A traditional Chinese medicine composition for inhibiting HPV infection, a preparation method thereof and application thereof

Abstract

The present application relates to the technical field of traditional Chinese medicine preparation, in particular to a traditional Chinese medicine composition for inhibiting HPV infection, a preparation method and application thereof. The traditional Chinese medicine composition is prepared from the following raw materials: smilax glabra, sophora flavescens, houttuynia cordata, honeycomb, dysphania, cnidium and brucea javanica; the preparation method comprises the following steps: (1) taking brucea javanica, crushing it and then preparing brucea javanica oil through supercritical extraction; (2) mixing cnidium and dysphania to obtain mixture A, adding ethanol for extraction, and then performing solid-liquid separation to obtain an alcohol extract; (3) mixing sophora flavescens, houttuynia cordata, smilax glabra and honeycomb to obtain mixture B, adding water for extraction, and then performing solid-liquid separation to obtain a water extract; (4) uniformly mixing the brucea javanica oil, the alcohol extract and the water extract to obtain the traditional Chinese medicine composition. The supercritical extraction adopts liquid carbon dioxide and a eutectic solvent, and the eutectic solvent is composed of a quaternary ammonium salt and an organic acid. The traditional Chinese medicine composition prepared by the present application has better HPV infection inhibition effect and lower side effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine preparation, in particular to a traditional Chinese medicine composition for inhibiting HPV infection and a preparation method and application thereof. BACKGROUND

[0002] Condyloma acuminata (CA) is a sexually transmitted disease mainly caused by human papillomavirus (HPV) infection, which is characterized by warty hyperplasia of skin and mucosa. The infection of CA is divided into three types, namely, latent infection, subclinical infection and clinical CA, and the subclinical infection is a transitional period, which can evolve into the other two conditions, depending on the immune function of the host.

[0003] So far, more than 200 types of HPV have been found, and according to the relationship between HPV infection and the risk of cervical cancer, they are divided into low-risk and high-risk types. The low-risk HPV mainly includes HPV6, HPV11, HPV30, HPV42 and HPV43, etc., which mainly cause benign lesions such as low-grade cervical intraepithelial neoplasia and genital warts. The high-risk HPV mainly includes HPV16, HPV18, HPV31, HPV33 and HPV52, etc., which mainly cause lesions such as cervical intraepithelial neoplasia, cervical cancer and external genital cancer. Although many types of HPV can cause CA, the vast majority of CA patients are infected with HPV6 and HPV11. CA is prone to recurrence and requires long-term repeated treatment, which seriously affects the quality of life of patients.

[0004] At present, the treatment methods for HPV in clinical practice are divided into Western medicine treatment, traditional Chinese medicine treatment and combination of traditional Chinese medicine and Western medicine. Western medicine treatment generally uses physical treatment such as laser, freezing, microwave and ozone fumigation, surgical treatment and drug treatment such as interferon and vaccine. Physical treatment and surgical treatment are easy to cause organic damage, and drug treatment focuses on the research of vaccine, mainly preventive vaccination, while therapeutic vaccine has not been realized.

[0005] Traditional Chinese medicine treatment adopts the mode of internal and external use or combination of the two. External preparations such as Flos Lonicerae, Folium Isatidis, Sophora flavescens, Cnidium monnieri, honeycomb, Calophyllum inophyllum and more than 10 kinds of traditional Chinese medicine compound preparation Pa Teling have been proved to be able to inhibit the expression of HPV related genes (such as E6, E7), affect the activity of Wnt / β-catenin signaling pathway, and thus inhibit the proliferation of HPV and the malignant transformation of cells.

[0006] Chinese patent CN100502920C discloses a traditional Chinese medicine preparation for treating condyloma acuminatum, which is composed of 15-60 parts of smilax glabra, 7.5-30 parts of sophora flavescens, 10-40 parts of houttuynia cordata, 7.5-30 parts of halloysite, 6-24 parts of dysosma versipellis, 7.5-30 parts of cnidium monnieri and 2.5-10 parts of brucea javanica. However, the preparation method of the traditional Chinese medicine preparation needs to be further improved so as to improve the HPV infection inhibition effect and further reduce the side effects.

[0007] Therefore, it is necessary to develop a preparation method and application of a traditional Chinese medicine composition for HPV infection inhibition which can solve the above technical problems. SUMMARY

[0008] The purpose of the present application is to overcome the deficiencies of the prior art and provide a traditional Chinese medicine composition for HPV infection inhibition which has better HPV infection inhibition effect and lower side effects, and a preparation method and application thereof.

[0009] The present application is realized by the following technical solutions:

[0010] The present application provides, in a first aspect, a preparation method of a traditional Chinese medicine composition for HPV infection inhibition, wherein the traditional Chinese medicine composition is prepared from the following raw materials: smilax glabra, sophora flavescens, houttuynia cordata, halloysite, dysosma versipellis, cnidium monnieri and brucea javanica.

[0011] The preparation method comprises the following steps:

[0012] (1) Take brucea javanica, crush it and then prepare brucea javanica oil by supercritical extraction;

[0013] (2) Mix cnidium monnieri and dysosma versipellis to obtain a mixture A, add ethanol to extract, separate the solid and liquid to obtain an alcohol extract;

[0014] (3) Mix sophora flavescens, houttuynia cordata, smilax glabra and halloysite to obtain a mixture B, add water to extract, separate the solid and liquid to obtain a water extract;

[0015] (4) Mix the brucea javanica oil, the alcohol extract and the water extract uniformly, and the traditional Chinese medicine composition for HPV infection inhibition is obtained.

[0016] In step (1), the solvent used in the supercritical extraction includes liquid carbon dioxide and a eutectic solvent, wherein the eutectic solvent is composed of a quaternary ammonium salt and an organic acid, and the molar ratio of the two is 0.5-2:1.

[0017] As an embodiment of the present application, the quaternary ammonium salt includes at least one of choline chloride and betaine.

[0018] As an embodiment of the present application, the organic acid includes at least one of lactic acid and malic acid.

[0019] As an embodiment of the present application, the ratio of the amount of the brucea javanica to the amount of the deep eutectic solvent in step (1) is 2 g / mL-4 g / mL.

[0020] As an embodiment of the present application, the flow rate of the liquid carbon dioxide in step (1) is 90-120 L / h.

[0021] As an embodiment of the present application, the pressure of the supercritical extraction in step (1) is 20-40 MPa, the temperature is 30-50℃, and the time is 1-2 h.

[0022] As an embodiment of the present application, the brucea javanica is crushed and then passed through a 20-60 mesh sieve in step (1).

[0023] As an embodiment of the present application, the Cnidium monnieri and Typhonium giganteum are extracted with 75-85% ethanol for 1-3 times in step (2), the extraction temperature is 75-85℃ for each time, the extraction time is 1-3 h for each time, and the amount of the ethanol is 6-10 times the mass of the mixture A for each time.

[0024] As an embodiment of the present application, the solid-liquid separation in step (2) is filtration.

[0025] As an embodiment of the present application, step (2) further comprises the following step: mixing the brucea javanica residue obtained after the supercritical extraction in step (1) with the Cnidium monnieri and Typhonium giganteum to obtain the mixture A.

[0026] As an embodiment of the present application, step (2) further comprises the following step: recovering the ethanol in the alcohol extract to obtain the alcohol extract.

[0027] As an embodiment of the present application, step (2) further comprises the following steps: recovering the ethanol in the alcohol extract to obtain a concentrated solution, diluting with water, adsorbing on a macroporous resin, collecting the eluate after elution with an eluent, and concentrating and drying to obtain the alcohol extract.

[0028] Preferably, the water is added to dilute the concentrated solution to 2-5 times the volume of the concentrated solution in the above step.

[0029] Preferably, the macroporous resin in the above step is at least one of D101 type macroporous resin and AB-8 type macroporous resin.

[0030] Preferably, the eluent in the above step comprises water, 25-35% ethanol, and 65-75% ethanol, and the water, 25-35% ethanol, and 65-75% ethanol are used for elution in sequence, the 65-75% ethanol eluate is collected, and the alcohol extract is obtained by concentrating and drying.

[0031] As an embodiment of the present application, the mixture B is obtained by mixing the Sophora flavescens, Houttuynia cordata, Smilax glabra and honeycomb in step (3).

[0032] As an embodiment of the present application, the solid-liquid separation in step (3) is at least one of standing and centrifugation.

[0033] As an embodiment of the present application, step (3) further comprises the following step: mixing the alcohol-extraction residue obtained after the solid-liquid separation in step (2) with the Sophora flavescens, Houttuynia cordata, Smilax glabra and honeycomb to obtain the mixture B.

[0034] As an embodiment of the present application, step (3) further comprises the following step: concentrating the water-extraction liquid to obtain the water-extraction thick paste.

[0035] As an embodiment of the present application, step (3) further comprises the following step: adding ethanol to the water-extraction liquid, standing, solid-liquid separation, and concentrating the liquid to obtain the water-extraction thick paste. Specifically, ethanol is added to the water-extraction liquid, the mixture is stood, the supernatant is filtered, and the filtrate is concentrated to obtain the water-extraction thick paste.

[0036] Preferably, step (3) further comprises the following step: adding ethanol to the water-extraction liquid to a content of 65-75%, standing, filtering the supernatant, and concentrating to a relative density of 1.30-1.35 at 60°C to obtain the water-extraction thick paste.

[0037] More preferably, step (3) further comprises the following step: adding ethanol to the water-extraction liquid to a content of 65-75%, standing at 4°C for 24 hours, filtering the supernatant (0.45 μm microfiltration membrane), and concentrating to a relative density of 1.30-1.35 at 60°C to obtain the water-extraction thick paste.

[0038] As an embodiment of the present application, step (4) is replaced by the following step: uniformly mixing the Calophyllum inophyllum oil, the alcohol-extraction thick paste and the water-extraction thick paste to obtain the product.

[0039] As an embodiment of the present application, the raw materials are used in the following amounts: Smilax glabra 15-60 parts, Sophora flavescens 7.5-30 parts, Houttuynia cordata 10-40 parts, honeycomb 7.5-30 parts, Tripterygium wilfordii Hook.f. 6-24 parts, Cnidium monnieri 7.5-30 parts, and Calophyllum inophyllum 2.5-10 parts.

[0040] The present application provides a traditional Chinese medicine composition prepared by the preparation method.

[0041] The present application provides the use of the traditional Chinese medicine composition in the preparation of a medicine for inhibiting HPV infection.

[0042] The present application has the following advantages:

[0043] The application optimizes the extraction process of brucea javanica oil, adopts liquid carbon dioxide and eutectic solvent for supercritical extraction, and further optimizes the composition of the eutectic solvent, so that the extracted brucea javanica oil and other medicinal ingredients are matched, the effect of resisting HPV infection is significantly improved, and the toxic side effects are further reduced.

[0044] The application adds a purification process on the basis of alcohol extraction or water extraction of each medicinal ingredient, so that the effect of resisting HPV infection of the medicine is further improved, and the toxic side effects are further reduced. DETAILED DESCRIPTION

[0045] The advantages and characteristics of the application will be more apparent with the description. However, these examples are only exemplary and do not constitute any limitation on the scope of the application. Those skilled in the art should understand that the details and forms of the technical solutions of the application can be modified or replaced without departing from the spirit and scope of the application, and such modifications and replacements all fall within the protection scope of the application.

[0046] Example 1

[0047] A traditional Chinese medicine composition for inhibiting HPV infection, the amount of each raw material is as follows according to weight fraction: 30 parts of smilax glabra, 15 parts of sophora flavescens, 20 parts of houttuynia cordata, 15 parts of honeycomb, 12 parts of dysphania, 15 parts of cnidium, and 5 parts of brucea javanica.

[0048] The preparation method of the traditional Chinese medicine composition comprises the following steps:

[0049] (1) Take brucea javanica, crush it, and pass it through a 40-mesh sieve to prepare brucea javanica oil by supercritical extraction. The solvent used in the supercritical extraction includes liquid carbon dioxide (flow rate 100 L / h) and eutectic solvent (the amount ratio of brucea javanica to eutectic solvent is 3 g / mL). The eutectic solvent is composed of choline chloride and lactic acid, and the molar ratio of the two is 1.2:1. The supercritical extraction pressure is 30 MPa, the temperature is 40℃, and the time is 1.5 h;

[0050] (2) Mix cnidium and dysphania, and extract them three times with 80% ethanol: the first time is 10 times the amount of ethanol for 1 h, the second time is 8 times the amount of ethanol for 1 h, and the third time is 6 times the amount of ethanol for 1 h. Combine the three extraction liquids and filter to obtain the alcohol extract;

[0051] (3) Mix sophora flavescens, houttuynia cordata, smilax glabra, and honeycomb, and decoct them three times with water: the first time is 10 times the amount of water for 2 hours, the second time is 8 times the amount of water for 1.5 hours, and the third time is 6 times the amount of water for 1 hour. Combine the decocted liquids, centrifuge, and obtain the water extract;

[0052] (4) mix the brucea javanica oil, the alcohol extract and the water extract evenly, and the product is obtained.

[0053] Example 2

[0054] A traditional Chinese medicine composition for inhibiting HPV infection, the raw material usage is as follows according to weight fraction: 15 parts of smilax glabra, 30 parts of sophora flavescens, 40 parts of houttuynia cordata, 7.5 parts of honeycomb, 6 parts of dysphania, 7.5 parts of cnidium and 2.5 parts of brucea javanica.

[0055] The preparation method of the traditional Chinese medicine composition comprises the following steps:

[0056] (1) take brucea javanica, crush it and pass it through a 20-mesh sieve, and then prepare brucea javanica oil through supercritical extraction, the solvent used in the supercritical extraction includes liquid carbon dioxide (flow rate 90 L / h) and a eutectic solvent (the usage ratio of brucea javanica to the eutectic solvent is 2 g / mL), the eutectic solvent is composed of betaine and malic acid, the molar ratio of the two is 0.5:1, the pressure of the supercritical extraction is 20 MPa, the temperature is 30℃, and the time is 1 h;

[0057] (2) mix cnidium and dysphania, and then extract them with 6 times the amount of 75% ethanol for 3 times, the extraction temperature of each time is 75℃, the extraction time of each time is 1 h, and then filter to obtain an alcohol extract;

[0058] (3) mix sophora flavescens, houttuynia cordata, smilax glabra and honeycomb, and then decoct them with 6 times the amount of water for 3 times, the decocting time of each time is 1.5 h, the decocting liquid is left to stand, and then centrifuged to obtain a water extract;

[0059] (4) mix the brucea javanica oil, the alcohol extract and the water extract evenly, and the product is obtained.

[0060] Example 3

[0061] A traditional Chinese medicine composition for inhibiting HPV infection, the raw material usage is as follows according to weight fraction: 60 parts of smilax glabra, 7.5 parts of sophora flavescens, 10 parts of houttuynia cordata, 30 parts of honeycomb, 24 parts of dysphania, 30 parts of cnidium and 10 parts of brucea javanica.

[0062] The preparation method of the traditional Chinese medicine composition comprises the following steps:

[0063] (1) take brucea javanica, crush it and pass it through a 60-mesh sieve, and then prepare brucea javanica oil through supercritical extraction, the solvent used in the supercritical extraction includes liquid carbon dioxide (flow rate 120 L / h) and a eutectic solvent (the usage ratio of brucea javanica to the eutectic solvent is 4 g / mL), the eutectic solvent is composed of choline chloride and malic acid, the molar ratio of the two is 2:1, the pressure of the supercritical extraction is 40 MPa, the temperature is 50℃, and the time is 2 h;

[0064] (2) Take the Cnidium and Typhonium mixed, add 10 times the amount of 85% ethanol extraction 1 times, extraction temperature is 85℃, extraction time is 3h, filter to get alcohol extract;

[0065] (3) Take the Sophora, Houttuynia cordata, Smilax glabra and honeycomb mixed, add 10 times the amount of water decoction 1 times, decoction time is 4.5h, the decoction liquid is placed, centrifugal, get water extract;

[0066] (4) The Bruguiera oil, alcohol extract and water extract are mixed uniformly, and the Bruguiera oil is obtained.

[0067] Example 4

[0068] The difference from example 1 is only in the preparation method, specifically, the drug residue obtained after each step is extracted in the next step, and the alcohol extract and water extract are concentrated, as follows:

[0069] The preparation method of the traditional Chinese medicine composition comprises the following steps:

[0070] (1) Take the Bruguiera, crush it through a 40-mesh sieve, and prepare Bruguiera oil by supercritical extraction. The solvent used in supercritical extraction includes liquid carbon dioxide (flow rate 100 L / h) and eutectic solvent (the ratio of Bruguiera to eutectic solvent is 3g / mL). The eutectic solvent is composed of choline chloride and lactic acid, and the molar ratio of the two is 1.2:1. The supercritical extraction pressure is 30MPa, the temperature is 40℃, and the time is 1.5h. The Bruguiera residue obtained after extraction is reserved;

[0071] (2) Take the Bruguiera residue, Cnidium and Typhonium mixed, use 80% ethanol to extract three times: the first time is 10 times the amount of ethanol extraction 1h, the second time is 8 times the amount of ethanol extraction 1h, the third time is 6 times the amount of ethanol extraction 1h, combine the three times of extraction liquid, filter to get alcohol extract and alcohol extract residue, recover ethanol in alcohol extract, get alcohol extract;

[0072] (3) Take the alcohol extract residue, Sophora, Houttuynia cordata, Smilax glabra and honeycomb mixed, water decoction three times: the first time is 10 times the amount of water decoction 2 hours, the second time is 8 times the amount of water decoction 1.5 hours, the third time is 6 times the amount of water decoction 1 hour, combine the decoction liquid, place it, centrifugal, get water extract, concentrate to 60℃ relative density 1.35, get water extract thick paste;

[0073] (4) Mix the Bruguiera oil, alcohol extract and water extract thick paste uniformly, and the Bruguiera oil is obtained.

[0074] Example 5

[0075] The difference from example 4 is only in the preparation method, specifically, the alcohol extract and water extract are further purified, as follows:

[0076] The preparation method of the traditional Chinese medicine composition comprises the following steps:

[0077] (1) Take the Calophyllum inophyllum, crush it, and pass it through a 40-mesh sieve. Then, prepare the Calophyllum inophyllum oil by supercritical extraction. The solvent used in the supercritical extraction includes liquid carbon dioxide (flow rate: 100 L / h) and a eutectic solvent (the ratio of the amount of Calophyllum inophyllum to the amount of the eutectic solvent is 3 g / mL). The eutectic solvent is composed of choline chloride and lactic acid, and the molar ratio of the two is 1.2:1. The pressure of the supercritical extraction is 30 MPa, the temperature is 40°C, and the time is 1.5 h. The residue of the Calophyllum inophyllum obtained after the extraction is retained.

[0078] (2) Mix the residue of the Calophyllum inophyllum, the Cnidium monnieri, and the Typhonium giganteum, and extract them three times with 80% ethanol. The first extraction is performed with 10 times the amount of ethanol for 1 h, the second extraction is performed with 8 times the amount of ethanol for 1 h, and the third extraction is performed with 6 times the amount of ethanol for 1 h. Then, combine the three extraction solutions, filter them to obtain the ethanol extraction solution and the ethanol extraction residue, recover the ethanol in the ethanol extraction solution, dilute it with water to 4 times the amount, and then adsorb it on a D101 macroporous resin. Then, elute the adsorbed solution with water, 30% ethanol, and 70% ethanol, respectively. Collect the 70% ethanol eluate, concentrate and dry it to obtain the ethanol extract.

[0079] (3) Mix the ethanol extraction residue, the Sophora flavescens, the Houttuynia cordata, the Smilax glabra, and the honeycomb, and decoct them three times with water. The first decoction is performed with 10 times the amount of water for 2 h, the second decoction is performed with 8 times the amount of water for 1.5 h, and the third decoction is performed with 6 times the amount of water for 1 h. Then, combine the decoction solutions, centrifuge them, and obtain the water extraction solution. Then, add ethanol to the water extraction solution to obtain an ethanol content of 70%, and then place the solution at 4°C for 24 h. Then, filter the supernatant (0.45 μm microfiltration membrane), concentrate it to a relative density of 1.35 at 60°C, and obtain the water extract thick paste.

[0080] (4) Mix the Calophyllum inophyllum oil, the ethanol extract, and the water extract thick paste uniformly, and obtain the traditional Chinese medicine composition.

[0081] Comparative Example 1

[0082] The difference between Comparative Example 1 and Example 1 is only that the solvent used in the supercritical extraction in step (1) of the preparation process is different. Specifically, only liquid carbon dioxide is used, and no eutectic solvent is used. The other conditions are the same.

[0083] Comparative Example 2

[0084] The difference between Comparative Example 2 and Example 1 is only that the composition of the eutectic solvent in step (1) of the preparation process is different. Specifically, the molar ratio of the quaternary ammonium salt to the organic acid is 0.3:1. The other conditions are the same.

[0085] Comparative Example 3

[0086] The difference between Comparative Example 3 and Example 1 is only that the composition of the eutectic solvent in step (1) of the preparation process is different. Specifically, the molar ratio of the quaternary ammonium salt to the organic acid is 2.5:1. The other conditions are the same.

[0087] Comparative Example 4

[0088] The difference from Example 1 is only in the composition of the eutectic solvent in step (1) in the preparation process, specifically, the organic acid is replaced by an equal amount of polyol hydrogen bond donor ethylene glycol.

[0089] Comparative Example 5

[0090] The difference from Example 5 is only in the different step parameters for further purification treatment of the alcohol extract and water extract, specifically as follows:

[0091] The preparation method of the traditional Chinese medicine composition comprises the following steps:

[0092] (1) Take Calophyllum inophyllum, crush it and pass it through a 40-mesh sieve, and then prepare Calophyllum inophyllum oil by supercritical extraction. The solvent used in the supercritical extraction includes liquid carbon dioxide (flow rate 100 L / h) and a eutectic solvent (the ratio of Calophyllum inophyllum to the eutectic solvent is 3 g / mL). The eutectic solvent is composed of choline chloride and lactic acid, and the molar ratio of the two is 1.2:1. The pressure of the supercritical extraction is 30 MPa, the temperature is 40°C, and the time is 1.5 h. The Calophyllum inophyllum residue obtained after extraction is retained.

[0093] (2) Mix the Calophyllum inophyllum residue, Cnidium monnieri and Typhonium giganteum, and extract them three times with 80% ethanol: the first time is 10 times the amount of ethanol for 1 h, the second time is 8 times the amount of ethanol for 1 h, and the third time is 6 times the amount of ethanol for 1 h. Combine the three extract solutions, filter to obtain the alcohol extract and alcohol extract residue, recover the ethanol in the alcohol extract, dilute with water to 4 times, adsorb on D101 macroporous resin, and elute with water, 20% ethanol and 60% ethanol in sequence. Collect the 60% ethanol eluate, concentrate and dry to obtain the alcohol extract.

[0094] (3) Mix the alcohol extract residue, Sophora flavescens, Houttuynia cordata, Smilax glabra and honeycomb, and decoct them three times with water: the first time is 10 times the amount of water for 2 hours, the second time is 8 times the amount of water for 1.5 hours, and the third time is 6 times the amount of water for 1 hour. Combine the decoction solutions, centrifuge to obtain the water extract. Add ethanol to a content of 60%, and let it stand at 4°C for 24 hours. Filter the supernatant (0.45 μm microfiltration membrane), concentrate to a relative density of 1.35 at 60°C to obtain the water extract thick paste.

[0095] (4) Mix the Calophyllum inophyllum oil, alcohol extract and water extract thick paste evenly to obtain the product.

[0096] Comparative Example 6

[0097] The difference from Example 5 is only in the different step parameters for further purification treatment of the alcohol extract and water extract, specifically as follows:

[0098] The preparation method of the traditional Chinese medicine composition comprises the following steps:

[0099] (1) Take brucea javanica, crush it and pass it through a 40-mesh sieve, and then prepare brucea javanica oil by supercritical extraction. The solvent used in the supercritical extraction includes liquid carbon dioxide (flow rate 100 L / h) and a eutectic solvent (the ratio of brucea javanica to the eutectic solvent is 3 g / mL). The eutectic solvent is composed of choline chloride and lactic acid, and the molar ratio of the two is 1.2:1. The supercritical extraction is carried out at a pressure of 30 MPa and a temperature of 40°C for 1.5 h. The residue obtained after extraction is brucea javanica residue.

[0100] (2) Take the brucea javanica residue, cnidium fruit, and typhonium giganteum, mix them together, and extract them three times with 80% ethanol: the first extraction is carried out with 10 times the amount of ethanol for 1 h, the second extraction is carried out with 8 times the amount of ethanol for 1 h, and the third extraction is carried out with 6 times the amount of ethanol for 1 h. Combine the three extracts, filter them to obtain an ethanol extract and an ethanol residue, recover the ethanol from the ethanol extract, dilute it with water to 4 times the amount, and then adsorb it on a D101 macroporous resin. Elute the adsorbed material with water, 40% ethanol, and 80% ethanol in sequence, collect the 80% ethanol eluate, and then concentrate and dry it to obtain an ethanol extract.

[0101] (3) Take the ethanol residue, sophora flavescens, houttuynia cordata, smilax glabra, and honeycomb, mix them together, and decoct them three times with water: the first decoction is carried out with 10 times the amount of water for 2 h, the second decoction is carried out with 8 times the amount of water for 1.5 h, and the third decoction is carried out with 6 times the amount of water for 1 h. Combine the decoctions, centrifuge them, and then obtain a water extract. Add ethanol to the water extract to a concentration of 80%, let it stand at 4°C for 24 h, filter the supernatant (0.45 μm microfiltration membrane), and then concentrate it to a relative density of 1.35 at 60°C to obtain a water extract thick paste.

[0102] (4) Mix the brucea javanica oil, the ethanol extract, and the water extract thick paste together to obtain the final product.

[0103] Test Example 1 Anti-HPV infection and cytotoxicity test

[0104] 1.1 Effect on the proliferation rate and survival rate of normal HaCaT cells and Hela cells

[0105] Before the experiment, the Chinese medicinal composition in Example 1-Example 3 and Comparative Example 1-Comparative Example 4 was pretreated for convenience. Specifically, the ethanol in the Chinese medicinal composition was recovered and then concentrated and dried to obtain an ethanol extract. The water extract in step (3) was concentrated to a relative density of 1.35 at 60°C to obtain a water extract thick paste. The brucea javanica oil, the ethanol extract, and the water extract thick paste were then mixed together to obtain the pretreated Chinese medicinal composition.

[0106] The medicine composition in Example 4 was pretreated, specifically, the alcohol extract was concentrated and dried after recovering ethanol in step (2) to obtain the alcohol extract; the jujube oil, the alcohol extract and the water extract were mixed to obtain the pretreated medicine composition.

[0107] The medicine compositions in Example 1-Example 3, Comparative Example 1-Comparative Example 4, Example 4-Example 5 and Comparative Example 5-Comparative Example 6 were dissolved in DMSO to obtain solutions with different concentrations, filtered, and the filtrate was the test liquid A, which was stored at low temperature for standby.

[0108] In order to intuitively compare the effects of the medicine compositions, the medicine compositions corresponding to 0.5 mg of crude drug were extracted according to Example 1-Example 5 and Comparative Example 1-Comparative Example 6, and 0.1 mL of DMSO was added for dissolution, filtered, and the filtrate was the test liquid B, which was stored at low temperature for standby.

[0109] 1.1.1 Effect on the proliferation rate of HaCaT cells

[0110] HaCaT cells were prepared into a cell suspension with a density of 0.5×10 6 / mL by using DMEM culture solution, inoculated into culture plates, 0.1 mL of the cell suspension was added to each well, 3 replicate wells were set for each test liquid A, and 3 replicate wells were set for each concentration of the test liquid A. The control group was only added with 0.1 mL of the cell suspension; the rest of the wells were added with 0.1 mL of the test liquid A of Example 1-Example 5 and Comparative Example 1-Comparative Example 6, and MTT was added after 24 h of culture, and then incubated for another 4 h, and the cell proliferation rate was calculated by colorimetry with an enzyme-linked immunoassay monitor, to obtain the IC 50 value (mg / mL) and CC 50 value (mg / mL) of the different test liquid A, the IC 50 value represents the concentration of the test liquid A when the proliferation inhibition rate is 50%, and the proliferation inhibition rate = (cell proliferation rate of the control group-cell proliferation rate of the drug-treated well) / cell proliferation rate of the control group*100%; the CC 50 value represents the concentration required for half of the cells to be toxic, i.e., the concentration of the test liquid A when half of the cells are killed.

[0111] HaCaT cells were prepared into a cell suspension with a density of 0.5×10 6Cell suspensions at a density of cells / mL were seeded into culture plates, with 0.1 mL of cell suspension per well. Each test drug solution B was used in triplicate. The control group received only 0.1 mL of cell suspension. The remaining wells were used as drug delivery wells, containing test drug solutions B from Examples 1-5 and Comparative Examples 1-6 (all solutions were added to maintain the same amount of raw drug). After culturing for 24 h, MTT was added, and the cells were cultured for another 4 h. Cell proliferation rate was calculated using an enzyme-linked immunosorbent assay (ELISA) monitor to obtain the cell proliferation inhibition rate (%) of different test drug solutions B.

[0112] The specific data results are shown in Table 1.

[0113] 1.1.2 Effect on HeLa cell proliferation rate

[0114] HeLa cells were cultured in DMEM medium to a concentration of 0.5 × 10⁶ cells / mL. 6 Cell suspensions at a density of 0.1 mL / mL were seeded into culture plates, with three replicates for each test drug solution A and three replicates for each concentration of test drug solution A. The control group received only 0.1 mL of cell suspension; the remaining wells were used as drug delivery wells, with 0.1 mL of test drug solution A from Examples 1-5 and Comparative Examples 1-6 added respectively. After culturing for 24 h, MTT was added, and the cells were cultured for another 4 h. Cell proliferation was calculated using an enzyme-linked immunosorbent assay (ELISA) monitor to obtain the IC50 values ​​for different test drug solutions A. 50 Value (mg / mL) and CC 50 Value (mg / mL).

[0115] HeLa cells were cultured in DMEM medium to a concentration of 0.5 × 10⁶ cells / mL. 6 Cell suspensions at a density of cells / mL were seeded into culture plates, with 0.1 mL of cell suspension per well. Each test drug solution B was used in triplicate. The control group received only 0.1 mL of cell suspension. The remaining wells were used as drug delivery wells, containing test drug solutions B from Examples 1-5 and Comparative Examples 1-6 (all solutions were added to maintain the same amount of raw drug). After culturing for 24 h, MTT was added, and the cells were cultured for another 4 h. Cell proliferation rate was calculated using an enzyme-linked immunosorbent assay (ELISA) monitor to obtain the cell proliferation inhibition rate (%) of different test drug solutions B.

[0116] The specific data results are shown in Table 1.

[0117] Table 1. Effects on normal HaCaT cells and HeLa cells

[0118]

[0119] 1.2 Effect on HPV18-positive HeLa E7 mRNA expression levels

[0120] HPV18 positive Hela cell strain was cultured in RPMI-1640 medium containing 10% fetal bovine serum, and the culture condition was as follows: 37°C, 5% carbon dioxide saturated humidity constant temperature box. Well grown cells were inoculated into culture plates, 7000-8000 cells per well, 0.1 mL of DMEM culture solution was added, the control group was not treated, and the rest of the wells were added with test solution B of Example 1-Example 5 and Comparative Example 1-Comparative Example 6 (all added, control crude drug amount is the same), and the expression of HPV18 E7 mRNA was determined by RT-PCR method after 72 hours, and the results are shown in Table 2.

[0121] Table 2 Influence on the expression of HPV18 positive Hela E7 mRNA

[0122]

[0123] The above detailed description is a specific description of one of the feasible embodiments of the present application, which is not used to limit the patent scope of the present application, and any equivalent implementation or change without departing from the present application shall be included in the scope of the technical scheme of the present application.

Claims

1. A preparation method of a traditional Chinese medicine composition for inhibiting HPV infection, characterized in that, The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 15-60 parts of smilax glabra, 7.5-30 parts of sophora flavescens, 10-40 parts of houttuynia cordata, 7.5-30 parts of honeycomb, 6-24 parts of dysphania, 7.5-30 parts of cnidium monnieri, and 2.5-10 parts of brucea javanica; The preparation method comprises the following steps: (1) taking brucea javanica, crushing it, and then preparing brucea javanica oil through supercritical extraction; (2) taking cnidium monnieri and dysphania, mixing them to obtain mixture A, adding ethanol for extraction, performing solid-liquid separation to obtain an ethanol extract; recovering ethanol in the ethanol extract to obtain a concentrated liquid, diluting the concentrated liquid with water, adsorbing the diluted liquid on a macroporous resin, collecting eluent after elution with an eluent, and then concentrating and drying the eluent to obtain an ethanol extract; (3) taking sophora flavescens, houttuynia cordata, smilax glabra, and honeycomb, mixing them to obtain mixture B, adding water for extraction, performing solid-liquid separation to obtain a water extract; adding ethanol to the water extract, standing, performing solid-liquid separation, and then concentrating the concentrated liquid to obtain a water extract thick paste; (4) uniformly mixing the brucea javanica oil, the ethanol extract, and the water extract thick paste to obtain the traditional Chinese medicine composition. In step (1), the solvent used in the supercritical extraction includes liquid carbon dioxide and a eutectic solvent, the eutectic solvent is composed of a quaternary ammonium salt and an organic acid, and the molar ratio of the two is 0.5-2:1; The quaternary ammonium salt includes at least one of choline chloride and betaine; and the organic acid includes at least one of lactic acid and malic acid. In step (2), the concentrated liquid is diluted with water to 2-5 times the volume of the concentrated liquid; the macroporous resin is at least one of D101 type macroporous resin and AB-8 type macroporous resin; and the eluent includes water, 25-35% ethanol, and 65-75% ethanol, and the eluent is sequentially water, 25-35% ethanol, and 65-75% ethanol, and the 65-75% ethanol eluent is collected, concentrated, and dried to obtain the ethanol extract; in step (3), the water extract is added with ethanol to an ethanol content of 65-75%, and then the supernatant is filtered to obtain the water extract thick paste.

2. The production method according to claim 1, characterized by, In step (1), the ratio of the amount of brucea javanica to the amount of the eutectic solvent is 2 g / mL-4 g / mL; the flow rate of the liquid carbon dioxide is 90-120 L / h; the pressure of the supercritical extraction is 20-40 MPa, the temperature is 30-50°C, and the time is 1-2 h.

3. The preparation method according to claim 1, characterized in that, In step (2), cnidium monnieri and dysphania are taken and extracted with 75-85% ethanol for 1-3 times, the extraction temperature is 75-85°C, the extraction time is 1-3 h, and the amount of ethanol used is 6-10 times the mass of mixture A; in step (2), the solid-liquid separation is performed by filtration; in step (3), sophora flavescens, houttuynia cordata, smilax glabra, and honeycomb are taken, and water is added for decoction and extraction for 1-3 times, the decoction time is 1.5-4.5 h, and the amount of water used is 6-10 times the mass of mixture B; in step (3), the solid-liquid separation is performed by at least one of standing and centrifugation.

4. The traditional Chinese medicine composition prepared by the preparation method of any one of claims 1-3.

5. The use of the traditional Chinese medicine composition of claim 4 in the preparation of a medicine for inhibiting HPV infection.

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