Application of berberine or berberine hydrochloride in preparation of medicine for resisting HPV infection related diseases

Abstract

The invention discloses application of berberine or berberine hydrochloride in preparation of drugs for resisting HPV infection related diseases. Preferably, the berberine is an extract of natural plants such as coptis chinensis, golden cypress, berberis poiretii schneid or Chinese mahonia. According to the application, the new application of the berberine or the hydrochloride thereof in the aspect of resisting the HPV virus is expanded, and the berberine or the hydrochloride thereof is used for preparing the preparation for resisting the HPV infection, so that diseases related to the HPV infection can be prevented or treated, and the virus is promoted to become negative. As a natural plant extract, berberine is high in safety and not easy to generate drug resistance, and has important clinical significance when being used for preparing Chinese patent medicine products for resisting HPV infection.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and specifically relates to the application of berberine or its hydrochloride in the preparation of drugs for treating HPV-related diseases. Background Technology

[0002] Human papillomavirus (HPV) belongs to the papillomaviridae family and is a type of epitheliophilic, non-enveloped, double-stranded DNA virus. HPV can invade the human body through damaged mucous membranes and the epidermis. Approximately 30-40 types of HPV can infect humans, and the incidence of female genital tract infections has been gradually increasing in recent years. Studies have shown that more than 80% of women will experience at least one HPV infection in their lifetime. Repeated infections with certain high-risk types may develop into precancerous lesions or even invasive cancers. Statistics show that 99.7% of cervical cancers are caused by HPV infection. The International Agency for Research on Cancer (IARC) indicates that HPV types 16 and 18 are the main types causing cervical cancer, accounting for nearly 70% of infections combined. HPV infection has become a serious public health problem threatening human health.

[0003] In my country, current Western medicine methods for combating HPV infection include prevention and treatment. While vaccines are available for HPV prevention, effective antiviral treatments are lacking, and the efficacy of commercially available antiviral gels is often uncertain. The inventors of this invention accidentally discovered that berberine or its hydrochloride has a very good inhibitory effect on HPV and can be used to treat HPV infection and promote viral clearance. Berberine, as a natural plant extract, has high safety, and its use in the preparation of traditional Chinese medicine products for combating HPV infection has significant clinical implications. Summary of the Invention

[0004] To achieve this objective, the present invention adopts the following technical solution:

[0005] In a first aspect, the present invention provides the use of berberine or its hydrochloride in the preparation of drugs for treating HPV infection-related diseases.

[0006] Preferably, the berberine is extracted from natural plants such as Coptis chinensis, Phellodendron chinense, Trifolium repens, or Mahonia fortunei.

[0007] Preferably, the extraction method of berberine is as follows: after crushing natural plants such as Coptis chinensis, Phellodendron chinense, Trichosanthes kirilowii, or Mahonia fortunei, berberine is extracted with ethanol.

[0008] Preferably, the berberine hydrochloride is prepared from berberine extracted from natural plants.

[0009] Preferably, HPV infection-related diseases include warts, recurrent respiratory papillomatosis, conjunctival papilloma, cervical cancer, or anal cancer.

[0010] Secondly, the present invention provides an anti-HPV infection preparation, wherein the raw materials for preparing the preparation include berberine or its hydrochloride and pharmaceutically acceptable excipients.

[0011] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of triethanolamine, carbomer, purified water, or alcohols having 2-8 carbon atoms in their molecular formula.

[0012] Preferably, the dosage form of the preparation includes gels, ointments, suppositories, solutions, films, or liniments.

[0013] Compared to existing technologies, this invention utilizes berberine or its hydrochloride to combat HPV infection, exhibiting good efficacy and a low likelihood of developing drug resistance. It shows promising application prospects in the development of drugs for the prevention or treatment of HPV. Attached Figure Description

[0014] Figure 1 The image shows the results of Berberin's in vitro anti-HPV activity and cytotoxicity assay. Detailed Implementation

[0015] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0016] Example 1

[0017] This embodiment provides a method for preparing berberine or its hydrochloride, comprising the following steps:

[0018] (1) After crushing Coptis chinensis, extract it with 75% ethanol by heat extraction;

[0019] (2) Dissolve the extract obtained in step (1) with dilute hydrochloric acid, remove impurities from the mother liquor, alkalize it to precipitate the precipitate and filter the precipitate;

[0020] (3) After washing with water and recrystallizing with ethanol, berberine was obtained;

[0021] (4) The berberine obtained in step (3) is neutralized with hydrochloric acid to obtain berberine hydrochloride.

[0022] Example 2

[0023] Detection of berberine's in vitro anti-HPV virus activity and cytotoxicity

[0024] 1. Experimental materials

[0025] (1) Test sample

[0026] The test sample, Berberin, was provided by Shanghai WuXi AppTec New Drug Development Co., Ltd., as shown in Table 1.

[0027] Table 1

[0028] Eight concentrations were tested in the sample, with duplicate wells.

[0029] The highest detection point concentration of Berberin in the test sample was 100,000 ng / ml. After a 2-fold serial dilution, the eight concentration points were 100,000 ng / ml, 50,000 ng / ml, 25,000 ng / ml, 12,500 ng / ml, 6,250 ng / ml, 3,125 ng / ml, 1,562.5 ng / ml, and 781.25 ng / ml, respectively.

[0030] (2) Virus-like particles (VLPs)

[0031] HPV16 and HPV18 GFP VLPs were provided by Shanghai WuXi AppTec Co., Ltd.

[0032] (3) Cells

[0033] 293FT (ATCC-CRL-3216) cells were provided by Shanghai WuXi AppTec Co., Ltd.

[0034] (4) Reagents and consumables

[0035] The key reagents and consumables used in this study were provided by Shanghai WuXi AppTec New Drug Development Co., Ltd., as shown in Table 2.

[0036] Table 2

[0037] (5) Instruments and equipment

[0038] The main instruments used in this study were a carbon dioxide incubator (Thermo Fisher Scientific-Heracell 240i) and a laser scanning microplate cell analyzer (TTP LabTech -Acumen® Cellista).

[0039] 2. Experimental Methods

[0040] (1) Experiment on the effect of in vitro on HPV virus activity

[0041] On day 1, 293FT cells were seeded into 96-well plates at a density of 4e+4 cells per well and cultured overnight in a 5% CO2, 37°C cell culture incubator.

[0042] On day 2, the virus and the test sample Berberin were diluted separately using serum-free medium. 50 μl of the diluted test sample and 50 μl of 100 TCID50 HPV16 VLP / 500 TCID50 HPV18 VLP were added to cell wells respectively. Cell controls (cells, without test sample and virus infection) and virus controls (cells infected with virus, without test sample treatment) were also set up.

[0043] The cells containing the added virus and test sample were placed in a 5% CO2, 37°C incubator and cultured for 3 days.

[0044] The antiviral activity of the test samples is expressed as the efficiency (%) of the samples at different concentrations in inhibiting viral infection of cells.

[0045] (2) In vitro cytotoxicity test

[0046] On day 1, 293FT cells were seeded into 96-well plates at a density of 4e+4 cells per well and cultured overnight in a 5% CO2, 37°C cell culture incubator.

[0047] On day 2, the Berberin test sample was diluted separately using serum-free medium. 50 μl of the diluted test sample and 50 μl of medium were added to cell wells free of virus infection. Cell control (cells, no test sample, and virus infection) and medium control (cell-free, no test sample, and virus infection) were also set up.

[0048] The cells with the added test sample were placed in a 5% CO2, 37°C incubator and cultured for 3 days.

[0049] The cytotoxicity of the test samples is expressed as the inhibition rate (%) of the activity of 293FT cells by the samples at different concentrations.

[0050] 3. Experimental Results

[0051] The results of the in vitro anti-HPV virus activity of the test samples are shown in Tables 3 and 4 below. Figure 1 .

[0052] Table 3. In vitro anti-HPV16 virus activity of the test samples

[0053] Table 4. In vitro anti-HPV18 virus activity of the test samples

[0054] As shown in Tables 3 and 4, the tested sample Berberin has a significant inhibitory effect on HPV16 and HPV18 viruses, with EC50 values ​​of 4440 nM and 3790 nM, respectively.

[0055] The in vitro cytotoxicity results of the test samples are shown in Table 5 and Figure 1 .

[0056] Table 5. Cytotoxicity of the test samples

[0057] As shown in Table 5, the half-maximal toxic concentration (CC50) of the test sample Berberin was 88275 nM. The cells could still maintain a high cell viability even at a high drug concentration, indicating that the test sample Berberin had low toxicity and could be used to prepare a safe anti-HPV drug.

[0058] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments in this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. Any modifications made within the scope of the inventive concept should be included within the protection scope of this invention.

Claims

1. Application of berberine or its hydrochloride in the preparation of drugs for treating HPV infection-related diseases.

2. The application according to claim 1, characterized in that, The berberine mentioned is extracted from natural plants such as Coptis chinensis, Phellodendron chinense, Trifolium repens, or Mahonia japonica.

3. The application according to claim 2, characterized in that, The extraction method of berberine is as follows: after crushing natural plants such as Coptis chinensis, Phellodendron chinense, Trichosanthes kirilowii or Mahonia fortunei, it is prepared by ethanol extraction.

4. The application according to claim 1 or 2, characterized in that, The berberine hydrochloride is prepared from berberine extracted from natural plants.

5. The application according to any one of claims 1-4, characterized in that, The HPV infection-related diseases include warts, recurrent respiratory papillomatosis, conjunctival papilloma, cervical cancer, or anal cancer.

6. An anti-HPV infection preparation, characterized in that, The raw materials for preparing the formulation include berberine or its hydrochloride and pharmaceutically acceptable excipients.

7. The formulation according to claim 6, characterized in that, The pharmaceutically acceptable excipients include any one or a combination of at least two of the following: triethanolamine, carbomer, purified water, or alcohols having 2-8 carbon atoms in their molecular formula.

8. The formulation according to claim 6 or 7, characterized in that, The dosage forms of the preparation include gels, ointments, suppositories, solutions, films, or liniments.

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