A compound traditional Chinese medicine composition for treating skin warts and condyloma and application thereof

Abstract

The application belongs to the technical field of traditional Chinese medicine, and particularly relates to a compound traditional Chinese medicine composition for treating skin warts and condyloma and application thereof. Raw materials of the compound traditional Chinese medicine composition comprise the following components: astilbin, sophoramine, houttuynia cordata, honeycomb, podophyllotoxin, cumin and brucea javanica. A preparation method comprises the following steps: first, extracting brucea javanica oil by pressing brucea javanica to obtain residues; then, adding houttuynia cordata, honeycomb, cumin and the residues into water to obtain decoction by boiling, adding tannase into the decoction to obtain enzyme hydrolysate by enzymolysis, inactivating, and finally, uniformly mixing and dispersing the enzyme hydrolysate, brucea javanica oil, astilbin, sophoramine and podophyllotoxin to obtain the compound traditional Chinese medicine composition. The application has the synergistic effect which is significantly better than that of the original traditional Chinese medicine prescription, and has a good application market prospect for treating skin warts and condyloma.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a compound traditional Chinese medicine composition for treating skin warts and condyloma acuminata and its application. Background Technology

[0002] Warts are benign skin lesions caused by human papillomavirus (HPV) infection of the skin and mucous membranes. Based on clinical manifestations and location, warts are classified into common warts, flat warts, plantar warts, condyloma acuminata, and epidermal dysplasia verruciformis, among others. The source of infection for warts is infected individuals and virus carriers, primarily caused by HPV types 1, 2, and 4. Wart lesions are painful to the touch, but may also be asymptomatic. Under a skin microscope, pale yellow papules, thickened keratin rings, and round papillary hyperkeratosis with small black dots formed by ruptured capillaries are visible. The course of the disease is closely related to the body's own immunity; those with weakened immune systems have a higher incidence. Some patients may experience spontaneous remission, but patients often seek treatment due to cosmetic concerns and pain. Current treatments for warts primarily focus on destroying the wart and enhancing local or systemic immunity, including medication, physical therapy, and surgical excision. However, these treatments suffer from long treatment courses, painful procedures, high infection rates, and high recurrence rates, leading to poor patient compliance.

[0003] Commonly used topical medications include imiquimod, a small-molecule immunomodulator with immunomodulatory and indirect antiviral effects. Its antiviral action works by stimulating the body's own immunity and inducing T-cell kinase activity to enhance cellular immunity, thereby generating an immune response against HPV-infected cells. Currently, it is mainly used clinically for the treatment of condyloma acuminata of the anus and external genitalia. The main drawbacks of this treatment method are the long treatment period, difficulty for patients to adhere to, and high treatment costs, increasing the financial burden on patients. However, it is less painful and invasive, easy to administer, and can be used at home and in children. Caution should be exercised when using this treatment in patients with skin allergies.

[0004] In addition, there is salicylic acid, a keratolytic agent that works by slowly destroying virus-infected cells without affecting the production of keratinocytes. The mild stimulation it produces can trigger an immune response. It slowly destroys the epidermis infected with HPV, causing local shedding or thinning of the stratum corneum. Its advantages include minimal side effects and no scarring after lesion healing, simple operation, and low treatment cost. Its disadvantages are slow onset of action and slight skin irritation; therefore, it should be discontinued if redness or swelling occurs during use, and it should not be used on the face or scalp to avoid skin pigmentation affecting appearance.

[0005] Other treatments include interferon and cantharidin. Recombinant human interferon has functions such as inhibiting tumor cell proliferation, antiviral activity, and regulating the human immune system. Interferon combined with other treatments is safe and effective for treating single, refractory common warts and can reduce the recurrence rate. Cantharidin, extracted from the traditional Chinese medicine cantharides, can cause hyperemia and blistering of the wart epidermis. Mild pain may occur when cantharidin treatment causes blisters to form on common warts; healing is scarless. If erosion occurs during treatment, treatment should be paused for a few days before resuming.

[0006] In addition, there are laser treatments and surgical treatments. Currently, there are many treatments for skin warts, but these methods often have drawbacks such as prolonged disease course, easy scarring, residual subclinical lesions or incomplete eradication of latent HPV virus, and easy recurrence. More importantly, the drugs have strong side effects and poor patient compliance.

[0007] Chinese invention patent application CN202411128022.6 discloses a traditional Chinese medicine composition for treating flat warts, belonging to the field of traditional Chinese medicine technology. According to the weight parts, it includes the following raw materials: 9-20 parts of Forsythia suspensa, 6-20 parts of Pogostemon cablin, 6-20 parts of Eupatorium fortunei, 9-20 parts of Prunella vulgaris, 10-30 parts of Coix lacryma-jobi, 9-20 parts of Lithospermum erythrorhizon, 9-20 parts of Polygonum cuspidatum, 9-20 parts of Paeonia suffruticosa, 10-30 parts of stir-fried white hyacinth bean, 9-30 parts of Poria cocos, 9-30 parts of Atractylodes macrocephala, 6-20 parts of Ligusticum chuanxiong, 6-20 parts of Citrus reticulata peel, and 6-15 parts of Glycyrrhiza uralensis. This traditional Chinese medicine composition treats flat warts by clearing heat and detoxifying, soothing the liver and regulating qi, tonifying the spleen and resolving dampness.

[0008] The aforementioned compositions may have complex flavors or uncertain efficacy. The physiological effects of drugs are closely related to their complex chemical components. Domestic and international research on their active ingredients mainly focuses on medicinal materials and indications, with limited in-depth research on the relationship between active ingredients and their activity. Precisely simplifying the formulation is crucial for elucidating the mechanism of action, and figuring out how to break down prescriptions to achieve higher efficacy is an urgent issue to be addressed in the field of traditional Chinese medicine. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention provides a compound traditional Chinese medicine composition for treating skin warts and condyloma acuminata, and its application.

[0010] To achieve the objectives of this invention, the following technical solution is adopted:

[0011] A compound traditional Chinese medicine composition for treating skin warts and condyloma acuminata, wherein the raw materials of the compound traditional Chinese medicine composition include the following components: astilbin, matrine, houttuynia cordata, bee honeycomb, podophyllotoxin, Cnidium monnieri and Brucea javanica.

[0012] Preferably, the raw materials of the compound traditional Chinese medicine composition, by weight, include the following components: astilbene 0.03-3.6 parts, matrine 0.003-1 parts, houttuynia cordata 10-30 parts, bee honeycomb 10-30 parts, podophyllotoxin 0.01-5 parts, cnidium monnieri 10-30 parts, and croton tiglium 1-20 parts.

[0013] Preferably, the raw materials of the compound traditional Chinese medicine composition, by weight, include the following components: astilbene 0.1-1.5 parts, matrine 0.1-0.9 parts, houttuynia cordata 15-25 parts, bee honeycomb 10-20 parts, podophyllotoxin 0.06-5 parts, cnidium monnieri 10-15 parts, and croton tiglium 5-15 parts.

[0014] Preferably, the mass ratio of astilbene, matrine, and podophyllotoxin is 1-3:1:1-3.

[0015] Preferably, the raw materials of the compound traditional Chinese medicine composition, by weight, include the following components: 1 part astilbene, 0.5 parts matrine, 20 parts houttuynia cordata, 15 parts bee honeycomb, 1 part podophyllotoxin, 10 parts Cnidium monnieri, and 10 parts Brucea javanica.

[0016] The second objective of this invention is to provide a method for preparing a compound traditional Chinese medicine composition for treating skin warts and condyloma acuminata, comprising the following steps:

[0017] (1) Press the croton seeds to extract croton oil, and obtain the residue;

[0018] (2) Add water to houttuynia cordata, bee honeycomb, cnidium monnieri and dregs, decoct to obtain decoction, add tanninase to the decoction to hydrolyze and inactivate, and obtain hydrolysate;

[0019] (3) The enzyme hydrolysate, croton oil, astilbene, matrine and podophyllotoxin are mixed and dispersed evenly to obtain the product.

[0020] Preferably, the amount of tanninase added in step (2) is 0.05-0.2% of the decoction mass.

[0021] Preferably, in step (2), the enzymatic hydrolysate is concentrated into a thick paste with a relative density of 1.30~1.35 at 50°C.

[0022] Preferably, the enzymatic hydrolysis temperature in step (2) is 45-50℃, the pH of the enzymatic hydrolysis is 4.5-6.0, and the hydrolysis time is 1.5-3h.

[0023] A third objective of this invention is to provide a pharmaceutical preparation for treating skin warts and condyloma acuminata, comprising the aforementioned compound traditional Chinese medicine composition and pharmaceutically acceptable excipients.

[0024] Preferably, the pharmaceutical preparation is a topical preparation, and the dosage form is selected from creams or tinctures.

[0025] Preferably, based on the mass of the compound traditional Chinese medicine composition, the pharmaceutically acceptable excipients include 20-50% polyethylene glycol 400, 20-50% polyethylene glycol 2000, 10-20% polyethylene glycol 4000, and 1-5% propylene glycol.

[0026] The fourth objective of this invention is to provide the application of the aforementioned compound traditional Chinese medicine composition or the aforementioned pharmaceutical preparation in the preparation of drugs for treating skin warts and condyloma acuminata.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] (1) The present invention combines the components, and its synergistic effect in treating condyloma and skin warts is significantly better than the original Chinese medicine prescription.

[0029] (2) This invention provides a compound composition of active ingredients and medicinal materials from traditional Chinese medicine, which is a simplified formula with synergistic effects among the components. It can effectively inhibit viral-induced BHK-21, MCF-7, and Hep-2 cell lesions and has a better effect on improving skin warts. The complex structure of active substances in traditional Chinese medicine makes it extremely difficult to elucidate their drug action mechanism and material basis, which is the biggest obstacle to their international recognition. This invention, through simplified formula decomposition, has developed a composition with better effects on improving skin warts and condyloma acuminata, which is a model of innovative research in traditional Chinese medicine.

[0030] (3) Previous research in this project found that herbal medicines such as houttuynia cordata contain hydrolyzable tannins. This invention releases active components by enzymatic hydrolysis and synergistically combines them with the formulation components, which can further effectively inhibit the proliferation of HPV11-HaCaT cells and HaCaT cells, as well as the expression of HPV11 E7 mRNA in HPV11-HaCaT cells, thereby improving its efficacy in treating condyloma acuminata. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments. All raw materials used are commercially available conventional raw materials; unless otherwise specified, the embodiments of the present invention are carried out under conventional conditions or conditions recommended by the manufacturer. The raw materials or excipients used, as well as the reagents or instruments whose manufacturers are not specified, are all conventional products that can be obtained commercially, and all raw materials used comply with the inspection requirements of each medicinal material in the Chinese Pharmacopoeia. The excipients comply with the requirements of the usage standards in Part IV of the Chinese Pharmacopoeia. The tannin enzyme supplier is Nanning Dongheng Huadao Biotechnology Co., Ltd., specification: 250u / g.

[0032] Unless otherwise stated, all percentages, ratios, proportions or % are by weight.

[0033] Example 1

[0034] This embodiment provides a compound traditional Chinese medicine composition for treating skin warts and condyloma acuminata. The raw materials, by weight, are:

[0035] Astilbene 1 part, matrine 0.5 parts, houttuynia cordata 20 parts, bee honeycomb 15 parts, podophyllotoxin 1 part, Cnidium monnieri 10 parts and Brucea javanica 10 parts.

[0036] The preparation method is as follows:

[0037] (1) Press the croton seeds to extract croton oil, and obtain the residue;

[0038] (2) Add 5 times the amount of water to houttuynia cordata, bee honeycomb, cnidium monnieri and dregs, soak for 30 minutes, decoct twice for 30 minutes each time, filter, combine the filtrates to obtain decoction, add 0.1% of tanninase by weight of decoction to decoction and mix evenly, enzymatically hydrolyze at 50℃ and pH5.0 for 2 hours, inactivate, filter to obtain enzymatic hydrolysate;

[0039] (3) Concentrate the enzymatic hydrolysate under reduced pressure to a thick paste with a relative density of 1.30~1.35 at 50℃, and then mix and disperse it evenly with Brucea javanica oil, astilbene, matrine and podophyllotoxin.

[0040] The above-mentioned compound traditional Chinese medicine composition was prepared into a topical cream. The preparation method is as follows:

[0041] Based on the quality of the compound Chinese medicine composition, 25% PEG400 and 5% propylene glycol were added to the compound Chinese medicine composition prepared above, heated to dissolve, and then a mixture of 15% PEG4000 and 20% PEG2000 after hot melting was added, stirred evenly, and cooled to a paste to make an ointment.

[0042] Example 2

[0043] This embodiment provides a compound traditional Chinese medicine composition for treating skin warts and condyloma acuminata. The raw materials, by weight, are:

[0044] Astilbene 0.1 parts, matrine 0.1 parts, houttuynia cordata 15 parts, bee honeycomb 10 parts, podophyllotoxin 0.06 parts, Cnidium monnieri 10 parts and Brucea javanica 5 parts.

[0045] The preparation method is as follows:

[0046] (1) Press the croton seeds to extract croton oil, and obtain the residue;

[0047] (2) Add 8 times the amount of water to houttuynia cordata, bee honeycomb, cnidium monnieri and dregs, soak for 30 minutes, decoct twice for 40 minutes each time, filter, mix the filtrates to obtain decoction, add 0.06% of tanninase by weight of decoction to decoction and mix evenly, enzymatically hydrolyze at 45℃ and pH 6.0 for 2 hours, inactivate, filter to obtain enzymatic hydrolysate;

[0048] (3) Concentrate the enzymatic hydrolysate under reduced pressure to a thick paste with a relative density of 1.30~1.35 at 50℃, and then mix and disperse it evenly with Brucea javanica oil, astilbene, matrine and podophyllotoxin.

[0049] The above-mentioned compound traditional Chinese medicine composition was prepared into a topical cream. The preparation method is as follows:

[0050] Based on the mass of the compound traditional Chinese medicine composition, 20% PEG400 and 2% propylene glycol were added to the prepared compound traditional Chinese medicine composition and heated to dissolve. Then, a mixture of 20% PEG4000 and 30% PEG2000 (after hot melting) was added, stirred evenly, and cooled to a paste-like consistency. This process produces an ointment.

[0051] Example 3

[0052] This embodiment provides a compound traditional Chinese medicine composition for treating skin warts and condyloma acuminata. The raw materials, by weight, are:

[0053] Astilbene 1.5 parts, matrine 0.9 parts, houttuynia cordata 25 parts, bee honeycomb 20 parts, podophyllotoxin 5 parts, Cnidium monnieri 15 parts and Brucea javanica 15 parts.

[0054] The preparation method is as follows:

[0055] (1) Press the croton seeds to extract croton oil, and obtain the residue;

[0056] (2) Add 8 times the amount of water to houttuynia cordata, bee honeycomb, cnidium monnieri and dregs, soak for 30 minutes, decoct for 1 hour, filter, mix the filtrate to obtain decoction, add 0.2% of tanninase by weight of decoction to decoction and mix evenly, enzymatically hydrolyze at 50℃ and pH 4.5 for 1.5 hours, inactivate, filter to obtain enzymatic hydrolysate;

[0057] (3) Concentrate the enzymatic hydrolysate under reduced pressure to a thick paste with a relative density of 1.30~1.35, and then mix and disperse it evenly with Brucea javanica oil, astilbene, matrine and podophyllotoxin.

[0058] The above-mentioned compound traditional Chinese medicine composition was prepared into a topical cream. The preparation method is as follows:

[0059] Based on the mass of the compound traditional Chinese medicine composition, 30% PEG400 and 5% propylene glycol were added to the prepared compound traditional Chinese medicine composition and heated to dissolve. Then, a mixture of 10% PEG4000 and 20% PEG2000 (after hot melting) was added, stirred evenly, and cooled to a paste-like consistency. This process produces an ointment.

[0060] Comparative Example 1

[0061] The only difference between this comparative example and Example 1 is that astilbene is replaced with scutellarin, and podophyllotoxin is replaced with podophyllotoxin. Details are as follows:

[0062] A compound traditional Chinese medicine composition, by weight, comprises the following raw materials:

[0063] One part of scutellarin, 0.5 parts of matrine, 20 parts of houttuynia cordata, 15 parts of bee honeycomb, 1 part of podophyllotoxin, 10 parts of Cnidium monnieri and 10 parts of Brucea javanica.

[0064] The preparation method is as follows:

[0065] (1) Press the croton seeds to extract croton oil, and obtain the residue;

[0066] (2) Add 5 times the amount of water to houttuynia cordata, bee honeycomb, cnidium monnieri and dregs, soak for 30 minutes, decoct twice for 30 minutes each time, filter, combine the filtrates to obtain decoction, add 0.1% of tanninase by weight of decoction to decoction and mix evenly, enzymatically hydrolyze at 50℃ and pH5.0 for 2 hours, inactivate, filter to obtain enzymatic hydrolysate;

[0067] (3) Concentrate the enzymatic hydrolysate under reduced pressure to a thick paste with a relative density of 1.30~1.35 at 50℃, and then mix and disperse it evenly with Brucea javanica oil, scutellarin, matrine and podophyllotoxin.

[0068] The above-mentioned compound traditional Chinese medicine composition was prepared into a topical cream. The preparation method is as follows:

[0069] Based on the quality of the compound Chinese medicine composition, 25% PEG400 and 5% propylene glycol were added to the compound Chinese medicine composition prepared above, heated to dissolve, and then a mixture of 15% PEG4000 and 20% PEG2000 after hot melting was added, stirred evenly, and cooled to a paste to make an ointment.

[0070] Comparative Example 2

[0071] The only difference between this comparative example and Example 1 is that matrine is replaced with oxymatrine, and podophyllotoxin is replaced with kaempferol and quercetin in a 1:1 mass ratio. Details are as follows:

[0072] A compound traditional Chinese medicine composition, by weight, comprises the following raw materials:

[0073] Astilbene 1 part, oxymatrine 0.5 parts, houttuynia cordata 20 parts, bee honeycomb 15 parts, kaempferol 0.5 parts, quercetin 0.5 parts, Cnidium monnieri 10 parts, and Brucea javanica 10 parts.

[0074] The preparation method is as follows:

[0075] (1) Press the croton seeds to extract croton oil, and obtain the residue;

[0076] (2) Add 5 times the amount of water to houttuynia cordata, bee honeycomb, cnidium monnieri and dregs, soak for 30 minutes, decoct twice for 30 minutes each time, filter, combine the filtrates to obtain decoction, add 0.1% of tanninase by weight of decoction to decoction and mix evenly, enzymatically hydrolyze at 50℃ and pH5.0 for 2 hours, inactivate, filter to obtain enzymatic hydrolysate;

[0077] (3) Concentrate the enzymatic hydrolysate under reduced pressure to a thick paste with a relative density of 1.30~1.35 at 50℃, and then mix and disperse it evenly with Brucea javanica oil, astilbene, oxymatrine, kaempferol and quercetin to obtain the final product.

[0078] The above-mentioned compound traditional Chinese medicine composition was prepared into a topical cream. The preparation method is as follows:

[0079] Based on the quality of the compound Chinese medicine composition, 25% PEG400 and 5% propylene glycol were added to the compound Chinese medicine composition prepared above, heated to dissolve, and then a mixture of 15% PEG4000 and 20% PEG2000 after hot melting was added, stirred evenly, and cooled to a paste to make an ointment.

[0080] Comparative Example 3

[0081] The only difference between this comparative example and Example 1 is that tanninase is removed, and enzymatic hydrolysis is performed using cellulase and pectinase in a 1:1 mass ratio. Details are as follows:

[0082] A compound traditional Chinese medicine composition, by weight, comprises the following raw materials:

[0083] Astilbene 1 part, matrine 0.5 parts, houttuynia cordata 20 parts, bee honeycomb 15 parts, podophyllotoxin 1 part, Cnidium monnieri 10 parts and Brucea javanica 10 parts.

[0084] The preparation method is as follows:

[0085] (1) Press the croton seeds to extract croton oil, and obtain the residue;

[0086] (2) Add 5 times the amount of water to Houttuynia cordata, bee honeycomb, Cnidium monnieri and dregs, soak for 30 minutes, decoct twice for 30 minutes each time, filter, combine the filtrates to obtain decoction, add 0.1% of the decoction mass of cellulase and pectin in a mass ratio of 1:1 to the decoction and mix evenly, enzymatically hydrolyze at 50℃ and pH 5.0 for 2 hours, inactivate, filter to obtain enzymatic hydrolysate; cellulase specification 10,000 u / g, pectinase specification 10,000-30,000 u / g, both suppliers are Nanning Dongheng Huadao Biotechnology Co., Ltd.;

[0087] (3) Concentrate the enzymatic hydrolysate under reduced pressure to a thick paste with a relative density of 1.30~1.35 at 50℃, and then mix and disperse it evenly with Brucea javanica oil, astilbene, matrine and podophyllotoxin.

[0088] The above-mentioned compound traditional Chinese medicine composition was prepared into a topical cream. The preparation method is as follows:

[0089] Based on the quality of the compound Chinese medicine composition, 25% PEG400 and 5% propylene glycol were added to the compound Chinese medicine composition prepared above, heated to dissolve, and then a mixture of 15% PEG4000 and 20% PEG2000 after hot melting was added, stirred evenly, and cooled to a paste to make an ointment.

[0090] Comparative Example 4

[0091] The positive control drug, a traditional Chinese medicine composition, is prescribed as follows:

[0092] Smilax glabra 30g, Sophora flavescens 15g, Houttuynia cordata 20g, Vespa nidus 15g, Podophyllum hexandrum 12g, Cnidium monnieri 15g and Brucea javanica 5g.

[0093] The preparation method is as follows:

[0094] (1) Press the croton seeds to extract croton seed oil, and obtain croton seed residue;

[0095] (2) The residues of Cnidium monnieri, Podophyllum hexandrum, and Brucea javanica were extracted three times with 80% ethanol. The first extraction was 1 hour with 10 times the amount of ethanol, the second extraction was 1 hour with 8 times the amount of ethanol, and the third extraction was 1 hour with 6 times the amount of ethanol. The three ethanol extracts were combined, filtered, and the ethanol was recovered from the filtrate to a thick paste to obtain the ethanol extract and residue A for later use.

[0096] (3) Mix the four herbs, Sophora flavescens, Houttuynia cordata, Smilax glabra, and Vespa nidus, with the above-mentioned dregs A, and decoct them three times: the first time is 2 hours and 10 times the amount of water, the second time is 1.5 hours and 8 times the amount of water, and the third time is 1 hour and 6 times the amount of water. Combine the decoctions, let them stand, centrifuge, and take the clear liquid and concentrate it to a thick paste with a relative density of 1.30~1.35 to obtain the water extract for use.

[0097] (4) The alcohol extract and water extract are mixed to obtain the product.

[0098] The above-mentioned traditional Chinese medicine composition was prepared into a topical cream, and the preparation method is as follows:

[0099] Add the alcohol extract and water extract to 25% PEG400 and 5% propylene glycol, heat to dissolve, then add the hot-melted mixture of 15% PEG4000 and 20% PEG2000, stir evenly, and cool to a paste-like consistency.

[0100] Experimental research

[0101] Experimental Example 1

[0102] The most commonly used method in in vitro antiviral drug research is the CPE method.

[0103] This invention investigates the inhibitory effect of the prepared compound traditional Chinese medicine composition on HSV-1 virus proliferation. First, the virus was inoculated into the wells of hamster kidney cells (BHK-21, MCF-7, and Hep-2 cells with 80% confluence) and incubated at 37°C for 1 hour. The supernatant was discarded, and the cells were washed with cell culture medium. The compound traditional Chinese medicine composition, diluted sequentially, was then added. A virus control group and a cell control group were also included. The cells were incubated at 37°C, and the cytopathic effect (CPE) was observed daily under a microscope. When the CPE in the virus control group reached "++++", the drug's effect was determined; when the CPE decreased to "++", "+", or "-", the drug was considered effective.

[0104] The results showed that the compound traditional Chinese medicine compositions prepared in each example and comparative example all had a certain degree of ability to inhibit virus-induced cytopathic effects. Among them, the compound traditional Chinese medicine compositions of Examples 1-3 had a better inhibitory effect on cytopathic effects than those of Comparative Examples 1-4. The specific results are shown in Tables 1-3 below.

[0105] Table 1. Results of the inhibitory effect of the compound traditional Chinese medicine composition on virus-induced BHK-21 cytopathic effects.

[0106]

[0107] Note: - indicates no obvious CPE was found, + indicates that CPE was present in approximately 25% of cells, and ++ indicates that CPE was present in approximately 50% of cells.

[0108] CPE, +++ indicates that CPE occurs in approximately 75% of cells, and ++++ indicates that CPE occurs in 100% of cells.

[0109] Table 2 Results of the inhibitory effect of the compound traditional Chinese medicine composition on MCF-7 cell pathogenesis

[0110]

[0111] Note: - indicates no obvious CPE was found, + indicates that CPE was present in approximately 25% of cells, and ++ indicates that CPE was present in approximately 50% of cells.

[0112] CPE, +++ indicates that CPE occurs in approximately 75% of cells, and ++++ indicates that CPE occurs in 100% of cells.

[0113] Table 3 Results of the inhibitory effect of compound traditional Chinese medicine composition on Hep-2 cell pathogenesis

[0114]

[0115] Note: - indicates no obvious CPE was found, + indicates that CPE was present in approximately 25% of cells, and ++ indicates that CPE was present in approximately 50% of cells.

[0116] CPE, +++ indicates that CPE occurs in approximately 75% of cells, and ++++ indicates that CPE occurs in 100% of cells.

[0117] Experimental Example 2: Study on the inhibitory effect of the compound composition of the present invention on the proliferation of HPV11-HaCaT cells by conventional MTT assay.

[0118] 2.1 Experimental cells, bacterial strains, and main reagents:

[0119] Tetramethylazole blue (MTT, 20090316 Sigma); human keratinocyte cell line (HaCaT), 96-well plate (GIBCO); multi-function analyzer (VICTOR×5). HPV11-positive Escherichia coli, purchased from ATCC, No. 45151.

[0120] 2.2. Drugs

[0121] The compound traditional Chinese medicine compositions prepared in Examples 1-3 and Comparative Examples 1-4.

[0122] 2.3 Preparation of HPV11-HaCaT cells

[0123] The cells were prepared using conventional recombinant cell transfection methods in the art, as detailed below:

[0124] The plasmid DNA of E. coli was extracted according to the instruction manual of the Invitrogen Midi Plasmid Extraction Kit. The HPV11-positive E. coli were cultured and amplified, and then the plasmid was extracted and purified. The full-length HPV11 gene was digested with the restriction endonuclease BamHI according to the instructions of the Promega BamHI Restriction Kit.

[0125] Following the instructions of the Invitrogen T4 DNA ligase kit, linear HPV11 DNA was self-circularized using T4 DNA ligase. The self-circularized HPV11 DNA was then purified using the Promega gel and PCR product recovery and purification kit to obtain circular HPV11 DNA.

[0126] HPV11-HaCaT cells were constructed through cell transfection:

[0127] 1) Culture the cells as usual, digest and centrifuge HaCaT cells that have grown to 80% confluence, and count them. (3 × 10⁶ cells) 5 The cells were seeded into six-well plates and incubated overnight at 37°C with 5% CO2. The old culture medium in the six-well plates was then aspirated and the cells were gently rinsed twice with serum-depleted culture medium.

[0128] 2) Take 1.2 μg of the previously prepared circular HPV11 DNA and 0.4 μg of PTK-neo DNA, add them to 100 μl of serum-depleted culture medium, mix gently, and label as Solution A. Add 8 μl of Lipofectamine TM Add 2000 μL of transfection reagent to 100 μL of serum-depleted culture medium, mix gently, label as solution B, and incubate at room temperature for 5 min. Gently mix solution A and solution B, and incubate at room temperature for 20 min to obtain the Lipofectamine-DNA mixture.

[0129] 3) Add 800 μL of serum-depleted culture medium to the Lipofectamine-DNA mixture and mix gently to obtain the mixture. Add 1 mL of this mixture dropwise to the surface of HaCaT cells from step 1), gently shake to distribute evenly, and set up a blank control group. A negative control group was also set up as follows: (1) Lipofectamine was added only. TM 2000, without HPV11 DNA and PTK-neoDNA. (2) Add Lipofectamine. TM 2000 and HPV11 DNA, without PTK-neoDNA, (3) with added Lipofectamine TM 2000 and PTK-neo DNA, without HPV11 DNA, (4) without Lipofectamine TM 2000, only HPV11 DNA and PTK-neo DNA were added, (5) no treatment was added (blank control). After being placed in a 37℃, 5% CO2 incubator for 6 hours, the medium was changed, DMEM complete medium was added again, and the culture was continued.

[0130] 4) When the transfected cells reach approximately 80% confluence, digest the cells, centrifuge, resuspend the cells, and transfer them to a new cell culture plate at a 1:10 ratio. Incubate overnight at 37°C with 5% CO2. Discard the medium and replace it with DMEM medium containing 500 μg / ml G418, and continue culturing. After approximately 5 days, most of the cells in the blank control wells will have died. Replace the medium with DMEM medium containing 250 μg / ml G418 and continue culturing.

[0131] 5) Once the resistant cell clones have formed, they are digested and merged, then transferred to DMEM medium containing 250 μg / ml G418.

[0132] Continue culturing. Perform the same treatment on the negative control group cells until all cells die.

[0133] 6) Prepare a cell cryopreservation solution using 45% fetal bovine serum, 45% DMEM medium, and 10% DMSO. Collect cells in good growth condition after selection, digest and centrifuge the cells, and resuspend them in the cell cryopreservation solution. Transfer the cell suspension to cryovials to obtain HPV11-HaCaT cells. Detect the presence of HPV11 DNA in the cells using FQ-PCR.

[0134] 2.4 Effects of Compound Traditional Chinese Medicine Composition on HPV11-HaCaT Cells and HaCaT Cell Proliferation Rate: Experimental Methods:

[0135] (1) Cell culture: HaCaT cells and HPV11-HaCaT cells were cultured in DMEM medium containing 10% newborn calf serum and cultured at 37°C, 5% CO2 and saturated humidity.

[0136] (2) Experimental grouping: HaCaT and HPV11-HaCaT cells in the logarithmic growth phase were divided into Example 1-3, Comparative Example 1-4 (with different concentrations of traditional Chinese medicine compositions prepared in Example 1-3 and Comparative Example 1-4), 0.1% DMSO solvent control group, and blank control group.

[0137] (3) Cell proliferation assay: HaCaT cells and HPV11-HaCaT cells were seeded in 96-well plates (2 × 10⁻⁶ cells / well). 4 Cells were cultured overnight in wells (1 cell / well), and then different concentrations of drug solution (200 μL / well) were added to each group: the final concentrations of each example and the comparative example were 8, 4, 2, 1, and 0.5 mg crude drug / mL (diluted with DMSO), respectively. The 0.1% DMSO solvent control group had a DMSO content of 0.1%, and the blank control group had an equal volume of culture medium. Each group had 3 replicates. After culturing for 24 h, 20 μL / well of 5 mg / mL MTT solution was added, and the cells were cultured for another 4 h. The culture medium was then discarded, and 150 μL / well of DMSO was added and shaken for 10 min in the dark to fully dissolve the blue-purple precipitate. The absorbance was measured at 550 nm using a microplate reader. The cell viability of different concentration groups was calculated as: Cell viability = (Experimental group A550 / Control group A550) × 100%. The mean cell viability of each group was calculated using the data from each well. Then, the relative expression levels of HPV11E7 mRNA at a drug concentration of 1 mg crude drug / ml in Examples 1-3 and Comparative Examples 1-4 were determined using RT-PCR. The experimental results are shown in Tables 4-11.

[0138] Table 4

[0139]

[0140] Table 5

[0141]

[0142] Table 6

[0143]

[0144] Table 7

[0145]

[0146] Table 8

[0147]

[0148] Table 9

[0149]

[0150] Table 10

[0151]

[0152] Table 11

[0153]

[0154] MTT assay uses MTT [(4,5-dimethylthiazole)2,5-diphenyltetrazol bromide] to stain cells. Changes in the optical density (OD value) can be detected to determine cell viability and understand the protective effect of the drug, thus enabling quantitative analysis of efficacy. Tables 4-10 show that the compound traditional Chinese medicine compositions in Examples 1-3 and Comparative Examples 1-4, at concentrations of 1-8 mg crude drug / ml, exhibited a certain degree of inhibitory effect on HPV11-HaCaT cells and HaCaT cells. Example 1 showed the best effect, with cell survival rates of 1-67% for HPV11-HaCaT cells. This demonstrates dose-dependent characteristics.

[0155] In Table 11, compared with the 0.1% DMSO solvent control group, the relative expression level of HPV11 E7 mRNA in each example group and the comparative example group showed a certain degree of decrease. The compound traditional Chinese medicine composition of the present invention is a simplified combination of the traditional Chinese medicine preparation of Comparative Example 4. After decomposition and combination, at the same dosage, its inhibitory effect on HPV11 E7 mRNA expression is comparable. The formula is concise and effective, and has good application prospects.

[0156] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. A compound traditional Chinese medicine composition for treating skin warts and condyloma acuminata, characterized in that, The raw materials of the compound traditional Chinese medicine composition are as follows, by weight: astilbene 0.1-1.5 parts, matrine 0.1-0.9 parts, houttuynia cordata 15-25 parts, bee honeycomb 10-20 parts, podophyllotoxin 0.06-5 parts, cnidium monnieri 10-15 parts and croton tiglium 5-15 parts.

2. The compound traditional Chinese medicine composition according to claim 1, characterized in that, The mass ratio of astilbene, matrine, and podophyllotoxin is 1-3:1:1-3.

3. The compound traditional Chinese medicine composition according to claim 1, characterized in that, The raw materials of the compound traditional Chinese medicine composition are as follows, by weight: 1 part astilbene, 0.5 parts matrine, 20 parts houttuynia cordata, 15 parts bee honeycomb, 1 part podophyllotoxin, 10 parts Cnidium monnieri and 10 parts croton tiglium.

4. A method for preparing a compound traditional Chinese medicine composition for treating skin warts and condyloma as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Press the croton seeds to extract croton oil, and obtain the residue; (2) Add water to houttuynia cordata, bee honeycomb, cnidium monnieri and dregs, decoct to obtain decoction, add tanninase to the decoction to hydrolyze and inactivate, and obtain hydrolysate; (3) The enzyme hydrolysate, croton oil, astilbene, matrine and podophyllotoxin are mixed and dispersed evenly to obtain the product.

5. A pharmaceutical preparation for treating skin warts and / or condyloma, characterized in that, It comprises the compound traditional Chinese medicine composition according to any one of claims 1-3 and pharmaceutically acceptable excipients.

6. The pharmaceutical preparation according to claim 5, characterized in that, The pharmaceutical preparation is for external use, and the dosage form is selected from creams or tinctures.

7. The pharmaceutical preparation according to claim 5, characterized in that, Based on the quality of the compound traditional Chinese medicine composition, the pharmaceutically acceptable excipients include 20-50% polyethylene glycol 400, 20-50% polyethylene glycol 2000, 10-20% polyethylene glycol 4000, and 1-5% propylene glycol.

8. The use of a compound traditional Chinese medicine composition according to any one of claims 1-3 or a pharmaceutical preparation according to any one of claims 5-7 in the preparation of a medicament for treating skin warts, condyloma acuminata and / or for treating HSV-1 virus infection.

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