Liquid functional dressing for preventing and treating cervical cancer, condyloma acuminatum and HPV infection and preparation method thereof

Abstract

The present invention provides a liquid functional dressing for preventing and treating cervical cancer, condyloma acuminatum and HPV infection and a preparation method thereof, wherein the liquid functional dressing comprises the following components in mass concentration: 0.05% to 0.1% anhydride-treated bovine β-lactoglobulin, 4 to 10% glycerol, 1 to 5% carbomer and water; the anhydride is selected from at least one of methylhexahydrophthalic anhydride, methyltetrahydrophthalic anhydride and succinic anhydride. The liquid functional dressing of the present invention is suitable for blocking HPV infection, and can effectively prevent and treat cervical cancer and condyloma acuminatum.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine and medical equipment, and in particular to a liquid functional dressing for preventing and treating cervical cancer, condyloma acuminatum and HPV infection and a preparation method thereof. Background Art

[0002] Cervical cancer is the most common malignant tumor of the female reproductive tract, and its main cause is related to persistent infection with human papillomavirus (HPV). Condyloma acuminatum (CA), also known as genital warts, is a sexually transmitted disease caused by HPV infection, with proliferative lesions in the anus and genitals as the main manifestation. The key to preventing and treating cervical cancer and condyloma acuminatum is to inhibit the replication and proliferation of HPV virus.

[0003] Human papilloma virus (HPV) is a double-stranded DNA virus with a diameter of 55nm and a spherical shell. It mainly infects the epithelium of the skin and mucous membranes, causing lesions. Anhydride-modified bovine β-lactoglobulin is a protein obtained after modification with anhydride. Studies have shown that anhydride-modified bovine β-lactoglobulin has a highly effective targeted inhibitory effect on HPV that causes cervical cancer and genital warts, and can effectively eliminate HPV viruses and block persistent HPV infection. However, the current therapeutic effect of anhydride-modified bovine β-lactoglobulin dressing products is limited and it is difficult to meet the treatment needs. Summary of the invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide a liquid functional dressing for preventing and treating cervical cancer, condyloma acuminatum and HPV infection and a preparation method thereof.

[0005] The technical solution of the present invention is as follows:

[0006] A liquid functional dressing for preventing and treating cervical cancer, condyloma acuminatum and HPV infection, comprising the following components in mass concentration: 0.05% to 0.1% acid anhydride bovine beta-lactoglobulin, 4 to 10% glycerol, 1 to 5% carbomer and water; the acid anhydride is selected from at least one of methylhexahydrophthalic anhydride, methyltetrahydrophthalic anhydride and succinic anhydride.

[0007] The research results of the present invention show that bovine β-lactoglobulin modified with acid anhydrides of different structures has significant differences in its destructive effect on HPV, among which methylhexahydrophthalic anhydride and methyltetrahydrophthalic anhydride have better effects than succinic anhydride. The combined modification effect of methyltetrahydrophthalic anhydride and succinic anhydride is better than other anhydrides.

[0008] Preferably, the KARAM is KARAM934.

[0009] Preferably, the acid anhydride is methyltetrahydrophthalic anhydride and succinic anhydride in a molar ratio of 1 to 2:1.

[0010] Preferably, the acid anhydride is methyltetrahydrophthalic anhydride and succinic anhydride in a molar ratio of 1:1.

[0011] Preferably, the method for preparing the anhydride-modified bovine β-lactoglobulin comprises the following steps:

[0012] (1) dissolving anhydride in dimethyl sulfoxide to obtain an anhydride solution with a concentration of 0.5-1 M;

[0013] (2) dissolving bovine β-lactoglobulin in sodium dihydrogen phosphate solution to make the final protein concentration be 15-20 mg / mL to obtain a protein solution;

[0014] (3) Add the anhydride solution to the protein solution, shake and mix, incubate for at least 1 hour, dialyze, filter and sterilize, and store for later use.

[0015] Preferably, the volume ratio of the anhydride solution to the protein solution is 1:3-5.

[0016] The present invention also provides a method for preparing the liquid functional dressing, comprising the following steps: uniformly dispersing carbomer in water, and after swelling, adding glycerol and anhydride-modified bovine beta-lactoglobulin at a temperature below 45° C., and stirring evenly to obtain the liquid functional dressing.

[0017] Compared with the prior art, the beneficial results of the present invention are:

[0018] The liquid functional dressing of the invention mainly consists of 0.05% to 0.1% acid anhydride bovine beta-lactoglobulin, 4 to 10% glycerol, 1 to 5% carbomer and water, and is suitable for blocking HPV infection of genitals and preventing and treating cervical cancer and condyloma acuminatum.

[0019] The liquid functional dressing product of the present invention has a destructive effect on the HPV virus that causes condyloma acuminatum and cervical cancer, effectively prevents and treats HPV, and has a rapid onset of effect.

[0020] The product of the invention has a simple formula and is beneficial to controlling production costs. DETAILED DESCRIPTION

[0021] In order to enable those skilled in the art to better understand the technical content of the present invention, the present invention is further described below in conjunction with specific embodiments.

[0022] Example 1

[0023] A liquid functional dressing for preventing and treating cervical cancer, condyloma acuminatum and HPV infection, comprising the following components in mass concentrations: 0.1% acid anhydride bovine beta-lactoglobulin, 10% glycerol, 1% carbomer and the balance water;

[0024] The preparation method of the acid anhydride-modified bovine β-lactoglobulin comprises the following steps:

[0025] (1) dissolving methylhexahydrophthalic anhydride in dimethyl sulfoxide to obtain a 1 M anhydride solution;

[0026] (2) dissolving bovine β-lactoglobulin in a 0.1 M sodium dihydrogen phosphate solution at pH 8.5 to obtain a protein solution with a final protein concentration of 15 mg / mL; adjusting the pH to 8.5-9 with a 5 M sodium hydroxide solution;

[0027] (3) Add the anhydride solution to the protein solution at a volume ratio of 1:5, shake and mix, incubate at 25°C for 1 hour, place in a 7.5 kDa dialysis bag and dialyze with pH 7.4 phosphate buffer for 24 hours, then filter and sterilize with a 0.45 um microporous filter membrane and store at 4°C for future use.

[0028] The preparation method of the liquid functional dressing comprises the following steps: dispersing carbomer evenly in water, swelling at room temperature for 6 hours, adding glycerol and anhydride-treated bovine beta-lactoglobulin at 25° C., and stirring evenly to obtain the liquid functional dressing.

[0029] Example 2

[0030] A liquid functional dressing for preventing and treating cervical cancer, condyloma acuminatum and HPV infection, comprising the following components in mass concentrations: 0.1% acid anhydride bovine beta-lactoglobulin, 10% glycerol, 1% carbomer and the balance water;

[0031] The preparation method of the acid anhydride-modified bovine β-lactoglobulin comprises the following steps:

[0032] (1) dissolving methyltetrahydrophthalic anhydride in dimethyl sulfoxide to obtain an anhydride solution with a concentration of 1 M;

[0033] (2) dissolving bovine β-lactoglobulin in a 0.1 M sodium dihydrogen phosphate solution at pH 8.5 to obtain a protein solution with a final protein concentration of 15 mg / mL; adjusting the pH to 8.5-9 with a 5 M sodium hydroxide solution;

[0034] (3) Add the anhydride solution to the protein solution at a volume ratio of 1:5, shake and mix, incubate at 25°C for 1 hour, place in a 7.5 kDa dialysis bag and dialyze with pH 7.4 phosphate buffer for 24 hours, then filter and sterilize with a 0.45 um microporous filter membrane and store at 4°C for future use.

[0035] The preparation method of the liquid functional dressing comprises the following steps: dispersing carbomer evenly in water, swelling at room temperature for 6 hours, adding glycerol and anhydride-treated bovine beta-lactoglobulin at 25° C., and stirring evenly to obtain the liquid functional dressing.

[0036] Example 3

[0037] A liquid functional dressing for preventing and treating cervical cancer, condyloma acuminatum and HPV infection, comprising the following components in mass concentrations: 0.1% acid anhydride bovine beta-lactoglobulin, 10% glycerol, 1% carbomer and water;

[0038] The preparation method of the acid anhydride-modified bovine β-lactoglobulin comprises the following steps:

[0039] (1) dissolving succinic anhydride in dimethyl sulfoxide to obtain a 1 M anhydride solution;

[0040] (2) dissolving bovine β-lactoglobulin in a 0.1 M sodium dihydrogen phosphate solution at pH 8.5 to obtain a protein solution with a final protein concentration of 15 mg / mL; adjusting the pH to 8.5-9 with a 5 M sodium hydroxide solution;

[0041] (3) Add the anhydride solution to the protein solution at a volume ratio of 1:5, shake and mix, incubate at 25°C for 1 hour, place in a 7.5 kDa dialysis bag and dialyze with pH 7.4 phosphate buffer for 24 hours, then filter and sterilize with a 0.45 um microporous filter membrane and store at 4°C for future use.

[0042] The preparation method of the liquid functional dressing comprises the following steps: dispersing carbomer evenly in water, swelling at room temperature for 6 hours, adding glycerol and anhydride-treated bovine beta-lactoglobulin at 25° C., and stirring evenly to obtain the liquid functional dressing.

[0043] Example 4

[0044] A liquid functional dressing for preventing and treating cervical cancer, condyloma acuminatum and HPV infection, comprising the following components in mass concentrations: 0.1% acid anhydride bovine beta-lactoglobulin, 10% glycerol, 1% carbomer and water;

[0045] The preparation method of the acid anhydride-modified bovine β-lactoglobulin comprises the following steps:

[0046] (1) dissolving methyltetrahydrophthalic anhydride and succinic anhydride in a molar ratio of 1:1 in dimethyl sulfoxide to obtain an anhydride solution with a concentration of 1 M;

[0047] (2) dissolving bovine β-lactoglobulin in a 0.1 M sodium dihydrogen phosphate solution at pH 8.5 to obtain a protein solution with a final protein concentration of 15 mg / mL; adjusting the pH to 8.5-9 with a 5 M sodium hydroxide solution;

[0048] (3) Add the anhydride solution to the protein solution at a volume ratio of 1:5, shake and mix, incubate at 25°C for 1 hour, place in a 7.5 kDa dialysis bag and dialyze with pH 7.4 phosphate buffer for 24 hours, then filter and sterilize with a 0.45 um microporous filter membrane and store at 4°C for future use.

[0049] The preparation method of the liquid functional dressing comprises the following steps: dispersing carbomer evenly in water, and after swelling, adding glycerol and anhydride-treated bovine beta-lactoglobulin at 25° C., and stirring evenly to obtain the liquid functional dressing.

[0050] Example 5

[0051] A liquid functional dressing for preventing and treating cervical cancer, condyloma acuminatum and HPV infection, comprising the following components in mass concentrations: 0.1% acid anhydride bovine beta-lactoglobulin, 10% glycerol, 1% carbomer and water;

[0052] The preparation method of the acid anhydride-modified bovine β-lactoglobulin comprises the following steps:

[0053] (1) dissolving methyltetrahydrophthalic anhydride and succinic anhydride in a molar ratio of 2:1 in dimethyl sulfoxide to obtain an anhydride solution with a concentration of 1 M;

[0054] (2) dissolving bovine β-lactoglobulin in a 0.1 M sodium dihydrogen phosphate solution at pH 8.5 to obtain a protein solution with a final protein concentration of 15 mg / mL; adjusting the pH to 8.5-9 with a 5 M sodium hydroxide solution;

[0055] (3) Add the anhydride solution to the protein solution at a volume ratio of 1:5, shake and mix, incubate at 25°C for 1 hour, place in a 7.5 kDa dialysis bag and dialyze with pH 7.4 phosphate buffer for 24 hours, then filter and sterilize with a 0.45 um microporous filter membrane and store at 4°C for future use.

[0056] The preparation method of the liquid functional dressing comprises the following steps: dispersing carbomer evenly in water, swelling at room temperature for 6 hours, adding glycerol and anhydride-treated bovine beta-lactoglobulin at 25° C., and stirring evenly to obtain the liquid functional dressing.

[0057] Example 6

[0058] A liquid functional dressing for preventing and treating cervical cancer, condyloma acuminatum and HPV infection, comprising the following components in mass concentrations: 0.1% acid anhydride bovine beta-lactoglobulin, 10% glycerol, 1% carbomer and water;

[0059] The preparation method of the acid anhydride-modified bovine β-lactoglobulin comprises the following steps:

[0060] (1) dissolving methylhexahydrophthalic anhydride and succinic anhydride in a molar ratio of 1:1 in dimethyl sulfoxide to obtain an anhydride solution with a concentration of 1 M;

[0061] (2) dissolving bovine β-lactoglobulin in a 0.1 M sodium dihydrogen phosphate solution at pH 8.5 to obtain a protein solution with a final protein concentration of 15 mg / mL; adjusting the pH to 8.5-9 with a 5 M sodium hydroxide solution;

[0062] (3) Add the anhydride solution to the protein solution at a volume ratio of 1:5, shake and mix, incubate at 25°C for 1 hour, place in a 7.5 kDa dialysis bag and dialyze with pH 7.4 phosphate buffer for 24 hours, then filter and sterilize with a 0.45 um microporous filter membrane and store at 4°C for future use.

[0063] The preparation method of the liquid functional dressing comprises the following steps: dispersing carbomer evenly in water, swelling at room temperature for 6 hours, adding glycerol and anhydride-treated bovine beta-lactoglobulin at 25° C., and stirring evenly to obtain the liquid functional dressing.

[0064] Example 7

[0065] A liquid functional dressing for preventing and treating cervical cancer, condyloma acuminatum and HPV infection, comprising the following components in mass concentrations: 0.05% acid anhydride bovine beta-lactoglobulin, 4% glycerol, 5% carbomer and water;

[0066] The preparation method of the acid anhydride-modified bovine β-lactoglobulin comprises the following steps:

[0067] (1) dissolving methyltetrahydrophthalic anhydride and succinic anhydride in a molar ratio of 1:1 in dimethyl sulfoxide to obtain an anhydride solution with a concentration of 1 M;

[0068] (2) dissolving bovine β-lactoglobulin in a 0.1 M sodium dihydrogen phosphate solution at pH 8.5 to obtain a protein solution with a final protein concentration of 15 mg / mL; adjusting the pH to 8.5-9 with a 5 M sodium hydroxide solution;

[0069] (3) Add the anhydride solution to the protein solution at a volume ratio of 1:5, shake and mix, incubate at 25°C for 1 hour, place in a 7.5 kDa dialysis bag and dialyze with pH 7.4 phosphate buffer for 24 hours, then filter and sterilize with a 0.45 um microporous filter membrane and store at 4°C for future use.

[0070] The preparation method of the liquid functional dressing comprises the following steps: dispersing carbomer evenly in water, and after swelling, adding glycerol and anhydride-treated bovine beta-lactoglobulin at 25° C., and stirring evenly to obtain the liquid functional dressing.

[0071] Example 8

[0072] A liquid functional dressing for preventing and treating cervical cancer, condyloma acuminatum and HPV infection, comprising the following components in mass concentrations: 0.1% acid anhydride bovine beta-lactoglobulin, 10% glycerol, 1% carbomer and water;

[0073] The preparation method of the acid anhydride-modified bovine β-lactoglobulin comprises the following steps:

[0074] (1) dissolving methyltetrahydrophthalic anhydride and succinic anhydride in a molar ratio of 1:1 in dimethyl sulfoxide to obtain an anhydride solution with a concentration of 0.5 M;

[0075] (2) dissolving bovine β-lactoglobulin in a 0.1 M sodium dihydrogen phosphate solution at pH 8.5 to obtain a protein solution with a final protein concentration of 15 mg / mL; adjusting the pH to 8.5-9 with a 5 M sodium hydroxide solution;

[0076] (3) Add the anhydride solution to the protein solution at a volume ratio of 1:5, shake and mix, incubate at 25°C for 1 hour, place in a 7.5 kDa dialysis bag and dialyze with pH 7.4 phosphate buffer for 24-48 hours, then filter and sterilize with a 0.45 um microporous filter membrane and store at 4°C for future use.

[0077] The preparation method of the liquid functional dressing comprises the following steps: dispersing carbomer evenly in water, and after swelling, adding glycerol and anhydride-treated bovine beta-lactoglobulin at 25° C., and stirring evenly to obtain the liquid functional dressing.

[0078] Example 9

[0079] A liquid functional dressing for preventing and treating cervical cancer, condyloma acuminatum and HPV infection, comprising the following components in mass concentrations: 0.1% acid anhydride bovine beta-lactoglobulin, 10% glycerol, 1% carbomer and water;

[0080] The preparation method of the acid anhydride-modified bovine β-lactoglobulin comprises the following steps:

[0081] (1) dissolving methyltetrahydrophthalic anhydride and succinic anhydride in a molar ratio of 1:1 in dimethyl sulfoxide to obtain an anhydride solution with a concentration of 1 M;

[0082] (2) dissolving bovine β-lactoglobulin in a 0.1 M sodium dihydrogen phosphate solution at pH 8.5 to obtain a protein solution with a final protein concentration of 20 mg / mL; adjusting the pH to 8.5-9 with a 5 M sodium hydroxide solution;

[0083] (3) Add the anhydride solution to the protein solution at a volume ratio of 1:5, shake and mix, incubate at 25°C for 1 hour, place in a 7.5 kDa dialysis bag and dialyze with pH 7.4 phosphate buffer for 24 hours, then filter and sterilize with a 0.45 um microporous filter membrane and store at 4°C for future use.

[0084] The preparation method of the liquid functional dressing comprises the following steps: dispersing carbomer evenly in water, and after swelling, adding glycerol and anhydride-treated bovine beta-lactoglobulin at 25° C., and stirring evenly to obtain the liquid functional dressing.

[0085] Example 10

[0086] A liquid functional dressing for preventing and treating cervical cancer, condyloma acuminatum and HPV infection, comprising the following components in mass concentrations: 0.1% acid anhydride bovine beta-lactoglobulin, 10% glycerol, 1% carbomer and water;

[0087] The preparation method of the acid anhydride-modified bovine β-lactoglobulin comprises the following steps:

[0088] (1) dissolving methyltetrahydrophthalic anhydride and succinic anhydride in a molar ratio of 1:1 in dimethyl sulfoxide to obtain an anhydride solution with a concentration of 1 M;

[0089] (2) dissolving bovine β-lactoglobulin in a 0.1 M sodium dihydrogen phosphate solution at pH 8.5 to obtain a protein solution with a final protein concentration of 15 mg / mL; adjusting the pH to 8.5-9 with a 5 M sodium hydroxide solution;

[0090] (3) Add the anhydride solution to the protein solution at a volume ratio of 1:3, shake and mix, incubate at 25°C for 1 hour, place in a 7.5 kDa dialysis bag and dialyze with pH 7.4 phosphate buffer for 24-48 hours, then filter and sterilize with a 0.45 um microporous filter membrane and store at 4°C for future use.

[0091] The preparation method of the liquid functional dressing comprises the following steps: dispersing carbomer evenly in water, and after swelling, adding glycerol and anhydride-treated bovine beta-lactoglobulin at 25° C., and stirring evenly to obtain the liquid functional dressing.

[0092] Experiment 1 In vitro pharmacodynamics test

[0093] 1.1 Preparation of condyloma acuminatum suspension

[0094] Fresh wart specimens (HPV6 / 11) were collected from the vulva of patients with condyloma acuminatum, homogenized, and added with normal saline to prepare condyloma acuminatum suspension.

[0095] 1.2 Interactions between drugs and warts

[0096] 50ul of the drug was mixed with 150ul of the condyloma acuminatum suspension and placed in a 37°C water bath for incubation. After 24h and 48h of incubation, samples were taken for fluorescence quantitative PCR detection. Normal saline was used as a negative control. The results are shown in Table 1, n=10, unit: copies / mL.

[0097] 1.3 Fluorescence quantitative PCR detection of HPV-DNA content

[0098] DNA was extracted according to the method provided by the kit, and five concentrations of HPV-DNA quantitative positive standard (5×10 8 / ml, 5×10 7 / ml, 5×10 6 / ml, 5×l0 5 / ml, 5×10 4 / ml), amplified and detected by fluorescent quantitative PCR, with the natural logarithm of the starting copy number as the horizontal axis and the cycle threshold as the vertical axis, to obtain a standard curve (r=0.9997), based on which the amplification quantity of the sample was quantified.

[0099] Culture for 24 hours Culture for 48 hours Negative control <![CDATA[6.7×10 7 ]]> <![CDATA[6.7×10 7 ]]> Experimental Group 1 (Example 1) <![CDATA[4.5×10 5 ]]> <![CDATA[2.6×10 4 <!-- 5 -->]]> Experimental Group 2 (Example 2) <![CDATA[3.0×10 5 ]]> <![CDATA[1.2×10 4 ]]> Experimental Group 3 (Example 3) <![CDATA[6.3×10 6 ]]> <![CDATA[4.2×10 4 ]]> Experimental Group 4 (Example 4) <![CDATA[3.3×10 4 ]]> <![CDATA[<10 3 ]]> Experimental Group 5 (Example 5) <![CDATA[5.7×10 4 ]]> <![CDATA[<10 3 ]]> Experimental Group 6 (Example 6) <![CDATA[4.8×10 5 ]]> <![CDATA[2.7×10 4 ]]>

[0100] The above results show that the liquid functional dressing product of the present invention has a destructive effect on HPV viruses that cause condyloma acuminatum and cervical cancer, and effectively prevents and treats HPV. Comparison of groups 1 to 3 shows that compared with succinic anhydride, methylhexahydrophthalic anhydride and methyltetrahydrophthalic anhydride have a significant inhibitory effect on HPV. Comparison of groups 1, 4, 5, and 6 shows that the destructive effect of methyltetrahydrophthalic anhydride and succinic anhydride combined anhydride on HPV viruses is significantly higher than that of other samples. The use effects of the samples of Examples 7 to 10 are similar to those of Example 4.

[0101] The above descriptions are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A liquid functional dressing for preventing and treating cervical cancer, condyloma acuminatum and HPV infection, characterized in that: The invention comprises the following components in mass concentration: 0.05% to 0.1% of acid anhydride bovine beta-lactoglobulin, 4% to 10% of glycerol, 1% to 5% of carbomer and water; the acid anhydride is methyltetrahydrophthalic anhydride and succinic anhydride in a molar ratio of 1 to 2:

1.

2. The liquid functional dressing according to claim 1, characterized in that: The acid anhydride is methyltetrahydrophthalic anhydride and succinic anhydride in a molar ratio of 1:

1.

3. The liquid functional dressing according to claim 1, characterized in that: The preparation method of the acid anhydride-modified bovine β-lactoglobulin comprises the following steps: (1) dissolving anhydride in dimethyl sulfoxide to obtain an anhydride solution with a concentration of 0.5-1 M; (2) dissolving bovine β-lactoglobulin in sodium dihydrogen phosphate solution to make the final protein concentration be 15-20 mg / mL to obtain a protein solution; (3) Add the anhydride solution to the protein solution, shake and mix, incubate for at least 1 hour, dialyze, filter and sterilize, and store for later use.

4. The liquid functional dressing according to claim 3, characterized in that: The volume ratio of the anhydride solution to the protein solution is 1:3-5.

5. The method for preparing the liquid functional dressing according to any one of claims 1 to 4, characterized in that: The following steps are involved: Disperse carbomer evenly in water, and after swelling, add glycerol and anhydride-modified bovine β-lactoglobulin at a temperature below 45°C, and stir evenly to obtain the product.