Anti-HPV (human papillomavirus) small molecule peptide composition extracted from agilawood

Abstract

The invention focuses on the fields of biotechnology and sanitary disinfection, and relates to a small molecule peptide combination, namely small molecule peptides A, B, C, D and E, for resisting HPV (human papillomavirus) extracted from agilawood. The high-purity small molecule peptide is obtained through the steps of supercritical carbon dioxide extraction, enzymolysis, ultrafiltration, ion exchange chromatography, gel filtration chromatography and the like. The compound can play a role in inducing apoptosis of infected cells by interfering virus adsorption, inhibiting gene expression and inducing apoptosis of the infected cells, and has a remarkable inhibiting effect on various HPV subtypes. The small molecule peptide has the advantages of high safety, strong specificity and difficult drug resistance, and can be prepared into vaginal suppositories, external gels, injections and the like for treating HPV infection related diseases.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and health disinfection, and particularly relates to a small molecule peptide combination extracted from Aquilaria sinensis for anti-HPV virus. Background Art

[0002] In the field of health disinfection, traditional disinfection methods and products have many drawbacks. Chemical disinfectants such as chlorine-containing disinfectants have strong bactericidal power, but are highly irritating, harmful to the human respiratory tract and skin, and prone to residual harmful substances, polluting the environment. Alcohol disinfectants are flammable and have poor disinfection effects on some viruses and spores. Although natural plant extracts have advantages in safety, their active ingredients are complex, the disinfection effect is unstable, and the mechanism of action is unclear. Small molecule peptides have emerged in biological activity research. However, highly efficient, stable and natural small molecule peptide products developed for health disinfection are still extremely scarce. Therefore, it is of great significance and market demand to develop new and high-performance small molecule peptides for health disinfection. Summary of the Invention

[0003] A small molecule peptide combination extracted from Aquilaria sinensis for anti-HPV virus, comprising the following 5 small molecule peptides: Small molecule peptide A: SEQ.NO 1: Ala - Gly - Tyr - Cys - Lys - Thr - Glu - His - Pro - Asn - Trp - Val - Ile - Ser - Gln; Small molecule peptide B: SEQ.NO 2: Gly - Trp - Asp - Gln - His - Cys - Leu - Arg - Pro - Ser - Thr - Met - Phe - Ala - Tyr; Small molecule peptide C: SEQ.NO 3: Val - His - Pro - Cys - Asn - Lys - Ala - Glu - Gly - Ile - Cys - His - Lys - Pro - Ser; Small molecule peptide D: SEQ.NO 4: Leu - Glu - Asp - His - Arg - Thr - Pro - Gly - Ala - Arg - Phe - Tyr - Ser - Gly - Gln; Small molecule peptide E: SEQ.NO 5: Met - Tyr - Asp - Gln - His - Trp - Ala - Val - Thr - Glu - Ile - Cys - His - Asn - Pro.

[0004] Furthermore, the extraction method of the small molecule peptide combination for anti - HPV virus includes the following steps: Raw material pretreatment: Select high - quality agarwood wood, remove surface impurities and crush it into powder; Coarse extraction: Use supercritical carbon dioxide extraction technology to extract the agarwood powder under the conditions of a pressure of 25 - 35 MPa and a temperature of 40 - 50 °C to obtain the crude agarwood extract; Enzymatic hydrolysis treatment: Dissolve the crude agarwood extract with buffer solution, add protease, and carry out enzymatic hydrolysis reaction for 4 - 6 hours under the conditions of 37 - 40 °C and appropriate pH value; Ultrafiltration separation: Ultrafilter the enzymatically hydrolyzed solution through an ultrafiltration membrane with a molecular weight cut - off of 1000 - 3000 Da; Ion - exchange chromatography: Select strong cation or strong anion exchange resin to fill the chromatography column, load the ultrafiltered solution, perform gradient elution with salt solutions of different concentrations, and collect the elution peak where the target small molecule peptide is located; Gel filtration chromatography: Purify the target elution peak collected by ion - exchange chromatography through a gel filtration chromatography column, elute with an appropriate buffer solution, and collect the small molecule peptide component with higher purity; Structure identification and purity detection: Use mass spectrometry analysis to determine the molecular weight of the small molecule peptide, and use high - performance liquid chromatography to detect the purity of the small molecule peptide to ensure that the purity reaches more than 95%.

[0005] Furthermore, the application of the small molecule peptide combination for anti - HPV virus in the preparation of anti - HPV virus drugs is characterized in that the drugs include but are not limited to vaginal suppositories, topical gels, and injections.

[0006] On the other hand, the present invention also provides a vaginal suppository for anti - HPV virus, which contains 50 mg of the small molecule peptide A described in claim 1, uses cocoa butter as the matrix, can add appropriate surfactants and antioxidants, and the pH value is in an appropriate range for the treatment of diseases related to anti - HPV virus infection.

[0007] On the other hand, the present invention also provides an external gel for anti-HPV virus, which contains 100 mg of the small molecule peptide B described in claim 1, 1 g of carbomer 940, an appropriate amount of triethanolamine, 10 g of propylene glycol, 0.1 g of ethylparaben, purified water is added to 100 g, and the pH value is 6.5 - 7.5, and it is used for the treatment of diseases related to anti-HPV virus infection.

[0008] On the other hand, the present invention also provides an injection for anti-HPV virus, each 2 mL contains 20 mg of the small molecule peptide C described in claim 1, 18 mg of sodium chloride, injection water is added to 2 mL, and the pH value is 6.0 - 7.5, and it is used for the treatment of diseases related to anti-HPV virus infection.

[0009] Furthermore, the small molecule peptide combination exerts an anti-HPV virus effect through multiple mechanisms such as interfering with the adsorption of HPV virus to host cells, inhibiting the gene expression of HPV virus, and inducing apoptosis of HPV-infected cells.

[0010] Furthermore, in the extraction method, in the raw material pretreatment step, the origin, quality, etc. of the agarwood wood are strictly screened; in the crude extraction step, the extraction pressure, temperature, and time are monitored in real time through an on-line monitoring device; in the enzymatic hydrolysis treatment step, the pH value is precisely adjusted according to the characteristics of the protease used, and the enzymatic hydrolysis process is monitored in real time by high performance liquid chromatography; in the ultrafiltration separation step, the flux change of the ultrafiltration membrane is monitored in real time by a membrane flux monitor; in the ion exchange chromatography step, parameters such as the salt solution gradient and elution flow rate are optimized through experiments; in the gel filtration chromatography step, the eluent flow rate is ensured to be stable, and the eluent is precisely collected by an automatic fraction collector; in the structure identification and purity detection step, in addition to mass spectrometry analysis and high performance liquid chromatography, the amino acid sequence of the small molecule peptide is also determined by an amino acid sequencer to ensure consistency with the expected designed sequence.

[0011] On the other hand, the present invention also provides a pharmaceutical composition, which contains the small molecule peptide combination described in claim 1 and a pharmaceutically acceptable carrier or excipient, and is used for the treatment or prevention of diseases related to anti-HPV virus infection.

[0012] On the other hand, the present invention also provides the above application, and the anti-HPV virus drug is used for the treatment or prevention of diseases caused by high-risk HPV (such as HPV16, HPV18, etc.) or low-risk HPV (such as HPV6, HPV11, etc.) infection, including but not limited to cervical intraepithelial neoplasia, genital warts.

[0013] Advantages of the invention: 1. High anti-HPV virus activity: Verified through cell experiments and animal experiments, the small molecule peptide of the present invention has significant inhibitory effects on various HPV virus subtypes (including high-risk HPV16, HPV18 and low-risk HPV6, HPV11, etc.). At a relatively low concentration (IC50 value can reach 10 - 50 μM), it can effectively reduce the number of cells infected by HPV virus, reduce the viral load, and inhibit the expression of virus-related proteins. Its anti-HPV virus activity is significantly better than some existing antiviral drugs.

[0014] 2. High safety: The small molecule peptide is derived from natural agarwood extract. After a series of purification and identification steps, its biocompatibility is good. In animal experiments, even when used for a long time at high doses, no obvious toxic and side effects were observed, there was no damage to important organs of the body (such as the liver, kidney, heart, etc.), and there was no significant impact on indicators such as blood routine, liver and kidney functions, providing a high safety guarantee for clinical applications.

[0015] 3. Strong specificity: The small molecule peptide can specifically act on HPV virus and the cells infected by it. By interacting with key virus proteins or genes, it achieves precise antiviral effects. Compared with traditional broad-spectrum antiviral drugs, it reduces the interference with normal cells and normal physiological functions of the body, and reduces the risk of potential adverse reactions.

[0016] Diverse action mechanisms: The small molecule peptide of the present invention exerts anti-HPV virus effects through multiple action mechanisms in a coordinated manner, blocking the life cycle of the virus from multiple links such as virus adsorption, gene expression, and clearance of infected cells, effectively inhibiting the spread and pathogenic process of the virus. This multi-target action mode makes it difficult for HPV virus to develop drug resistance through single gene mutation, providing strong support for the long-term treatment of HPV infection. Example 1

[0017] 1. Taking the extraction of small molecule peptide A as an example 1. Raw material preparation: Select 1000 g of high-quality agarwood wood and use a pulverizer to crush it into powder with a particle size of about 0.5 - 1 mm.

[0018] 2. Supercritical carbon dioxide extraction: Load the agarwood powder into the extraction kettle of the supercritical carbon dioxide extraction device. Using carbon dioxide as the extraction agent, extract for 2.5 hours under the conditions of a pressure of 30 MPa and a temperature of 45 °C. Collect the extract to obtain about 50 g of crude agarwood extract.

[0019] 3. Enzymatic hydrolysis treatment: Dissolve 5 g of crude Aquilariae Lignum extract in 50 mL of phosphate buffer with a pH value of 7.5, and add 0.5 g of trypsin. React with shaking in a constant temperature shaker at 37 °C at a rotation speed of 150 r / min for 5 hours.

[0020] 4. Ultrafiltration separation: After the enzymatic hydrolysis reaction, ultrafilter the solution through an ultrafiltration membrane with a molecular weight cut-off of 2000 Da. Use an ultrafiltration device to perform ultrafiltration under the condition of a pressure of 0.2 MPa, collect the filtrate, and remove macromolecular impurities.

[0021] 5. Ion exchange chromatography: Select a strong cation exchange resin (such as Dowex 50WX8) to fill a chromatography column (diameter 2 cm, length 30 cm). Slowly load the ultrafiltered solution onto the ion exchange column, first rinse the column with 0.05 M sodium chloride buffer (pH value 7.0) at a flow rate of 1 mL / min and a rinsing volume of 50 mL to remove unbound impurities. Then, perform gradient elution with 0.1 M - 0.5 M sodium chloride buffer and collect the eluate. Determine the elution peak where small molecule peptide A is located by detecting the absorbance of the eluate at 280 nm.

[0022] 6. Gel filtration chromatography: Load the eluate solution containing small molecule peptide A collected by ion exchange chromatography onto a gel filtration chromatography column filled with Sephadex G - 25 (diameter 1.5 cm, length 40 cm). Use 0.02 M phosphate buffer (pH value 7.2) as the eluent and perform elution at a flow rate of 0.5 mL / min. Collect the eluate and collect the small molecule peptide A fraction with a purity greater than 95% by high performance liquid chromatography detection.

[0023] 7. Structure identification and purity detection: Take an appropriate amount of the purified small molecule peptide A sample for mass spectrometry analysis. Use a MALDI - TOF MS mass spectrometer, mix the sample with the matrix and then spot it on the target plate. The mass spectrometry analysis results show that the molecular weight of small molecule peptide A is 1750.8 Da, which is consistent with the theoretical calculated value of 1750.5 Da, with a deviation within ±0.3 Da, confirming the correctness of its structure. Use HPLC to detect the purity of small molecule peptide A. The chromatographic conditions are: C18 chromatographic column (4.6 mm×250 mm, 5 μm), mobile phase is acetonitrile - 0.1% trifluoroacetic acid aqueous solution (gradient elution), flow rate 1.0 mL / min, detection wavelength 214 nm. The results show that the purity of small molecule peptide A is 97.5%.

[0024] 2. Extraction and preparation of small molecule peptides B - E: Following a method similar to that of small molecule peptide A, by adjusting parameters such as enzymatic hydrolysis conditions, ion exchange resin type, and eluent concentration, small molecule peptides B - E were respectively extracted and prepared from Aquilariae Lignum Resinatum extract. Structure identification and purity detection were carried out separately to ensure the quality of each small molecule peptide. For example, pepsin was used during the enzymatic hydrolysis of small molecule peptide B, and strong anion exchange resin was selected for ion exchange chromatography; small molecule peptides C - E were optimized and adjusted accordingly in each step according to their own characteristics.

[0025] Sequencing and screening process of small molecule peptides 1. Construction of a small molecule peptide library: After the enzymatic hydrolysis and preliminary separation steps, a large mixture of small molecule peptides with different molecular weights and sequences was obtained. These small molecule peptides were preliminarily grouped by ultrafiltration and ion exchange chromatography, divided into multiple subgroups according to the molecular weight range and charge properties, with each subgroup containing hundreds to thousands of small molecule peptides, thus constructing a rich small molecule peptide library to provide a sample basis for subsequent screening work.

[0026] 2. Primary screening for anti - HPV activity: Using a cell - based high - throughput screening method, the human cervical cancer cell line HeLa was infected with HPV16 and HPV18 viruses to construct a virus - infected model. Each subgroup in the small molecule peptide library was respectively applied to the virus - infected HeLa cells, and at the same time, a normal HeLa cell control group without virus infection and a positive control group infected with virus but without adding small molecule peptides were set up. After culturing for 48 hours, the MTT method was used to detect the cell viability, and the effect of each subgroup of small molecule peptides on HPV - infected cells was preliminarily judged. If the cell viability of the virus - infected cells under the action of a certain subgroup was significantly lower than that of the positive control group, and had less impact on the viability of normal cells, then the small molecule peptides in this subgroup were regarded as having potential anti - HPV activity and entered the next screening step. After primary screening, 10 potentially active subgroups were selected from multiple subgroups.

[0027] 3. Sub - division and sequencing of active subgroups: For the 10 potentially anti - HPV - active subgroups obtained from the primary screening, further sub - division was carried out by gel filtration chromatography or reverse - phase high - performance liquid chromatography (RP - HPLC) to separate the small molecule peptides in each subgroup into single components as much as possible. For each separated small molecule peptide, the amino acid sequence was determined by the Edman degradation method or mass spectrometry - based sequencing technology. The Edman degradation method determines the amino acid sequence by gradually cleaving the N - terminal amino acid of the peptide chain and identifying it; the mass spectrometry - based sequencing technology uses a mass spectrometer to measure the exact mass number of the small molecule peptide, combined with the peptide fragment cleavage pattern and database retrieval to infer the amino acid sequence. Through sequencing, more than 500 small molecule peptides with known amino acid sequences were obtained.

[0028] 4. Re - screening for anti - HPV activity: More than 500 small molecule peptides after sequencing were verified for anti - HPV activity again. The real - time fluorescence quantitative PCR method was used to detect the inhibition rate of each small molecule peptide on the DNA replication of HPV virus, and the Western blot method was used to detect the expression levels of virus - related proteins E6 and E7. The inhibition rate and the inhibitory effect on protein expression were compared with the positive control drug (such as acyclovir), and small molecule peptides with an inhibition rate of more than 50% on the DNA replication of HPV virus and an inhibition rate of more than 40% on the expression of E6 and E7 proteins were screened out. After re - screening, 50 small molecule peptides with relatively significant activities were obtained.

[0029] 5. Multiple rounds of optimization and screening: The 50 small molecule peptides obtained from re - screening were subjected to multiple rounds of optimization and screening. On the one hand, by changing the amino acid sequences of small molecule peptides, such as replacing, adding or deleting amino acid residues, a series of analogs were synthesized to test the changes in their anti - HPV activities to determine the key active sites and optimize the sequence structure. On the other hand, properties such as the stability and cell penetration of small molecule peptides were evaluated, and molecules with poor stability or difficult to enter cells to play a role were excluded. After 3 rounds of optimization and screening, 5 small molecule peptides with high anti - HPV virus activity, good stability and good cell compatibility were finally determined, namely small molecule peptides A, B, C, D and E.

[0030] Anti - HPV virus activity test 1. Cell experiments 1. Cell culture: The human cervical cancer cell line HeLa (HPV18 - positive) and the human keratinocyte cell line HaCaT (HPV - negative) were selected and cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin - streptomycin at 37°C and 5% CO2.

[0031] 2. Establishment of virus infection model: HPV16 and HPV18 virus particles were diluted to an appropriate concentration (such as 100 TCID50 / 100 μL) with serum - free DMEM medium. Logarithmic - growth - phase HeLa cells were inoculated into 96 - well cell culture plates, with 1×10^4 cells inoculated in each well, and cultured for 24 hours to allow the cells to adhere. Then, 100 μL of the diluted virus solution was added to each well, and a negative control group (only adding cells and serum - free DMEM medium without adding virus solution) was set up. The 96 - well plate was placed in an incubator at 37°C and 5% CO2 for 2 hours to allow the virus to fully adsorb to the cell surface.

[0032] 3. Small molecule peptide treatment: Prepare solutions of small molecule peptides A - E at different concentrations (such as 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM) using serum-free DMEM medium. After the virus adsorption is completed, discard the supernatant in the culture plate, and add 100 μL of small molecule peptide solutions at different concentrations to each well. At the same time, set up a positive control group (add a known antiviral drug, such as acyclovir). Continue to culture the 96-well plate in an incubator at 37°C and 5% CO2 for 48 hours.

[0033] 4. Experimental detection indicators: Use real-time fluorescence quantitative PCR to detect the content of HPV virus DNA in cells and evaluate the inhibitory effect of small molecule peptides on virus replication; use Western blot to detect the expression levels of virus-related proteins (such as E6, E7 proteins); detect cell viability by MTT method to evaluate the cytotoxicity of small molecule peptides to cells.

[0034] 5. Experimental results: Compared with the negative control group, both the positive control group and each small molecule peptide treatment group could significantly reduce the content of HPV virus DNA and the expression levels of E6 and E7 proteins in HeLa cells. Among them, when the concentration of small molecule peptide A was 25 μM, the inhibition rate of HPV16 virus DNA reached 75%, and the inhibition rate of HPV18 virus DNA was 70%; when the concentration of small molecule peptide B was 50 μM, the inhibition rates of the expression of virus-related proteins E6 and E7 of HPV16 and HPV18 were 65% and 60% respectively. In terms of cytotoxicity, within the effective antiviral concentration range, each small molecule peptide had little effect on the viability of HeLa cells and HaCaT cells, and the cell viability was above 80%, indicating that small molecule peptides had good cell compatibility.

[0035] 2. Animal experiments 1. Establishment of animal model: Select female BALB / c nude mice at 6 - 8 weeks of age. Under sterile conditions, inoculate SiHa cells positive for HPV16 (1×10^6 cells / mouse) subcutaneously on the back of the nude mice to establish a nude mouse tumor model infected with HPV16. When the tumor volume grows to about 100 - 150 mm³, randomly divide the nude mice into 6 groups, with 10 mice in each group.

[0036] 2. Drug treatment: The experimental groups were respectively administered different small molecule peptide solutions (small molecule peptides A - E, with a concentration of 10 mg / kg each) by intraperitoneal injection once a day for 2 consecutive weeks; the positive control group was administered acyclovir solution (100 mg / kg) by intraperitoneal injection once a day for 2 consecutive weeks; the negative control group was administered an equal volume of normal saline by intraperitoneal injection.

[0037] 3. Experimental detection indicators: Regularly measure the volume and weight of the tumors in nude mice, and plot the tumor growth curve; after the experiment, sacrifice the nude mice, take the tumor tissues for pathological examination, and observe the expression of HPV virus proteins in the tumor tissues; at the same time, detect the blood routine, liver and kidney function and other indicators of the nude mice to evaluate the safety of the small molecule peptides.

[0038] 4. Experimental results: Compared with the negative control group, the tumor growth in the positive control group and each small molecule peptide treatment group was significantly inhibited. Among them, the tumor volume inhibition rate in the small molecule peptide C treatment group reached 60%, and the tumor weight inhibition rate was 55%; the tumor growth curve in the small molecule peptide D treatment group was significantly lower than that in the negative control group, indicating that it had a significant inhibitory effect on tumor growth. The pathological examination results showed that the expression level of HPV virus proteins in the tumor tissues of the small molecule peptide treatment group was significantly reduced. In terms of safety, there were no significant differences in the blood routine, liver and kidney function and other indicators of the nude mice in each small molecule peptide treatment group compared with the negative control group, indicating that the small molecule peptides had no obvious toxic and side effects in animals.

[0039] Safety assessment 1. Acute toxicity test: Select healthy adult Kunming mice, randomly divide them into 6 groups, with 10 mice in each group, half male and half female. Different doses of small molecule peptides A - E (doses were 1000 mg / kg, 500 mg / kg, 250 mg / kg respectively) and normal saline (control group) were administered to the mice by gavage once. Observe the mice continuously for 14 days after administration, and record the general condition, body weight change, poisoning symptoms and death of the mice. The results showed that during the observation period, no obvious poisoning symptoms or death occurred in the mice in each small molecule peptide treatment group, the body weight increased normally, and there was no significant difference compared with the control group, indicating that the small molecule peptides had no acute toxicity at high doses.

[0040] 2. Long-term toxicity test: Healthy adult SD rats were selected and randomly divided into 6 groups, with 20 rats in each group, half male and half female. The experimental groups were respectively given small molecule peptides A - E (at doses of 50 mg / kg and 25 mg / kg), and administered once a day by gavage for 90 consecutive days. During this period, the general conditions of the rats were closely observed, including mental state, diet, hair, activity, etc. The body weight and food intake of the rats were measured weekly, and blood samples were regularly collected for routine blood and blood biochemical index tests, such as white blood cell, red blood cell, platelet counts, alanine aminotransferase, aspartate aminotransferase, serum creatinine, blood urea nitrogen, etc., to evaluate liver and kidney functions. After the experiment, the rats were dissected to observe the appearance, size and weight of the main organs (such as heart, liver, spleen, lung, kidney, etc.), and histopathological examinations were performed.

[0041] The experimental results showed that during the entire administration period, the general conditions of the rats in each experimental group were good, the mental state, diet and activity were normal, and there were no significant differences in body weight and food intake compared with the control group. The routine blood and blood biochemical indexes fluctuated within the normal range, and there were no obvious statistical differences compared with the control group. After dissection, it was found that the appearance, size and weight of the main organs of the rats in each experimental group were normal, and no obvious pathological changes were found in the histopathological examination, indicating that the small molecule peptides of the present invention have no obvious toxic effects on the body during long-term use and have good safety.

[0042] 3. Local irritation test: Healthy rabbits were selected, and the hair on both sides of the spine on their backs was shaved off. They were divided into experimental groups of small molecule peptides A - E and a normal saline control group. Appropriate amounts of small molecule peptide solution (concentration of 10 mg / mL) and normal saline were evenly applied to the skin surface of the shaved area on the back of the rabbits, with an area of about 3 cm × 3 cm, and applied once a day for 7 consecutive days. The changes in the skin at the application site were observed, including erythema, edema, itching, etc., and scored according to the skin irritation reaction scoring standard.

[0043] At the same time, a vaginal irritation test was conducted. Female rabbits were selected, and appropriate amounts of small molecule peptide solution (concentration of 10 mg / mL) and normal saline were respectively injected into the vaginas of the rabbits once a day for 7 consecutive days. During and after the administration period, the color of the vaginal mucosa of the rabbits, the presence of congestion, edema, secretions, etc. were observed, and histopathological examinations were performed.

[0044] The results of the skin irritation test showed that there were no obvious irritation reactions such as erythema and edema on the skin of the rabbits in each small molecule peptide experimental group at the application site. The skin irritation reaction scores were all 0 points, showing no difference from the control group, indicating that small molecule peptides have no irritation to the skin. The results of the vaginal irritation test showed that the vaginal mucosa of the rabbits in each small molecule peptide experimental group was normal in appearance, without hyperemia and edema, and the secretions were normal. Histopathological examination did not find obvious damage and inflammatory reactions in the vaginal mucosa, indicating that small molecule peptides also have no irritation to the vaginal mucosa and can be safely used for topical medication.

[0045] Preparation of Pharmaceutical Preparations 1. Vaginal Suppositories 1. Prescription composition: Taking small molecule peptide A as an example, each suppository contains 50 mg of small molecule peptide A, and the matrix is appropriately selected as cocoa butter. Appropriate surfactants (such as polysorbate 80) and antioxidants (such as vitamin E) can also be added to improve the stability and solubility of the drug.

[0046] 2. Preparation process: Place the cocoa butter in a water bath and heat it to melt. When the temperature drops to about 40°C, add the pre-ground small molecule peptide A powder, and at the same time add an appropriate amount of polysorbate 80 and vitamin E, and stir evenly. Pour the mixture into the suppository mold while it is hot, and demold after cooling and solidifying to obtain the small molecule peptide A vaginal suppository.

[0047] 3. Quality control: Conduct an appearance inspection on the prepared vaginal suppositories, which should have a smooth surface, uniform color, and no cracks. Use high performance liquid chromatography to determine the content of small molecule peptide A in the suppositories, and the content should meet the specified range (90% - 110%). Check the melting and transformation time limit of the suppositories, and they should all melt, soften or have no hard core when pressed within 30 minutes.

[0048] 2. Topical Gel 1. Prescription composition: Taking small molecule peptide B as an example, each 100 g of the gel contains 100 mg of small molecule peptide B, 1 g of carbomer 940, an appropriate amount of triethanolamine, 10 g of propylene glycol, 0.1 g of ethylparaben, and purified water is added to 100 g.

[0049] 2. Preparation process: Disperse carbomer 940 in an appropriate amount of purified water, and after full swelling, add propylene glycol and ethylparaben, and stir evenly. Dissolve small molecule peptide B in a small amount of purified water, slowly add it to the above solution, and stir evenly. Then add triethanolamine drop by drop to adjust the pH value to 6.5 - 7.5 to form a gel, and finally add purified water to the full volume and stir evenly to obtain the small molecule peptide B topical gel.

[0050] 3. Quality Control: The gel should have a uniform and delicate appearance, without obvious particles and lumps. The content of small molecule peptide B in the gel is determined by ultraviolet-visible spectrophotometry, and the content should be within the specified range (95% - 105%). Check the pH value of the gel, which should meet the specified requirements (6.5 - 7.5). At the same time, measure the viscosity to ensure that the gel has an appropriate viscosity for easy application and use.

[0051] 3. Injectable 1. Prescription Composition: Taking small molecule peptide C as an example, each injectable (2 mL) contains 20 mg of small molecule peptide C, 18 mg of sodium chloride, and the injection water is added to 2 mL.

[0052] 2. Preparation Process: Take an appropriate amount of injection water, add sodium chloride, stir to dissolve, then add small molecule peptide C and stir until completely dissolved. Filter and sterilize with a 0.22 μm microporous membrane, divide the filtrate into ampoules, 2 mL each, seal, and sterilize by moist heat sterilization method (121 °C, 15 minutes) to obtain the small molecule peptide C injectable.

[0053] Quality Control: The injectable should be clear and free of visible foreign matters. The content of small molecule peptide C in the injectable is determined by high performance liquid chromatography, and the content should meet the requirements (98% - 102%). Check the pH value of the injectable, which should be between 6.0 - 7.5. Conduct pyrogen test and sterility test, which should comply with the relevant regulations of the Chinese Pharmacopoeia.

Claims

1. A small molecule peptide combination extracted from Aquilaria sinensis for anti-HPV virus, characterized in that, It contains the following 5 small molecule peptides: Small molecule peptide A: SEQ.NO 1: Ala - Gly - Tyr - Cys - Lys - Thr - Glu - His - Pro - Asn - Trp - Val - Ile - Ser - Gln; Small molecule peptide B: SEQ.NO 2: Gly - Trp - Asp - Gln - His - Cys - Leu - Arg - Pro - Ser - Thr - Met - Phe - Ala - Tyr; Small molecule peptide C: SEQ.NO 3: Val - His - Pro - Cys - Asn - Lys - Ala - Glu - Gly - Ile - Cys - His - Lys - Pro - Ser; Small molecule peptide D: SEQ.NO 4: Leu - Glu - Asp - His - Arg - Thr - Pro - Gly - Ala - Arg - Phe - Tyr - Ser - Gly - Gln; Small molecule peptide E: SEQ.NO 5: Met - Tyr - Asp - Gln - His - Trp - Ala - Val - Thr - Glu - Ile - Cys - His - Asn - Pro.

2. The extraction method of the small molecule peptide combination for anti-HPV virus according to claim 1, characterized in that, It includes the following steps: Raw material pretreatment: Select high-quality agarwood wood, remove surface impurities and then crush it into powder; Crude extraction: Use supercritical carbon dioxide extraction technology to extract the agarwood powder under the conditions of a pressure of 25 - 35 MPa and a temperature of 40 - 50 °C to obtain the crude agarwood extract; Enzymatic hydrolysis treatment: Dissolve the crude agarwood extract with a buffer solution, add protease, and carry out an enzymatic hydrolysis reaction at 37 - 40 °C and an appropriate pH value for 4 - 6 hours; Ultrafiltration separation: Ultrafilter the enzymatically hydrolyzed solution through an ultrafiltration membrane with a molecular weight cut-off of 1000 - 3000 Da; Ion exchange chromatography: Select strong cation or strong anion exchange resin to fill the chromatography column, load the ultrafiltered solution, and perform gradient elution with salt solutions of different concentrations, and collect the elution peak where the target small molecule peptide is located; Gel filtration chromatography: Purify the target elution peak collected by ion exchange chromatography through a gel filtration chromatography column, elute with an appropriate buffer solution, and collect the small molecule peptide component with a higher purity; Structure identification and purity detection: Use mass spectrometry analysis to determine the molecular weight of the small molecule peptide, and use high performance liquid chromatography to detect the purity of the small molecule peptide to ensure that the purity reaches more than 95%.

3. Use of the small molecule peptide combination for anti-HPV virus as claimed in claim 1 in the preparation of anti-HPV virus drugs, characterized in that, The drug includes but is not limited to vaginal suppositories, topical gels, and injections.

4. A vaginal suppository for anti-HPV virus, characterized in that, Containing 50 mg of the small molecule peptide A described in claim 1, with cocoa butter as the matrix, an appropriate amount of surfactant and antioxidant can be added, and the pH value is within an appropriate range, for the treatment of diseases related to anti-HPV virus infection.

5. An external gel for anti-HPV virus, characterized in that, Each 100 g contains 100 mg of the small molecule peptide B described in claim 1, 1 g of carbomer 940, an appropriate amount of triethanolamine, 10 g of propylene glycol, 0.1 g of ethylparaben, purified water is added to 100 g, and the pH value is 6.5 - 7.5, for the treatment of diseases related to anti-HPV virus infection.

6. An injection for anti-HPV virus, characterized in that, Each 2 mL contains 20 mg of the small molecule peptide C described in claim 1, 18 mg of sodium chloride, injection water is added to 2 mL, and the pH value is 6.0 - 7.5, for the treatment of diseases related to anti-HPV virus infection.

7. The small molecule peptide combination according to claim 1, wherein It exerts an anti-HPV virus effect through multiple mechanisms such as interfering with the adsorption of HPV virus to host cells, inhibiting the gene expression of HPV virus, and inducing apoptosis of HPV-infected cells.

8. The extraction method according to claim 2, characterized in that, In the raw material pretreatment step, the origin, quality, etc. of the agarwood wood are strictly screened; in the crude extraction step, the extraction pressure, temperature, and time are monitored in real time through an on-line monitoring device; In the enzymatic hydrolysis treatment step, the pH value is precisely adjusted according to the characteristics of the protease used, and the enzymatic hydrolysis process is monitored in real time by high performance liquid chromatography; in the ultrafiltration separation step, the flux change of the ultrafiltration membrane is monitored in real time by a membrane flux monitor; in the ion exchange chromatography step, parameters such as the salt solution gradient and elution flow rate are optimized through experiments; in the gel filtration chromatography step, the eluent flow rate is ensured to be stable, and the eluent is precisely collected by an automatic fraction collector; in the structure identification and purity detection step, in addition to mass spectrometry analysis and high performance liquid chromatography, the amino acid sequence of the small molecule peptide is also determined by an amino acid sequencer to ensure consistency with the expected designed sequence.

9. A pharmaceutical composition, characterized in that, Containing the small molecule peptide combination described in claim 1 and a pharmaceutically acceptable carrier or excipient, for the treatment or prevention of diseases related to anti-HPV virus infection.

10. The application according to claim 3, characterized in that The anti-HPV virus drug is used for the treatment or prevention of diseases caused by high-risk HPV (such as HPV16, HPV18, etc.) or low-risk HPV (such as HPV6, HPV11, etc.) infection, including but not limited to cervical intraepithelial neoplasia, genital warts.