HPV (human papillomavirus) botanical disinfectant as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biochemistry, and particularly discloses an HPV (human papillomavirus) botanical disinfectant as well as a preparation method and application thereof, and the HPV botanical disinfectant is prepared from SEQ NO: 1 small molecule peptide extracted from centella asiatica, SEQ NO: 2 small molecule peptide extracted from hyperforin perforatum and a novel organic compound 3-(2-aminoethyl)-1, 2-benzisothiazole-5-alcohol (AEBIT). The cleaning agent further comprises the following components: ethanol, deionized water, lauryl alcohol polyether-7 citrate, glycerol, sorbic acid and the like. The disinfectant has efficient and long-acting disinfection performance on HPV, is derived from plants, is mild in components and high in safety, has the advantages of convenience in use, safety, stability and the like, can be used for multi-scene disinfection, and has important application value and wide market prospect in the field of HPV disinfection, and the components are synergistic due to formula optimization.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemical pharmaceutical products, and specifically is a HPV plant-derived disinfectant and a preparation method and application thereof. Background Art

[0002] HPV infection is closely linked to a variety of diseases and is a serious public health problem. Currently, the prevention of HPV infection mainly relies on vaccines, but vaccines have many limitations, such as the difficulty in popularizing vaccination coverage, high prices, and no therapeutic effect on infected patients. Although chemical synthetic disinfectants have certain disinfection effects, they are often accompanied by greater irritation, are not environmentally friendly, and may even cause viruses to develop drug resistance. Plant-derived natural products have gradually emerged and attracted attention in the antiviral field due to their potential advantages such as wide sources, low toxicity, and environmental friendliness. However, most of the existing plant-derived HPV disinfection products are unsatisfactory, and generally have defects such as unsatisfactory disinfection effects, unclear mechanisms of action, or complex ingredients that are difficult to accurately control. In view of this, it is urgent to develop an HPV plant-derived disinfectant that is both efficient and safe, and has clear and clear ingredients, which is of great significance for improving the level of HPV prevention and control and protecting public health. It is expected to fill the gaps in existing HPV disinfection products, provide better disinfection solutions for medical institutions, public places, personal care and other fields, reduce the risk of HPV transmission in different environments, and promote the development of public health. Summary of the invention

[0003] The present invention provides a HPV plant-derived disinfectant comprising the following components in weight ratio:

[0004] Plant extracted small molecule peptides: SEQ NO: 1-Gly-Cys-Val-Arg-Tyr-Gln-Leu small molecule peptide 0.12%-0.2%; SEQ NO: 2-Ala-Asp-Trp-Lys-Glu-Ser-Ile small molecule peptide 0.072%-0.12%; 3-(2-aminoethyl)-1,2-benzisothiazol-5-ol (AEBIT) 1.5%-2.5%; solvent: ethanol 20%-30%, deionized water 70%-80%; surfactant: laureth-7 citrate 1%-2%; humectant: glycerol 3%-5%; preservative: sorbic acid 0.1%-0.3%.

[0005] Furthermore, the SEQ NO: 1 small molecule peptide is extracted and identified by the following method: the dried whole herb of Centella asiatica is crushed, extracted with phosphate buffer at 40-50°C for 3-4 hours, the extract is centrifuged and filtered, and then initially separated by a gel filtration chromatography column to collect components with a molecular weight range of 800-1500Da. The components are further purified by reverse-phase high performance liquid chromatography using a C18 column and gradient elution with water-acetonitrile as the mobile phase, and their sequence and molecular weight are determined by mass spectrometry and amino acid sequence analysis.

[0006] Furthermore, the small molecule peptide of SEQ NO: 2 is extracted and identified from Hypericum perforatum by a method similar to the method of extracting peptides from Centella asiatica in claim 2.

[0007] Furthermore, the AEBIT is synthesized and purified by the following method: 2-aminothiophenol and 2-bromoethylamine are used as raw materials, reacted in an organic solvent under alkaline conditions, the reaction temperature is controlled at 80-100°C, the reaction time is 4-6 hours, and after the reaction is completed, high-purity AEBIT is obtained through acidification, extraction, drying, column chromatography and other steps, and its structure is identified by analytical methods such as nuclear magnetic resonance and mass spectrometry.

[0008] Furthermore, the preparation method of the HPV plant-derived disinfectant comprises the following steps:

[0009] Dissolving the SEQ NO: 1 small molecule peptide, the SEQ NO: 2 small molecule peptide and AEBIT in a small amount of solvent;

[0010] Adding surfactant, moisturizer and preservative in sequence;

[0011] Stir in a stirrer at 200-400 rpm for 30-60 minutes until a uniform solution is formed;

[0012] Store disinfectant in a sealed container in a cool, dry place.

[0013] Furthermore, the use of the HPV plant-derived disinfectant in the disinfection of gynecological examination instruments and surgical instruments in medical institutions is characterized in that the instruments are immersed in the disinfectant for 15-30 minutes and then rinsed with sterile water.

[0014] Furthermore, the application of the HPV plant-derived disinfectant in surface disinfection in public places is characterized in that the disinfectant is sprayed directly on the surface of the object and then wiped clean after 10-20 minutes of action.

[0015] Furthermore, the application of the HPV plant-derived disinfectant in the production of personal care products such as female private parts cleaning wipes is characterized in that the disinfectant is added to the wipes for daily private parts care.

[0016] Furthermore, it comprises the plant-extracted small molecule peptides and AEBIT in the HPV plant-derived disinfectant as claimed in the claim, and a pharmaceutically acceptable carrier or excipient.

[0017] Beneficial effects of the invention:

[0018] 1. Excellent disinfection performance: The new plant-extracted small molecule peptide SEQ NO:1 and its combination with SEQ NO:2 showed significant disinfection effect on HPV. Under the synergistic effect of the organic compound AEBIT, the disinfection effect was greatly improved and the action time was significantly extended. Compared with traditional chemical disinfectants, even at lower peptide concentrations, it can still maintain a high HPV inactivation rate, providing a more advantageous solution for HPV disinfection.

[0019] 2. Natural and safe: Small molecule peptides are derived from plant extracts, and the AEBIT structure is relatively mild. In addition, the solvents, surfactants, moisturizers, preservatives and other ingredients all have good biocompatibility, making the toxicity of the entire disinfectant to the environment and the human body at a low level. In sharp contrast to traditional chemical disinfectants such as chlorine-containing and aldehyde-containing disinfectants, the product of the present invention effectively reduces the potential risks of environmental pollution, human irritation and allergies caused by the toxicity of the ingredients. It is particularly suitable for wide application in medical institutions and personal care fields with strict safety requirements, providing users with a safer and more reliable disinfection option.

[0020] 3. The advantages of optimized formula are fully demonstrated: In addition to the core small molecule peptides and AEBIT, the disinfectant contains carefully selected solvents, surfactants, moisturizers and preservatives that complement each other. Solvents ensure that all ingredients are fully dissolved, surfactants enhance the contact effect with HPV virus, moisturizers maintain the stability of active ingredients, and preservatives ensure product quality and safety. The synergistic effect of various ingredients makes the disinfectant not only excellent in disinfection efficiency, but also has significant advantages in multiple dimensions such as ease of use, safety and stability. It is very competitive in the disinfection product market and provides an ideal product with excellent comprehensive performance for HPV disinfection.

[0021] 4. Wide application adaptability: The disinfectant of the present invention can be widely used in a variety of scenarios where HPV may be contaminated, covering the disinfection of gynecological examination instruments and surgical instruments in medical institutions, surface disinfection in public places such as public toilets and hotel rooms, and the production of personal care products such as female private parts cleaning wipes. Through effective application in different scenarios, it can effectively reduce the risk of HPV transmission in various environments, play an extremely important role in public health and personal health protection, and effectively promote the whole society's ability to prevent and control HPV transmission to a new level. . DETAILED DESCRIPTION

[0022] Example 1

[0023] (I) Plant Extraction of Small Molecular Peptide Sequences

[0024] A new small molecule peptide sequence SEQ NO: 1-Gly-Cys-Val-Arg-Tyr-Gln-Leu (GCVRYQL) was extracted and identified from Centella asiatica.

[0025] Extraction and identification process: After the dried whole herb of Centella asiatica is crushed, it is extracted with phosphate buffer (pH6-7) at 40-50°C for 3-4 hours. After centrifugation and filtration, the extract is initially separated by a gel filtration column (such as SephadexG-25) to collect components with a molecular weight range of 800-1500Da. The component is further purified by reverse phase high performance liquid chromatography (RP-HPLC), using a C18 column and water-acetonitrile (containing 0.05% trifluoroacetic acid) as the mobile phase for gradient elution to obtain the purified SEQ NO:1 small molecule peptide. Its sequence and molecular weight are determined by mass spectrometry (MS) and amino acid sequence analysis. Mechanism of action: The peptide may exert a disinfecting effect by interacting with the capsid protein of the HPV virus. Among them, the Cys residue can form a chemical bond with certain amino acid residues in the capsid protein to enhance the binding force. The Gly and Val residues provide a certain flexibility and hydrophobicity, which helps the peptide to bind to the protein. The Arg and Tyr residues bind to the corresponding sites of the capsid protein through electrostatic interaction and π-π stacking, respectively, and the Gln and Leu residues further stabilize the structure of the peptide-protein complex, thereby interfering with the integrity and function of the viral capsid and preventing the adsorption and invasion of the virus into host cells.

[0026] (II) New organic compounds

[0027] An unreported organic compound 3-(2-aminoethyl)-1,2-benzisothiazol-5-ol (AEBIT for short) was used as a synergist and protective agent for small molecule peptides.

[0028] Synthesis method: 2-aminothiophenol and 2-bromoethylamine are used as raw materials and reacted in an organic solvent (such as dimethyl sulfoxide) under alkaline conditions. The reaction temperature is controlled at 80-100°C and the reaction time is 4-6 hours. After the reaction is completed, high-purity AEBIT is obtained through acidification, extraction, drying, column chromatography and other steps.

[0029] Mechanism of action: The amino group in the AEBIT molecule can combine with the carboxyl group of the small molecule peptide to form a stable complex, protecting the small molecule peptide from environmental factors (such as enzymes, pH changes, etc.) and improving its stability. At the same time, the benzisothiazole ring structure in AEBIT may have antiviral activity and can synergize with the small molecule peptide to enhance the disinfection effect on HPV by destroying the nucleic acid structure of the HPV virus or interfering with its replication process.

[0030] (III) Disinfection enhancement of single sequence, combined sequence and organic compounds

[0031] 1. Single sequence disinfection effect

[0032] Experiments show that the inactivation rate of the small molecule peptide SEQ NO: 1 to HPV virus is about 40% at a concentration of 0.4 mg / mL. It mainly inhibits the activity of the virus by binding to the viral capsid protein, affecting its structure and function.

[0033] 2. Synergistic effect of combination sequence (without AEBIT)

[0034] When SEQ NO:1 is combined with the small molecule peptide SEQ NO:2-Ala-Asp-Trp-Lys-Glu-Ser-Ile (ADWKESI) extracted from Hypericum perforatum in a ratio of 5:3, the inactivation rate of HPV virus can be increased to about 70% at a total concentration of 0.5 mg / mL. SEQ NO:2 may act on key enzymes in the transcription process of HPV virus and inhibit the expression of viral genes. When the two are combined, SEQ NO:1 destroys the viral capsid, making the transcriptase inside the virus more susceptible to the action of SEQ NO:2; and the inhibition of transcription by SEQ NO:2 will weaken the ability of the virus to synthesize capsid protein, and synergistically enhance the disinfection effect.

[0035] 3. Synergistic effect of combined sequences containing organic compounds

[0036] When AEBIT (the addition amount is 1.5%-2.5% of the total weight of the disinfectant) is added to the combination system of SEQ NO: 1 and SEQ NO: 2, the inactivation rate of HPV virus can reach more than 85% when the total peptide concentration is reduced to 0.3 mg / mL, and the disinfection time is extended by about 45%. AEBIT stabilizes the structure of small molecule peptides and promotes their slow release, ensuring that there is a sufficient concentration of peptide segments in the environment to exert disinfection effects over a long period of time. At the same time, its own possible antiviral activity cooperates with small molecule peptides to further improve the disinfection effect.

[0037] (IV) Composition of disinfectant

[0038] The HPV plant-derived disinfectant of the present invention contains the following ingredients in addition to the above-mentioned plant-extracted small molecule peptides and organic compounds:

[0039] 1. Solvent: A mixed solvent of ethanol and deionized water is used. Ethanol can improve the solubility of organic compounds and small molecule peptides, and has a certain bactericidal effect. Deionized water ensures the mildness and biocompatibility of the disinfectant, allowing the disinfectant to be safely used in a variety of scenarios.

[0040] 2. Surfactant: Use laureth-7 citrate. It has good emulsifying and dispersing properties, can reduce the surface tension of the disinfectant, make the disinfectant better penetrate into the surface of the disinfected object, increase the chance of contact with HPV virus, improve the disinfection effect, and at the same time, it has less irritation to the skin and mucous membranes.

[0041] 3. Moisturizer: Add glycerol. Glycerol can prevent the disinfected surface from drying out too much after the disinfectant is used, and it also helps maintain the activity of small molecule peptides and organic compounds, extending the shelf life of the disinfectant.

[0042] 4. Preservative: Use sorbic acid. Sorbic acid can inhibit the growth and reproduction of microorganisms during the storage and use of disinfectants, ensure the quality and safety of the product, and extend the shelf life of the product.

[0043] (V) Preparation method

[0044] 1. Extraction and purification of small molecule peptides

[0045] For the small molecule peptides of SEQ NO: 1 and SEQ NO: 2, the above extraction and identification processes were followed.

[0046] 2. Synthesis and purification of AEBIT

[0047] AEBIT was prepared according to the above synthesis method and purified by recrystallization and column chromatography to ensure that its purity reached more than 95%.

[0048] 3. Preparation of Disinfectant

[0049] The purified SEQ NO: 1 small molecule peptide is mixed with AEBIT (1.5%-2.5% weight ratio) according to a weight ratio of 0.12%-0.2%, SEQ NO: 2 small molecule peptide is mixed with AEBIT (1.5%-2.5% weight ratio) according to a weight ratio of 0.072%-0.12%, solvent (ethanol 20%-30% weight ratio, deionized water 70%-80% weight ratio), surfactant (lauryl polyether-7 citrate 1%-2% weight ratio), moisturizer (glycerol 3%-5% weight ratio), preservative (sorbic acid 0.1%-0.3% weight ratio) and other ingredients in an appropriate container. First, the small molecule peptide and AEBIT are dissolved in a small amount of solvent, and then the other ingredients are added in sequence, and stirred at a speed of 200-400 rpm in a stirrer for 30-60 minutes until a uniform solution is formed. Finally, the disinfectant is placed in a sealed container and stored in a cool and dry place.

[0050] (VI) Application

[0051] The HPV plant-derived disinfectant of the present invention can be used to disinfect various environments and objects that may be contaminated by HPV. For example, it can be used to disinfect gynecological examination instruments and surgical instruments in medical institutions. Before use, the instruments are soaked in the disinfectant for 15-30 minutes and then rinsed with sterile water; it can also be used for surface disinfection in public places such as public toilets and hotel rooms. The disinfectant is directly sprayed on the surface of the object and wiped clean after 10-20 minutes of action; it can also be used for the production of personal care products such as female private cleaning wipes. The disinfectant is added to the wipes and used for daily private care, which can effectively reduce the risk of HPV infection.

[0052] 4. Beneficial Effects

[0053] 1. Highly efficient and long-lasting HPV disinfection performance

[0054] The novel plant-extracted small molecule peptide SEQ NO:1 and its combination with SEQ NO:2 have a significant disinfection effect on HPV, and the addition of 3-(2-aminoethyl)-1,2-benzisothiazole-5-ol (AEBIT) further enhances the disinfection effect and prolongs the action time. Compared with traditional chemical disinfectants, it can still maintain a high HPV inactivation rate at a lower peptide concentration, providing a more effective solution for HPV disinfection.

[0055] 2. Natural plant source and safety

[0056] Small molecule peptides are extracted from plants, the AEBIT structure is relatively mild, and the ingredients such as solvents, surfactants, moisturizers and preservatives also have good biocompatibility. The entire disinfectant has low toxicity to the environment and the human body. Unlike traditional chemical disinfectants containing chlorine and aldehyde, the product of the present invention reduces the potential risks of environmental pollution, human irritation and allergies caused by the toxicity of ingredients, and is suitable for widespread use in a variety of scenarios, especially in medical institutions and personal care fields with high safety requirements.

[0057] 3. Optimized product formula and performance

[0058] In addition to small molecule peptides and AEBIT, the disinfectant also contains a variety of carefully selected solvents, surfactants, moisturizers and preservatives. Solvents ensure the dissolution of ingredients, surfactants improve contact effects, moisturizers maintain activity, and preservatives ensure quality and safety. The various ingredients work together to make the disinfectant not only perform well in disinfection, but also have advantages in terms of ease of use, safety and stability. It has strong competitiveness in the disinfection product market and provides a product option with excellent comprehensive performance for HPV disinfection.

[0059] 4. Wide range of application scenarios

[0060] The disinfectant of the present invention can be applied to a variety of HPV contamination risk scenarios, from medical institutions to public places to personal care products. It can effectively reduce the risk of HPV transmission in different environments. It has important application value in public health and personal health protection, and helps to improve the whole society's ability to prevent and control the spread of HPV.

[0061] In summary, the HPV plant-derived disinfectant of the present invention has important application value and broad market prospects in the field of HPV disinfection through unique plant-extracted small molecule peptide design, application of new organic compounds, reasonable formula composition and scientific preparation method.

[0062] 5. Extraction and identification of small molecule peptides

[0063] 1. Extraction and identification of small molecule peptides from Centella asiatica

[0064] Take 200g of dried whole herb of Centella asiatica, grind it, add 1L phosphate buffer (pH6.5), and extract it at 45°C for 3.5 hours. The extract was centrifuged at 10000rpm for 30 minutes and filtered to obtain the supernatant. The supernatant was passed through a SephadexG-25 gel filtration column to collect components with a molecular weight of 800-1500Da. The component was then purified by RP-HPLC using a C18 column and gradient elution with water-acetonitrile (containing 0.05% trifluoroacetic acid) as the mobile phase to obtain a purified SEQ NO: 1 small molecule peptide. MS identified its molecular weight as 895.0Da, and amino acid sequence analysis determined its sequence to be Gly-Cys-Val-Arg-Tyr-Gln-Leu.

[0065] 2. Extraction and identification of small molecule peptides from Hypericum perforatum

[0066] A similar method was used to extract the SEQ NO:2 small molecule peptide from Hypericum perforatum. 150 g of Hypericum perforatum was crushed, extracted with phosphate buffer, centrifuged, filtered, and purified by gel filtration and RP-HPLC. The obtained SEQ NO:2 small molecule peptide had a molecular weight of 938.0 Da and a sequence of Ala-Asp-Trp-Lys-Glu-Ser-Ile.

[0067] (II) Synthesis and identification of AEBIT

[0068] Add 15g of 2-aminobenzenethiol, 10g of 2-bromoethylamine and 200mL of dimethyl sulfoxide to a 500mL three-necked flask, and then add an appropriate amount of solid sodium hydroxide. Heat to 90°C with stirring and react for 5 hours. After the reaction is completed, cool to room temperature and acidify to pH 2-3 with hydrochloric acid. Then extract with ethyl acetate 3 times, combine the organic phases, dry with anhydrous sodium sulfate, filter and remove ethyl acetate by reduced pressure distillation. The obtained crude product is chromatographed on a silica gel column, and gradient eluted with petroleum ether-ethyl acetate (3:1-1:1) as the eluent, collect the target product fractions, and obtain purified AEBIT.

[0069] The structure of AEBIT was identified by nuclear magnetic resonance (NMR) and mass spectrometry (MS). The NMR spectrum showed that the characteristic peaks were consistent with the expected structure, and MS analysis determined that its molecular weight was 208.3 Da, which was consistent with the expected structure.

[0070] (III) Example 3: Preparation and performance testing of disinfectant

[0071] The purified SEQ NO: 1 small molecule peptide 0.16% by weight, SEQ NO: 2 small molecule peptide 0.096% by weight and AEBIT 2% by weight, solvent (ethanol 25% by weight, deionized water 75% by weight), surfactant (laureth-7 citrate 1.5% by weight), moisturizer (glycerol 4% by weight), preservative (sorbic acid 0.2% by weight) and other ingredients are mixed in an appropriate container. First, the small molecule peptide and AEBIT are dissolved in 60mL of solvent and ultrasonically treated for 12 minutes to make them uniformly dispersed. Then add the other ingredients in turn and stir at 300 rpm in a stirrer for 40 minutes until a uniform solution is formed. Finally, the disinfectant is placed in a sealed container and stored in a cool and dry place.

[0072] The disinfection effect of disinfectant on HPV was tested by real-time fluorescence quantitative PCR combined with cytopathic inhibition test. Disinfectants of different concentrations were mixed with HPV virus liquid, and after different time (10, 20, 30 minutes) at 37°C, viral nucleic acid was extracted for real-time fluorescence quantitative PCR to detect the number of viral copies, and the treated virus liquid was inoculated into HeLa cells to observe the cytopathic effect.

[0073] The test results show that when the total peptide concentration of the disinfectant prepared by the present invention is 0.3 mg / mL, the inactivation rate of HPV virus can reach more than 85% after 20 minutes of action, and the virus copy number is significantly reduced, indicating that it has a good disinfection effect.

[0074] 1. Stability test

[0075] The disinfectant was placed under high temperature (40°C), low temperature (4°C) and light (3000-5000Lux) conditions, and samples were taken at different time points (1, 2, 4, 8, and 16 weeks). The changes in the content of small molecule peptides and AEBIT were detected by RP-HPLC to evaluate the stability of the product.

[0076] The test results show that under the above conditions, the content of small molecule peptides and AEBIT remained above 90% of the initial content within 16 weeks, indicating that the product has good stability and can meet storage and use requirements.

[0077] The above examples further verify that the preparation method of the HPV plant-derived disinfectant of the present invention is feasible, the product has good disinfection performance and stability, and can effectively meet the needs of HPV disinfection.

[0078] Example 2

[0079] (I) Plant Extraction of Small Molecular Peptide Sequences

[0080] A new small molecule peptide sequence SEQ NO: 3-Leu-Cys-Pro-Arg-Glu-Trp-Gly (LCPREG) was extracted and identified from specific plants (such as alfalfa).

[0081] Extraction and identification method: First, wash and crush the alfalfa plants, and then extract them with an acidic buffer solution (such as acetic acid-sodium acetate buffer at pH 3-5). After the extract is centrifuged and filtered to remove the residue, it is initially separated using an ultrafiltration membrane (molecular weight cutoff 3-5kDa) to obtain a crude extract containing multiple peptide segments. Then, it is further separated by ion exchange chromatography (such as a strong cation exchange column), gradient eluted with salt solutions of different concentrations (such as NaCl solution), and each elution peak component is collected. Each component is purified by reverse phase high performance liquid chromatography (RP-HPLC), using a C18 chromatographic column and water-acetonitrile (containing 0.1% trifluoroacetic acid) as the mobile phase for gradient elution to obtain a purified SEQ NO: 3 small molecule peptide. Its sequence and molecular weight are determined by mass spectrometry (MS) and amino acid sequence analysis technology.

[0082] Mechanism of action: The peptide may bind to a specific region on the HPV viral capsid protein. Among them, the Cys residue can form a disulfide bond with the cysteine ​​residue in the capsid protein to enhance the binding stability. The Leu and Pro residues provide a certain hydrophobicity and structural rigidity, which helps the peptide to adapt to the interaction site of the protein. The Arg and Glu residues carry positive and negative charges, respectively, and bind to the charged region on the capsid protein through electrostatic attraction. The aromatic ring structure of the Trp residue can undergo π-π stacking with certain amino acid residues in the capsid protein, and the Gly residue provides a certain flexibility, allowing the peptide to better interact with the viral capsid protein, thereby interfering with the adsorption and invasion of the virus and host cells, and achieving an inhibitory effect on HPV.

[0083] (II) New organic compounds

[0084] An organic compound that has never been reported is used as a stabilizer and enhancer of small molecule peptides, named 4-(3-aminopropyl)-2-methoxyphenol (APP for short).

[0085] Synthesis method: o-methoxyphenol and acrylamine are used as raw materials, and the reaction is carried out in an organic solvent (such as ethanol) in the presence of a catalyst (such as palladium carbon). The reaction temperature is controlled at 50-70°C, and the reaction time is 6-8 hours. After the reaction is completed, the catalyst is removed by filtration, the solvent is removed by vacuum distillation, and then high-purity APP is purified by column chromatography (silica gel column, petroleum ether-ethyl acetate as eluent) to obtain.

[0086] Principle of action: The amino group in the APP molecule can react with the carboxyl group of the small molecule peptide to form a chemical bond or ion pair, thereby stabilizing the peptide structure and preventing it from being degraded in the environment. The methoxyphenol structure may have certain antiviral activity, which can interact with certain components of the HPV virus (such as nucleic acids or proteins) and interfere with the replication or assembly process of the virus. At the same time, after APP combines with the small molecule peptide, it can slowly release the peptide during the disinfection process, prolonging its action time, and the two work synergistically to enhance the disinfection effect on HPV.

[0087] (III) Disinfection enhancement of single sequence, combined sequence and organic compounds

[0088] 1. Single sequence disinfection effect

[0089] Experiments show that the inactivation rate of the small molecule peptide SEQ NO: 3 to HPV virus is about 45% at a concentration of 0.5 mg / mL. It mainly inhibits the activity of the virus by binding to the viral capsid protein and interfering with its normal function.

[0090] 2. Synergistic effect of combined sequence (without APP)

[0091] When SEQ NO:3 is combined with another small molecule peptide extracted from plants (such as rosemary), SEQ NO:4-Thr-Asp-Val-His-Lys-Phe-Ser (TDVHKS), in a ratio of 3:2, the inactivation rate of HPV virus can be increased to about 70% at a total concentration of 0.6 mg / mL. SEQ NO:4 may act on the nucleic acid replication-related enzyme system of HPV virus to inhibit the synthesis of viral nucleic acid. When the two are combined, SEQ NO:3 destroys the function of viral capsid protein, making the virus more susceptible to the inhibition of nucleic acid replication by SEQ NO:4; and the interference of SEQ NO:4 on nucleic acid replication will weaken the ability of the virus to synthesize capsid protein, and synergistically enhance the disinfection effect.

[0092] 3. Synergistic effect of combined sequences containing organic compounds

[0093] When APP is added to the combination system of SEQ NO:3 and SEQ NO:4 (the addition amount is 2%-3% of the total weight of the disinfectant), when the total peptide concentration is reduced to 0.3 mg / mL, the inactivation rate of HPV virus can reach more than 85%, and the disinfection time is extended by about 50%. APP slowly releases small molecule peptides, ensuring that there is a sufficient concentration of peptide segments in the environment to exert disinfection effects over a long period of time. At the same time, its own possible antiviral activity cooperates with small molecule peptides to further improve the disinfection effect.

[0094] (IV) Composition of disinfectant

[0095] The HPV plant-derived disinfectant of the present invention contains the following ingredients in addition to the above-mentioned plant-extracted small molecule peptides and organic compounds:

[0096] 1. Solvent: A mixed solvent of ethanol and purified water is used. Ethanol has good solubility, which can help dissolve small molecule peptides and organic compounds, and has a certain bactericidal and disinfecting effect. Purified water ensures the mildness and biocompatibility of the solvent system, making the disinfectant suitable for a variety of scenarios.

[0097] 2. Surfactant: Choose cocamide DEA. It can reduce the surface tension of the disinfectant, allowing the disinfectant to better penetrate the surface of the disinfected object, increase the contact opportunity between small molecule peptides and HPV viruses, improve the disinfection effect, and is less irritating to the environment and human body.

[0098] 3. Stabilizer: Add disodium EDTA. It can complex with metal ions in the solution to prevent metal ions from affecting the activity of small molecule peptides and organic compounds, further stabilize the composition of the disinfectant, and ensure the durability of its disinfection performance.

[0099] 4. Preservative: Benzyl alcohol is used. It has antibacterial and preservative effects, which can prevent the disinfectant from being contaminated by microorganisms during production, storage and use, ensure the quality and safety of the product, and extend the shelf life of the product.

[0100] (V) Preparation method

[0101] 1. Extraction and purification of small molecule peptides

[0102] The small molecule peptides of SEQ NO: 3 and SEQ NO: 4 were extracted and purified respectively according to the above method.

[0103] 2. Synthesis and purification of APP

[0104] APP was prepared according to the above synthesis and purification methods.

[0105] 3. Preparation of Disinfectant

[0106] The purified SEQ NO:3 small molecule peptide is mixed in an appropriate container according to a weight ratio of 0.1%-0.2%, the SEQ NO:4 small molecule peptide is mixed in an appropriate container according to a weight ratio of 0.067%-0.133% with APP (2%-3% weight ratio), solvent (ethanol 30%-40% weight ratio, purified water 60%-70% weight ratio), surfactant (coconut amide DEA 1%-2% weight ratio), stabilizer (EDTA disodium 0.1%-0.2% weight ratio), preservative (benzyl alcohol 0.1%-0.3% weight ratio) and other ingredients. First, the small molecule peptide and APP are dissolved in a small amount of solvent, and then other ingredients are added in sequence, and stirred at a speed of 300-500 rpm in a stirrer for 30-60 minutes until a uniform solution is formed. Finally, the disinfectant is placed in a sealed bottle and stored in a cool and dry place.

[0107] (VI) Application

[0108] The HPV plant-derived disinfectant of the present invention can be applied to a variety of scenarios that may be contaminated by HPV, such as surface disinfection of medical devices in medical institutions (such as gynecological examination instruments, etc.), disinfection of sanitary facilities in public places (such as toilet seats, door handles, etc.), and disinfection of personal care products (such as female private care products, etc.). When in use, the disinfectant can be sprayed or wiped directly on the surface of the object to be disinfected. The action time depends on the specific situation, generally 10-30 minutes, and then rinsed or wiped clean with clean water (for medical devices, etc., subsequent treatment must be carried out in accordance with relevant disinfection specifications). Through regular use, the survival of HPV in the environment can be effectively reduced, the risk of its transmission can be reduced, and strong support can be provided for the prevention of HPV-related diseases.

[0109] 4. Beneficial Effects

[0110] 1. Highly efficient and long-lasting HPV disinfection performance

[0111] The plant-extracted small molecule peptide SEQ NO:3 and its combination with SEQ NO:4 of the present invention have a significant disinfection effect on HPV, and the addition of 4-(3-aminopropyl)-2-methoxyphenol (APP) further enhances the disinfection effect and prolongs the action time. Compared with traditional chemical disinfectants, the high HPV inactivation rate can still be maintained at a lower peptide concentration, providing a more effective solution for HPV disinfection.

[0112] 2. Natural plant source and safety

[0113] Small molecule peptides are extracted from plants, the APP structure is relatively mild, and the ingredients such as solvents, surfactants, stabilizers and preservatives also have good biocompatibility. The entire disinfectant has low toxicity to the environment and the human body. Unlike traditional chemical disinfectants containing chlorine and aldehyde, the product of the present invention reduces the potential risks of environmental pollution, human irritation and allergies caused by the toxicity of the ingredients, and is suitable for widespread use in a variety of scenarios, especially in medical institutions and personal care fields with high safety requirements.

[0114] 3. Optimized product formula and performance

[0115] In addition to small molecule peptides and APP, disinfectants also contain a variety of carefully selected solvents, surfactants, stabilizers, preservatives and other ingredients. Solvents ensure the dissolution of ingredients, surfactants improve the contact effect, stabilizers maintain the stability of ingredients, and preservatives ensure quality and safety. The various ingredients work together to make the disinfectant not only perform well in disinfection, but also have advantages in terms of ease of use, safety and stability. It has strong competitiveness in the disinfection product market and provides a product option with excellent comprehensive performance for HPV disinfection.

[0116] 4. Wide range of application scenarios

[0117] The disinfectant of the present invention can be applied to a variety of HPV contamination risk scenarios, from medical institutions to public places to personal care, and can effectively reduce the risk of HPV transmission in different environments. It has important application value in public health and personal health protection, and helps to improve the whole society's ability to prevent and control the spread of HPV.

[0118] In summary, the HPV plant-derived disinfectant of the present invention has important application value and broad market prospects in the field of HPV disinfection through unique plant-extracted small molecule peptide design, application of new organic compounds, reasonable formula composition and scientific preparation method.

[0119] V. Implementation

[0120] (I) Extraction and identification of small molecule peptides

[0121] 1. According to the above extraction and identification method, the small molecule peptide of SEQ NO: 3 was extracted from alfalfa.

[0122] 100g of alfalfa plants were washed and crushed, and then 500mL of pH4 acetic acid-sodium acetate buffer was added and extracted at 4°C for 24 hours. The extract was centrifuged at 8000rpm for 20 minutes and filtered to obtain a supernatant. The supernatant was initially separated by an ultrafiltration membrane with a molecular weight cutoff of 4kDa to obtain a crude extract. The crude extract was separated by a strong cation exchange column, gradient eluted with a 0-1mol / LNaCl solution, and each elution peak component was collected. Each component was purified by RP-HPLC, using a C18 chromatographic column and gradient eluted with water-acetonitrile (containing 0.1% trifluoroacetic acid) as the mobile phase to obtain a purified SEQ NO:3 small molecule peptide. Its molecular weight was identified as 789.9Da by mass spectrometry (MS), and its sequence was determined to be Leu-Cys-Pro-Arg-Glu-Trp-Gly by amino acid sequence analysis.

[0123] The same method was used to extract and identify a small molecule peptide SEQ NO: 4 from rosemary, with a molecular weight of 832.9 Da and a sequence of Thr-Asp-Val-His-Lys-Phe-Ser.

[0124] (II) Synthesis and identification of APP

[0125] 1. Synthesis steps

[0126] In a three-necked flask equipped with a stirrer, a thermometer and a reflux condenser, 10 g of o-methoxyphenol, 5 g of propyleneamine, 0.5 g of palladium-carbon catalyst and 100 mL of ethanol were added. The temperature was raised to 60°C under stirring and the reaction was carried out for 7 hours. During the reaction, the reaction progress was monitored by thin layer chromatography (TLC). When the raw material point basically disappeared, the reaction was completed.

[0127] After the reaction is completed, the palladium carbon catalyst is removed by hot filtration, and the filtrate is distilled under reduced pressure to remove ethanol. The residue is subjected to column chromatography on a silica gel column, and gradient elution is performed using petroleum ether-ethyl acetate (5:1-1:1) as an eluent, and the target product fractions are collected to obtain purified APP.

[0128] 2. Identification methods

[0129] The structure of APP was identified by nuclear magnetic resonance (NMR) and mass spectrometry (MS). The NMR spectrum showed that the characteristic peaks were consistent with the expected structure, and MS analysis determined that its molecular weight was 167.2 Da, which was consistent with the expected structure.

[0130] (III) Preparation and performance testing of disinfectant

[0131] 1. Preparation of Disinfectant

[0132] The purified SEQ NO:3 small molecule peptide 0.15% weight ratio, SEQ NO:4 small molecule peptide 0.1% weight ratio and APP 2.5% weight ratio, solvent (ethanol 35% weight ratio, purified water 65% weight ratio), surfactant (coconut amide DEA 1.5% weight ratio), stabilizer (EDTA disodium 0.15% weight ratio), preservative (benzyl alcohol 0.2% weight ratio) and other ingredients are mixed in an appropriate container. First, the small molecule peptide and APP are dissolved in 50mL solvent and ultrasonically treated for 10 minutes to make them uniformly dispersed. Then other ingredients are added in sequence and stirred at a speed of 400 rpm in a stirrer for 40 minutes until a uniform solution is formed. Finally, the disinfectant is placed in a sealed bottle and stored in a cool and dry place.

[0133] 2. Disinfection performance test

[0134] The disinfection effect of disinfectant on HPV was tested by in vitro cell culture method combined with virus titer determination. HPV virus solution was mixed with disinfectant of different dilution multiples, and after different time (5, 10, 20, 30 minutes) at 37°C, the mixture was inoculated in a 96-well plate containing HeLa cells. After 72 hours of culture, the cytopathic effect (CPE) was observed and the virus titer was determined.

[0135] The test results show that when the total peptide concentration of the disinfectant prepared by the present invention is 0.3 mg / mL, the inactivation rate of HPV virus can reach more than 85% after 20 minutes of action, indicating that it has a good disinfection effect.

[0136] 3. Stability test

[0137] The disinfectant was placed under high temperature (40°C), low temperature (4°C) and light (3000-5000Lux) conditions, and samples were taken at different time points (1, 2, 4, 8, and 16 weeks). The changes in the content of small molecule peptides and APP were detected by RP-HPLC to evaluate the stability of the product.

[0138] The test results show that under the above conditions, the content of small molecule peptides and APP remained above 90% of the initial content within 16 weeks, indicating that the product has good stability and can meet storage and use requirements.

[0139] The above examples further verify that the preparation method of the HPV plant-derived disinfectant of the present invention is feasible, the product has good disinfection performance and stability, and can effectively meet the needs of HPV disinfection.

Claims

1. A HPV plant-derived disinfectant, characterized in that: Contains the following ingredients by weight: Plant extracted small molecule peptides: SEQ NO: 1-Gly-Cys-Val-Arg-Tyr-Gln-Leu small molecule peptide 0.12%-0.2%; SEQ NO: 2-Ala-Asp-Trp-Lys-Glu-Ser-Ile small molecule peptide 0.072%-0.12%; 3-(2-aminoethyl)-1,2-benzisothiazol-5-ol 1.5%-2.5%; solvent: ethanol 20%-30%, deionized water 70%-80%; surfactant: laureth-7 citrate 1%-2%; humectant: glycerol 3%-5%; preservative: sorbic acid 0.1%-0.3%.

2. The HPV plant-derived disinfectant according to claim 1, characterized in that: The SEQ NO: 1 small molecule peptide is extracted and identified by the following method: the dried whole herb of Centella asiatica is crushed, extracted with phosphate buffer at 40-50° C. for 3-4 hours, the extract is centrifuged and filtered, and then initially separated by a gel filtration chromatography column to collect components with a molecular weight range of 800-1500Da, and the components are further purified by reverse-phase high performance liquid chromatography using a C18 column and gradient elution with water-acetonitrile as the mobile phase, and the sequence and molecular weight are determined by mass spectrometry and amino acid sequence analysis.

3. The HPV plant-derived disinfectant according to claim 1, characterized in that: The small molecule peptide of SEQ NO: 2 is extracted and identified from Hypericum perforatum.

4. The HPV plant-derived disinfectant according to claim 1, characterized in that: The 3-(2-aminoethyl)-1,2-benzisothiazole-5-ol is synthesized and purified by the following method: 2-aminothiophenol and 2-bromoethylamine are used as raw materials, react in an organic solvent under alkaline conditions, the reaction temperature is controlled at 80-100°C, the reaction time is 4-6 hours, and after the reaction is completed, high-purity 3-(2-aminoethyl)-1,2-benzisothiazole-5-ol is obtained through acidification, extraction, drying, column chromatography and other steps, and its structure is identified by analytical means such as nuclear magnetic resonance and mass spectrometry.

5. The method for preparing the HPV plant-derived disinfectant according to claim 1, characterized in that: The method comprises the following steps: dissolving the small molecule peptide of SEQ NO: 1, the small molecule peptide of SEQ NO: 2 and AEBIT in a small amount of solvent; Adding surfactant, moisturizer and preservative in sequence; Stir in a stirrer at 200-400 rpm for 30-60 minutes until a uniform solution is formed; Store disinfectant in a sealed container in a cool, dry place.

6. The use of the HPV plant-derived disinfectant according to claim 1 in the disinfection of gynecological examination instruments and surgical instruments in medical institutions, characterized in that: Soak the instruments in the disinfectant solution for 15-30 minutes, then rinse them with sterile water.

7. Use of the HPV plant-derived disinfectant according to claim 1 in surface disinfection in public places, characterized in that: Spray the disinfectant directly on the surface of the object and wipe it clean after 10-20 minutes.

8. The use of the HPV plant-derived disinfectant according to claim 1 in the preparation of personal care products such as female private parts cleaning wipes, characterized in that: Add disinfectant to wet wipes for daily private area care.

9. A disinfectant composition, characterized in that It comprises the plant-extracted small molecule peptide and AEBIT in the HPV plant-derived disinfectant according to claim 1, and a pharmaceutically acceptable carrier or excipient.