Application of venenum bufonis and bufalin in preparation of herpes simplex virus inhibitor

Abstract

The invention provides application of venenum bufonis and bufalin in preparation of a herpes simplex virus inhibitor, herpes simplex virus is HSV-1 or acyclovir drug-resistant virus strain HSV-1 / 153, a disease caused by HSV infection is herpes keratitis or herpes progenitalis disease infection, and the invention further discloses a pharmaceutical composition. Venenum bufonis and bufalin are used as active components or main active components, and a pharmaceutically acceptable carrier is supplemented.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to new uses of toad venom and bufalin, specifically to the use of toad venom and bufalin in preparing herpes simplex virus inhibitors, and more specifically to the use of toad venom and bufalin in preparing drugs for treating infectious diseases caused by herpes simplex virus type I. Background Art

[0002] Herpes simplex keratitis (HSK) is an infectious corneal disease caused by HSV-1. It is characterized by high morbidity, difficulty in treatment, and a high recurrence rate. In severe cases, it can lead to blindness and is currently one of the leading causes of blindness worldwide. Humans are the only natural reservoir for this virus, and HSV-1 infection is highly prevalent. According to statistics, 50%-80% of the world's population is a carrier of HSV-1. Each year, 1.5 million new cases of keratitis caused by HSV infection occur, including 40,000 patients with vision loss or blindness. Therefore, HSK is a major public health and social issue.

[0003] HSV-1 infection is widespread in humans. Most people are carriers. Once infected, the virus remains dormant in neurons for life. When the immune system is weakened, HSV-1 can attack and cause inflammation. HSV-1 infects the cornea, causing viral keratitis. HSV-1-induced corneal scarring (HSK) is the leading cause of corneal scarring and corneal opacity leading to blindness worldwide.

[0004] Currently, the preferred clinical treatment for HSK is acyclovir (ACV) and similar broad-spectrum antiviral drugs. While initially effective in mild cases, these drugs can only temporarily suppress HSV-1 replication in the affected area and fail to eliminate the virus. Furthermore, they are ineffective against viruses that remain dormant in the nerves, leading to a high risk of relapse. Furthermore, long-term use of acyclovir, a broad-spectrum antiviral, can lead to drug resistance, compromising the effectiveness of subsequent treatment. Severe HSK patients require corneal transplantation to restore vision, but the limited availability of corneal donors does not address the needs of all patients. Furthermore, there is a risk of relapse after corneal transplantation. Finally, there is currently no effective vaccine against HSV infection. Therefore, there is an urgent need to identify new drugs with different pharmacological actions to address these challenges.

[0005] The traditional Chinese medicine (TCM) toad venom is a dried secretion from the skin and parotid glands of the Bufo bufo gargarizans or Bufo gargarizans, members of the Toad family. The main component of toad venom is bufalin, which has pharmacological activities such as cardiotonic, pressor, anesthetic, analgesic, antibacterial, immune-enhancing, anti-tumor, and anti-SARS-CoV-2. Traditional Chinese medicine preparations of toad venom are now widely used in clinical anti-tumor treatment, with some therapeutic effects against lung cancer, leukemia, liver cancer, gastric cancer, and gynecological malignancies. However, a drawback of toad venom is that its limited focus on anti-tumor effects has limited its clinical applications. Summary of the Invention

[0006] The present invention aims to provide the use of venom of toad and bufalin in the preparation of herpes simplex virus inhibitors.

[0007] The specific purpose of the present invention is to provide the use of toad venom and bufalin in the preparation of drugs for treating infectious diseases caused by herpes simplex virus type I.

[0008] The present invention also aims to provide a pharmaceutical composition, which contains venom of toad or bufalin as active ingredients and is supplemented with a pharmaceutically acceptable carrier.

[0009] The specific technical solutions are:

[0010] Application of toad venom in the preparation of herpes simplex virus inhibitors.

[0011] Application of bufalin in the preparation of herpes simplex virus inhibitors.

[0012] Use of bufalin as the sole ingredient in the preparation of herpes simplex virus inhibitors.

[0013] The venom of toad is used in the preparation of a herpes simplex virus inhibitor, wherein the herpes simplex virus is HSV-1 or an acyclovir-resistant virus strain HSV-1 / 153.

[0014] The application of bufalin in the preparation of a herpes simplex virus inhibitor, wherein the herpes simplex virus is HSV-1 or an acyclovir-resistant virus strain HSV-1 / 153.

[0015] The invention relates to the use of bufalin as the sole ingredient in the preparation of a herpes simplex virus inhibitor, wherein the herpes simplex virus is HSV-1 or an acyclovir-resistant virus strain HSV-1 / 153.

[0016] The use of bufalin as the sole ingredient in the preparation of a herpes simplex virus inhibitor, wherein the disease caused by HSV infection is one of herpes keratitis and genital herpes.

[0017] The invention discloses an application of bufalin as the sole ingredient in the preparation of a herpes simplex virus inhibitor, which is a drug for treating herpes keratitis or genital herpes.

[0018] A pharmaceutical composition comprises the bufalin as an active ingredient and is supplemented with a pharmaceutically acceptable carrier.

[0019] The use of bufalin as the sole ingredient in the preparation of a herpes simplex virus inhibitor is prepared by the following steps:

[0020] (1) Take dried toad venom, wash it, soak it in 8-10 times the weight of injection water for 0.5-24 hours, grind it, add ethanol to make the ethanol content of the extract reach 50-90% by volume, and then let it stand at 10-30°C for 1-6 days;

[0021] (2) The extract solution obtained in step (1) was filtered, and the filtrate was concentrated until there was no alcohol taste, and water for injection was added and stirred evenly, and the mixture was refrigerated at 0-5°C for 30-60 hours, and the filtrate was separated by filtration and concentrated to obtain bufalin, molecular formula C 24 H 34 O4.

[0022] Currently, there are no reports domestically or internationally on the use of toad venom and bufalin in inhibiting HSV-1 or HSV-2, nor on their use in the preparation of products for the prevention and / or treatment of herpes simplex keratitis. Furthermore, there are no reports on their use in inhibiting diseases caused by infection with acyclovir-resistant herpes simplex virus strains HSV-1 / 153. The present invention, through the concept of "new uses for old drugs," evaluates the in vitro and in vivo efficacy of toad venom and bufalin in resisting HSV-1 infection, and in treating diseases caused by infection with acyclovir-resistant virus strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings of the embodiments are briefly introduced below.

[0024] The drawings described below only relate to some embodiments of the present invention, but are not intended to limit the present invention.

[0025] In the attached figure:

[0026] Figure 1 The figure shows the real-time quantitative polymerase chain reaction (qPCR) assay showing that different concentrations of toad venom extract inhibit the expression of the viral glycoprotein gd-1 gene of herpes simplex virus type 1; Chan Su (toad venom extract); Vehicle: control group; Viral gd-1: viral glycoprotein gd-1 gene of herpes simplex virus type 1; ARPE-19: human retinal epithelial cells;

[0027] Figure 2 This figure shows that different concentrations of toad venom extract inhibit the expression of herpes simplex virus type 1 viral glycoprotein gD-1; Chan Su (toad venom extract); Vehicle: control group; gD-1: herpes simplex virus type 1 viral glycoprotein; ARPE-19: human retinal epithelial cells; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; KDa: protein molecular weight;

[0028] Figure 3 The figure shows the ability of different concentrations of toad venom extract to inhibit the replication of herpes simplex virus type 1 progeny viruses; Chan Su (toad venom extract); Vehicle: control group; Viral titers: virus titer;

[0029] Figure 4 Shows that different concentrations of toad venom extract inhibit the expression of the viral glycoprotein gd-1 gene of the acyclovir-resistant virus strain HSV-1 / 153; Chan Su (toad venom extract); ARPE-19: human retinal epithelial cells; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; gD-1: viral glycoprotein of herpes simplex virus type 1; ACV: acyclovir; μM: working concentration of the drug; KDa: protein molecular weight;

[0030] Figure 5 It shows that different concentrations of toad venom extract inhibit the expression of viral glycoprotein gD-1 of acyclovir-resistant virus strain HSV-1 / 153;

[0031] Figure 6 It shows that different concentrations of toad venom extract reduce the viral plaques of acyclovir-resistant virus strain HSV-1 / 153;

[0032] Figure 7 The results show that different concentrations of toad venom extract can inhibit the proliferation of progeny viruses of acyclovir-resistant virus strain HSV-1 / 153.

[0033] Figure 8 The flowchart of the mouse experiment of intraperitoneal injection of toad venom extract to treat HSK is shown;

[0034] Figure 9 The results show that the treatment of HSK mice with toad venom extract can prevent and treat the weight loss of mice caused by viral infection; HSK: herpes simplex virus keratitis model group; Chansu treatment: toad venom extract treatment group; GCV treatment: ganciclovir treatment group;

[0035] Figure 10This study shows that treating HSK mice with venom of toad extract can prevent and treat eye disease progression caused by viral infection in mice. Disease Score: disease score, with higher values indicating higher disease severity. HSK: herpes simplex virus keratitis model group; Chansu treatment: venom of toad extract treatment group; GCV treatment: ganciclovir treatment group.

[0036] Figure 11 This study shows that treating HSK mice with venom of toad extract can prevent and treat corneal epithelial thickening and corneal inflammation caused by viral infection. Mock: control group (healthy mice); HSK: herpes simplex virus keratitis model group; Chansu treatment: venom of toad extract treatment group; GCV treatment: ganciclovir treatment group.

[0037] Figure 12 This figure shows that treating HSK mice with toad venom extract can prevent and treat corneal epithelial damage caused by viral infection. Mock: control group (healthy mice); HSK: herpes simplex virus keratitis model group; Chansu treatment: toad venom extract treatment group; GCV treatment: ganciclovir treatment group; Light: photos of mice taken under a stereomicroscope; Fluorescein sodium: fluorescein sodium staining to reveal corneal damage areas; and photos of mouse eyes taken under a slit lamp. The depth of staining and the size of the area represent the severity of corneal damage.

[0038] Figure 13 The data show that the treatment of HSK mice with Toad Venenum Extract can prevent and treat the corneal epithelial damage area caused by viral infection in mice; Relative epithelial defect area: relative area of corneal epithelial damage in mice;

[0039] Figure 14 It shows that treating HSK mice with Toad Venenum Bufonis extract can reduce the viral titers in corneal swabs of mice caused by viral infection; Viral titers in corneal swabs: Viral titers in corneal swabs of mice;

[0040] Figure 15 The results show that subconjunctival injection of toad venom extract in the treatment of HSK mice caused by HSV-1 / 153 infection can prevent and treat the weight loss of mice caused by viral infection; Vehicle: herpes simplex virus keratitis model mouse group; Chansu t: toad venom extract-treated HSK mouse group; ACV: acyclovir-treated HSK mouse group;

[0041] Figure 16The results show that subconjunctival injection of toad venom extract in the treatment of HSK caused by HSV-1 / 153 infection in mice can prevent and treat the progression of eye diseases caused by viral infection in mice; Vehicle: herpes simplex virus keratitis model mouse group; Chansu: toad venom extract-treated HSK mouse group; ACV: acyclovir-treated HSK mouse group;

[0042] Figure 17 The results show that subconjunctival injection of toad venom extract in the treatment of HSK mice caused by HSV-1 / 153 infection can prevent and treat corneal epithelial damage caused by viral infection in mice; Vehicle: herpes simplex virus keratitis model group; Chansu: toad venom extract-treated HSK mouse group; ACV: acyclovir-treated HSK mouse group;

[0043] Figure 18 This figure shows that subconjunctival injection of toad venom extract in treating HSV-1 / 153-infected mice with HSK can prevent and treat viral infection-induced corneal epithelial damage in mice. Relative epithelial defect area: Relative area of corneal epithelial defect area in mice; Vehicle: Herpes simplex virus keratitis model group; Chansu: Toad venom extract-treated HSK mouse group; ACV: Acyclovir-treated HSK mouse group; Light: Photos of mice taken under a stereomicroscope; Fluorescein sodium: Fluorescein sodium staining to reveal corneal damage areas; and photos of mouse eyes taken under a slit lamp. The depth of staining and the size of the area represent the severity of corneal damage.

[0044] Figure 19 The results show that subconjunctival injection of toad venom extract in treating HSV-1 / 153-infected mice can significantly reduce the viral titer in eye swabs; Vehicle: herpes simplex virus keratitis model group; Chansu: toad venom extract-treated HSK mouse group; ACV: acyclovir-treated HSK mouse group;

[0045] Figure 20 It shows that subconjunctival injection of bufalin to treat HSK mice can prevent and treat weight loss caused by viral infection in mice; HSK: herpes simplex virus keratitis model group; bufalin treatment: bufalin-treated HSK mouse group; ACV treatment: acyclovir-treated HSK mouse group;

[0046] Figure 21The results show that subconjunctival injection of bufalin to treat HSK mice can prevent and treat the progression of eye diseases caused by viral infection in mice; HSK: herpes simplex virus keratitis model group; bufalin treatment: bufalin-treated HSK mouse group; ACV treatment: acyclovir-treated HSK mouse group;

[0047] Figure 22 The results showed that the drug could prevent and treat viral infection-induced corneal epithelial thickening and corneal inflammation in mice. Mock: control group (healthy mice); HSK: herpes simplex virus keratitis model group; bufalin treatment: bufalin-treated HSK mice; ACV treatment: acyclovir-treated HSK mice.

[0048] Figure 23 It shows that subconjunctival injection of bufalin to treat HSK mice can significantly reduce the virus titer in mouse eye swabs; HSK: herpes simplex virus keratitis model group; bufalin treatment: bufalin-treated HSK mouse group; ACVtreatment: acyclovir-treated HSK mouse group. DETAILED DESCRIPTION

[0049] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] Example 1: Preparation of Toad Venenum Extract

[0051] Take 2g of fine toad venom powder (purchased from Jiangsu Pujin Pharmaceutical Co., Ltd.), add 10-fold the amount of water, and reflux to extract for 2 hours. Filter the filtrate, add ethanol to a concentration of 75%, let it stand overnight, filter, recover the ethanol, add water to 1000mL, and filter. The toad venom extracts used in the following examples were all prepared using this method.

[0052] Example 2: Preparation of Bufalin

[0053] (1) Take 1000 g of dried toad venom (purchased from Jiangsu Pujin Pharmaceutical Co., Ltd.), wash it, soak it in 9 times the mass of injection water for 12 h, grind it, add ethanol to make the ethanol content of the extract reach 75% by volume, and then let it stand at 20°C for 5 days;

[0054] (2) The extract solution obtained in step (1) was filtered, and the filtrate was concentrated until there was no alcohol taste, and water for injection was added and stirred evenly, and the mixture was refrigerated at 0-5°C for 45 hours, and the filtrate was separated by filtration and concentrated to obtain bufalin, molecular formula C 24 H 34 O4.

[0055] Example 3: Evaluation of the inhibitory activity of Venenum Toadum extract on HSV-1 at the cellular level

[0056] ARPE-19 cells infected with HSV-1 F strain (MOI=1) were treated with venom of toad extract (2μg / mL) at doses ranging from 0μL, 10μL, 20μL, to 30μL for 24 hours. The expression of gD-1 mRNA and gD-1 protein was detected by qPCR and Western blot, respectively. The TCID50 assay was used to determine the effect of venom of toad extract post-treatment on HSV-1-infected ARPE progeny viruses.

[0057] The specific steps are:

[0058] Exponentially growing ARPE-19 cells were seeded in 96-well cell culture plates so that each well contained 1×10 4 cells and cultured them in 100 μL of 10% FBS DMEM medium. The culture conditions were constant temperature, constant humidity and 5% CO2 at 37°C. After 24 hours, the HSV-1 virus particles stored at -80°C were thawed on ice and infected ARPE-19 cells at a titer of MOI=1. After 2 hours, ARPE-19 was treated with toad venom extract (2 μg / μL) to a concentration of 20 μg, 40 μg and 60 μg in the cell culture medium. The culture was continued for 24 hours, and the cells were lysed with TRizol to harvest RNA. The expression of HSV-1 gD-1 mRNA and gD-1 protein was detected by qPCR and WB, respectively. The results showed that treatment with toad venom extract could significantly inhibit the replication of HSV-1 ( Figure 1 and Figure 2 The TCID50 method was used to measure the proliferation of the supernatant progeny virus. TCID50 was tested on Vero cells and it was found that the toad venom extract at 10μL, 20μL and 30μL could significantly reduce the proliferation of the progeny virus ( Figure 3 ).

[0059] Example 4: Toad Venenum Extract Inhibits Replication of Clinical Acyclovir-Resistant Virus Strain HSV-1 / 153 at the Cellular Level

[0060] ARPE-19 cells infected with the drug-resistant HSV-1 / 153 strain (MOI=1) were treated with venom of toad extract (2 μg / mL) at concentrations ranging from 0 μL, 10 μL, 20 μL to 30 μL for 24 hours. The expression of gD-1 mRNA and gD-1 protein was detected by qPCR and Western blot, respectively. ARPE-19 cells infected with HSV-1 / 153 were treated with different concentrations of venom of toad extract or ACV (1.0 μM), and then the expression of gD-1 protein was detected by Western blot and gD-1 mRNA was detected by qPCR. Finally, the TCID50 assay was used to determine the effect of venom of toad extract post-treatment on the progeny of HSV-1-infected ARPE-19 viruses.

[0061] The specific steps are:

[0062] Different concentrations of toad venom extract (2 μg / μL) or ACV (1.0 μM) were added to HSV-1 / 153-infected ARPE-19 cells for intervention. Western blotting, qPCR, crystal violet plaque analysis, and TCID50 assays were used to investigate the effects of toad venom extract and ACV on HSV-1 / 153 gD-1 protein and gD-1 mRNA expression. The results showed that toad venom extract treatment significantly inhibited HSV-1 / 153 replication. Figure 4 and Figure 5 The proliferation of the supernatant progeny virus was measured on Vero cells by crystal violet plaque and TCID50 methods. It was found that the toad venom extract at 10 μL, 20 μL and 30 μL significantly reduced the proliferation of the progeny virus ( Figure 6 and Figure 7 ), but ACV had no inhibitory effect on HSV-1 / 153 progeny. Conclusion: HSV-1 / 153 is an ACV-resistant strain, so 1.0 μM ACV had little effect on HSV-1 / 153 gD-1 protein and mRNA expression. However, 10 μL of toad venom extract significantly inhibited HSV-1 / 153 gD-1 protein and mRNA expression, more effectively than ACV.

[0063] Example 5: Evaluation of the inhibitory activity of Venenum Toadum extract on HSV-1 in mice

[0064] Anesthetized C57BL / 6J mice (6 weeks old, male) were placed on a stencil, and the corneal epithelium was scratched with a needle. The corneas were immediately infected with 1×10⁶ PFU of HSV-1 F strain. Following HSV-1 infection, mice were treated intraperitoneally with PBS, toad venom extract (100 μL, 200 μg, n=6), or GCV (100 μL, 200 μg, n=6) every other day for three consecutive treatments. Body weights and disease progression scores were recorded from day 0 to day 7. On day 7, corneal fluorescein staining with cobalt blue was performed to determine corneal surface damage, and hematoxylin and eosin (H&E) staining was used to assess corneal integrity, thickness, and inflammation. Tear fluid was obtained from the mice on day 7, and HSV-1 progeny titers were determined.

[0065] The specific steps are:

[0066] Six-week-old male C57BL / 6J mice were selected and anesthetized with sodium pentobarbital by intraperitoneal injection. The right eye was fully exposed. Under a stereomicroscope, a "well" scratch was made on the cornea using a 1 mL syringe needle. The mice were immediately infected with HSV-1 F (1×106 PFU), and HSV-1 infection was assisted by gently lowering and lowering the eyelids. Four hours later, HSK mice were treated with 200 μg / mouse of toad venom extract by intraperitoneal injection. GCV (200 μg / mouse) served as a control group ( Figure 8 The body weight information of mice was collected and recorded from day 0 to day 7 ( Figure 9 From day 0 to day 7, the clinical symptoms of mice were collected and recorded for disease scoring ( Figure 8 H&E staining was used to observe the integrity and thickness of the cornea of each group of mice on the 7th day ( Figure 9 ), corneal fluorescein staining was performed using cobalt blue light on the 3rd and 5th days to determine the damage to the corneal surface ( Figure 12 and Figure 13 TCID50 was used to measure the proliferation of progeny viruses in mouse eye swabs on the 3rd, 5th and 7th days. The results showed that treatment with toad venom extract could significantly inhibit HSV-1 infection in the mouse cornea ( Figure 14 ).

[0067] Example 6: Evaluation of the activity of toad venom extract in inhibiting ACV-resistant virus strain HSV-1 / 153 in mice

[0068] Anesthetized C57BL / 6J mice (6 weeks old, male) were placed on a stencil, and the corneal epithelium was scratched with a needle. The corneas were immediately infected with 1×10⁶ PFU of HSV-1 / 153. Following HSV-1 infection, mice were treated with subconjunctival injections of PBS, toad venom extract (10 μL, 20 μg, n=6), or ACV (10 μL, 20 μg, n=6) every other day for three consecutive treatments. Body weights and disease progression scores were recorded from day 0 to day 7. On day 7, corneal fluorescein staining with cobalt blue was performed to determine corneal surface damage, and hematoxylin and eosin (H&E) staining was used to assess corneal integrity, thickness, and inflammation. Tear fluid was obtained from the mice on day 7, and HSV-1 / 153 progeny viral titers were measured.

[0069] The specific steps are:

[0070] Six-week-old male C57BL / 6J mice were selected and anesthetized by intraperitoneal injection of sodium pentobarbital, and the right eye was fully exposed. A "well" scratch was made on the cornea with a 1 mL syringe needle under a stereomicroscope. Then, HSV-1 / 153 (1×106 PFU) was immediately infected, and HSV-1 infection was assisted by gently lowering and lowering the eyelids. After 4 hours, the HSK mice were treated with toad venom extract (20 μg / mouse) by subconjunctival injection, and ACV (20 μg / mouse) was used as the control group. The treatment was given once every other day for 3 consecutive times. The weight information of the mice was collected and recorded from day 0 to day 7 ( Figure 15 From day 0 to day 7, the clinical symptoms of mice were collected and recorded for disease scoring ( Figure 16 On the 7th day, corneal fluorescein staining was performed using cobalt blue light to determine the damage to the corneal surface ( Figure 17 and Figure 18 The proliferation of progeny viruses in mouse eye swabs was determined by TCID50. The results showed that treatment with toad venom extract could significantly inhibit HSV-1 / 153 infection in the mouse cornea ( Figure 19 ).

[0071] Example 7: Evaluation of the activity of bufalin in inhibiting HSV-1 in mice

[0072] To prepare the bufalin preparation, prepare the bufalin prepared by the method in Example 2 and add 10% DMSO, 40% PEG-300, 5% Tween-80, and 45% PBS. Anesthetized C57BL / 6J male mice (6 weeks old) were placed on a stencil, and the corneal epithelium was punctured with a needle. The corneas of the mice were immediately infected with 1×10⁶ PFU of HSV-1 F strain. Following HSV-1 infection, mice were treated with subconjunctival injections of PBS, bufalin (0.1 mg / kg, 10 μL), or ACV (0.1 mg / kg, 10 μL) every other day for three consecutive treatments. From day 0 to day 7, mouse body weights and disease progression scores were recorded. Corneal integrity, thickness, and inflammation were assessed using hematoxylin and eosin (H&E) staining. Eye swabs were obtained on day 7 and HSV-1 progeny titers were determined.

[0073] The specific steps are:

[0074] Six-week-old male C57BL / 6J mice were selected and anesthetized by intraperitoneal injection of sodium pentobarbital, and the right eye was fully exposed. A "well" scratch was made on the cornea with a 1 mL syringe needle under a stereomicroscope. Then, HSV-1 F (1×106 PFU) was immediately infected, and HSV-1 infection was assisted by gently lowering and lowering the eyelids. After 4 hours, 1.8 μg / mouse of tofavin (dissolved in 10% DMSO+40% PEG300+5% Tween80+45% PBS) was used to treat HSK mice by subconjunctival injection, and ACV (1.8 μg / mouse) was used as the control group. Once every other day for 3 consecutive times. The weight information of the mice was collected and recorded from day 0 to day 7 ( Figure 20 From day 0 to day 7, the clinical symptoms of mice were collected and recorded for disease scoring ( Figure 21 On the 7th day, H&E staining was used to observe the integrity and thickness of the cornea of each group of mice ( Figure 22 TCID50 was used to measure the proliferation of progeny viruses in mouse eye swabs. The results showed that bufalin treatment could significantly inhibit HSV-1 infection in the mouse cornea ( Figure 23 ).

[0075] In this study, the toad venom extract demonstrated superior antiviral activity and safety against the ACV-resistant HSV-1 / 153 strain at extremely low dosages compared to the traditional antiviral drug acyclovir (ACV). In ARPE-19 cells, the toad venom extract significantly inhibited HSV-1 / 153 strain replication. Therefore, the toad venom extract exhibits excellent antiviral activity against HSV-1 and is highly safe at therapeutic doses, making it suitable for use as an antiviral agent. Treatment with bufalin significantly inhibited HSV-1 infection in the mouse cornea.

[0076] Example 8: Take 10 g of bufalin and add physiological saline to prepare bufalin injection.

[0077] Example 9: 10 g of bufalin was added with physiological saline to prepare bufalin freeze-dried powder.

[0078] The embodiments of the present invention are presented for purposes of illustration and description and are not intended to be exhaustive or to limit the invention to the disclosed forms. Many modifications and variations will be apparent to those skilled in the art. The embodiments are chosen and described in order to better illustrate the principles of the invention and its practical application and to enable those skilled in the art to understand the invention and design various embodiments with various modifications as suited for specific applications.

Claims

1. Application of Toad Venenum in the preparation of herpes simplex virus inhibitors.

2. Application of bufalin in the preparation of herpes simplex virus inhibitors.

3. Use of bufalin as the sole ingredient in the preparation of herpes simplex virus inhibitors.

4. The use of the venom of toad in preparing a herpes simplex virus inhibitor according to claim 1, wherein: The herpes simplex virus was HSV-1 or acyclovir-resistant strain HSV-1 / 153.

5. The use of bufalin in the preparation of herpes simplex virus inhibitors according to claim 2, characterized in that: The herpes simplex virus was HSV-1 or acyclovir-resistant strain HSV-1 / 153.

6. Use of bufalin as the sole ingredient in the preparation of herpes simplex virus inhibitors as claimed in claim 3, characterized in that: The herpes simplex virus was HSV-1 or acyclovir-resistant strain HSV-1 / 153.

7. Use of bufalin as the sole ingredient in the preparation of a herpes simplex virus inhibitor as claimed in claim 6, characterized in that: HSV infection causes herpes keratitis or genital herpes.

8. The use of bufalin as the sole ingredient in the preparation of a herpes simplex virus inhibitor according to claim 7, characterized in that: Herpes simplex virus inhibitors are a type of drug used to treat infections such as herpes keratitis or genital herpes.

9. A pharmaceutical composition, characterized in that The bufalin as claimed in claim 3 is used as the active ingredient and is supplemented with a pharmaceutically acceptable carrier.

10. The use of bufalin as the sole ingredient in the preparation of herpes simplex virus inhibitors according to claim 3, characterized in that: Bufalin was prepared by the following steps: (1) Take dried toad venom, wash it, soak it in 8-10 times the weight of injection water for 0.5-24 hours, grind it, add ethanol to make the ethanol content of the extract reach 50-90% by volume, and then let it stand at 10-30°C for 1-6 days; (2) The extract solution obtained in step (1) was filtered, and the filtrate was concentrated until there was no alcohol taste, and water for injection was added and stirred evenly, and the mixture was refrigerated at 0-5°C for 30-60 hours, and the filtrate was separated by filtration and concentrated to obtain bufalin, molecular formula C 24 H 34 O4.

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