (KSHV, also known as human herpesvirus type 8 (HHV-8), which can infect immunocompromised individuals. Herpes viruses persist in a latent state within the host's peripheral nervous system. Like deeply buried landmines, sudden changes in temperature and humidity, ultraviolet exposure, chemotherapy and other immunosuppressive interventions, hormonal fluctuations, altered circadian rhythms, and mood disorders can disrupt the dynamic equilibrium between the virus and the host's immune system, triggering outbreaks of the herpes virus. Herpes simplex virus type 1 (HSV-1) is one of the representative strains of herpes virus.

[0003] Cucurbitacins are tetracyclic triterpenoid secondary metabolites found in Cucurbitaceae plants, including Cassia truncatula and Radix Glehniae, as well as other species of commonly used medicinal plants in the folk cultivar Cucurbitaceae. They have a variety of potential medical uses, including anti-tumor, anti-inflammatory, and analgesic effects. Radix Glehniae and Radix Glehniae are commonly used in folk medicine to treat herpes zoster, carbuncles, boils, and other ailments.

[0004] However, there are currently no studies on the potential of cucurbitacin compounds to treat herpes simplex virus infections. Natural products offer numerous advantages, including minimal side effects, low cost, multiple targets, and multiple pathways, and hold great promise for clinical application. The search for cheaper, less toxic, and more effective natural medicines for the treatment of herpes simplex virus infections remains of great clinical value. Summary of the Invention

[0005] To address the above-mentioned technical problems in treating herpes virus infection, the present invention provides the use of cucurbitacin compounds in the preparation of a drug for treating herpes virus infection. Studies have found that cucurbitacin compounds, particularly cucurbitacin I, cucurbitacin B, and cucurbitacin IIA, can effectively inhibit herpes virus infection at the cellular level.

[0006] In one aspect, the present invention provides a use of a cucurbitacin compound in the preparation of a medicament for treating herpes virus infection.

[0007] Specifically, the cucurbitacin compound is selected from cucurbitacin I, cucurbitacin IIA, cucurbitacin B or pharmaceutically acceptable salts thereof.

[0008] Specifically, the herpes virus includes varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), Epstein-Barr virus (EBV), human cytomegalovirus (CMV), human herpes virus type 6 (HHV-6), human herpes virus type 7 (HHV-7) or Kaposi's sarcoma herpesvirus (KSHV, also known as human herpesvirus type 8 HHV-8) that can infect immunocompromised patients. Preferably, the herpes virus is herpes simplex virus type 1.

[0009] Specifically, the drug contains pharmaceutically acceptable excipients or ingredients.

[0010] Specifically, the drug is an oral preparation, an injection, an external preparation or a sustained-release preparation.

[0011] Specifically, the dosage forms of the drug include granules, ordinary tablets, dispersible tablets, effervescent tablets, orally disintegrating tablets, lozenges, chewable tablets, capsules, soft capsules, microcapsules, pills, powders, dropping pills, sustained-release preparations, controlled-release preparations, oral liquid preparations and injections.

[0012] On the other hand, the present invention also provides a pharmaceutical composition for treating herpes virus infection, wherein the active ingredient of the pharmaceutical composition is the above-mentioned cucurbitacin compound.

[0013] Specifically, the cucurbitacin compound is selected from cucurbitacin I, cucurbitacin IIA, cucurbitacin B or pharmaceutically acceptable salts thereof.

[0014] Specifically, the chemical structural formula of cucurbitacin I is: ; .

[0015] Specifically, the chemical structural formula of cucurbitacin IIA is: ; .

[0016] Specifically, the chemical structural formula of cucurbitacin B is: ; .

[0017] Specifically, the herpes virus includes varicella-zoster virus, herpes simplex virus type 1, herpes simplex virus type 2, Epstein-Barr virus, human cytomegalovirus, human herpes virus type 6, human herpes virus type 7 or Kaposi's sarcoma herpes virus that can infect immunodeficient patients. Preferably, the herpes virus is herpes simplex virus type 1.

[0018] Specifically, the composition includes one or more pharmaceutically acceptable excipients.

[0019] Specifically, the composition is prepared into an oral preparation, an injection, an external preparation or a sustained-release preparation.

[0020] Compared with the prior art, the present invention has the following technical effects: (1) The present invention studies more than 90 monomer compounds in the aqueous extract of fresh herb of Radix Glehniae and finds that cucurbitacin compounds, especially cucurbitacin I, cucurbitacin B and cucurbitacin IIA, can effectively inhibit herpes virus infection at the cellular level.

[0021] (2) The present invention can provide more low-toxic and highly effective drugs for the treatment of herpes virus infection. Through research, the present invention found that cucurbitacin compounds, especially cucurbitacin I, cucurbitacin B and cucurbitacin IIA, can effectively inhibit HSV-1 infection in Vero cells while maintaining the viability of the corresponding cells. The effective dose window is wide and has better drug potential. Cucurbitacin compounds are a type of tetracyclic triterpenoid compound naturally present in plants of the Cucurbitaceae family. They are widely present in Cucurbitaceae and other species of folk medicinal plants such as snow dandelion and scutellaria baicalensis. At present, cucurbitacin I, cucurbitacin B and cucurbitacin IIA are mainly used in the development of drugs for anti-cancer and anti-inflammatory purposes. The present invention discovered for the first time the role of cucurbitacin compounds in the prevention and treatment of herpes virus infection. Cucurbitacin compounds are expected to become a low-toxic and highly effective drug. The present invention provides a new solution for the treatment of herpes virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This study analyzed the anti-herpes simplex virus type 1 activity of 90 monomeric compounds in the aqueous extract of Radix Codonopsis pilosulae.

[0023] Figure 2 It is the cytotoxicity of the nine monomer components contained in Radix Glehniae and their inhibitory activity against HSV-1 infection. DETAILED DESCRIPTION

[0024] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of any conflict with any incorporated document, the content of this specification shall prevail.

[0027] Example 1: Cucurbitacin-containing anti-herpes virus ingredients in Radix Glehniae In this example, the aqueous extract of fresh herb of Radix Corydalis was analyzed by liquid chromatography-mass spectrometry to identify 90 obtainable monomeric compounds, and the inhibitory effects of the above components on HSV-1 infection in Vero cells were analyzed using high-content immunofluorescence assay.

[0028] The whole herb of Radix Angelicae Sinensis was collected from Meilin Town, Jieyang City, Guangdong Province, cut into small pieces less than 5 cm, crushed with a grinder, soaked in 10 times deionized water, decocted and extracted, and allowed to stand at 4°C for 24 hours and centrifuged at 3000 rpm for 15 minutes. The supernatant was spray-dried to prepare the Radix Angelicae Sinensis water extract.

[0029] The chemical components of the aqueous extract of Radix Glehniae were analyzed using a high-resolution liquid chromatography-mass spectrometry (Quadrupole-TOF LC-MS / MS system, resulting in the identification of 90 accessible monomeric compounds. Their anti-HSV activity was then evaluated using a Vero cell-based HSV-1 infection model.

[0030] Well-grown Vero cells were plated in 96-well plates and allowed to adhere for 24 hours until 90%-95% confluency was achieved. HSV-1 virus was diluted to a 100-fold half-cell infectious dose (TCID50) using serum-free DMEM (Gibco). After aspirating the original culture medium from each well, 50 μL of the HSV-1 virus working solution was added to infect the Vero cells. A control group was treated with 50 μL of serum-free DMEM and incubated at 37°C in a CO2 incubator for 2 hours. During this time, 200 μM dilutions of the 90 monomeric compounds were prepared in DMEM supplemented with 10% fetal bovine serum (Gibco) and sterilized using a 0.22 μm sterile microporous filter. Two hours after infection, 50 μL of the monomeric compound dilutions were added to the infected cell solution in the corresponding well and gently tapped to mix thoroughly, resulting in a final compound concentration of 100 μM in each well.

[0031] After incubating the cell culture plates at 37°C in a 5% CO2 incubator for 48 hours, immunofluorescence labeling was performed using HSV-1 and HSV-2 gD antibodies (abcam, AB6507) and goat anti-mouse fluorescent secondary antibodies (abcam, AB150113). DAPI was used to stain cell nuclei as an internal control. HSV-1 infection rates were analyzed using the CQ1 High-Content Cytometry System. HSV-1 infection rate = (total fluorescence intensity of HSV-1 gD protein in the test wells / total fluorescence intensity of DAPI in the cell nucleus) / (average total fluorescence intensity of HSV-1 gD protein in the infection model group for each well / average total fluorescence intensity of DAPI in the cell nucleus in the infection model group for each well) × 100%.

[0032] The anti-herpes simplex virus type 1 activity of 90 monomeric compounds in the aqueous extract of Radix Glehniae was analyzed. Figure 1 shown.

[0033] Immunofluorescence analysis of HSV-1 infection rates revealed that at a concentration of 100 μM, the nine compounds were able to reduce the HSV-1 infection rate in Vero cells to below 10%, while the infection rates of the other 83 compounds in the treated wells were all greater than 10%. Therefore, the nine compounds numbered 10, 43, 46, 53, 56, 71, 78, 82, and 88 exhibited HSV-1 inhibitory activity. Among them, compound 10 is cucurbitacin I, compound 43 is cucurbitacin IIA, compound 46 is cucurbitacin B, compound 53 is cucurbitacin B, compound 56 is fingolimod hydrochloride, compound 71 is emodin, compound 78 is ursolic acid, compound 82 is biochanin A, and compound 88 is aescin.

[0034] Example 2: Cytotoxicity of 9 monomeric compounds and their inhibitory effects on herpes simplex virus type 1 infection The anti-HSV-1 activity of nine monomeric compounds from the aqueous extract of Radix Corydalis was analyzed using an HSV-1-infected Vero cell model. Two hours after infection, 200 μM of the monomeric compounds, equal in volume to the viral suspension, were added and gently tapped to mix, resulting in a final concentration of 100 μM. After incubation at 37°C in a CO2 incubator for 48 hours, the effects of the monomeric compounds on cell viability were assessed using the CCK-8 assay. Immunofluorescence labeling of HSV-1 gD protein was used to analyze the inhibitory effect of the monomeric compounds on HSV-1 infection in Vero cells.

[0035] The calculation formula is as follows: cell viability (%) = (absorbance at 450 nm of compound-intervention wells - average absorbance at 450 nm of blank culture medium) / (average absorbance at 450 nm of normal control group - average absorbance at 450 nm of blank culture medium) × 100%; HSV-1 infection inhibition rate (%) = [1 – (total fluorescence intensity of HSV-1 gD protein in compound-intervention wells / cell viability in compound-intervention wells) / (average total fluorescence intensity of HSV-1 gD protein in HSV-1 infection model wells / cell viability in HSV-1 infection model wells)] × 100%.

[0036] The chemical structures of the nine monomeric compounds and their respective inhibitory effects on HSV-1 infection in Vero cells are shown in Figure 2. Figure 2 shown.

[0037] The results showed that only six monomeric compounds, namely cucurbitacin I, cucurbitacin II A, cucurbitacin B, lulutonic acid, emodin and biochanin A, could inhibit HSV-1 infection while maintaining cell viability above 50% (relative to the normal control group). 50 EC50 is the drug concentration that causes a 50% decrease in cell viability. 50 EC: is the half effective concentration, which indicates the drug concentration that reduces the viral infection rate by 50%. 50 and CC 50 It is the key data for in vitro antiviral activity screening, proving that the drug has antiviral activity at an achievable concentration and that the drug does not cause toxicity to cells at this concentration; it excludes the possibility that the measured in vitro antiviral activity is caused by host cell death. 50 With EC 50 The higher the ratio, the stronger the antiviral effect of the drug.

[0038] EC of cucurbitacin I 50 <0.78 μM, CC 50 Greater than 100 μM; EC50 of cucurbitacin IIA = 27.32 μM, CC 50 Greater than 100 μM, EC of passelate acid50 =52.24μM, CC 50 Greater than 100 μM; EC of cucurbitacin B 50 <0.78 μM, CC 50 Greater than 100 μM; EC of emodin 50 =16.37 μM, CC 50 Greater than 100 μM; EC of biochanin A 50 =48.18μM, CC 50 Greater than 100μM.

[0039] According to the above results, it can be seen that cucurbitacin I, cucurbitacin IIA and cucurbitacin B can effectively inhibit herpes virus HSV-1 infection at the cellular level, especially cucurbitacin I and cucurbitacin B, which can inhibit HSV-1 infection in Vero cells at concentrations below 10 μM, and have a wide effective dose window, and have better drug potential.

[0040] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of a cucurbitacin compound in the preparation of a medicament for treating herpes virus infection.

2. The use according to claim 1, characterized in that The cucurbitacin compound is selected from cucurbitacin I, cucurbitacin IIA, cucurbitacin B or pharmaceutically acceptable salts thereof.

3. The use according to claim 2, characterized in that The herpes virus includes varicella-zoster virus, herpes simplex virus type 1, herpes simplex virus type 2, Epstein-Barr virus, human cytomegalovirus, human herpes virus type 6, human herpes virus type 7 or Kaposi's sarcoma herpes virus that can infect immunodeficient patients. Preferably, the herpes virus is herpes simplex virus type 1.

4. The use according to any one of claims 1 to 3, characterized in that The drug contains pharmaceutically acceptable excipients or ingredients.

5. The use according to claim 4, characterized in that The medicine is an oral preparation, an injection, an external preparation or a sustained-release preparation.

6. A pharmaceutical composition for treating herpes virus infection, characterized in that The active ingredient of the pharmaceutical composition is a cucurbitacin compound.

7. The pharmaceutical composition according to claim 6, characterized in that The cucurbitacin compound is selected from cucurbitacin I, cucurbitacin IIA, cucurbitacin B or pharmaceutically acceptable salts thereof.

8. The pharmaceutical composition according to claim 7, characterized in that The herpes virus includes varicella-zoster virus, herpes simplex virus type 1, herpes simplex virus type 2, Epstein-Barr virus, human cytomegalovirus, human herpes virus type 6, human herpes virus type 7 or Kaposi's sarcoma herpes virus that can infect immunodeficient patients. Preferably, the herpes virus is herpes simplex virus type 1.

9. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition includes one or more pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition is prepared into an oral preparation, an injection, an external preparation or a sustained-release preparation.

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