Compound traditional Chinese medicine composition for treating skin wart and condyloma and application of compound traditional Chinese medicine composition

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a compound traditional Chinese medicine composition for treating skin warts and condyloma and application thereof. The compound traditional Chinese medicine composition is prepared from the following raw material components: astilbin, sophocarpidine, herba houttuyniae, honeycomb, podophyllotoxin, fructus cnidii and brucea javanica. The preparation method comprises the following steps: firstly, squeezing the brucea javanica to extract brucea javanica oil to obtain decoction dregs; the preparation method comprises the following steps: adding water into herba houttuyniae, nidus vespae, fructus cnidii and medicine residues, decocting to obtain decoction, adding tannase into the decoction for enzymolysis to obtain enzymatic hydrolysate, inactivating, and finally mixing and dispersing the enzymatic hydrolysate, brucea javanica oil, astilbin, sophocarpidine and podophyllotoxin uniformly to obtain the medicine. All the components are compatible, and the synergistic effect is remarkably superior to that of an original traditional Chinese medicine prescription; the traditional Chinese medicine composition has a good application market prospect for treating skin warts and condyloma.

Description

Technical Field

[0001] The invention 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 an application thereof. Background Art

[0002] Warts are benign skin lesions caused by infection of the skin and mucous membranes by the human papillomavirus (HPV). Based on their clinical manifestations and location, warts are categorized as common warts, flat warts, plantar warts, condyloma acuminata, and epidermodysplasia verruciformis. Warts are transmitted by infected individuals and carriers, and are primarily caused by HPV types 1, 2, and 4. Wart lesions are painful to touch, but may also be asymptomatic. Microscopic examination of the skin reveals yellowish papules, thickened keratin rings, and rounded papillary keratinous hyperplasias, interspersed with small black spots caused by capillary rupture and bleeding. The course of the disease is closely related to the body's immune system, with a higher incidence in those with compromised immunity. While some patients may resolve spontaneously, patients often seek treatment due to cosmetic reasons and pain. Current treatments for warts primarily focus on destroying the warts and enhancing local or systemic immunity, including medications, physical therapy, and surgical excision. However, these treatments are associated with a long course of disease, painful procedures, high infection rates, and recurrence, leading to poor patient compliance.

[0003] Common topical medications include imiquimod, a small molecule immunomodulator with immunomodulatory and indirect antiviral effects. Its antiviral effects are achieved by stimulating the body's own immune system and inducing T cell factor activity, thereby enhancing cellular immunity and generating an immune response against HPV-infected cells. Currently, it is primarily used clinically to treat anal and genital warts. The main drawbacks of this treatment are its long treatment cycle, difficulty in patient adherence, and high cost, which increases the financial burden on patients. However, it is painless, invasive, and easy to administer, making it suitable for home use and for children. It should be used with caution in patients with skin allergies.

[0004] Another treatment option is salicylic acid, a keratin exfoliant that slowly destroys virus-infected cells without affecting keratinocyte production. The resulting mild stimulation stimulates an immune response. It slowly destroys the HPV-infected epidermis, causing local stratum corneum shedding or thinning. Its advantages lie in scar-free healing, minimal side effects, ease of use, and low cost. However, its disadvantages are slow onset and mild skin irritation. Therefore, if redness, swelling, or irritation occurs, discontinue use. It should also be avoided on the face and head to avoid unwanted pigmentation that could affect appearance.

[0005] Interferon and cantharidin are also available. Recombinant human interferon has the ability to inhibit tumor cell proliferation, fight viruses, and regulate the human immune system. Interferon combined with other treatments is safe and effective for treating single, refractory common warts and can reduce recurrence rates. Cantharidin, extracted from the traditional Chinese medicine Mylabris blister, can cause congestion and blistering of the wart epidermis. Blisters formed during cantharidin treatment of common warts are mildly painful but heal without scarring. If erosions occur during treatment, pause treatment for a few days before resuming.

[0006] There are also laser and surgical treatments. Currently, there are many treatments for skin warts, but many of these methods have drawbacks, such as a persistent course, increased scarring, residual subclinical lesions, incomplete elimination of latent HPV, and a high risk of recurrence. More importantly, the drugs can have significant side effects and poor patient compliance.

[0007] Chinese invention patent application CN202411128022.6 discloses a traditional Chinese medicine composition for treating flat warts, which belongs to the technical field of traditional Chinese medicine. The composition comprises the following raw materials in parts by weight: 9-20 parts of Forsythia suspensa, 6-20 parts of Patchouli, 6-20 parts of Cymbopogon citratus, 9-20 parts of Prunella Vulgaris, 10-30 parts of Coix seeds, 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 lentils, 9-30 parts of Poria cocos, 9-30 parts of Atractylodes macrocephala, 6-20 parts of Chuanxiong, 6-20 parts of dried tangerine peel and 6-15 parts of Licorice. The traditional Chinese medicine composition treats flat warts by clearing away heat and detoxifying, soothing the liver and regulating qi, tonifying spleen qi, and strengthening the spleen and eliminating dampness. These combinations can have complex flavors or unclear efficacy. However, the physiological effects of a drug are closely related to its complex chemical composition. Research on its active ingredients, both domestically and internationally, has primarily focused on the medicinal materials and indications, with little in-depth study of the relationship between active ingredients and activity. Accurately simplifying the compositional formula is crucial for elucidating the mechanism of action, and understanding how to break down the formula to achieve higher efficacy remains a pressing challenge in the field of Traditional Chinese Medicine. Summary of the Invention

[0008] In view of the deficiencies in the prior art, the present invention provides a compound Chinese medicine composition for treating skin warts and condyloma and its application.

[0009] In order to achieve the purpose of the present invention, the technical solutions adopted are as follows: A compound traditional Chinese medicine composition for treating skin warts and condyloma. The raw materials of the compound traditional Chinese medicine composition include the following components: astilbin, matrine, houttuynia cordata, honeycomb, podophyllotoxin, cnidium monnieri and javanica javanica.

[0010] Preferably, the raw materials of the compound Chinese medicine composition include the following components in parts by weight: 0.03-3.6 parts of astilbin, 0.003-1 part of matrine, 10-30 parts of houttuynia cordata, 10-30 parts of honeycomb, 0.01-5 parts of podophyllotoxin, 10-30 parts of cnidium monnieri and 1-20 parts of Brucea javanica.

[0011] Preferably, the raw materials of the compound Chinese medicine composition include the following components in parts by weight: 0.1-1.5 parts of astilbin, 0.1-0.9 parts of matrine, 15-25 parts of houttuynia cordata, 10-20 parts of honeycomb, 0.06-5 parts of podophyllotoxin, 10-15 parts of cnidium monnieri and 5-15 parts of javanica javanica.

[0012] Preferably, the mass ratio of astilbin, matrine and podophyllotoxin is 1-3:1:1-3.

[0013] Preferably, the raw materials of the compound Chinese medicine composition include the following components in parts by weight: 1 part of astilbin, 0.5 parts of matrine, 20 parts of Houttuynia cordata, 15 parts of honeycomb, 1 part of podophyllotoxin, 10 parts of Cnidium monnieri and 10 parts of Brucea javanica.

[0014] The second object of the present invention is to provide a method for preparing a compound Chinese medicine composition for treating skin warts and condyloma, comprising the following steps: (1) Pressing the Brucea javanica to extract Brucea javanica oil to obtain medicinal residue; (2) Add water to Houttuynia cordata, beehive, Cnidium monnieri and medicinal residues, and boil to obtain a decoction. Add tannin enzyme to the decoction to deactivate it and obtain an enzymatic solution; (3) The enzymatic hydrolysate, Brucea javanica oil, astilbin, matrine and podophyllotoxin are mixed and dispersed evenly.

[0015] Preferably, the amount of tannase added in step (2) is 0.05-0.2% of the mass of the decoction.

[0016] Preferably, in step (2), the enzymatic hydrolysate is concentrated into a thick paste having a relative density of 1.30 to 1.35 at 50°C.

[0017] Preferably, the enzymatic hydrolysis temperature in step (2) is 45-50°C, the enzymatic hydrolysis pH is 4.5-6.0, and the enzymatic hydrolysis time is 1.5-3 hours.

[0018] The third object of the present invention is to provide a pharmaceutical preparation for treating skin warts and condyloma, comprising the compound Chinese medicine composition and pharmaceutically acceptable excipients.

[0019] Preferably, the pharmaceutical preparation is an external preparation, and the dosage form is selected from a cream or a tincture.

[0020] Preferably, based on the mass of the compounded traditional Chinese medicine composition, the pharmaceutically acceptable excipients include polyethylene glycol 400 20-50%, polyethylene glycol 2000 20-50%, polyethylene glycol 4000 10-20%, and propylene glycol 1-5%.

[0021] The fourth object of the present invention is to provide an application of the compound Chinese medicine composition or the pharmaceutical preparation in the preparation of medicines for treating skin warts and condyloma.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention combines the components in a coordinated manner, and the synergistic effect of treating condyloma and skin warts is significantly better than the original Chinese medicine prescription.

[0023] (2) The present invention provides a compound composition of traditional Chinese medicine active ingredients and medicinal materials, which is a simplified formula with synergistic compatibility of various components. It can effectively inhibit BHK-21, MCF-7 and Hep-2 cell pathological changes caused by viruses, and has a better effect in improving skin warts. The complex structure of active substances in traditional Chinese medicine leads to great difficulties in explaining its drug action mechanism and material basis, which is the biggest obstacle affecting its recognition by the international community. The present invention has developed a composition with better effect in improving skin warts and condyloma by simplifying the formula, which is a model for innovative research in traditional Chinese medicine.

[0024] (3) Preliminary studies of this project have found that herbs such as Houttuynia cordata contain hydrolyzed tannins. The present invention 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, by enzymatically hydrolyzing them to release active components that can be synergistically combined with the ingredients of the formula to improve the efficacy of the drug in treating condyloma. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with specific embodiments. The following raw materials are all commercially available conventional raw materials; in the embodiments of the present invention, if specific conditions are not specified, they are all carried out in accordance with conventional conditions or conditions recommended by the manufacturer, and the raw materials or excipients used, as well as the reagents or instruments used without indicating the manufacturer, are all conventional products that can be obtained through commercial purchase, and the raw materials used all meet the requirements of the inspection items of various medicinal materials in the Chinese Pharmacopoeia, and the excipients meet the requirements of the use standards of the fourth part of the Chinese Pharmacopoeia. Among them, the supplier of tannase is Nanning Dongheng Huadao Biotechnology Co., Ltd., specification: 250u / g.

[0026] Unless otherwise indicated, all percentages, ratios, proportions or % are by weight.

[0027] Example 1 This embodiment provides a compound Chinese medicine composition for treating skin warts and condyloma. The raw materials are, by weight: 1 part of astilbin, 0.5 part of matrine, 20 parts of herba houttuyniae, 15 parts of honeycombs, 1 part of podophyllotoxin, 10 parts of fructus cnidii and 10 parts of fructus javanica.

[0028] The preparation method is as follows: (1) Pressing the Brucea javanica to extract Brucea javanica oil to obtain medicinal residue; (2) Add 5 times the amount of water to Houttuynia cordata, beehive, Cnidium monnieri and the medicinal residue, soak for 30 minutes, decoct twice, each time for 30 minutes, filter, combine the filtrates to obtain a decoction, add 0.1% of the decoction weight of tannase to the decoction, mix evenly, incubate at 50℃, pH 5.0 for 2 hours, inactivate, and filter to obtain the enzymatic solution; (3) The enzymatic hydrolysate is concentrated under reduced pressure to a thick paste with a relative density of 1.30-1.35 at 50°C, and then mixed with Brucea javanica oil, astilbin, matrine and podophyllotoxin and dispersed evenly to obtain the paste.

[0029] The above compounded Chinese medicine composition is prepared into an external preparation cream. The preparation method is as follows: Based on the mass 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 hot-melt mixture of 15% PEG4000 and 20% PEG2000 was added, stirred evenly, and cooled to a paste to prepare an ointment.

[0030] Example 2 This embodiment provides a compound Chinese medicine composition for treating skin warts and condyloma. The raw materials are, by weight: 0.1 part of astilbin, 0.1 part of matrine, 15 parts of herba houttuyniae, 10 parts of honeycombs, 0.06 part of podophyllotoxin, 10 parts of fructus cnidii and 5 parts of fructus javanica.

[0031] The preparation method is as follows: (1) Pressing the Brucea javanica to extract Brucea javanica oil to obtain medicinal residue; (2) Add 8 times the amount of water to Houttuynia cordata, beehive, Cnidium monnieri and the medicinal residue, soak for 30 minutes, decoct twice, each time for 40 minutes, filter, mix the filtrates to obtain a decoction, add 0.06% of the decoction weight of tannase to the decoction, mix evenly, incubate at 45°C, pH 6.0 for 2 hours, inactivate, and filter to obtain the enzymatic hydrolyzate; (3) The enzymatic hydrolysate is concentrated under reduced pressure to a thick paste with a relative density of 1.30-1.35 at 50°C, and then mixed with Brucea javanica oil, astilbin, matrine and podophyllotoxin and dispersed evenly to obtain the paste.

[0032] The above compounded Chinese medicine composition is prepared into an external preparation cream. The preparation method is as follows: Based on the mass of the compounded Chinese medicine composition, 20% PEG400 and 2% propylene glycol were added to the compounded Chinese medicine composition prepared above, heated to dissolve, and then a hot-melt mixture of 20% PEG4000 and 30% PEG2000 was added. The mixture was stirred evenly and cooled until a paste was formed. This was prepared into a paste.

[0033] Example 3 This embodiment provides a compound Chinese medicine composition for treating skin warts and condyloma. The raw materials are, by weight: 1.5 parts of astilbin, 0.9 part of matrine, 25 parts of herba houttuyniae, 20 parts of honeycombs, 5 parts of podophyllotoxin, 15 parts of fructus cnidii and 15 parts of fructus javanica.

[0034] The preparation method is as follows: (1) Pressing the Brucea javanica to extract Brucea javanica oil to obtain medicinal residue; (2) Add 8 times the amount of water to Houttuynia cordata, beehive, Cnidium monnieri and the residue, soak for 30 minutes, decoct for 1 hour, filter, mix the filtrate to obtain a decoction, add 0.2% of the decoction weight of tannase to the decoction, mix evenly, incubate at 50°C, pH 4.5 for 1.5 hours, inactivate, and filter to obtain the enzymatic solution; (3) The enzymatic hydrolysate is concentrated under reduced pressure to a thick paste with a relative density of 1.30-1.35, and then mixed with Brucea javanica oil, astilbin, matrine and podophyllotoxin and dispersed evenly to obtain the paste.

[0035] The above compounded Chinese medicine composition is prepared into an external preparation cream. The preparation method is as follows: Based on the mass of the compounded Chinese medicine composition, 30% PEG400 and 5% propylene glycol were added to the compounded Chinese medicine composition prepared above, heated to dissolve, and then a hot-melt mixture of 10% PEG4000 and 20% PEG2000 was added. The mixture was stirred evenly and cooled until it became a paste. This was prepared into a paste.

[0036] Comparative Example 1 The only difference between this comparative example and Example 1 is that astilbin is replaced by cyperitoside, and podophyllotoxin is replaced by picropodophyllotoxin. The details are as follows: A compound Chinese medicine composition, the raw materials are calculated by weight: 1 part of cyperus glutinosin, 0.5 part of matrine, 20 parts of herba houttuyniae, 15 parts of honeycombs, 1 part of picropodophyllotoxin, 10 parts of fructus cnidii and 10 parts of fructus javanica.

[0037] The preparation method is as follows: (1) Pressing the Brucea javanica to extract Brucea javanica oil to obtain medicinal residue; (2) Add 5 times the amount of water to Houttuynia cordata, beehive, Cnidium monnieri and the medicinal residue, soak for 30 minutes, decoct twice, each time for 30 minutes, filter, combine the filtrates to obtain a decoction, add 0.1% of the decoction weight of tannase to the decoction, mix evenly, incubate at 50℃, pH 5.0 for 2 hours, inactivate, and filter to obtain the enzymatic solution; (3) The enzymatic hydrolysate is concentrated under reduced pressure to a thick paste with a relative density of 1.30-1.35 at 50°C, and then mixed with Brucea javanica oil, cyperus glycoside, matrine and picropodophyllotoxin and dispersed evenly to obtain the paste.

[0038] The above compounded Chinese medicine composition is prepared into an external preparation cream. The preparation method is as follows: Based on the mass 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 hot-melt mixture of 15% PEG4000 and 20% PEG2000 was added, stirred evenly, and cooled to a paste to prepare an ointment.

[0039] Comparative Example 2 The only difference between this comparative example and Example 1 is that matrine is replaced by oxymatrine, and podophyllotoxin is replaced by kaempferol and quercetin in a mass ratio of 1:1. The details are as follows: A compound Chinese medicine composition, the raw materials are calculated by weight: 1 part of astilbin, 0.5 part of oxymatrine, 20 parts of herba houttuyniae, 15 parts of honeycomb, 0.5 part of kaempferol, 0.5 part of quercetin, 10 parts of fructus cnidii and 10 parts of fructus javanica.

[0040] The preparation method is as follows: (1) Pressing the Brucea javanica to extract Brucea javanica oil to obtain medicinal residue; (2) Add 5 times the amount of water to Houttuynia cordata, beehive, Cnidium monnieri and the medicinal residue, soak for 30 minutes, decoct twice, each time for 30 minutes, filter, combine the filtrates to obtain a decoction, add 0.1% of the decoction weight of tannase to the decoction, mix evenly, incubate at 50℃, pH 5.0 for 2 hours, inactivate, and filter to obtain the enzymatic solution; (3) The enzymatic hydrolysate is concentrated under reduced pressure to a thick paste with a relative density of 1.30-1.35 at 50°C, and then mixed and dispersed evenly with Brucea javanica oil, astilbin, oxymatrine, kaempferol and quercetin.

[0041] The above compounded Chinese medicine composition is prepared into an external preparation cream. The preparation method is as follows: Based on the mass 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 hot-melt mixture of 15% PEG4000 and 20% PEG2000 was added, stirred evenly, and cooled to a paste to prepare an ointment.

[0042] Comparative Example 3 The only difference between this comparative example and Example 1 is that tannase is removed and cellulase and pectinase are used in a mass ratio of 1:1 for enzymatic hydrolysis. The details are as follows: A compound Chinese medicine composition, the raw materials are calculated by weight: 1 part of astilbin, 0.5 part of matrine, 20 parts of herba houttuyniae, 15 parts of honeycombs, 1 part of podophyllotoxin, 10 parts of fructus cnidii and 10 parts of fructus javanica.

[0043] The preparation method is as follows: (1) Pressing the Brucea javanica to extract Brucea javanica oil to obtain medicinal residue; (2) Add 5 times the amount of water to Houttuynia cordata, honeycomb, Cnidium monnieri and medicinal residues, soak for 30 minutes, decoct twice, each time for 30 minutes, filter, combine the filtrates to obtain a decoction, add 0.1% of the decoction weight of cellulase and pectin in a mass ratio of 1:1 to the decoction, mix evenly, incubate at 50°C, pH 5.0 for 2 hours, inactivate, and filter to obtain an enzymatic solution; cellulase, specification 10,000 u / g, pectinase, specification 10,000-30,000 u / g, both suppliers are Nanning Dongheng Huadao Biotechnology Co., Ltd. (3) The enzymatic hydrolysate is concentrated under reduced pressure to a thick paste with a relative density of 1.30-1.35 at 50°C, and then mixed with Brucea javanica oil, astilbin, matrine and podophyllotoxin and dispersed evenly to obtain the paste.

[0044] The above compounded Chinese medicine composition is prepared into an external preparation cream. The preparation method is as follows: Based on the mass 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 hot-melt mixture of 15% PEG4000 and 20% PEG2000 was added, stirred evenly, and cooled to a paste to prepare an ointment.

[0045] Comparative Example 4 Positive control drug, the prescription of Chinese medicine composition is: 30g of Smilax glabra, 15g of Sophora flavescens, 20g of Houttuynia cordata, 15g of Beehive, 12g of Illicium verum, 15g of Cnidium monnieri and 5g of Brucea javanica.

[0046] The preparation method is as follows: (1) Pressing the Brucea javanica to extract Brucea javanica oil to obtain Brucea javanica residue; (2) The residues of Cnidium monnieri, Illicium verum and Brucea javanica were mixed and extracted three times with 80% ethanol. The first extraction was 10 times the amount of ethanol for 1 hour, the second extraction was 8 times the amount of ethanol for 1 hour, and the third extraction was 6 times the amount of ethanol for 1 hour. The three alcohol extracts were combined and filtered. The filtrate was used to recover ethanol until a thick paste was obtained. The alcohol extract and residue A were then used for standby.

[0047] (3) Sophora flavescens, Houttuynia cordata, Smilax glabra, and Beehives are mixed with the above-mentioned medicinal residue A and decocted three times in water: the first time is decocted in 10 times the amount of water for 2 hours, the second time is decocted in 8 times the amount of water for 1.5 hours, and the third time is decocted in 6 times the amount of water for 1 hour. The combined decoctions are allowed to stand and centrifuged. The clear liquid is concentrated to a thick paste with a relative density of 1.30-1.35 to obtain the aqueous extract for use.

[0048] (4) Mix the alcohol extract and the water extract.

[0049] The above-mentioned Chinese medicine composition is prepared into an external preparation cream, and the preparation method is as follows: Add 25% PEG400 and 5% propylene glycol to the alcohol extract and the water extract, heat to dissolve, then add the hot-melt mixture of 15% PEG4000 and 20% PEG2000, stir evenly, and cool until it becomes a paste.

[0050] Experimental studies Experimental Example 1 In the process of studying the antiviral activity of drugs in vitro, the most commonly used method is the CPE method.

[0051] The present invention tests the inhibitory effect of the prepared compound traditional Chinese medicine composition on HSV-1 viral proliferation. First, the virus was inoculated into 80% confluent hamster kidney cell culture wells (BHK-21, MCF-7, and Hep-2). The cells were allowed to adsorb 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 to a certain degree, was then added. A control group (virus control group) without drug and a control group (cell control group) were also established. The cells were cultured in a 37°C incubator, and the cytopathic effect (CPE) was observed daily under a microscope. The drug was considered effective when the CPE in the virus control group reached "++++." Effectiveness was determined when the CPE in the virus control group decreased to "++," "+," or "-."

[0052] The results showed that the compound Chinese medicine compositions prepared in each embodiment and comparative embodiment all had a certain degree of ability to inhibit virus-induced cytopathic effects. Among them, the compound Chinese medicine compositions of embodiments 1-3 had better cytopathic effects than comparative embodiments 1-4. The specific results are shown in Tables 1-3 below.

[0053] Table 1 Results of the inhibition of virus-induced BHK-21 cell pathological effect by the compound Chinese medicine composition

[0054] Note: - indicates no obvious CPE was found, + indicates about 25% of cells showed CPE, ++ indicates about 50% of cells showed CPE CPE, +++ means that approximately 75% of the cells showed CPE, and ++++ means that 100% of the cells showed CPE. Table 2 Results of inhibition of MCF-7 cell pathological effect by compound Chinese medicine composition

[0055] Note: - indicates no obvious CPE was found, + indicates about 25% of cells showed CPE, ++ indicates about 50% of cells showed CPE CPE, +++ means that approximately 75% of the cells showed CPE, and ++++ means that 100% of the cells showed CPE. Table 3 Results of the inhibition of Hep-2 cell pathological effect by the compound Chinese medicine composition

[0056] Note: - indicates no obvious CPE was found, + indicates about 25% of cells showed CPE, ++ indicates about 50% of cells showed CPE CPE, +++ means that approximately 75% of the cells showed CPE, and ++++ means that 100% of the cells showed CPE. Experimental Example 2 Conventional MTT method to determine the inhibitory effect of the composite composition of the present invention on the proliferation of HPV11-HaCaT cells 2.1 Experimental cells, strains and main reagents: Methyl tetrazolium blue (MTT, 20090316, Sigma); human keratinocyte cell line (HaCaT), 96-well plate (GIBCO); multifunctional analyzer (VICTOR×5); HPV11-positive Escherichia coli, purchased from ATCC, No. 45151.

[0057] 2.2.Medications The compound Chinese medicine compositions prepared in Examples 1-3 and Comparative Examples 1-4.

[0058] 2.3 Preparation of HPV11-HaCaT cells The preparation is carried out using conventional recombinant cell transfection methods in the art, as follows: E. coli plasmid DNA was extracted according to the operating instructions of the Invitrogen Midi plasmid extraction kit, the HPV11-positive E. coli was cultured and amplified, and then the plasmid was extracted and purified; and the full-length HPV11 gene was cut with the restriction endonuclease BamHI according to the operating instructions of the Promega BamHI enzyme digestion kit.

[0059] According to 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 purified using the instructions of the Promega gel and PCR product recovery and purification kit to obtain circular HPV11 DNA.

[0060] Cell transfection to construct HPV11-HaCaT cells: 1) Culture cells as usual, digest and centrifuge HaCaT cells grown to 80% confluence, and count them. 5 The cells were inoculated into each well of a six-well plate and cultured overnight in a 37°C, 5% CO2 incubator. The old culture medium in the six-well plate was discarded, and the cells were gently rinsed twice with reduced serum culture medium.

[0061] 2) Take 1.2ug of the circular HPV11 DNA and 0.4ug of PTK-neo DNA prepared above, add them to 100ul of reduced serum medium, mix gently, and mark it as solution A. TM Add 2000 μL of transfection reagent to 100 μL of reduced serum medium, mix gently, label this solution B, and let it stand at room temperature for 5 minutes. Gently mix solution A and solution B and let it stand at room temperature for 20 minutes to obtain the Lipofectamine-DNA mixture.

[0062] 3) Add 800ul of reduced serum medium to the Lipofectamine-DNA mixture and mix gently to obtain a mixed solution. Add 1ml of this mixed solution dropwise to the surface of the HaCaT cells in step 1) and shake gently to ensure even distribution. Set up a blank control group. At the same time, set up a negative control as follows: (1) Add only Lipofectamine TM 2000, without HPV11 DNA and PTK-neoDNA. (2) Add Lipofectamine TM 2000 and HPV11 DNA, without PTK-neoDNA, (3) adding 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 incubation at 37°C and 5% CO2 for 6 h, the medium was changed and DMEM complete medium was added again for further culture.

[0063] 4) When the transfected cells reach approximately 80% confluency, digest the cells and centrifuge. Resuspend the cells and transfer them to a new cell culture plate at a 1:10 ratio. Incubate overnight at 37°C in a 5% CO2 incubator. Discard the culture medium and replace it with DMEM supplemented with 500 μg / ml G418. Continue culturing. After approximately 5 days, most cells in the blank control wells will die. Replace the medium with DMEM supplemented with 250 μg / ml G418 and continue culturing.

[0064] 5) When resistant cell clones are formed, digest and merge them and transfer them into DMEM medium containing 250ug / ml G418 Continue culturing. Perform the same treatment on the negative control group cells until all cells die.

[0065] 6) Prepare a freezing solution containing 45% fetal bovine serum, 45% DMEM medium, and 10% DMSO. Collect well-growing, screened cells, digest and centrifuge them, and resuspend them in the freezing solution. Transfer the cell suspension to a cryovial to obtain HPV11-HaCaT cells. Detect the presence of intracellular HPV11 DNA using FQ-PCR.

[0066] 2.4 Effect of the compound Chinese medicine composition on HPV11-HaCaT cells and HaCaT cell proliferation rate Experimental methods: (1) Cell culture: HaCaT cells and HPV11-HaCaT cells were cultured in DMEM medium containing 10% newborn calf serum at 37°C, 5% CO2, and saturated humidity.

[0067] (2) Experimental grouping: HaCaT and HPV11-HaCaT cells in the logarithmic growth phase were divided into Examples 1-3, Comparative Examples 1-4 (with different concentrations of the Chinese medicine compositions prepared in Examples 1-3 and Comparative Examples 1-4), a 0.1% DMSO solvent control group, and a blank control group.

[0068] (3) Cell proliferation assay: HaCaT cells and HPV11-HaCaT cells were inoculated into 96-well plates (2 × 10 4Cells were cultured overnight at 200 μl / well of each drug solution at different concentrations. The final concentrations for each Example and Comparative Example group were 8, 4, 2, 1, and 0.5 mg of the crude drug / mL (diluted in DMSO), respectively. The 0.1% DMSO vehicle control group contained 0.1% DMSO, and the blank control group received an equal amount of culture medium. Three replicates were set up for each group. After 24 hours of culture, 20 μl / well of 5 mg / mL MTT solution was added. Culture was continued for another 4 hours, the culture medium was discarded, and 150 μl / well of DMSO was added and mixed in the dark for 10 minutes with shaking to fully dissolve the blue-purple precipitate. The absorbance was measured at 550 nm using a microplate reader. Cell survival rates were calculated for the different concentration groups: cell survival rate = A550 of experimental group / A550 of control group × 100%. The mean cell survival rate of each group was calculated using each well, and then the relative expression of HPV11E7 mRNA was determined using RT-PCR at a drug concentration of 1 mg crude drug / ml in Examples 1-3 and Comparative Examples 1-4. The experimental results are shown in Tables 4 to 11.

[0069] Table 4

[0070] Table 5

[0071] Table 6

[0072] Table 7

[0073] Table 8

[0074] Table 9

[0075] Table 10

[0076] Table 11

[0077] The MTT assay uses MTT (4,5-dimethylthiazole) 2,5-diphenyltetrazolium bromide) to stain cells. By measuring changes in optical density (OD), cell viability can be assessed, understanding the protective effects of the drug and enabling quantitative analysis of its efficacy. Tables 4-10 show that the combined Chinese herbal compositions of Examples 1-3 and Comparative Examples 1-4 exhibited a certain degree of inhibitory effect on HPV11-HaCaT cells and HaCaT cells at concentrations of 1-8 mg of crude drug / ml. Example 1 showed the best effect, with cell viability rates of 1-67% against HPV11-HaCaT cells, respectively. This exhibited a dose-dependent effect.

[0078] In Table 11, the relative expression of HPV11 E7 mRNA in each Example group and Comparative Example group showed a certain degree of decrease compared to the 0.1% DMSO vehicle control group. The composite Chinese medicine composition of the present invention is a streamlined combination of the Chinese medicine preparation of Comparative Example 4. After the combined combination, at the same dosage, it has a comparable inhibitory effect on HPV11 E7 mRNA expression, demonstrating its simplicity and effectiveness, and has promising application prospects.

[0079] The above detailed description is a specific description of one feasible embodiment of the present invention. This embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not depart from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A compound Chinese medicine composition for treating skin warts and condyloma, characterized in that: The raw materials of the compound traditional Chinese medicine composition include the following components: astilbin, matrine, houttuynia cordata, honeycomb, podophyllotoxin, cnidium monnieri and javanica javanica.

2. The compound Chinese medicine composition according to claim 1, characterized in that The raw materials of the compound Chinese medicine composition include the following components by weight: 0.03-3.6 parts of astilbin, 0.003-1 part of matrine, 10-30 parts of houttuynia cordata, 10-30 parts of honeycomb, 0.01-5 parts of podophyllotoxin, 10-30 parts of cnidium monnieri and 1-20 parts of Brucea javanica.

3. The compound Chinese medicine composition according to claim 1, characterized in that The raw materials of the compound Chinese medicine composition include the following components by weight: 0.1-1.5 parts of astilbin, 0.1-0.9 parts of matrine, 15-25 parts of houttuynia cordata, 10-20 parts of honeycomb, 0.06-5 parts of podophyllotoxin, 10-15 parts of cnidium monnieri and 5-15 parts of javanica javanica.

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

5. The compound Chinese medicine composition according to claim 1, characterized in that The raw materials of the compound Chinese medicine composition include the following components by weight: 1 part of astilbin, 0.5 part of matrine, 20 parts of houttuynia cordata, 15 parts of honeycomb, 1 part of podophyllotoxin, 10 parts of cnidium monnieri and 10 parts of javanica javanica.

6. A method for preparing the compound Chinese medicine composition for treating skin warts and condyloma according to any one of claims 1 to 5, characterized in that: The steps include: (1) Pressing the Brucea javanica to extract Brucea javanica oil to obtain medicinal residue; (2) Add water to Houttuynia cordata, beehive, Cnidium monnieri and medicinal residues, and boil to obtain a decoction. Add tannin enzyme to the decoction to deactivate it and obtain an enzymatic solution; (3) The enzymatic hydrolysate, Brucea javanica oil, astilbin, matrine and podophyllotoxin are mixed and dispersed evenly.

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

8. The pharmaceutical preparation according to claim 6, characterized in that The pharmaceutical preparation is an external preparation, and the dosage form is selected from cream or tincture.

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

10. Use of the compound Chinese medicine composition according to any one of claims 1 to 5 or the pharmaceutical preparation according to any one of claims 7 to 9 in the preparation of a medicament for treating skin warts and / or condylomata.

Images