Anti-HPV (human papillomavirus) earthworm extract carrier gel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to anti-HPV (human papillomavirus) earthworm extract carrier gel as well as a preparation method and application thereof. The preparation method comprises the following steps: stirring and hydrating an earthworm extract and a lipid layer to obtain lipid vesicles, then carrying out expansion, ultrasonic treatment and extrusion to obtain an earthworm extract carrier, and finally adding pH-adjusted carbomer and uniformly mixing to obtain the anti-HPV earthworm extract carrier gel. The Carbomer gel is compounded with the earthworm extract carrier, so that the inhibiting effect on HPV (human papillomavirus) is improved, cervical cancer cells can be promoted to shrink and die, the gel is combined with the earthworm extract carrier, the viscosity of the earthworm extract carrier is further improved, the fitting property of the earthworm extract carrier and the inner wall of the vagina is improved, and the effect of treating cervical cancer is achieved. Therefore, the effect of preventing and treating HPV virus infection is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and in particular relates to an anti-HPV earthworm extract transfersome gel and a preparation method and application thereof. Background Art

[0002] Cervical cancer is one of the most common malignant tumors in women and has become a major threat to the health and life of women worldwide. Studies have found that almost all cases of cervical cancer are related to human papillomavirus (HPV) infection. High-risk HPV infection is the main risk factor for cervical cancer, among which HPV types 16 and 18 are closely related to cervical cancer. Since there is currently no specific drug or effective treatment plan, finding new anti-HPV drugs is of great significance for preventing cervical cancer and improving the treatment effect of cervical cancer patients.

[0003] Medicinal earthworms are called earthworms in Chinese medicine. They have a unique chemical composition, such as high unsaturated fatty acid content, relatively high abundance of trace elements such as Fe, Mn, Cu, Zn and Se, as well as earthworms, earthworm antipyretic alkali and purine, choline, cholesterol and other active ingredients. Studies have found that earthworm extracts have a direct inhibitory effect on cancer cells in vitro, and can regulate the proliferation and differentiation of spleen B cells in tumor-bearing mice in vivo, and enhance the specific immune function of tumor-bearing mice. However, in clinical applications, the limited efficacy directly limits the use of earthworm extracts, especially in anti-HPV. Summary of the invention

[0004] Based on the above technical background, the main purpose of the present invention is to provide an anti-HPV earthworm extract transfersome gel and its preparation method and application, so as to overcome the shortcomings of the prior art.

[0005] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0006] The first aspect of the present invention is to provide a method for preparing an anti-HPV transfersome gel of earthworm extract, the preparation method comprising the following steps:

[0007] Step 1, centrifuging the body cavity fluid of sexually mature earthworms, collecting the supernatant, filtering, and freeze-drying to obtain earthworm extract;

[0008] Step 2, dissolving lecithin and cholesterol in a solvent, evaporating the solvent under reduced pressure to obtain a lipid layer, dissolving the earthworm extract obtained in step 1 in water and stirring evenly, then adding the lipid layer to hydrate to obtain lipid vesicles, and then swelling, ultrasonicating, and extruding to obtain earthworm extract transfersomes;

[0009] Step 3: Add the earthworm extract transfersome prepared in step 2 into the gel and mix well to obtain the anti-HPV earthworm extract transfersome gel.

[0010] In step 1,

[0011] Preferably, the body cavity fluid of the sexually mature earthworm is centrifuged at 0-4°C;

[0012] Preferably, the collected supernatant is filtered through a 0.20-0.25 μm microporous filter membrane.

[0013] In step 2,

[0014] Preferably, the edge activator is dissolved together with lecithin and cholesterol in a solvent; or,

[0015] Dissolve the edge activator and the earthworm extract obtained in step 1 in water and stir evenly.

[0016] Preferably, the mass ratio of the lecithin, cholesterol, edge activator and earthworm extract is (5-9):1:(0.6-1.4):1.

[0017] Preferably, egg yolk lecithin and cholesterol are dissolved in ethanol, and then the solvent is evaporated under reduced pressure at 45-55° C. to obtain a lipid layer.

[0018] Preferably, the earthworm extract obtained in step 1 is dissolved in water at room temperature, and then added to the lipid layer for hydration at 35-45° C. and a stirring speed of 70-80 rpm for 1-2 hours to obtain lipid vesicles.

[0019] Preferably, the lipid vesicles are expanded at room temperature for 1 to 3 hours, then sonicated in an ice bath for 20 to 30 minutes, and then the vesicles are sequentially extruded through microporous filter membranes of 0.70 to 0.90 μm, 0.40 to 0.50 μm, and 0.20 to 0.25 μm.

[0020] In step 3,

[0021] Preferably, the gel is Carbomer 980;

[0022] Water is added to the carbomer to completely swell it, and the pH is adjusted to 6.0-6.5, and then the earthworm extract transfersome prepared in step 2 is added at a mass ratio of 1:1 and mixed.

[0023] The second aspect of the present invention is to provide an anti-HPV earthworm extract transfersome gel prepared according to the preparation method described in the first aspect of the present invention.

[0024] The third aspect of the present invention is to provide a use of the anti-HPV earthworm extract transfersome gel described in the second aspect of the present invention in the preparation of drugs for preventing and treating cervical cancer infected with HPV.

[0025] The beneficial effects of the present invention are:

[0026] (1) The present invention loads earthworm extract into a transfersome and compounds it with a gel. The obtained earthworm extract transfersome gel can not only promote the shrinkage and death of cervical cancer cells, but also effectively enter the cells to inhibit the expression of HPV virus. Cell experiments have shown that after acting on SiHa cells and Hela cells, it can significantly reduce the expression level of HPV mRNA gene, thereby achieving the purpose of preventing and treating HPV virus infection in the human body.

[0027] In the present invention, the earthworm extract is first dispersed in water, and then an edge activator is added for mixing, which can improve the encapsulation rate of the earthworm extract. Carbomer gel with a pH value of 6.0-6.5 is mixed with the earthworm extract transfer body, which can further improve the viscosity of the earthworm extract transfer body, improve the fit between the earthworm extract transfer body and the inner wall of the vagina, and is conducive to prolonging the retention time of the drug (earthworm extract transfer body) in the vagina, improving the efficacy time and effect of the earthworm extract, further improving the effect of preventing and treating HPV virus infection, and at the same time can reduce the number of drug administrations and improve the patient's compliance with medication, thereby improving the application of earthworm extract in anti-HPV.

[0028] (2) The preparation method of the present invention is simple, the raw materials are easily available, and industrial large-scale production can be achieved. The transfersome gel has good application prospects in the prevention and treatment of cervical cancer drugs infected with HPV. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The actual photograph (a) and the transmission electron microscopy characterization image (b) of the earthworm extract transfersome prepared in Example 6 are shown;

[0030] Figure 2 A photo of the transfersome gel prepared in Example 12 is shown;

[0031] Figure 3 Photograph (a) and microscope photograph (b) showing the effect of the transfersome gel prepared in Example 12 on animal skin irritation;

[0032] Figure 4 Microscope photos showing the effects of the blank control group, earthworm solution group, and earthworm transfersome gel group on SiHha cell morphology;

[0033] Figure 5 Microscope photos showing the effects of the blank control group, earthworm solution group, and earthworm transfersome gel group on the morphology of Hela cells;

[0034] Figure 6 The bar charts show the effects of the earthworm solution group and earthworm transfersome gel group on the expression of HPV16 E6 and E7 mRNA;

[0035] Figure 7 The bar graph shows the effects of the earthworm solution group and the earthworm transfersome gel group on the expression of HPV18 E6 and E7 mRNA. DETAILED DESCRIPTION

[0036] The present invention will be described in detail below, and the characteristics and advantages of the present invention will become clearer and more distinct with the description.

[0037] The first aspect of the present invention is to provide a method for preparing an anti-HPV transfersome gel of earthworm extract, the preparation method comprising the following steps:

[0038] Step 1, centrifuging the body cavity fluid of sexually mature earthworms, collecting the supernatant, filtering, and freeze-drying to obtain earthworm extract;

[0039] Step 2, dissolving lecithin and cholesterol in a solvent, evaporating the solvent under reduced pressure to obtain a lipid layer, dissolving the earthworm extract obtained in step 1 in water and stirring evenly, then adding the lipid layer to hydrate to obtain lipid vesicles, and then swelling, ultrasonicating, and extruding to obtain earthworm extract transfersomes;

[0040] Step 3: Add the earthworm extract transfersome prepared in step 2 into the gel and mix well to obtain the anti-HPV earthworm extract transfersome gel.

[0041] The above steps are described in detail below.

[0042] In step 1, the earthworm coelomic fluid is prepared by the following steps:

[0043] Select sexually mature (the reproductive zone is a sign of sexual maturity) earthworms, wash them, and place them on filter paper soaked in phosphate buffered saline (PBS) for two days to allow them to spit out all the dirt in their abdomens to avoid unnecessary contamination during the collection of body cavity fluid.

[0044] Place the washed fresh earthworms in a clean beaker to stimulate the earthworms to contract and eject yellow viscous liquid, i.e., coelomic fluid, from their body surfaces. Preferably, the stimulation method may be a 6V electrical stimulator.

[0045] The coelomic fluid of sexually mature earthworms is centrifuged at 0-4°C, preferably at 4°C.

[0046] The collected supernatant is filtered through a 0.20-0.25 μm microporous membrane, preferably a 0.22 μm microporous membrane.

[0047] In step 2, during the preparation process of step 2, an edge activator is also added, and the edge activator is preferably Tween 80 and sodium cholate mixed in a mass ratio of 1:1. The mixture of the edge activator Tween 80 and sodium cholate is dissolved in a solvent with lecithin and cholesterol, or the mixture of the edge activator Tween 80 and sodium cholate is dissolved in water with the earthworm extract obtained in step 1 and stirred evenly. Preferably, the edge activator Tween 80 and sodium cholate are dissolved in water with the earthworm extract obtained in step 1 and stirred evenly.

[0048] The experiment found that after the edge activator and the earthworm extract were mixed before preparation, the obtained earthworm extract transfer body was more transparent, had no particle precipitation, and had a higher encapsulation rate.

[0049] The lecithin is preferably egg yolk lecithin.

[0050] The mass ratio of the lecithin, cholesterol, edge activator and earthworm extract is (5-9):1:(0.6-1.4):1.

[0051] Preferably, the mass ratio of the lecithin, cholesterol, edge activator and earthworm extract is 5:1:1.2:1.

[0052] Egg yolk lecithin and cholesterol were dissolved in ethanol, and then the solvent was evaporated under reduced pressure at 45-55°C.

[0053] Preferably, egg yolk lecithin and cholesterol are dissolved in ethanol, and then the solvent is evaporated under reduced pressure at 50° C. to form a uniform lipid layer, which is then dried overnight under vacuum conditions to completely evaporate the solvent.

[0054] The earthworm extract obtained in step 1 is dissolved in ultrapure water at room temperature, and then added to the lipid layer for hydration at 35-45° C. and a stirring speed of 70-80 rpm for 1-2 hours to obtain lipid vesicles.

[0055] Preferably, the earthworm extract obtained in step 1 is dissolved in ultrapure water at 22-25° C., and then added to the lipid layer for hydration at 40° C. and a stirring speed of 75 rpm for 1 hour to obtain lipid vesicles.

[0056] The lipid vesicles were expanded at room temperature for 1-3 h, then sonicated in an ice bath for 20-30 min, and then extruded through microporous filter membranes of 0.70-0.90 μm, 0.40-0.50 μm, and 0.20-0.25 μm in sequence.

[0057] Preferably, the lipid vesicles are expanded at room temperature for 2 h, then sonicated in an ice bath for 25 min, and then the vesicles are sequentially extruded through microporous filter membranes of 0.80 μm, 0.45 μm, and 0.22 μm.

[0058] In step 3, the gel is preferably Carbomer 980.

[0059] Ultrapure water is added to the carbomer to completely swell it, the pH is adjusted to 6.0-6.5, and then the earthworm extract transfersome prepared in step 2 is added at a mass ratio of 1:1 and slowly mixed to obtain an anti-HPV earthworm extract transfersome gel.

[0060] Transfersomes are modified liposomes that not only have the advantages of traditional liposomes such as good biocompatibility and low toxicity, but also have good deformability due to the addition of edge activators, and can pass through pores several times smaller than themselves, making them one of the ideal carriers for transdermal drug delivery. Studies have also found that transfersomes can effectively accommodate most drugs regardless of their structure, polarity, or size. More importantly, the flexible vesicle structure of transfersomes It is beneficial to enhance cell uptake, but there are problems such as poor adhesion when applied alone to the skin and mucosal surfaces. Although the vagina itself does not have glands, it produces mucus to form a mucus layer, which hinders the contact between the drug and the target tissue. The gel can swell rapidly in the aqueous environment of the vagina and adhere tightly to the vaginal mucosa. Therefore, the gel combined with the transfer body will significantly improve the above problems, which is beneficial to prolong the retention time of the drug in the vagina, improve the therapeutic effect, reduce the number of dosing, and improve the patient's compliance with medication.

[0061] The second aspect of the present invention is to provide an anti-HPV earthworm extract transfersome gel prepared according to the preparation method described in the first aspect of the present invention.

[0062] The third aspect of the present invention is to provide a use of the anti-HPV earthworm extract transfersome gel described in the second aspect of the present invention in the preparation of drugs for preventing and treating cervical cancer infected with HPV.

[0063] Example

[0064] The present invention is further described below by specific examples, which are limited to illustrating the present invention but not to limiting the scope of the present invention. The raw materials used in the examples of the present invention were all purchased.

[0065] Example 1

[0066] Select sexually mature (the reproductive zone is a sign of sexual maturity) earthworms, wash them, and place them on filter paper soaked in phosphate buffered saline (PBS) for two days to allow them to vomit out all the dirt in their abdomens to avoid unnecessary contamination during the collection of coelomic fluid. Place the washed fresh earthworms in a clean beaker and stimulate them with a homemade 6V electric stimulator. The earthworms will contract and spray out a yellow viscous liquid from the body surface, which is the coelomic fluid. After the collected coelomic fluid is centrifuged at 4°C, the supernatant is collected, and then sterilized with a 0.22μm microporous filter membrane, freeze-dried to obtain freeze-dried powder, which is the earthworm extract, and stored at -20°C for use. The protein content is 80.3% as measured by the BCA method.

[0067] Example 2

[0068] Weigh 45 mg of egg yolk lecithin, 5 mg of cholesterol, 2.5 mg of Tween 80 and 2.5 mg of sodium cholate in a dry round-bottom flask, add an appropriate amount of ethanol to dissolve them, use a rotary evaporator to evaporate the solvent under reduced pressure at 50°C, form a uniform lipid layer on the wall of the flask, and dry it overnight under vacuum to completely evaporate the organic solvent. Dissolve 5 mg of earthworm extract in 5 mL of ultrapure water at room temperature (22-25°C), add the above aqueous solution to the deposited lipid layer film at 40°C and 75 rpm, hydrate for 1 hour to obtain lipid vesicles; fully expand at room temperature (22-25°C) for 2 hours, then ultrasonically treat in an ice bath for 25 minutes, 3 seconds "on", 3 seconds "off". Finally, the obtained vesicle system is sequentially extruded through 0.80μm, 0.45μm and 0.22μm microporous filter membranes to obtain earthworm extract transfer bodies.

[0069] Example 3

[0070] Weigh 35 mg egg yolk lecithin, 5 mg cholesterol, 2.5 mg Tween 80 and 2.5 mg sodium cholate in a dry round-bottom flask, add an appropriate amount of ethanol to dissolve them, use a rotary evaporator to evaporate the solvent under reduced pressure at 50°C, form a uniform lipid layer on the wall of the flask, and dry it overnight under vacuum to completely evaporate the organic solvent. Dissolve 5 mg of earthworm extract in 5 mL of ultrapure water at room temperature (22-25°C), add the above aqueous solution to the deposited lipid layer film at 40°C and 75 rpm, hydrate for 1 hour to obtain lipid vesicles; fully expand at room temperature (22-25°C) for 2 hours, then ultrasonically treat in an ice bath for 25 minutes, 3s "on", 3s "off". Finally, the obtained vesicle system is sequentially extruded through 0.80μm, 0.45μm and 0.22μm microporous filter membranes to obtain earthworm extract transfer bodies.

[0071] Example 4

[0072] Weigh 25 mg of egg yolk lecithin, 5 mg of cholesterol, 2.5 mg of Tween 80 and 2.5 mg of sodium cholate in a dry round-bottom flask, add an appropriate amount of ethanol to dissolve them, and use a rotary evaporator to evaporate the solvent under reduced pressure at 50°C. After a uniform lipid layer is formed on the wall of the flask, dry it overnight under vacuum to completely evaporate the organic solvent. Dissolve 5 mg of earthworm extract in 5 mL of ultrapure water at room temperature (22-25°C), add the above aqueous solution to the deposited lipid layer film at 40°C and 75 rpm, hydrate for 1 hour, and obtain lipid vesicles; fully expand at room temperature (22-25°C) for 2 hours, then ultrasonically treat in an ice bath for 25 minutes, 3s "on", 3s "off". Finally, the obtained vesicle system is sequentially extruded through 0.80μm, 0.45μm and 0.22μm microporous filter membranes to obtain earthworm extract transfer bodies.

[0073] Example 5

[0074] Weigh 25 mg of egg yolk lecithin and 5 mg of cholesterol into a dry round-bottom flask, add an appropriate amount of ethanol to dissolve them, use a rotary evaporator to evaporate the solvent under reduced pressure at 50°C, and after a uniform lipid layer is formed on the wall of the flask, dry it overnight under vacuum to completely evaporate the organic solvent. Dissolve 5 mg of earthworm extract, 2.5 mg of Tween 80 and 2.5 mg of sodium cholate in 5 mL of ultrapure water at room temperature (22-25°C), add the above aqueous solution to the deposited lipid layer film at 40°C and 75 rpm, hydrate for 1 hour to obtain lipid vesicles; fully expand at room temperature (22-25°C) for 2 hours, then ultrasonically treat in an ice bath for 25 minutes, 3 seconds "on", 3 seconds "off". Finally, the obtained vesicle system is sequentially extruded through 0.80μm, 0.45μm and 0.22μm microporous filter membranes to obtain earthworm extract transfer bodies.

[0075] Example 6

[0076] Weigh 25 mg of egg yolk lecithin and 5 mg of cholesterol into a dry round-bottom flask, add an appropriate amount of ethanol to dissolve them, use a rotary evaporator to evaporate the solvent under reduced pressure at 50°C, and after a uniform lipid layer is formed on the wall of the flask, dry it overnight under vacuum to completely evaporate the organic solvent. Dissolve 5 mg of earthworm extract, 3 mg of Tween 80 and 3 mg of sodium cholate in 5 mL of ultrapure water at room temperature (22-25°C), add the above aqueous solution to the deposited lipid layer film at 40°C and 75 rpm, hydrate for 1 hour to obtain lipid vesicles; fully expand at room temperature (22-25°C) for 2 hours, then ultrasonically treat in an ice bath for 25 minutes, 3s "on", 3s "off". Finally, the obtained vesicle system is sequentially extruded through 0.80μm, 0.45μm and 0.22μm microporous filter membranes to obtain earthworm extract transfersomes.

[0077] The photos and transmission electron microscopy photos of the earthworm extract transfersomes are shown in Figure 1 shown. Figure 1 In a, the earthworm extract transfersomes appear as a light green, nearly colorless, translucent solution with a clear appearance and no precipitation or flocs. Figure 1 In b, under transmission electron microscopy, the earthworm extract transfersomes are basically spherical, with round particles and uniform distribution without aggregation.

[0078] Example 7

[0079] Weigh 25 mg of egg yolk lecithin and 5 mg of cholesterol into a dry round-bottom flask, add an appropriate amount of ethanol to dissolve them, use a rotary evaporator to evaporate the solvent under reduced pressure at 50°C, and after a uniform lipid layer is formed on the wall of the flask, dry it overnight under vacuum to completely evaporate the organic solvent. Dissolve 5 mg of earthworm extract, 3.5 mg of Tween 80 and 3.5 mg of sodium cholate in 5 mL of ultrapure water at room temperature (22-25°C), add the above aqueous solution to the deposited lipid layer film at 40°C and 75 rpm, hydrate for 1 hour to obtain lipid vesicles; fully expand at room temperature (22-25°C) for 2 hours, then ultrasonically treat in an ice bath for 25 minutes, 3s "on", 3s "off". Finally, the obtained vesicle system is sequentially extruded through 0.80μm, 0.45μm and 0.22μm microporous filter membranes to obtain earthworm extract transfer bodies.

[0080] Example 8

[0081] Weigh 25 mg of egg yolk lecithin and 5 mg of cholesterol into a dry round-bottom flask, add an appropriate amount of ethanol to dissolve them, and use a rotary evaporator to evaporate the solvent under reduced pressure at 50°C. After a uniform lipid layer is formed on the wall of the flask, dry it overnight under vacuum to completely evaporate the organic solvent. Dissolve 5 mg of earthworm extract, 2 mg of Tween 80 and 2 mg of sodium cholate in 5 mL of ultrapure water at room temperature (22-25°C), add the above aqueous solution to the deposited lipid layer film at 40°C and 75 rpm, hydrate for 1 hour, and obtain lipid vesicles; fully expand at room temperature (22-25°C) for 2 hours, then ultrasonically treat in an ice bath for 25 minutes, 3 seconds "on", 3 seconds "off". Finally, the obtained vesicle system is sequentially extruded through 0.80μm, 0.45μm and 0.22μm microporous filter membranes to obtain earthworm extract transfer bodies.

[0082] Example 9

[0083] Weigh 25 mg of egg yolk lecithin and 5 mg of cholesterol into a dry round-bottom flask, add an appropriate amount of ethanol to dissolve them, and use a rotary evaporator to evaporate the solvent under reduced pressure at 50°C. After a uniform lipid layer is formed on the wall of the flask, dry it overnight under vacuum to completely evaporate the organic solvent. Dissolve 5 mg of earthworm extract, 1.5 mg of Tween 80 and 1.5 mg of sodium cholate in 5 mL of ultrapure water at room temperature (22-25°C), add the above aqueous solution to the deposited lipid layer film at 40°C and 75 rpm, hydrate for 1 hour, and obtain lipid vesicles; fully expand at room temperature (22-25°C) for 2 hours, then ultrasonically treat in an ice bath for 25 minutes, 3 seconds "on", 3 seconds "off". Finally, the obtained vesicle system is sequentially extruded through 0.80μm, 0.45μm and 0.22μm microporous filter membranes to obtain earthworm extract transfersomes.

[0084] Example 10

[0085] Weigh 50 mg of egg yolk lecithin and 10 mg of cholesterol into a dry round-bottom flask, add an appropriate amount of ethanol to dissolve them, and use a rotary evaporator to evaporate the solvent under reduced pressure at 50°C. After a uniform lipid layer is formed on the wall of the flask, dry it overnight under vacuum to completely evaporate the organic solvent. Dissolve 10 mg of earthworm extract, 6 mg of Tween 80 and 6 mg of sodium cholate in 5 mL of ultrapure water at room temperature (22-25°C), add the above aqueous solution to the deposited lipid layer film at 40°C and 75 rpm, hydrate for 1 hour, and obtain lipid vesicles; fully expand at room temperature (22-25°C) for 2 hours, then ultrasonically treat in an ice bath for 25 minutes, 3 seconds "on", 3 seconds "off". Finally, the obtained vesicle system is sequentially extruded through 0.80μm, 0.45μm and 0.22μm microporous filter membranes to obtain earthworm extract transfersomes.

[0086] Embodiment 11

[0087] Weigh 12.5 mg egg yolk lecithin and 2.5 mg cholesterol into a dry round-bottom flask, add an appropriate amount of ethanol to dissolve them, and use a rotary evaporator to evaporate the solvent under reduced pressure at 50°C. After a uniform lipid layer is formed on the wall of the flask, dry it overnight under vacuum to completely evaporate the organic solvent. Dissolve 2.5 mg earthworm extract, 1.5 mg Tween 80 and 1.5 mg sodium cholate in 5 mL ultrapure water at room temperature (22-25°C), add the above aqueous solution to the deposited lipid layer film at 40°C and 75 rpm, hydrate for 1 hour, and obtain lipid vesicles; fully expand at room temperature (22-25°C) for 2 hours, then ultrasonically treat in an ice bath for 25 minutes, 3s "on", 3s "off". Finally, the obtained vesicle system is sequentially extruded through 0.80μm, 0.45μm and 0.22μm microporous filter membranes to obtain earthworm extract transfer bodies.

[0088] Example 12

[0089] After 1% Carbomer 980 is completely swollen with distilled water, triethanolamine is added dropwise to adjust its pH value to 6.0-6.5, and then the mixture is slowly mixed with the transfersome of Example 6 at a mass ratio of 1:1 under gentle stirring to obtain a transfersome gel.

[0090] The photo of the transfersome gel is shown in Figure 2 As shown, from Figure 2 It can be seen that the transfersome gel has an appearance of nearly colorless gel, has no particles or lumps visible to the naked eye, is relatively easy to pick up, has moderate viscosity, and has a fine and uniform texture.

[0091] Example 13

[0092] After 1% Carbomer 980 is completely swollen with distilled water, triethanolamine is added dropwise to adjust its pH value to 6.0-6.5, and then the mixture is slowly mixed with the transfersome of Example 10 at a mass ratio of 1:1 under gentle stirring to obtain a transfersome gel.

[0093] Embodiment 14

[0094] After 1% Carbomer 980 is completely swollen with distilled water, triethanolamine is added dropwise to adjust its pH value to 6.0-6.5, and then the mixture is slowly mixed with the transfersome of Example 11 at a mass ratio of 1:1 under gentle stirring to obtain a transfersome gel.

[0095] Experimental example

[0096] Experimental Example 1 Particle size, PDI, Zeta potential and encapsulation efficiency test

[0097] The particle size, PDI, Zeta potential and encapsulation efficiency of the earthworm extract transfersomes prepared in Examples 2-11 were tested respectively. The test results are shown in Table 1:

[0098] Table 1 Particle size, PDI, Zeta potential and encapsulation efficiency of Examples 2-11

[0099]

[0100] As can be seen from Table 1, the particle size of the earthworm extract transfersomes prepared in Examples 2-11 is between 83nm and 100nm, indicating that the particle size of the earthworm extract transfersomes of the present invention is small. The earthworm extract transfersomes prepared in Example 6 have the highest encapsulation efficiency, which can reach 71.3%, and its average particle size is (89.8±0.9)nm, and the average Zeta potential is (-38.9±1.1)mV.

[0101] Experimental Example 2 Test on skin irritation of earthworm extract transfersome gel

[0102] Experimental animals: Healthy SPF-grade ICR mice (female, weighing 20-25 g) were provided by the Experimental Animal Center of Shanxi Medical University.

[0103] Select healthy male ICR mice with intact skin, anesthetize them with 20% urethane (dose of 5 mL / kg), remove the hair on the abdomen, and remove the hair in an area of ​​2 cm×2 cm. Fix the limbs on the mouse board. Take 0.5 g of the transfersome gel prepared in Example 12 of the present invention and apply it to the hairless skin surface on one side, and apply blank transfersome gel on the hairless skin on the other side as a control (blank transfersome gel without drug). After 12 hours, remove the covering, wash the skin surface with warm water, observe the skin reaction at the application site, kill and remove the skin at the administration site, carefully remove the fat tissue, and stain with HE. Observe whether the tissue cells are abnormal under an optical microscope. Figure 3 As shown, Figure 3 a is a photo of mouse skin after the experiment. Figure 3 b is a microscope picture of mouse skin after the experiment.

[0104] Compared with the blank control, no erythema, swelling or inflammation was observed on the skin surface of the experimental animals ( Figure 3 a), under a microscope ( Figure 3 b), it can be seen that the epidermis has a complete tissue structure; the dermis has staggered collagen fiber bundles and some skin appendages; the subcutaneous tissue is mainly composed of loose connective tissue and adipose tissue; no obvious inflammatory cell infiltration is observed, indicating that the earthworm extract transfersome gel of the present invention is non-irritating to the skin.

[0105] Experimental Example 3: Effect of transfersome gel of earthworm extract on HPV clearance

[0106] 1. Experimental Methods

[0107] 1. Cell culture

[0108] Human cervical cancer SiHa cells and human cervical cancer Hela cells were revived and inoculated in 5 mL of DMEM complete medium and transferred to 25 cm 2 Place the cells in a culture bottle and culture at 37°C, 5% CO2 until the logarithmic growth phase for later use.

[0109] 2. CCK-8 method to detect cell survival rate

[0110] SiHa and Hela cells growing in logarithmic phase were taken and 5×10 310 cells were inoculated in a 96-well cell culture plate, and a solvent control group, a transfersome gel group of earthworm extract (the transfersome gel prepared in Example 12 was used, referred to as the earthworm transfersome gel group, ultrapure water was added to the transfersome gel and mixed, and transfersome gel aqueous solutions with concentrations of 10 μg / mL, 20 μg / mL, and 25 μg / mL were obtained respectively), an earthworm extract solution group (the earthworm extract prepared in Example 1 was dissolved in ultrapure water to prepare an earthworm extract aqueous solution with a concentration of 1 mg / mL, referred to as the earthworm solution group, ultrapure water was added to the earthworm extract aqueous solution and mixed, and earthworm extract aqueous solutions with concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 500 μg / mL were obtained respectively), and cells without treatment were blank control group (the culture medium was DMEM containing 1% DMSO (dimethyl sulfoxide)), and each group had 6 replicate wells. The cell survival was determined by CCK-8 method. If the cell survival rate was ≥80%, it was considered to have no toxic effect on the cells.

[0111] 3. Detection of apoptosis rate of cervical cancer cells by flow cytometry

[0112] SiHa and Hela cells in logarithmic growth phase were collected and cultured at 2×10 5 / well were inoculated into 6-well plates, and after the cells adhered to the wall, a solvent control group, an earthworm extract transfersome gel group (the transfersome gel prepared in Example 12 was used, ultrapure water was added to the transfersome gel and mixed, and transfersome gel aqueous solutions with concentrations of 10 μg / mL, 20 μg / mL, and 25 μg / mL were obtained, respectively), and an earthworm extract solution group (the earthworm extract prepared in Example 1 was dissolved in ultrapure water to prepare an earthworm extract aqueous solution with a concentration of 1 mg / mL, and ultrapure water was added to the earthworm extract aqueous solution and mixed, and earthworm extract aqueous solutions with concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, and 250 μg / mL were obtained, respectively), and the untreated cells were the culture medium control group (untreated here means: only culture medium was used for culture, and the culture medium was DMEM containing 1% DMSO), with 6 replicate wells in each group. After 24 hours, the cells were collected, and 500 μL of Binding Buffer was added to resuspend the cells. 5 μL of Annexin V-FITC and 2 μL of PI were added, and the cells were incubated at room temperature in the dark for 15 minutes. The apoptosis was detected by flow cytometry.

[0113] 4. HPV16 E6 / E7 mRNA gene and HPV18 E6 / E7 mRNA gene expression detection

[0114] The cell treatment method was the same as step 3. After 24 hours of treatment, the total RNA of each well was extracted and reverse transcribed. The changes in the gene expression levels of HPV16 and HPV18 E6 and E7 mRNA were determined by real-time quantitative fluorescence detection technology (q-PCR).

[0115] 2. Experimental Results

[0116] 1. Effects of earthworm extract and transfersome gel on proliferation of Hela cells

[0117] After SiHa and Hela cells were given different concentrations of SiHa earthworm extract and transfersome gel, SiHa and Hela cells were observed under a microscope. Figure 4 The test results of the earthworm extract and transfersome gel on the proliferation of SiHa and Hela cells are shown in Table 2.

[0118] Table 2 Effects of earthworm extract and transfersome gel on proliferation of SiHa and Hela cells

[0119]

[0120]

[0121] Note: n=6, * indicates P<0.05 compared with the control group, ** indicates P<0.01 compared with the control group, *** indicates P<0.001 compared with the control group, **** indicates P<0.0001 compared with the control group; the control group here refers to: in the earthworm extract solution, it refers to the solution group with an earthworm extract concentration of 0 (i.e., the blank control group described below), and in the earthworm extract transfersome gel group, it refers to the group with an earthworm extract transfersome gel concentration of 0 (i.e., the solvent control group described below).

[0122] From Table 2 and Figure 4 It can be seen that in the earthworm extract solution group, when the concentration was 10 μg / mL and below, the cell growth was not significantly inhibited. When the concentration increased to 100 μg / mL, the cell survival rate was significantly reduced, the proliferation rate was significantly slowed down, the cell fragments increased, the gaps between cells increased, and some cells were vacuolated ( Figure 4 ). When administered in the form of transfersome gel, different concentrations can change cell morphology and cause cell death to varying degrees; when the concentration is 20μg / mL, the cell survival rate is reduced by more than half, and when the concentration increases to 25μg / mL, the cell survival rate is about 10% or even less. This shows that the transfersome gel of earthworm extract can significantly inhibit the growth of SiHa and Hela cells when the concentration is one order of magnitude lower than that of earthworm extract solution, greatly improving the activity and effect of earthworm extract.

[0123] 2. Effects of earthworm extract and transfersome gel on apoptosis of cervical cancer cells

[0124] The test results of the earthworm extract and transfersome gel on SiHa and Hela cell apoptosis are shown in Table 3. Figure 5 shown.

[0125] Table 3 Effects of earthworm extract and transfersome gel on apoptosis rate of SiHa and Hela cells

[0126]

[0127] Note: n=6, * indicates P<0.05 compared with the control group, ** indicates P<0.01 compared with the control group, *** indicates P<0.001 compared with the control group, **** indicates P<0.0001 compared with the control group; the control group here refers to: in the earthworm extract solution, it refers to the solution group with an earthworm extract concentration of 0 (i.e., the blank control group described below), and in the earthworm extract transfersome gel group, it refers to the group with an earthworm extract transfersome gel concentration of 0 (i.e., the solvent control group described below).

[0128] From Table 3 and Figure 5 It can be seen that compared with the blank control group, different concentrations of earthworm extract solution groups had significant differences in the number of apoptotic and necrotic cells of SiHa cells (P<0.01); while for Hela cells, there was no significant difference in the number of apoptotic and necrotic cells compared with the blank control group at 50μg / mL (P>0.05), and there was a significant difference at concentrations of 100μg / mL and above compared with the blank control group (P<0.01).

[0129] The concentrations of earthworm extract transfersome gel group had significant differences in the number of early and late apoptotic and necrotic cells in SiHa cells compared with the solvent control group (P<0.01), and there was a concentration-effect relationship. When treating Hela cells, compared with the solvent control group, the number of apoptotic and necrotic cells in the 20μg / mL and above concentration groups was significantly different (P<0.01), while there was no significant difference in the 10μg / mL concentration group.

[0130] Through comparative analysis, compared with the earthworm extract solution group, the earthworm extract transfersome gel group had a similar or even stronger apoptosis rate on the two cancer cells when the concentration was one order of magnitude lower.

[0131] 3. Effects of earthworm extract and transfersome gel on HPV16 and HPV18 mRNA expression

[0132] After administration of different doses of earthworm extract and transfersome gel, total RNA was extracted from SiHa and Hela cells, and q-PCR was performed to determine the mRNA expression of HPV16 and HPV18 E6 and E7. The results are shown in Tables 4 and Figure 6 , Figure 7 shown.

[0133] Table 4 Effects of earthworm extract and transfersome gel on HPV16 and HPV18 E6 / E7 mRNA expression

[0134]

[0135]

[0136] Note: n=6, * indicates P<0.05 compared with the control group, ** indicates P<0.01 compared with the control group, *** indicates P<0.001 compared with the control group, **** indicates P<0.0001 compared with the control group; the control group here refers to: in the earthworm extract solution, it refers to the solution group with an earthworm extract concentration of 0 (i.e., the blank control group described below), and in the earthworm extract transfersome gel group, it refers to the group with an earthworm extract transfersome gel concentration of 0 (i.e., the solvent control group described below).

[0137] From Table 4 and Figure 6 , Figure 7 It can be seen that compared with untreated cells, both the earthworm extract solution group and the earthworm extract transfersome gel group could change the morphology of SiHa cells and significantly reduce the mRNA expression levels of key oncogenes E6 and E7 in SiHa cells (Table 4 and Figure 6 It can be seen that when the concentration of the earthworm extract transfersome gel is 20 μg / mL, E6 and E7 are basically not expressed, indicating that SiHa cells basically do not grow at this time. The earthworm extract solution group and the earthworm extract transfersome gel group can also change the morphology of Hela cells and significantly reduce the mRNA expression levels of key oncogenes E6 and E7 in Hela cells (Table 4 and Figure 7 ), but compared with SiHa cells, the effect of earthworm extract solution group and earthworm extract transfersome gel group on the mRNA expression levels of E6 and E7 in Hela cells was not as great as that on SiHa cells. When the concentration of earthworm extract transfersome gel was 20 μg / mL, the expression levels of E6 and E7 were 0.792 and 0.692, and when the concentration increased to 25 μg / mL, the expression levels of E6 and E7 were 0.365 and 0.400, indicating that earthworm extract transfersome gel has different effects on different cancer cells, and has a greater effect on HPV16-positive cells. When the concentration is increased, it can also inhibit the growth of HPV 18-positive cells and promote their apoptosis.

[0138] From Table 4 and Figure 6 , Figure 7 It can be seen that after the earthworm extract is made into a transfersome gel in the present invention, compared with the earthworm extract solution, its effect of reducing the mRNA expression levels of key oncogenes E6 and E7 in SiHa and Hela cells can be significantly improved (the concentration is reduced by one order of magnitude), indicating that the earthworm extract transfersome gel described in the present invention has a good inhibitory effect on HPV virus.

[0139] It can be seen that the earthworm extract transfersome gel prepared by the present invention can not only promote the shrinkage and death of cervical cancer cells, but also effectively enter the cells to inhibit the expression of HPV virus. The combination of the earthworm extract and the transfersome gel of the present invention can be used to resist HPV. Cell experiments have shown that it can significantly reduce the expression level of HPVmRNA gene after acting on SiHa cells and Hela cells.

[0140] The present invention has been described in detail above in conjunction with specific implementations and exemplary examples, but these descriptions cannot be understood as limiting the present invention. Those skilled in the art understand that, without departing from the spirit and scope of the present invention, a variety of equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its implementation methods, all of which fall within the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing an anti-HPV transfersome gel of earthworm extract, It is characterized in that The preparation method comprises the following steps: Step 1, centrifuging the body cavity fluid of sexually mature earthworms, collecting the supernatant, filtering, and freeze-drying to obtain earthworm extract; Step 2, dissolving lecithin and cholesterol in a solvent, evaporating the solvent under reduced pressure to obtain a lipid layer, dissolving the earthworm extract obtained in step 1 in water and stirring evenly, then adding the lipid layer to hydrate to obtain lipid vesicles, and then swelling, ultrasonicating, and extruding to obtain earthworm extract transfersomes; Step 3: Add the earthworm extract transfersome prepared in step 2 into the gel and mix well to obtain the anti-HPV earthworm extract transfersome gel.

2. The preparation method according to claim 1, It is characterized in that In step 1, The coelomic fluid of sexually mature earthworms was centrifuged at 0-4°C; The collected supernatant was filtered through a 0.20-0.25 μm microporous membrane.

3. The preparation method according to claim 1, It is characterized in that In step 2, Dissolve the edge activator together with lecithin and cholesterol in a solvent; or Dissolve the edge activator and the earthworm extract obtained in step 1 in water and stir evenly.

4. The preparation method according to claim 3, It is characterized in that In step 2, The mass ratio of the lecithin, cholesterol, edge activator and earthworm extract is (5-9):1:(0.6-1.4):

1.

5. The preparation method according to claim 1, It is characterized in that In step 2, Egg yolk lecithin and cholesterol are dissolved in ethanol, and then the solvent is evaporated under reduced pressure at 45-55°C to obtain a lipid layer.

6. The preparation method according to claim 1, It is characterized in that In step 2, The earthworm extract obtained in step 1 is dissolved in ultrapure water at room temperature, and then added to the lipid layer for hydration at 35-45° C. and a stirring speed of 70-80 rpm for 1-2 hours to obtain lipid vesicles.

7. The preparation method according to claim 1, It is characterized in that In step 2, The lipid vesicles were expanded at room temperature for 1-3 h, then sonicated in an ice bath for 20-30 min, and then extruded through microporous filter membranes of 0.70-0.90 μm, 0.40-0.50 μm, and 0.20-0.25 μm in sequence.

8. The preparation method according to claim 1, It is characterized in that In step 3, The gel is Carbomer 980; Ultrapure water is added to the carbomer to completely swell it, the pH is adjusted to 6.0-6.5, and then the earthworm extract transfersome prepared in step 2 is added at a mass ratio of 1:1 to mix.

9. An anti-HPV earthworm extract transfersome gel prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the anti-HPV earthworm extract transfersome gel according to claim 9 in the preparation of drugs for preventing and treating cervical cancer infected by HPV.

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