Dendrobium extract delivery gel against HPV and its preparation method and application

Abstract

The application belongs to the technical field of medicine, and particularly relates to an anti-HPV earthworm extract delivery body gel and a preparation method and application thereof. The preparation method comprises the following steps: stirring and hydrating earthworm extract and a lipid layer to obtain lipid vesicles, then performing inflation, ultrasonic treatment and extrusion to obtain earthworm extract delivery bodies, and finally adding a carbomer after adjusting pH and uniformly mixing to obtain the anti-HPV earthworm extract delivery body gel. The application improves the inhibitory effect on HPV viruses by compounding the carbomer gel with the earthworm extract delivery body, can promote the shrinkage and death of cervical cancer cells, further improves the viscosity of the earthworm extract delivery body by combining the gel with the earthworm extract delivery body, improves the adhesion of the earthworm extract delivery body and the inner wall of the vagina, and thus improves the effect of preventing and treating HPV virus infection.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an anti-HPV earthworm extract delivery gel, its preparation method, and its application. Background Technology

[0002] Cervical cancer is one of the most common malignant tumors in women and has become a major threat to women's health worldwide. Studies have found that almost all cervical cancer cases are related to human papillomavirus (HPV) infection, with high-risk HPV infection being a major risk factor for cervical cancer. HPV types 16 and 18 are particularly closely associated with the development of cervical cancer. Since there are currently no specific drugs or effective treatments, finding new anti-HPV drugs is of great significance for preventing cervical cancer and improving treatment outcomes for cervical cancer patients.

[0003] Earthworms, known as "dilong" in traditional Chinese medicine, possess a unique chemical composition, including high levels of unsaturated fatty acids, relatively abundant trace elements such as Fe, Mn, Cu, Zn, and Se, as well as various active ingredients such as lumbromine, earthworm antipyretic alkaloids, purines, choline, and cholesterol. Studies have found that earthworm extract has 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, enhancing the specific immune function of these mice. However, in clinical applications, the limited efficacy of earthworm extract directly restricts its use, especially in anti-HPV applications. Summary of the Invention

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

[0005] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

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

[0007] Step 1: Centrifuge the body cavity fluid of sexually mature earthworms, collect the supernatant, filter, and freeze-dry to obtain earthworm extract;

[0008] Step 2: Dissolve lecithin and cholesterol in a solvent, evaporate the solvent under reduced pressure to obtain a lipid layer, dissolve the earthworm extract obtained in Step 1 in water and stir evenly, then add the lipid layer to hydrate and obtain lipid vesicles, then expand, sonicate and squeeze to obtain the earthworm extract delivery body.

[0009] Step 3: Add the earthworm extract delivery body obtained in Step 2 to the gel and mix well to obtain the anti-HPV earthworm extract delivery body gel.

[0010] In step 1,

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

[0012] Preferably, the collected supernatant is filtered through a microporous membrane with a diameter of 0.20–0.25 μm.

[0013] In step 2,

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

[0015] The edge activator and the earthworm extract obtained in step 1 were dissolved in water and stirred until homogeneous.

[0016] Preferably, the mass ratio of 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 a lipid layer is added for hydration at 35-45°C and 70-80 rpm for 1-2 hours to obtain lipid vesicles.

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

[0020] In step 3,

[0021] Preferably, the gel is carbomer 980;

[0022] Add water to the carbomer until it swells completely, adjust the pH to 6.0-6.5, and then add it to the earthworm extract delivery body prepared in step 2 at a mass ratio of 1:1 for mixing.

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

[0024] A third aspect of the present invention is to provide the application of the earthworm extract delivery gel described in the second aspect of the present invention in the preparation of a cervical cancer drug for the prevention and treatment of HPV infection.

[0025] The beneficial effects of this invention are as follows:

[0026] (1) In this invention, earthworm extract is loaded into a delivery body and combined with a gel. The resulting earthworm extract delivery body gel can not only promote the shrinkage and death of cervical cancer cells, but also effectively enter the cells to inhibit HPV virus expression. 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 humans.

[0027] In this invention, earthworm extract is first dispersed in water, and then an edge activator is added and mixed to improve the encapsulation efficiency of the earthworm extract. Mixing carbomer gel with a pH of 6.0-6.5 with the earthworm extract delivery system further increases the viscosity of the earthworm extract delivery system, improves its adhesion to the vaginal wall, and helps prolong the retention time of the drug (earthworm extract delivery system) in the vagina, thus increasing the duration and effectiveness of the earthworm extract's efficacy. This further enhances the prevention and treatment of HPV infection, while also reducing the frequency of administration and improving patient compliance, thereby increasing the application of earthworm extract in anti-HPV treatment.

[0028] (2) The preparation method described in this invention is simple, the raw materials are readily available, and it can be industrialized on a large scale. This delivery gel has good application prospects in drugs for the prevention and treatment of cervical cancer caused by HPV infection. Attached Figure Description

[0029] Figure 1 Photograph (a) and transmission electron microscopy characterization (b) of the earthworm extract delivery system prepared in Example 6 are shown.

[0030] Figure 2 A photograph of the delivery gel prepared in Example 12 is shown.

[0031] Figure 3 Photograph (a) and micrograph (b) show the effect of the delivery gel prepared in Example 12 on the skin irritation of animals;

[0032] Figure 4 Microscopic images showing the effects of the blank control group, earthworm solution group, and earthworm carrier gel group on the morphology of SiHha cells;

[0033] Figure 5 Microscopic images showing the effects of the blank control group, the earthworm solution group, and the earthworm delivery gel group on the morphology of HeLa cells;

[0034] Figure 6 A bar chart showing the effects of earthworm solution group and earthworm delivery body gel group on HPV16 E6 and E7 mRNA expression is shown.

[0035] Figure 7 The bar chart shows the effects of earthworm solution and earthworm delivery gel on HPV18 E6 and E7 mRNA expression. Detailed Implementation

[0036] The present invention will now be described in detail, and its features and advantages will become clearer and more apparent from these descriptions.

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

[0038] Step 1: Centrifuge the body cavity fluid of sexually mature earthworms, collect the supernatant, filter, and freeze-dry to obtain earthworm extract;

[0039] Step 2: Dissolve lecithin and cholesterol in a solvent, evaporate the solvent under reduced pressure to obtain a lipid layer, dissolve the earthworm extract obtained in Step 1 in water and stir evenly, then add the lipid layer to hydrate and obtain lipid vesicles, then expand, sonicate and squeeze to obtain the earthworm extract delivery body.

[0040] Step 3: Add the earthworm extract delivery body obtained in Step 2 to the gel and mix well to obtain the anti-HPV earthworm extract delivery body gel.

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

[0042] In step 1, the earthworm cavity fluid is prepared through the following steps:

[0043] Select sexually mature earthworms (the reproductive organs are a marker of sexual maturity), wash them, and place them on filter paper soaked in phosphate-buffered saline (PBS) for two days to allow them to expel the soil from their abdomens, thus avoiding unnecessary contamination during the collection of body cavity fluid.

[0044] Place the cleaned, live earthworms in a clean beaker to stimulate them to contract and eject a yellow, viscous liquid, i.e., coelomic fluid, from their body surface. Preferably, stimulation can be performed using a 6V electrical stimulator.

[0045] Centrifuge the body cavity fluid of sexually mature earthworms at 0–4°C, preferably at 4°C.

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

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

[0048] Experiments have shown that mixing the edge activator with earthworm extract before preparation results in a more transparent earthworm extract delivery system with no particulate precipitation and a higher encapsulation rate.

[0049] The lecithin is preferably egg yolk lecithin.

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

[0051] Preferably, the mass ratio of 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. The mixture is then dried under vacuum overnight to allow the solvent to evaporate completely.

[0054] The earthworm extract obtained in step 1 was dissolved in ultrapure water at room temperature, and then a lipid layer was added for hydration at 35-45°C and 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 a lipid layer is added at 40°C and 75 rpm for hydration 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 the vesicles were sequentially squeezed through microporous membranes of 0.70–0.90 μm, 0.40–0.50 μm, and 0.20–0.25 μm.

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

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

[0059] Add ultrapure water to the carbomer until it swells completely, adjust the pH to 6.0-6.5, and then slowly mix the earthworm extract delivery body obtained in step 2 at a mass ratio of 1:1 to obtain the anti-HPV earthworm extract delivery body gel.

[0060] The delivery system is a modified liposome that not only possesses the advantages of traditional liposomes, such as good biocompatibility and low toxicity, but also exhibits excellent deformability due to the addition of an edge activator. It can pass through channels several times smaller than its own size, making it an ideal carrier for transdermal drug delivery formulations. Studies have also found that the delivery system can effectively accommodate the vast majority of drugs, regardless of their structure, polarity, or size. More importantly, the delivery system's flexible vesicle structure... While enhancing cellular uptake, gels, when applied alone to skin and mucous membranes, suffer from poor adhesion. Although the vagina itself lacks glands, it produces mucus forming a layer that hinders drug contact with target tissues. Gels, however, swell rapidly in the aqueous environment of the vagina, adhering tightly to the vaginal mucosa. Therefore, gels combined with delivery systems significantly improve these issues, prolonging drug retention time in the vagina, enhancing therapeutic efficacy, reducing dosing frequency, and improving patient compliance.

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

[0062] A third aspect of the present invention is to provide the application of the earthworm extract delivery gel described in the second aspect of the present invention in the preparation of a cervical cancer drug for the prevention and treatment of HPV infection.

[0063] Example

[0064] The present invention is further illustrated below with specific examples. These embodiments are merely illustrative and not intended to limit the scope of the invention. All raw materials used in the embodiments of the present invention are commercially available.

[0065] Example 1

[0066] Sexually mature earthworms (identified by the genital patch) were selected, washed, and placed on filter paper soaked in phosphate-buffered saline (PBS) for two days to allow them to expel the soil from their abdomens, thus avoiding unnecessary contamination during the collection of coelomic fluid. The washed, live earthworms were placed in a clean beaker and stimulated with a homemade 6V electrical stimulator. The earthworms contracted, expelling a yellow, viscous liquid from their bodies—this was the coelomic fluid. The collected coelomic fluid was centrifuged at 4°C, and the supernatant was collected. After sterilization through a 0.22μm microporous membrane, it was freeze-dried to obtain a lyophilized powder, which is the earthworm extract, stored at -20°C. BCA analysis showed a protein content of 80.3%.

[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 into a dry round-bottom flask, add an appropriate amount of ethanol to dissolve them, and evaporate the solvent under reduced pressure at 50 °C using a rotary evaporator. After forming a uniform lipid layer on the flask wall, dry under vacuum overnight 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 membrane at 40 °C and 75 rpm, hydrate for 1 h to obtain lipid vesicles; allow them to fully expand at room temperature (22-25 °C) for 2 h, then sonicate in an ice bath for 25 min, with a 3-second "on" and 3-second "off" cycle. Finally, squeeze the obtained vesicle system sequentially through microporous membranes of 0.80 μm, 0.45 μm, and 0.22 μm to obtain the earthworm extract delivery body.

[0069] Example 3

[0070] Weigh 35 mg of egg yolk lecithin, 5 mg of cholesterol, 2.5 mg of Tween 80, and 2.5 mg of sodium cholate into a dry round-bottom flask, add an appropriate amount of ethanol to dissolve them, and evaporate the solvent under reduced pressure at 50 °C using a rotary evaporator. After forming a uniform lipid layer on the flask wall, dry under vacuum overnight 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 membrane at 40 °C and 75 rpm, and hydrate for 1 h to obtain lipid vesicles. Allow them to fully expand at room temperature (22-25 °C) for 2 h, and then sonicate them in an ice bath for 25 min, with the on end for 3 seconds and the off end for 3 seconds. Finally, squeeze the obtained vesicle system sequentially through microporous membranes of 0.80 μm, 0.45 μm, and 0.22 μm to obtain the earthworm extract delivery body.

[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 into a dry round-bottom flask, add an appropriate amount of ethanol to dissolve them, and evaporate the solvent under reduced pressure at 50 °C using a rotary evaporator. After a uniform lipid layer forms on the flask wall, dry under vacuum overnight 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 membrane at 40 °C and 75 rpm, hydrate for 1 h to obtain lipid vesicles; allow them to fully expand at room temperature (22-25 °C) for 2 h, then sonicate in an ice bath for 25 min, with a 3-second "on" and 3-second "off" cycle. Finally, sequentially squeeze the obtained vesicle system through microporous membranes of 0.80 μm, 0.45 μm, and 0.22 μm to obtain the earthworm extract delivery body.

[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, and evaporate the solvent under reduced pressure at 50 °C using a rotary evaporator. After forming a uniform lipid layer on the flask wall, dry under vacuum overnight 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 membrane at 40 °C and 75 rpm, and hydrate for 1 h to obtain lipid vesicles. Allow them to fully expand at room temperature (22-25 °C) for 2 h, and then sonicate them in an ice bath for 25 min, with the on end for 3 seconds and the off end for 3 seconds. Finally, squeeze the obtained vesicle system sequentially through microporous membranes of 0.80 μm, 0.45 μm, and 0.22 μm to obtain the earthworm extract delivery body.

[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, and evaporate the solvent under reduced pressure at 50 °C using a rotary evaporator. After forming a uniform lipid layer on the flask wall, dry under vacuum overnight 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 membrane at 40 °C and 75 rpm, and hydrate for 1 h to obtain lipid vesicles. Allow them to fully expand at room temperature (22-25 °C) for 2 h, and then sonicate them in an ice bath for 25 min, with the on end for 3 seconds and the off end for 3 seconds. Finally, squeeze the obtained vesicle system sequentially through microporous membranes of 0.80 μm, 0.45 μm, and 0.22 μm to obtain the earthworm extract delivery system.

[0077] The photograph and transmission electron microscope image of the earthworm extract delivery system are as follows: Figure 1 As shown. Figure 1 In sample a, the earthworm extract delivery system appears as a pale green, nearly colorless, translucent solution, clear in appearance, without any precipitate or flocculent matter. Figure 1 In section b, the earthworm extract delivery system under transmission electron microscopy is basically spherical, with rounded particles, uniform distribution, and no 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, and evaporate the solvent under reduced pressure at 50 °C using a rotary evaporator. After forming a uniform lipid layer on the flask wall, dry under vacuum overnight 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 membrane at 40 °C and 75 rpm, and hydrate for 1 h to obtain lipid vesicles. Allow them to fully expand at room temperature (22-25 °C) for 2 h, and then sonicate them in an ice bath for 25 min, with the sonication timed 3 s on and 3 s off. Finally, squeeze the obtained vesicle system sequentially through microporous membranes of 0.80 μm, 0.45 μm, and 0.22 μm to obtain the earthworm extract delivery system.

[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 evaporate the solvent under reduced pressure at 50 °C using a rotary evaporator. After a uniform lipid layer forms on the flask wall, dry under vacuum overnight 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 membrane at 40 °C and 75 rpm, and hydrate for 1 h to obtain lipid vesicles. Allow them to fully expand at room temperature (22-25 °C) for 2 h, and then sonicate them in an ice bath for 25 min, with the sonication timed 3 s on and 3 s off. Finally, squeeze the obtained vesicle system sequentially through microporous membranes of 0.80 μm, 0.45 μm, and 0.22 μm to obtain the earthworm extract delivery system.

[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 evaporate the solvent under reduced pressure at 50 °C using a rotary evaporator. After a uniform lipid layer forms on the flask wall, dry under vacuum overnight 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 membrane at 40 °C and 75 rpm, and hydrate for 1 h to obtain lipid vesicles. Allow them to fully expand at room temperature (22-25 °C) for 2 h, and then sonicate them in an ice bath for 25 min, with the sonication timed 3 s on and 3 s off. Finally, squeeze the obtained vesicle system sequentially through microporous membranes of 0.80 μm, 0.45 μm, and 0.22 μm to obtain the earthworm extract delivery system.

[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 evaporate the solvent under reduced pressure at 50 °C using a rotary evaporator. After a uniform lipid layer forms on the flask wall, dry under vacuum overnight 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 membrane at 40 °C and 75 rpm, and hydrate for 1 h to obtain lipid vesicles. Allow them to fully expand at room temperature (22-25 °C) for 2 h, and then sonicate them in an ice bath for 25 min, with the on end for 3 seconds and the off end for 3 seconds. Finally, squeeze the obtained vesicle system sequentially through microporous membranes of 0.80 μm, 0.45 μm, and 0.22 μm to obtain the earthworm extract delivery body.

[0086] Example 11

[0087] Weigh 12.5 mg of egg yolk lecithin and 2.5 mg of cholesterol into a dry round-bottom flask, add an appropriate amount of ethanol to dissolve them, and evaporate the solvent under reduced pressure at 50 °C using a rotary evaporator. After a uniform lipid layer forms on the flask wall, dry under vacuum overnight to completely evaporate the organic solvent. Dissolve 2.5 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 membrane at 40 °C and 75 rpm, and hydrate for 1 h to obtain lipid vesicles. Allow them to fully expand at room temperature (22-25 °C) for 2 h, then sonicate in an ice bath for 25 min, with a 3-second "on" and 3-second "off" cycle. Finally, sequentially squeeze the obtained vesicle system through microporous membranes of 0.80 μm, 0.45 μm, and 0.22 μm to obtain the earthworm extract delivery system.

[0088] Example 12

[0089] After completely swelling 1% Carbomer 980 in distilled water, triethanolamine was added dropwise to adjust the pH to 6.0-6.5. Then, it was slowly mixed with the transporter from Example 6 at a 1:1 mass ratio under gentle stirring to obtain a transporter gel.

[0090] A photograph of the transporter gel is shown below. Figure 2 As shown, from Figure 2 As can be seen, the delivery gel has a nearly colorless gel appearance, with no visible particles or lumps, is easy to pick up, has moderate viscosity, and a fine and uniform texture.

[0091] Example 13

[0092] After completely swelling 1% Carbomer 980 in distilled water, triethanolamine was added dropwise to adjust the pH to 6.0-6.5. Then, it was slowly mixed with the transporter from Example 10 at a 1:1 mass ratio under gentle stirring to obtain a transporter gel.

[0093] Example 14

[0094] After completely swelling 1% Carbomer 980 in distilled water, triethanolamine was added dropwise to adjust the pH to 6.0-6.5. Then, it was slowly mixed with the transporter from Example 11 at a 1:1 mass ratio under gentle stirring to obtain a transporter gel.

[0095] Experimental Example

[0096] Experimental Example 1: Particle Size, PDI, Zeta Potential, and Encapsulation Efficiency Tests

[0097] The particle size, PDI, Zeta potential, and encapsulation efficiency of the earthworm extract delivery systems prepared in Examples 2-11 were tested respectively, and 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 shown in Table 1, the particle size of the earthworm extract transporters prepared in Examples 2-11 is between 83 nm and 100 nm, indicating that the earthworm extract transporters of the present invention have small particle sizes. The earthworm extract transporter prepared in Example 6 has the highest encapsulation efficiency, reaching 71.3%, with an average particle size of (89.8 ± 0.9) nm and an average Zeta potential of (-38.9 ± 1.1) mV.

[0101] Experiment Example 2: Skin Irritation Test of Earthworm Extract Delivery Gel

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

[0103] Healthy male ICR mice with intact skin were selected and anesthetized with 20% urethane (5 mL / kg). The abdominal hair was removed to an area of ​​2 cm × 2 cm, and the limbs were fixed to a board. 0.5 g of the delivery gel prepared in Example 12 of this invention was applied to one side of the hairless skin, while a blank delivery gel (without the drug) was applied to the other side as a control. After 12 hours, the covering was removed, the skin surface was washed with warm water, and the skin reaction at the application site was observed. The mice were then euthanized, and the skin at the application site was removed. Adipose tissue was carefully removed, stained with hematoxylin and eosin (HE), and the tissue cells were observed under a light microscope for any abnormalities. Figure 3 As shown, Figure 3 a is a photograph of the mouse's skin after the experiment. Figure 3 b is a microscopic image 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 is evident that the epidermal tissue structure is intact; the dermis has interlaced 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 delivery gel of the present invention is non-irritating to the skin.

[0105] Experimental Example 3: The effect of earthworm extract delivery gel on HPV clearance

[0106] I. Experimental Methods

[0107] 1. Cell Culture

[0108] Human cervical cancer SiHa cells and human cervical cancer HeLa cells were revived and seeded in 5 mL LDM complete medium, then transferred to a 25 cm⁻¹ culture medium. 2 The culture was cultured in a culture flask at 37°C and 5% CO2 until the logarithmic growth phase, and then used for later use.

[0109] 2. CCK-8 assay for cell viability

[0110] SiHa and HeLa cells in logarithmic growth phase were harvested and divided into two groups of 5 × 10⁶ cells per well. 3Cells were seeded in 96-well cell culture plates, with the following groups: a solvent control group, a *Pheretima aspergillum* extract delivery gel group (using the delivery gel prepared in Example 12, referred to as the *Pheretima aspergillum* delivery gel group, where ultrapure water was added to the delivery gel and mixed to obtain aqueous solutions with concentrations of 10 μg / mL, 20 μg / mL, and 25 μg / mL, respectively), a *Pheretima aspergillum* extract solution group (using the *Pheretima aspergillum* extract prepared in Example 1 dissolved in ultrapure water to prepare an aqueous solution with a concentration of 1 mg / mL, referred to as the *Pheretima aspergillum* solution group, where ultrapure water was added to the *Pheretima aspergillum* extract aqueous solution and mixed to obtain aqueous solutions with concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 500 μg / mL, respectively), and an untreated control group (using DMEM culture medium containing 1% DMSO). Each group had 6 replicates. Cell viability was determined using the CCK-8 assay. If the cell survival rate is ≥80%, it is considered to have no toxic effect on cells.

[0111] 3. Flow cytometry detection of cervical cancer cell apoptosis rate

[0112] SiHa and HeLa cells in logarithmic growth phase were harvested at a concentration of 2 × 10⁻⁶. 5 The cells were seeded at a density of / wells into 6-well plates. After cell attachment, the following groups were set up: a solvent control group, an earthworm extract delivery gel group (using the delivery gel prepared in Example 12, adding ultrapure water to the delivery gel and mixing to obtain delivery gel aqueous solutions with concentrations of 10 μg / mL, 20 μg / mL, and 25 μg / mL, respectively), an earthworm extract solution group (using the earthworm extract prepared in Example 1 dissolved in ultrapure water to prepare an earthworm extract aqueous solution with a concentration of 1 mg / mL, adding ultrapure water to the earthworm extract aqueous solution and mixing to obtain earthworm extract aqueous solutions with concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, and 250 μg / mL, respectively), and untreated cells as a culture medium control group (untreated cells here mean: cultured only in culture medium, which is DMEM containing 1% DMSO). Each group had 6 replicates. After 24 hours, cells were collected, resuspended in 500 μL Binding Buffer, and then incubated with 5 μL Annexin V-FITC and 2 μL LPI at room temperature in the dark for 15 minutes. Apoptosis was then detected by flow cytometry.

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

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

[0115] II. Experimental Results

[0116] 1. Effects of earthworm extract and delivery gel on HeLa cell proliferation

[0117] SiHa and HeLa cells were treated with different concentrations of SiHa earthworm extract and delivery gel, and then observed under a microscope. Figure 4 As shown in Table 2, the test results of earthworm extract and delivery gel on the proliferation of SiHa and HeLa cells are presented.

[0118] Table 2. Effects of earthworm extract and delivery gel on the 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 the solution group with a concentration of 0 earthworm extract in the earthworm extract solution (i.e., the blank control group mentioned below), and the group with a concentration of 0 earthworm extract delivery gel in the earthworm extract delivery gel group (i.e., the solvent control group mentioned below).

[0122] From Table 2 and Figure 4 It can be seen that in the earthworm extract solution group, cell growth was not significantly inhibited at concentrations of 10 μg / mL and below. However, when the concentration increased to 100 μg / mL, cell viability decreased significantly, and phenomena such as a significant slowdown in proliferation rate, increased cell debris, increased intercellular spaces, and some cells appearing vacuolated were observed. Figure 4 When administered in the form of a delivery gel, different concentrations altered cell morphology and induced cell death to varying degrees. At a concentration of 20 μg / mL, cell survival decreased by more than half; at 25 μg / mL, cell survival was approximately 10% or even lower. This indicates that the earthworm extract delivery gel, at a concentration one order of magnitude lower than that of the earthworm extract solution, significantly inhibited the growth of SiHa and HeLa cells, greatly enhancing the activity and efficacy of the earthworm extract.

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

[0124] The results of the tests on apoptosis of SiHa and HeLa cells by earthworm extract and delivery gel are shown in Table 3. Figure 5 As shown.

[0125] Table 3. Effects of earthworm extract and delivery 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 the solution group with a concentration of 0 earthworm extract in the earthworm extract solution (i.e., the blank control group mentioned below), and the group with a concentration of 0 earthworm extract delivery gel in the earthworm extract delivery gel group (i.e., the solvent control group mentioned 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 showed significant differences in the number of apoptotic and necrotic cells in SiHa cells (P<0.01); while for HeLa cells, at 50 μg / mL, there was no significant difference in the number of apoptotic and necrotic cells compared with the blank control group (P>0.05), but at concentrations of 100 μg / mL and above, there were significant differences compared with the blank control group (P<0.01).

[0129] The concentrations of earthworm extract delivery gel significantly affected the number of early and late apoptotic and necrotic cells in SiHa cells compared to the solvent control group (P<0.01), exhibiting a concentration-response relationship. When treating HeLa cells, compared to the solvent control group, the 20 μg / mL and higher concentration groups showed significant differences in the number of apoptotic and necrotic cells (P<0.01), while the 10 μg / mL concentration group showed no significant difference.

[0130] Comparative analysis showed that, compared with the earthworm extract solution group, the earthworm extract delivery gel group had similar or even stronger apoptosis rates against the two cancer cells at a concentration one order of magnitude lower.

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

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

[0133] Table 4. Effects of earthworm extract and delivery 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 the solution group with a concentration of 0 earthworm extract in the earthworm extract solution (i.e., the blank control group mentioned below), and the group with a concentration of 0 earthworm extract delivery gel in the earthworm extract delivery gel group (i.e., the solvent control group mentioned 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 delivery gel group can alter the morphology of SiHa cells and significantly reduce the mRNA expression levels of key oncogenes E6 and E7 in SiHa cells (Table 4 and 1). Figure 6 As can be seen, when the concentration of the earthworm extract delivery gel was 20 μg / mL, E6 and E7 were basically not expressed, indicating that SiHa cells did not grow at this concentration. The earthworm extract solution group and the earthworm extract delivery gel group also altered HeLa cell morphology and significantly reduced the mRNA expression levels of the key oncogenes E6 and E7 in HeLa cells (Table 4 and...). Figure 7 However, compared with SiHa cells, the effects of earthworm extract solution and earthworm extract delivery gel on the mRNA expression levels of E6 and E7 in HeLa cells were not as significant as those on SiHa cells. When the concentration of earthworm extract delivery gel was 20 μg / mL, the expression levels of E6 and E7 were 0.792 and 0.692, respectively. When the concentration was increased to 25 μg / mL, the expression levels of E6 and E7 were 0.365 and 0.400, respectively. This indicates that the earthworm extract delivery gel has different effects on different cancer cells, with a greater effect on HPV16 positive cells. When the concentration is increased, it can also inhibit the growth of HPV18 positive cells and promote their apoptosis.

[0138] From Table 4 and Figure 6 , Figure 7 It is evident that, compared to earthworm extract solution, the earthworm extract gel prepared by this invention significantly enhances the effect of reducing the mRNA expression levels of key oncogenes E6 and E7 in SiHa and HeLa cells (concentration is reduced by an order of magnitude), indicating that the earthworm extract gel of this invention has a good inhibitory effect on HPV virus.

[0139] Therefore, the earthworm extract delivery gel prepared in this invention can not only promote the shrinkage and death of cervical cancer cells, but also effectively enter cells to inhibit HPV virus expression. The combination of earthworm extract and delivery gel in this invention can be used for anti-HPV purposes. Cell experiments have shown that it can significantly reduce the expression level of HPV mRNA gene after acting on SiHa cells and HeLa cells.

[0140] The present invention has been described in detail above with reference to specific embodiments and exemplary examples; however, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the invention, and all such modifications and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims.

Claims

1. A method for preparing an anti-HPV earthworm extract delivery gel, characterized in that, The preparation method includes the following steps: Step 1: Centrifuge the body cavity fluid of sexually mature earthworms, collect the supernatant, filter, and freeze-dry to obtain earthworm extract; The earthworm body cavity fluid is prepared through the following steps: Select sexually mature earthworms, wash them, and place them on filter paper soaked in phosphate buffer for two days to allow them to expel the soil from their abdomens. Place the cleaned live earthworms in a clean beaker, and use a 6V electric stimulator to stimulate the earthworms to contract and spray out a yellow, viscous liquid from their body surface, which is the earthworm's body cavity fluid. Step 2: Dissolve lecithin and cholesterol in a solvent, evaporate the solvent under reduced pressure to obtain a lipid layer, dissolve the earthworm extract obtained in Step 1 in water and stir until homogeneous, then add the lipid layer to hydrate and obtain lipid vesicles. After expansion, ultrasonic treatment, and extrusion, the earthworm extract delivery body is obtained. In the process of preparing the earthworm extract delivery body, an edge activator is also added. The edge activator is Tween 80 and sodium cholate mixed in a mass ratio of 1:

1. The edge activator is dissolved together with lecithin and cholesterol in a solvent; or, the edge activator and the earthworm extract obtained in step 1 are dissolved in water and stirred evenly. Step 3: Add the earthworm extract delivery body obtained in Step 2 to the gel and mix evenly to obtain the anti-HPV earthworm extract delivery body gel. The gel is Carbomer 980. Add ultrapure water to the carbomer to completely swell it and adjust the pH to 6.0-6.

5.

2. The preparation method according to claim 1, characterized in that, In step 1, Centrifuge the body cavity fluid of sexually mature earthworms at 0–4°C; The collected supernatant was filtered through a microporous membrane with a diameter of 0.20–0.25 μm.

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

1.

4. The preparation method according to claim 1, characterized in that, In step 2, Egg yolk lecithin and cholesterol were dissolved in ethanol, and then the solvent was evaporated under reduced pressure at 45–55 °C to obtain the lipid layer.

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

6. The preparation method according to claim 1, 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 the vesicles were sequentially squeezed through microporous membranes of 0.70–0.90 μm, 0.40–0.50 μm, and 0.20–0.25 μm.

7. The preparation method according to claim 1, characterized in that, In step 3, The earthworm extract delivery body obtained in step 2 was added at a mass ratio of 1:1 and mixed.

8. An anti-HPV earthworm extract delivery gel prepared by the preparation method according to any one of claims 1 to 7.

9. The use of the anti-HPV earthworm extract delivery gel according to claim 8 in the preparation of cervical cancer drugs for the prevention and treatment of HPV infection.

Images