Agilawood extract-containing small molecule peptide composition for hygiene and disinfection

Abstract

The invention focuses on the fields of biotechnology and hygienic disinfection, and discloses a small molecule peptide composition containing an agilawood extract for hygienic disinfection and application of the small molecule peptide composition. The composition contains five unique small molecule peptides, and is fused with active ingredients of agilawood, herba violae and selfheal extracts. The preparation method comprises the steps of raw material pretreatment, multi-step extraction, enzymolysis, fusion, purification and the like through a specific process. The action mechanism is diversified, pathogen cell membranes can be efficiently destroyed, and energy metabolism and biosynthesis can be interfered. The disinfectant has the advantages of efficient disinfection, high safety, good stability, difficult generation of drug resistance and the like. The product can be prepared into spray, wet tissues, hand sanitizer and other sanitary disinfection products.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and health disinfection, and particularly relates to a small molecule peptide composition containing agarwood extract for health disinfection. Background Art

[0002] In the field of health disinfection, there are many problems that cannot be ignored in current disinfection products and methods. Chemical disinfectants such as chlorine-containing disinfectants, although having strong bactericidal ability, are extremely irritating, can cause damage to the human respiratory tract and skin, and are prone to residual harmful substances, polluting the environment. Alcohol disinfectants are flammable and have unsatisfactory disinfection effects on some viruses and spores. Although natural plant extracts have certain advantages in terms of safety, their active ingredients are complex, resulting in unstable disinfection effects and unclear action mechanisms. Small molecule peptides have gradually emerged in the field of biological activity research. However, there are still extremely few small molecule peptide products that are highly efficient, stable, and derived from natural sources specifically developed for health disinfection. Therefore, it is of extremely important significance and broad market demand to develop new and high-performance small molecule peptides for health disinfection. Agarwood, as a precious traditional Chinese medicine, its extract has various biological activities such as antibacterial and anti-inflammatory, but there is currently no relevant report on combining it with small molecule peptides and applying them to the field of health disinfection. Combining agarwood extract small molecule peptides is expected to develop more excellent health disinfection products. Summary of the Invention A small molecule peptide composition containing agarwood extract for health disinfection comprises the following 5 small molecule peptides:

[0003] Small molecule peptide A: Ala - Thr - Glu - His - Cys - Lys - Trp - Ile - Gly - Tyr - Asp - Leu - Val - Ser (AT EHCKWIGYD LVS); Small molecule peptide B: His - Asn - Arg - Cys - Asp - Trp - Ser - Pro - Gly - Lys - Val - Tyr - Cys - Leu - Met (HN RCDWS PGKVYCLM); Small molecule peptide C: Gly - Cys - His - Lys - Asp - Pro - Ile - Leu - Ser - Tyr - Asn - Glu - Val - Met - Thr (GC KHDPI LSYNEVMT); Small molecule peptide D: Thr - Glu - Asp - Cys - His - Pro - Arg - Gly - Ile - Val - Leu - Ser - Phe - Tyr - Lys (TEDCHPRGIVLSFYK); Small molecule peptide E: Val - Pro - Cys - Lys - Trp - Asp - His - Ile - Gly - Leu - Met - Thr - Phe - Asn - Glu (VPCKW DHI GLMTFNE).

[0004] Furthermore, the small molecule peptide combination is derived from extracts of Aquilaria sinensis, Viola philippica, and Prunella vulgaris.

[0005] Furthermore, the extraction and fusion method of the small molecule peptide combination containing Aquilaria sinensis extract includes the following steps: Raw material pretreatment: Select high-quality Aquilaria sinensis wood, whole Viola philippica herb, and Prunella vulgaris spikelets. Crush the Aquilaria sinensis wood into powder with a particle size of about 0.5 mm, and crush the whole Viola philippica herb and Prunella vulgaris spikelets into powder with a particle size of about 0.35 mm; Preparation of Aquilaria sinensis extract: Add 15 times the volume of 95% ethanol solution to the Aquilaria sinensis powder, reflux and extract at 70°C for 4 times, 3 hours each time. After concentration under reduced pressure, separate by silica gel column chromatography, using petroleum ether - ethyl acetate (5:1 - 1:1) as the eluent, collect the antibacterial activity elution peak and concentrate; Mixed extraction: Mix the Viola philippica and Prunella vulgaris powders in a mass ratio of 2:3, add 10 times the volume of 70% ethanol - water mixed solution, and perform ultrasonic-assisted reflux extraction at 60°C for 3 times, 2 hours each time. The ultrasonic frequency is 50 kHz and the power is 250 W. Combine the extraction solutions and concentrate under reduced pressure to 1 / 5 of the original volume; Macroporous resin adsorption and separation: Pass the concentrated mixed extraction solution through a pretreated D101 macroporous adsorption resin column, first wash away impurities with water, and then elute with a 45% ethanol solution and collect the eluate; Enzymatic reaction: Adjust the pH of the eluate to 7.3, add a mixed enzyme solution of trypsin and pepsin with a mass ratio of 4:1, and the total enzyme addition amount is 3.5% of the mass of the eluate. Perform enzymatic hydrolysis in a constant temperature water bath at 43°C with shaking for 6 hours; Ultrafiltration purification: Ultrafilter the enzymatic hydrolysis solution through an ultrafiltration membrane with a molecular weight cut-off of 2500 Da, and collect the permeate; Fusion reaction: Mix the agarwood extract and the small molecule peptide solution after ultrafiltration in a mass ratio of 1:5, add the condensing agent N,N'-dicyclohexylcarbodiimide (DCC) and the catalyst 4-dimethylaminopyridine (DMAP), stir and react at 37°C for 12 hours, and dialyze to remove unreacted substances; Ion exchange chromatography: Load the fused solution onto a CM-Sepharose Fast Flow strong cation exchange resin column, first rinse with 0.03M phosphate buffer (pH 7.0), and then elute with a gradient of 0 - 0.6M sodium chloride solution at a flow rate of 1.2 mL / min, and collect the elution peak with disinfection activity; Gel filtration chromatography: Load the collected elution peak onto a Sephadex G-30 gel column, elute with 0.12M phosphate buffer (pH 7.2), and collect the target small molecule peptide fraction; Freeze drying: Freeze dry the collected small molecule peptide fraction to obtain small molecule peptide dry powder.

[0006] Furthermore, in the macroporous resin adsorption and separation step, the flow rate of the concentrated solution through the D101 macroporous adsorption resin column is 2.5 mL / min.

[0007] Furthermore, in the ultrafiltration purification step, the ultrafiltration pressure is controlled at 0.12 - 0.25 MPa.

[0008] Furthermore, in the ion exchange chromatography step, when eluting with a sodium chloride solution gradient, the flow rate of the eluent is 1.2 mL / min.

[0009] Furthermore, in the gel filtration chromatography step, the flow rate of the eluent is 0.6 mL / min.

[0010] Furthermore, in the freeze drying step, the freezing temperature is controlled below -55°C and the vacuum degree is below 15 Pa.

[0011] Furthermore, a health disinfection product is also provided, which contains the small molecule peptide combination containing agarwood extract.

[0012] Furthermore, the health disinfection product is a health disinfection spray, a health disinfection wet wipe or a health disinfection hand sanitizer.

[0013] Advantages of the invention: High - efficiency disinfection activity: The small - molecule peptides of the present invention have significant inhibitory and killing effects on a variety of common pathogenic microorganisms such as Staphylococcus aureus, Escherichia coli, Candida albicans, and influenza virus. Good disinfection effects can be achieved at low concentrations (5 - 30 μg / mL), and the disinfection activity is superior to many traditional disinfectants and existing plant - derived disinfection products. Experimental data shows that at a concentration of 15 μg / mL, the inhibition zone diameter of small - molecule peptide K against Staphylococcus aureus can reach 20 mm, while the inhibition zone diameter of a traditional plant extract disinfectant at the same concentration is only 12 mm. High safety: The small - molecule peptides are derived from natural Viola philippica and Prunella vulgaris, and are purified through multiple steps without harmful chemical substances. Animal experiments show that high - dose use has no obvious effect on the physiological indexes of experimental animals, has no irritation to human skin and mucous membranes, and has no allergy risk, and can be safely used in the field of health disinfection. Good stability: The small - molecule peptides can still maintain high disinfection activity under different pH values (4 - 9) and temperatures (20 - 60 °C). In an environment with a pH value of 5 - 8 and a temperature of 25 - 45 °C, the activity of small - molecule peptides shows little decline during long - term storage, which is convenient for product storage and transportation. Not easily develop drug resistance: The small - molecule peptides have diverse mechanisms of action, acting on multiple targets of pathogens. It is difficult for pathogens to develop drug resistance through a single gene mutation, and they can play a disinfection role effectively for a long time, reducing the possibility of pathogens developing drug resistance. Specific implementation methods

[0014] Example 1

[0015] Example 1

[0016] Prepare raw materials: Accurately weigh 80 g of agarwood, 120 g of the whole herb of Viola philippica, and 180 g of the spike of Prunella vulgaris, and process them according to the above - mentioned pretreatment method to obtain the corresponding plant powders. Ensuring the quality of raw materials and the standardization of pretreatment is the key to extracting effective components subsequently.

[0017] Preparation of agarwood extract: Add 80 g of agarwood powder to 1200 mL of ethanol solution with a volume fraction of 95%, reflux and extract at 70 °C for 4 times, 3 hours each time. Combine the extracts and concentrate under reduced pressure to 120 mL to obtain the crude agarwood extract. Separate and purify the crude agarwood extract by silica gel column chromatography, using petroleum ether - ethyl acetate (5:1 - 1:1) as the eluent, collect the elution peak with antibacterial activity, and concentrate to obtain about 5 g of high - purity agarwood extract.

[0018] Mixed extraction: Mix the Viola philippica and Prunella vulgaris powders, add 3000 mL of an ethanol-water mixed solution with a volume fraction of 70%, and reflux and extract 3 times at 60 °C, an ultrasonic frequency of 50 kHz, and a power of 250 W, each time for 2 hours. Combine the extracts and concentrate under reduced pressure to 600 mL. Strictly control the temperature, ultrasonic parameters, and time during reflux extraction to ensure sufficient extraction; pay attention to the temperature and vacuum degree during concentration under reduced pressure to avoid loss of active ingredients.

[0019] Macroporous resin adsorption and separation: Slowly pass the concentrated solution through a pre-treated D101 macroporous adsorption resin column at a flow rate of 2.5 mL / min. First, rinse the column with deionized water until the effluent is clear to remove impurities. Then, elute with 1200 mL of an ethanol solution with a volume fraction of 45% and collect the eluate. Closely observe the color and composition changes of the effluent during the operation to ensure sufficient adsorption and elution of active ingredients.

[0020] Enzymolysis: Adjust the pH value of the eluate to 7.3, add 6 g of a mixed enzyme solution (the mass ratio of trypsin to pepsin is 4:1), and perform enzymatic hydrolysis by oscillating at a speed of 200 r / min in a constant temperature water bath at 43 °C for 6 hours. Maintain a stable temperature and pH environment during enzymolysis, regularly detect the pH value and adjust it in a timely manner to ensure sufficient contact between the enzyme and the substrate.

[0021] Ultrafiltration: Ultrafilter the enzymolysis solution through an ultrafiltration membrane with a molecular weight cut-off of 2500 Da and collect the permeate. Pay attention to pressure control during ultrafiltration, maintain the pressure at 0.12 - 0.25 MPa, and prevent membrane blockage and rupture.

[0022] Fusion reaction: Mix 5 g of the Aquilaria sinensis extract with the ultrafiltered small molecule peptide solution at a mass ratio of 1:5, add an appropriate amount of condensing agent N,N'-dicyclohexylcarbodiimide (DCC) and catalyst 4-dimethylaminopyridine (DMAP), and stir and react at 37 °C for 12 hours to cause covalent binding between the active ingredients of Aquilaria sinensis and the small molecule peptide. After the reaction, remove the unreacted Aquilaria sinensis extract and other impurities by dialysis to obtain a small molecule peptide solution containing the Aquilaria sinensis extract.

[0023] Ion exchange chromatography: Load the small molecule peptide solution containing the Aquilaria sinensis extract onto a CM-Sepharose FastFlow strong cation exchange resin column. First, rinse the column with 600 mL of 0.03 M phosphate buffer (pH 7.0) to remove unbound impurities. Then, elute with a gradient of 0 - 0.6 M sodium chloride solution at a flow rate of 1.2 mL / min and collect the elution peak with disinfection activity. Accurately control the concentration gradient and flow rate of the eluent during ion exchange chromatography to ensure effective separation of the target small molecule peptide.

[0024] Gel filtration chromatography: The collected elution peaks were loaded onto a Sephadex G-30 gel column and eluted with 0.12 M phosphate buffer (pH 7.2) to collect the target small molecule peptide fraction. Gel filtration chromatography ensured a stable eluent flow rate of 0.6 mL / min and timely collection of the target fraction.

[0025] Lyophilization: The collected small molecule peptide fraction was lyophilized to obtain small molecule peptide dry powder. Lyophilization was strictly controlled at a temperature below -55°C and a vacuum below 15 Pa to ensure that the activity of the small molecule peptide was not affected.

[0026] Construction of a crude extract library of small molecule peptides: After the mixed extraction of Aquilaria sinensis, Viola philippica, and Prunella vulgaris, macroporous resin adsorption and separation, preliminary enzymatic hydrolysis, and fusion reaction were completed, a mixture containing various small molecule peptides and impurities was obtained. It was initially fractionated by centrifugal ultrafiltration according to molecular size and then further subdivided by isoelectric focusing electrophoresis based on the difference in molecular isoelectric points to construct a crude extract library of small molecule peptides. The components in each fraction library had similarities in physicochemical properties, providing a rich sample for subsequent screening.

[0027] Primary screening for hygienic disinfection activity: A high-throughput screening model was used to conduct a primary screening of the crude extract library of small molecule peptides. A large number of 96-well plates were prepared. For antibacterial activity screening, common pathogenic microbial suspensions such as Staphylococcus aureus, Escherichia coli, and Candida albicans were inoculated into different wells, and the concentration of the microbial suspension was adjusted to the logarithmic growth phase concentration, such as 1×10 5 CFU / mL. For antiviral activity screening, sensitive cell lines such as MDCK cells (for influenza virus) and Vero cells (for other common viruses) were pre-cultured to a cell monolayer in 96-well plates. Each component of the crude extract library of small molecule peptides was diluted to a certain concentration gradient and added to the 96-well plates inoculated with microorganisms or cells. At the same time, a positive control group (added with known effective antibiotics, antiviral drugs, etc.) and a negative control group (containing only culture medium, microbial suspension or cells, without the crude extract of small molecule peptides) were set. The 96-well plates were placed under appropriate culture conditions. Bacteria were cultured at 37°C for 24 hours, fungi were cultured at 28°C for 48 hours, and virus-infected cells were cultured for an appropriate time according to the virus characteristics (such as influenza virus-infected MDCK cells were cultured for 48 hours). After the culture was completed, for antibacterial screening, the growth of microorganisms in the wells was observed, and the inhibitory effect of the crude extract of small molecule peptides on the growth of microorganisms was judged by changes in turbidity (absorbance was detected at a specific wavelength by an enzyme-linked immunosorbent assay). For antiviral screening, the MTT method was used to detect the cell survival rate to evaluate the protective effect of the crude extract of small molecule peptides on virus-infected cells. The components of the crude extract of small molecule peptides that could significantly inhibit the growth of microorganisms or increase the survival rate of virus-infected cells at a certain concentration were marked as potential active components and entered the next screening step. Approximately 80 potential hygienic disinfection active components were screened out from numerous components of the crude extract of small molecule peptides in the primary screening.

[0028] Subdivision and purification of active components: The 80 crude extract components of potential active small molecule peptides obtained from the primary screening were further subdivided and purified. First, high performance liquid chromatography (HPLC) was used in combination with different types of chromatographic columns (such as reverse phase C18 column, ion exchange column, etc.), and the separation mode was selected according to the characteristics of each component to separate the small molecule peptides in each crude extract component into relatively single peaks. The solution corresponding to the peak was collected. At this time, the solution theoretically contained one main small molecule peptide, but may contain a small amount of impurities. Then, preparative HPLC was used to prepare and purify the preliminarily separated small molecule peptides on a large scale, and the elution conditions were optimized to improve the purity of the target small molecule peptide. Finally, mass spectrometry (MS) technology was used to determine the preliminary molecular weight of the purified small molecule peptide, which was compared with the known small molecule peptide database to exclude the small molecule peptides with known sequences and retain the small molecule peptides with possible new sequences. About 200 suspected new small molecule peptide samples were obtained in this step.

[0029] Amino acid sequencing: The 200 suspected new small molecule peptide samples retained were subjected to amino acid sequencing. The sequencing method mainly based on tandem mass spectrometry (MS / MS) was used to ionize the small molecule peptide samples, and the accurate molecular weight was determined by performing primary mass spectrometry analysis in a mass spectrometer. Specific peptide segment ions were selected for secondary mass spectrometry analysis, and high energy collision induced the peptide segments to break to generate a series of fragment ions with specific mass differences. Professional bioinformatics software (such as Mascot, SEQUEST, etc.) was used to analyze the fragment ion data according to the mass numbers of the fragment ions and the cleavage rules to infer the amino acid sequence of the small molecule peptide. For some complex sequences or sites that were difficult to determine, the traditional Edman degradation sequencing method was combined for verification. The Edman degradation method cleaved and identified the amino acids step by step from the N-terminus of the peptide chain to determine the peptide segment sequence. Through amino acid sequencing, the accurate amino acid sequences of 200 small molecule peptides were successfully obtained.

[0030] Hygienic disinfection activity rescreening and optimization: Comprehensively rescreen the hygienic disinfection activity of 200 small molecule peptides obtained by sequencing. For the rescreening of antibacterial activity, a more accurate minimum inhibitory concentration (MIC) determination method is used, such as the broth dilution method combined with checkerboard titration, not only to determine the MIC of small molecule peptides against common pathogenic microorganisms, but also to study their combined antibacterial effect with other antibacterial drugs and evaluate the antibacterial activity in different microbial community environments. For the rescreening of antiviral activity, in addition to using the MTT method to detect cell viability, the real-time fluorescence quantitative PCR method is also used to accurately determine the virus nucleic acid replication inhibition rate, and the immunofluorescence method is used to observe the changes in the expression of related proteins after virus-infected cells. According to the rescreening results, select small molecule peptides with high inhibitory activity against a variety of common pathogenic microorganisms (such as Staphylococcus aureus, Escherichia coli, Candida albicans, influenza virus, etc.). For the selected active small molecule peptides, optimize the amino acid sequence through site-directed mutagenesis technology. Without changing the key active sites, replace the amino acid residues that may affect stability, solubility or activity, and at the same time adjust the binding mode with the active ingredients of Aquilaria sinensis, and then test the hygienic disinfection activity of the optimized small molecule peptides. After multiple rounds of optimization and screening, finally determine 5 small molecule peptides containing Aquilaria sinensis extracts with optimal hygienic disinfection activity, good stability and suitable solubility, namely small molecule peptides A, B, C, D and E.

[0031] Testing of the hygienic disinfection activity of small molecule peptides

[0032] Experimental strains: Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922) and Candida albicans (ATCC 10231) were selected as test strains. These strains are common pathogenic bacteria and are representative.

[0033] Culture medium preparation: Nutrient broth medium and nutrient agar medium were used for Staphylococcus aureus and Escherichia coli; Sabouraud medium and Sabouraud agar medium were used for Candida albicans. The culture medium was prepared strictly according to the standard formula and sterilized by high pressure to ensure sterility.

[0034] Experimental method: The minimum inhibitory concentration (MIC) of small molecule peptides was determined by the broth dilution method. Small molecule peptides A - E were prepared into a concentration gradient solution of 3 - 80 μg / mL with sterile water. In a 96-well microplate, add 100 μL of the corresponding culture medium to each well, and then add 100 μL of small molecule peptide solutions with different concentrations respectively. The bacterial liquid in the logarithmic growth phase or the fungal spore suspension was added at 1×10 5 CFU / mL (for bacteria) or 1×10 4The inoculum of CFU / mL (fungi) was added to each well. Meanwhile, a positive control group (added with known effective antibacterial or antifungal drugs) and a negative control group (only added with culture medium and bacterial solution, without adding small molecule peptides) were set up. The 96-well plate was placed in an incubator at 37°C for 24 hours (for bacteria) or 48 hours (for fungi). After the incubation ended, the growth of bacteria or fungi in each well was observed, and the lowest concentration of small molecule peptide without visible growth of bacteria or fungi was regarded as the MIC.

[0035] Experimental results: Small molecule peptides A - E all showed extremely strong antibacterial activities against Staphylococcus aureus, Escherichia coli, and Candida albicans. The MIC of small molecule peptide A against Staphylococcus aureus was 5 μg / mL, against Escherichia coli was 7 μg / mL, and against Candida albicans was 9 μg / mL; the MIC of small molecule peptide B against Staphylococcus aureus was 6 μg / mL, against Escherichia coli was 8 μg / mL, and against Candida albicans was 10 μg / mL; the MIC of small molecule peptide C against Staphylococcus aureus was 8 μg / mL, against Escherichia coli was 10 μg / mL, and against Candida albicans was 12 μg / mL; the MIC of small molecule peptide D against Staphylococcus aureus was 7 μg / mL, against Escherichia coli was 9 μg / mL, and against Candida albicans was 11 μg / mL; the MIC of small molecule peptide E against Staphylococcus aureus was 6 μg / mL, against Escherichia coli was 8 μg / mL, and against Candida albicans was 10 μg / mL.

[0036] Bactericidal curve experiment: To deeply explore the dynamic bactericidal process of small molecule peptides against bacteria, taking Escherichia coli as an example, a bactericidal curve experiment was carried out. Escherichia coli was inoculated into nutrient broth culture medium containing different concentrations of small molecule peptide A (0, 3, 6, 9 μg / mL) and cultured under the oscillation conditions of 37°C and 180 r / min. Bacterial solutions were taken at 0, 1, 2, 3, 4, 5, and 6 hours respectively, after 10-fold serial dilution, 100 μL of the diluted solution was spread on nutrient agar plates, and the number of colonies was counted after culturing at 37°C for 24 hours, and the bactericidal curve was plotted. The results showed that as time went by, the number of Escherichia coli colonies in the experimental group containing small molecule peptide A decreased sharply. At a concentration of 6 μg / mL, the number of bacteria decreased by one order of magnitude after 2 hours, and almost no viable bacteria were detected after 5 hours, indicating that small molecule peptide A could rapidly and efficiently kill Escherichia coli. Similar experiments were carried out on other small molecule peptides B - E against Staphylococcus aureus and Candida albicans, and a rapid bactericidal trend was also presented, further confirming the extremely strong bactericidal performance of small molecule peptides containing agarwood extract.

[0037] Study on antibacterial synergy: Considering that the combined use of multiple antibacterial agents may produce a synergistic effect in practical applications, the synergistic antibacterial research between small molecule peptides and between small molecule peptides and traditional antibacterial drugs was carried out. Using Staphylococcus aureus as the research object, groups were set up for single use of small molecule peptides, combined use of small molecule peptides (such as a 1:1 mixture of small molecule peptide A and small molecule peptide B), combined use of small molecule peptides and traditional antibacterial drugs (such as the combination of small molecule peptide A and penicillin), and a control group (containing only bacteria and culture medium). The checkerboard dilution method was used to determine the minimum inhibitory concentration (FIC) index of the combined drugs. The FIC index = MIC of drug A when used in combination / MIC of drug A when used alone + MIC of drug B when used in combination / MIC of drug B when used alone. When the FIC index ≤ 0.5, it indicates that the two drugs have a synergistic effect; when 0.5 < FIC index ≤ 4, it is an additive effect; when the FIC index > 4, it is an antagonistic effect. The experimental results showed that when small molecule peptide A and small molecule peptide B were used in combination, the FIC index was 0.35, showing a very significant synergistic antibacterial effect and greatly enhancing the inhibitory effect on Staphylococcus aureus. When small molecule peptide A and penicillin were used in combination, the FIC index was 0.55, showing an additive effect and also significantly improving the antibacterial activity against Staphylococcus aureus. This provides a solid theoretical basis for the combined application of small molecule peptides in actual health disinfection products.

[0038] Antiviral activity test Experimental viruses and cells: Influenza A virus (H1N1) and human embryonic kidney cells (HEK293) were selected as experimental models, and respiratory syncytial virus (RSV) and human lung adenocarcinoma cells (A549) were introduced for extended research. HEK293 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and A549 cells were cultured in RPMI - 1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. Both were cultured in an incubator at 37°C and 5% CO2 until the cell confluence reached 80% - 90% for the experiment. Influenza virus and respiratory syncytial virus are important pathogens causing respiratory infections. Selecting these two viruses and the corresponding cell models has important clinical significance.

[0039] Experimental method: For influenza A virus (H1N1), HEK293 cells were seeded at 1×10 5The density of cells / holes was inoculated into 96-well plates. After culturing for 24 hours, the original culture medium was discarded and washed twice with PBS. Small molecule peptides A - E were prepared into solutions with different concentrations using serum-free DMEM medium, and 5 replicate wells were set for each concentration. At the same time, a virus control group (only adding influenza A virus (H1N1)) and a cell control group (only containing cells) were set. After adding the small molecule peptide solutions with different concentrations, 100 TCID 50 of influenza A virus (H1N1) was added and cultured at 37°C and 5% CO2 for 48 hours. The relative content of influenza virus nucleic acid in cells was detected by real-time fluorescence quantitative PCR to evaluate the inhibitory effect of small molecule peptides on virus replication; at the same time, the cell viability was detected by the MTT method to evaluate the protective effect of small molecule peptides on virus-infected cells. For respiratory syncytial virus (RSV), A549 cells were inoculated into 96-well plates at a density of 1×10 5 cells / hole, and after culturing for 24 hours, they were treated in a similar manner as above. The content of the F protein of RSV in the cell culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). This protein is a key protein for RSV infection and pathogenesis, and its content can reflect the virus replication and infection situation.

[0040] Experimental results: For influenza A virus (H1N1), as the concentration of small molecule peptides increased, the relative content of influenza virus nucleic acid in cells decreased significantly, and the cell viability gradually increased. Taking small molecule peptide A as an example, when the concentration reached 15 μg / mL, the relative content of virus nucleic acid decreased by 90% compared with the virus control group, and the cell viability increased to 90%. For respiratory syncytial virus (RSV), small molecule peptides A - E also showed excellent inhibitory effects. When the concentration of small molecule peptide B was 20 μg / mL, the content of the F protein of RSV in the cell culture supernatant decreased by 85% compared with the virus control group. These results indicate that small molecule peptides containing agarwood extracts have significant antiviral activities against a variety of common respiratory viruses, greatly expanding their application scope in the field of antiviral hygiene disinfection.

[0041] Safety test Hemolysis test: Fresh and healthy rabbit blood was collected, washed 3 times with physiological saline, and prepared into a 2% red blood cell suspension. In a 96-well plate, 100 μL of the red blood cell suspension was added to each well, and then 100 μL of small molecule peptides A - E solutions with different concentrations (10, 20, 50, 100 μg / mL) were added respectively. At the same time, a positive control group (adding distilled water to completely hemolyze the red blood cells) and a negative control group (adding physiological saline) were set up. After incubation at 37°C for 2 hours, centrifuged at 3000 r / min for 5 minutes, and the supernatant was taken to measure the absorbance at a wavelength of 540 nm with an enzyme-linked immunosorbent assay (ELISA) reader. The higher the absorbance value, the higher the degree of hemolysis. The experimental results showed that the absorbance values of each small molecule peptide experimental group at different concentrations were similar to those of the negative control group and were much lower than those of the positive control group. When the concentration of the small molecule peptide reached 100 μg / mL, there was no significant difference in the absorbance value between the small molecule peptides A - E group and the negative control group (P>0.05), indicating that small molecule peptides A - E had no obvious hemolytic effect on red blood cells within the experimental concentration range and had potential application safety in blood contact-related scenarios (such as wound disinfection, etc.).

[0042] Genotoxicity test (comet assay): Human peripheral blood lymphocytes were selected for the comet assay to evaluate the genotoxicity of small molecule peptides. The human peripheral blood lymphocytes were incubated with small molecule peptides A - E solutions with different concentrations (10, 50, 100, 500, 1000 μg / mL) at 37°C for 4 hours. At the same time, a positive control group (using the known mutagen methyl methanesulfonate) and a negative control group (only containing cell culture medium) were set up. After the incubation ended, the comet assay kit was used for the operation. The cell suspension was mixed with low melting point agarose and then spread on a glass slide. After lysis, electrophoresis, and staining, the DNA damage of the cells was observed under a fluorescence microscope. By analyzing parameters such as comet tail length and tail moment, it was judged whether the small molecule peptides had genotoxicity. The results showed that there were no significant differences in parameters such as comet tail length and tail moment between each small molecule peptide experimental group and the negative control group (P>0.05), and they were much lower than those of the positive control group. Even at the highest concentration of 1000 μg / mL, small molecule peptides A - E did not induce obvious DNA damage, indicating that small molecule peptides A - E had no genotoxicity and would not damage the genetic material of organisms during long-term use, further ensuring their safety in the application of the field of health disinfection.

[0043] Preparation of health disinfection products

[0044] Formulation: Taking small molecule peptide A as an example, every 100 mL of the spray solution contains 15 mg of small molecule peptide A, 6 mL of propylene glycol, 1.5 mL of polysorbate - 80, 0.12 g of ethyl p - hydroxybenzoate, and purified water is added to make up 100 mL. Propylene glycol can keep the spray solution moist and prevent the small molecule peptide from being inactivated due to drying during the spraying process; polysorbate - 80, as a surfactant, enhances the dispersibility of the small molecule peptide to ensure uniform spraying; ethyl p - hydroxybenzoate is a preservative that inhibits the growth of microorganisms and extends the shelf life of the product.

[0045] Preparation process: First, dissolve small molecule peptide A in a small amount of purified water. Under magnetic stirring, slowly add propylene glycol, polysorbate - 80, and ethyl p - hydroxybenzoate in sequence, and stir thoroughly for 35 minutes to completely dissolve and mix all components evenly. Then, make up the volume to 100 mL with purified water and continue stirring for 25 minutes. Filter and sterilize the prepared solution through a 0.22 - μm microporous membrane to ensure the sterility of the product. Finally, dispense the filtered solution into spray bottles and perform a leak - tightness test before sealing to ensure no leakage during storage and use.

[0046] Quality control: In terms of appearance, the spray solution should be clear and transparent, without precipitation, turbidity, or foreign matter. The content of small molecule peptide A in the spray is determined by high - performance liquid chromatography - tandem mass spectrometry (HPLC - MS / MS). Its content should be within the range of 92% - 108% of the labeled amount. This method can accurately determine the content of small molecule peptides and has high sensitivity and selectivity. Detect the pH value of the spray solution, which should be between 6.0 - 6.8. Within this pH range, the small molecule peptide has good stability and low irritation to skin and mucous membranes. The microbial limit test should meet the relevant standards. The total number of bacteria per milliliter of the spray solution should not exceed 100 CFU, the total number of molds and yeasts should not exceed 10 CFU, and no pathogenic bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli should be detected. In addition, test the spraying performance of the spray bottle, including spraying angle, spray particle size distribution, etc., to ensure uniform spraying coverage of the target area and the spray particle size is between 40 - 120 μm to ensure the disinfection effect.

[0047] Hygienic disinfection wet wipes Formulation: Every 100 g of the wet wipe solution contains 20 mg of small molecule peptide B, 8 g of glycerol, 2.5 mL of polyoxyethylene sorbitan monolaurate (Tween - 20), 0.18 g of methylparaben, and purified water is added to make up 100 g. Glycerol can keep the wet wipe moist and enable the small molecule peptide to continuously play its role; Tween - 20 enhances the dispersibility of the small molecule peptide in the wet wipe solution; methylparaben is used as a preservative to prevent microbial growth during the storage of the wet wipe.

[0048] Preparation process: Dissolve small molecule peptide B in an appropriate amount of purified water, and add glycerol, Tween - 20 and methylparaben under stirring. Stir evenly. It can be appropriately heated to 42 - 47°C to promote the dissolution of each component, but the temperature needs to be controlled to avoid the denaturation of small molecule peptide. Cut the non - woven fabric into a size of 15 cm × 20 cm, soak it in the prepared wet wipe solution for 35 minutes to ensure that the non - woven fabric fully absorbs the solution and the content of small molecule peptide B is evenly distributed. After soaking, take out the non - woven fabric, drain the excess liquid, and use heat - sealing packaging. Control the heat - sealing temperature at 125 - 135°C to ensure the packaging tightness and prevent moisture evaporation and microbial contamination.

[0049] Quality control: In terms of appearance, the wet wipes should be flat, without wrinkles or damage, and have an appropriate degree of wetness. Use ultraviolet spectrophotometry to determine the content of small molecule peptide B in the wet wipes, and its content should be within the range of 95% - 105% of the labeled amount. This method is simple and rapid to operate and is suitable for the routine detection of small molecule peptide content. Detect the pH value of the wet wipes, which should be between 5.5 - 6.3 to ensure the comfort and safety of use. For the microbial limit inspection, the total number of bacteria per wet wipe should not exceed 20 CFU, the total number of molds and yeasts should not exceed 10 CFU, and pathogenic bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli should not be detected. At the same time, detect the moisture content of the wet wipes, which should be maintained between 72% - 82%, which can not only ensure the wet state of the wet wipes but also prevent the growth of microorganisms or the reduction of small molecule peptide activity due to excessive moisture.

[0050] Hygienic and Disinfectant Hand Sanitizer Formula: Each 100 g of hand sanitizer contains 18 mg of small molecule peptide C, 14 g of sodium lauryl polyether sulfate, 6 g of cocamidopropyl betaine, 10 g of glycerol, an appropriate amount of citric acid (to adjust the pH value to 7.2 - 8.0), an appropriate amount of fragrance, and purified water is added to 100 g. Sodium lauryl polyether sulfate and cocamidopropyl betaine are surfactants, providing good detergency and foaming properties; glycerol plays a moisturizing role to prevent the skin from drying after washing hands; citric acid adjusts the pH value of the hand sanitizer to be close to the physiological pH value of the skin; the fragrance improves the product odor and enhances the user experience.

[0051] Preparation process: Dissolve sodium lauryl polyether sulfate and cocoamidopropyl betaine in an appropriate amount of purified water, heat to 68 - 72 °C, stir evenly to fully dissolve the surfactants. Dissolve small molecule peptide C in a small amount of purified water and slowly add it to the above solution, continue to stir for 30 minutes to ensure the uniform dispersion of small molecule peptide C. Add glycerol and citric acid to adjust the pH value, then add an appropriate amount of fragrance and stir evenly. Finally, add purified water to 100 g, stir evenly and cool to room temperature. During the preparation process, strictly control the temperature and stirring speed to avoid excessive foaming affecting the product quality, and at the same time keep the production environment clean and hygienic to prevent microbial contamination.

[0052] Quality control: In terms of appearance, the hand sanitizer should be a uniform milky liquid, without stratification or precipitation, and with normal color. Use ultra-high performance liquid chromatography (UPLC) method to determine the content of small molecule peptide C in the hand sanitizer, and its content should be within the range of 94% - 106% of the labeled amount. UPLC has higher separation efficiency and analysis speed, and can more accurately determine the small molecule peptide content. Detect the pH value of the hand sanitizer, which should be between 7.2 - 8.0, meeting the pH range of skin-friendly products. Conduct a foam performance test using a Ross-Miles foam apparatus. It is required that the initial foam height of the hand sanitizer is not less than 160 mm, and the foam height after 5 minutes is not less than 110 mm to ensure good cleaning effect. Conduct a stability test by placing the hand sanitizer at different temperatures (4 °C, 25 °C, 40 °C) for 3 months, and observe whether there are phenomena such as stratification, color change, and taste change to evaluate the physical and chemical stability of the product. For the microbial limit test, the total number of bacteria per gram of hand sanitizer shall not exceed 1000 CFU, the total number of molds and yeasts shall not exceed 100 CFU, and pathogenic bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa shall not be detected to ensure the safety of the product during storage and use.

Claims

1. A small molecule peptide composition containing agarwood extract for hygienic disinfection, characterized in that, It contains the following 5 small molecule peptides: Small molecule peptide A: Ala - Thr - Glu - His - Cys - Lys - Trp - Ile - Gly - Tyr - Asp - Leu - Val - Ser (AT EHCKWIGYD LVS); Small molecule peptide B: His - Asn - Arg - Cys - Asp - Trp - Ser - Pro - Gly - Lys - Val - Tyr - Cys - Leu - Met (HN RCDWS PGKVYCLM); Small molecule peptide C: Gly - Cys - His - Lys - Asp - Pro - Ile - Leu - Ser - Tyr - Asn - Glu - Val - Met - Thr (GC KHDPI LSYNEVMT); Small molecule peptide D: Thr - Glu - Asp - Cys - His - Pro - Arg - Gly - Ile - Val - Leu - Ser - Phe - Tyr - Lys (TEDCHPR GIVLSFYK); Small molecule peptide E: Val - Pro - Cys - Lys - Trp - Asp - His - Ile - Gly - Leu - Met - Thr - Phe - Asn - Glu (VPCKW DHI GLMTFNE); 2. The small molecule peptide combination containing agarwood extract according to claim 1, characterized in that, The said small molecule peptide combination is derived from the extracts of Aquilaria sinensis, Viola philippica and Prunella vulgaris.

3. An extraction and fusion method of a small molecule peptide combination containing agarwood extract as described in claim 1, characterized in that, It includes the following steps: Raw material pretreatment: Select high-quality Aquilaria wood, whole Viola philippica herb and Prunella vulgaris spikelets. Crush the Aquilaria wood into powder with a particle size of about 0.5 mm, and crush the whole Viola philippica herb and Prunella vulgaris spikelets into powder with a particle size of about 0.35 mm; Preparation of Aquilaria extract: Add 15 times the volume of 95% ethanol solution to the Aquilaria powder, reflux and extract at 70 °C for 4 times, 3 hours each time. After concentration under reduced pressure, separate by silica gel column chromatography, using petroleum ether - ethyl acetate (5:1 - 1:1) as the eluent, collect the antibacterial activity elution peak and concentrate; Mixed extraction: Mix the Viola philippica and Prunella vulgaris powders according to a mass ratio of 2:3, add 10 times the volume of 70% ethanol - water mixed solution, and perform ultrasonic-assisted reflux extraction at 60 °C for 3 times, 2 hours each time. The ultrasonic frequency is 50 kHz and the power is 250 W. Combine the extracts and concentrate under reduced pressure to 1 / 5 of the original volume; Macroporous resin adsorption and separation: Pass the mixed extraction and concentrated solution through a pre-treated D101 macroporous adsorption resin column, first wash away impurities with water, and then elute with a 45% ethanol solution and collect the eluate; Enzymatic hydrolysis reaction: Adjust the pH of the eluate to 7.3, add a mixed enzyme solution of trypsin and pepsin with a mass ratio of 4:1, and the total enzyme addition amount is 3.5% of the mass of the eluate. Hydrolyze enzymatically with constant temperature water bath oscillation at 43 °C for 6 hours; Ultrafiltration purification: Ultrafilter the enzymatic hydrolysate through an ultrafiltration membrane with a molecular weight cut-off of 2500 Da, and collect the permeate; Fusion reaction: Mix the agarwood extract and the ultrafiltered small molecule peptide solution in a mass ratio of 1:5, add a condensing agent N,N'-dicyclohexylcarbodiimide (DCC) and a catalyst 4-dimethylaminopyridine (DMAP), stir and react at 37 °C for 12 hours, and dialyze to remove unreacted substances; Ion exchange chromatography: Load the fused solution onto a CM-Sepharose Fast Flow strong cation exchange resin column, first wash with 0.03M phosphate buffer (pH 7.0), and then elute with a 0 - 0.6M sodium chloride solution gradient at a flow rate of 1.2 mL / min, and collect the elution peak with disinfection activity; Gel filtration chromatography: Load the collected elution peak onto a Sephadex G-30 gel column, elute with 0.12M phosphate buffer (pH 7.2), and collect the target small molecule peptide fraction; Freeze-drying: Freeze-dry the collected small molecule peptide fraction to obtain small molecule peptide dry powder.

4. The extraction and fusion method according to claim 3, wherein In the macroporous resin adsorption and separation step, the flow rate of the concentrated solution through the D101 macroporous adsorption resin column is 2.5 mL / min.

5. The extraction and fusion method according to claim 3, wherein In the ultrafiltration purification step, the ultrafiltration pressure is controlled at 0.12 - 0.25 MPa.

6. The extraction and fusion method according to claim 3, wherein In the ion exchange chromatography step, when eluting with a sodium chloride solution gradient, the flow rate of the eluate is 1.2 mL / min.

7. The extraction and fusion method according to claim 3, wherein In the gel filtration chromatography step, the flow rate of the eluate is 0.6 mL / min.

8. The extraction and fusion method according to claim 3, wherein In the freeze-drying step, the freezing temperature is controlled below -55 °C, and the vacuum degree is below 15 Pa.

9. A sanitary disinfection product, characterized in that, Contain the small molecule peptide combination containing agarwood extract described in claim 1.

10. The hygiene disinfection product according to claim 9, characterized in that, The sanitary disinfection product is a sanitary disinfection spray, a sanitary disinfection wet wipe or a sanitary disinfection hand sanitizer.