An antibacterial product of an olea europaea leaf base

Abstract

The present application relates to an antibacterial product based on olive leaf extract, belonging to the technical field of antibacterial care. The product is based on olive leaf extract rich in polyphenol active ingredients, combined with bacteriostatic polypeptides derived from Xenopus laevis as the core active component, to achieve synergistic antibacterial effect. Studies have shown that the combination has rapid and strong inhibitory and killing ability on common pathogenic microorganisms such as Staphylococcus aureus and Candida albicans, and is significantly better than single component. In vitro test results show that the product can achieve strong bacteriostatic and bactericidal effect in a short time, and has certain sustained bacteriostatic ability. Compared with traditional antibiotics or single plant extract, the present application improves the antibacterial efficiency and reduces the risk of drug resistance through the synergistic mechanism of "natural plant matrix + functional polypeptide", and has good safety and microecological friendliness, and is suitable for daily care and infection protection of female reproductive system.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial products technology, specifically relating to an antibacterial product based on olive leaf matrix. Background Technology

[0002] Gynecological infections are among the most common and prevalent health problems affecting the female reproductive system, primarily including bacterial vaginosis, fungal vaginitis, and trichomoniasis. These diseases typically manifest as abnormal vaginal discharge, vulvar itching, burning sensation, and odor, impacting women's quality of life and potentially leading to pelvic inflammatory disease, infertility, and pregnancy-related complications. Therefore, their prevention and treatment hold significant clinical and public health importance.

[0003] Currently, gynecological infectious diseases are mostly treated with antibacterial or antifungal Western medicines, such as metronidazole and clotrimazole. Although these drugs have good antibacterial or bactericidal effects in the short term, long-term or repeated use can easily lead to an imbalance of the normal vaginal flora, disrupting the original acidic environment (pH 3.8–4.5) and microecological homeostasis of the vagina. At the same time, they may also induce drug resistance in pathogenic microorganisms, thus leading to recurrent disease or decreased efficacy.

[0004] In recent years, active ingredients derived from natural plants have received widespread attention in the development of antibacterial and anti-inflammatory products due to their multi-component, multi-target effects and relatively low toxicity. For example, olive leaves are rich in polyphenolic active substances such as oleuropein and hydroxytyrosol, which have good antibacterial, anti-inflammatory, and antioxidant activities. In traditional Chinese medicine, herbs such as Sophora flavescens, Phellodendron chinense, and Cnidium monnieri are often used to clear heat and dry dampness, kill parasites and relieve itching, and as an adjunct treatment for gynecological inflammation. Based on this, existing technologies have explored the use of compound extracts from various plants to prepare gynecological washes or external care preparations to leverage the comprehensive antibacterial effects of natural ingredients.

[0005] However, existing antibacterial agents based on plant extracts still have certain limitations. On the one hand, the synergistic mechanism between different plant active ingredients is not yet clear, resulting in limited overall antibacterial efficacy. On the other hand, some stubborn pathogenic microorganisms associated with gynecological infections, such as drug-resistant bacteria or microorganisms with biofilm-forming capabilities, often cannot achieve ideal inhibition or elimination effects by relying solely on natural plant extracts, thus affecting the stable efficacy of the product.

[0006] Therefore, how to introduce functional components with stronger antibacterial activity into the natural plant extract system and achieve synergistic enhancement through reasonable combination to improve the inhibitory ability against a variety of pathogenic microorganisms, especially stubborn microorganisms, while maintaining good safety and microecological friendliness, has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] This invention provides an antibacterial product based on olive leaf matrix, specifically an antibacterial product prepared by adding antimicrobial peptides to olive leaf extract as a matrix, thereby making it more effective as an antibacterial care product for the female reproductive system.

[0008] The present invention first provides a polypeptide with the amino acid sequence VLKCKRFFKVCKLR (SEQ ID NO: 1).

[0009] Furthermore, the polypeptide is prepared from the African clawed frog;

[0010] The present invention also provides the use of the aforementioned polypeptide in the preparation of products that inhibit pathogenic microorganisms;

[0011] The pathogenic microorganisms mentioned are pathogenic microorganisms of the female reproductive system;

[0012] More specifically, the pathogenic microorganisms mentioned are Staphylococcus aureus and Candida albicans.

[0013] In another aspect, the present invention provides an antibacterial product, wherein the active component contains the aforementioned polypeptide;

[0014] Furthermore, the antibacterial product is an antibacterial care solution prepared by combining olive leaf matrix extract and the above-mentioned polypeptide;

[0015] The olive leaf matrix extract is prepared by adding olive leaves to an ethanol solution for reflux extraction, filtering the extract, concentrating the filtrate, adsorbing the concentrate onto a macroporous adsorption resin column, and eluting sequentially with deionized water, 30% ethanol, and 50% ethanol; collecting the 50% ethanol eluent, concentrating under reduced pressure, and spray drying.

[0016] Furthermore, other antibacterial components may be added to the antibacterial product.

[0017] The antibacterial product based on olive leaf matrix provided by this invention uses olive leaf matrix and antibacterial polypeptide as active components, which enables it to have a rapid and effective inhibitory effect on Candida albicans and Staphylococcus aureus. Attached Figure Description

[0018] Figure 1 Graph of fine purification of peptide by reversed-phase high-performance liquid chromatography (RP-HPLC);

[0019] Figure 2 : Mass spectrometry identification results of polypeptides;

[0020] Figure 3 The results of the antibacterial experiment are shown in the figure. A is the negative control (PBS), B is the control group (without peptides), and C is the experimental group (containing peptides). Detailed Implementation

[0021] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0022] Example 1: Preparation and Screening of Antimicrobial Peptides

[0023] This invention involves the targeted preparation and screening of antibacterial peptides from specific biological sources that exhibit high activity against common gynecological pathogens and can synergistically interact with olive leaf extract matrix.

[0024] 1. Preparation of crude antibacterial polypeptide extract

[0025] 1.1 Immunostimulation and Tissue Collection: Fifty healthy adult African clawed frogs (Xenopus laevis) were selected, using their skin secretions rich in antimicrobial peptides as the biological source. Each frog was subcutaneously injected with 0.2 mL of a mixed antigen solution (heat-inactivated Staphylococcus aureus ATCC 6538 and Candida albicans ATCC 10231, both at a concentration of 1×10⁻⁶) on its back. 9 Specific immune stimulation was performed using CFU / mL. After 72 hours, skin secretions were collected using a mild electrical stimulation method (3V, 5s pulse stimulation, 10s interval, 5 times in total) and immediately mixed with a pre-cooled 0.1% trifluoroacetic acid (TFA) solution containing 1 mM benzyl sulfonyl fluoride (PMSF) at a ratio of 1:3 (v / v).

[0026] 1.2 Preparation of crude extract: The above mixture was placed in an ice-water bath and homogenized three times at 10,000 rpm using a tissue homogenizer, each time for 30 seconds, with a 1-minute interval. The homogenate was centrifuged at 12,000 × g for 40 minutes at 4°C. The supernatant was carefully collected and filtered through a 0.45 μm aqueous filter membrane to obtain a crude extract containing antimicrobial peptides.

[0027] 1.3 Separation and purification:

[0028] a. Cation exchange chromatography: Load the crude extract onto a pre-equilibrated SP Sepharose Fast Flow column (10 mL bed volume) with 20 mM ammonium acetate buffer (pH 5.5). Wash unbound components with 5 column volumes of equilibration buffer, then perform linear gradient elution (total gradient volume 200 mL) using the same buffer containing 0.1 M to 1.0 M NaCl at a flow rate of 1.0 mL / min. Collect the eluted fractions in 5 mL tubes using an automated fraction collector.

[0029] b. Initial activity screening: Prepare a bacterial suspension (approximately 1×10⁻⁶) from a fresh culture of Candida albicans (ATCC 10231). 6The elution fraction (CFU / mL) was spread onto Sabouraud agar plates. Four sterile Oxford cups were evenly placed on each plate, and 100 μL of each elution fraction was added to each plate, with sterile water as a negative control. After incubation at 28°C for 48 hours, the diameter of the inhibition zone was measured. Active fractions with inhibition zones > 8 mm in diameter were combined, frozen at -80°C, and concentrated to approximately 2 mL using a vacuum freeze dryer.

[0030] c. Reversed-phase high-performance liquid chromatography (RP-HPLC) purification: The concentrated fraction with antibacterial activity was filtered through a 0.22 μm filter membrane and then purified by RP-HPLC. Chromatographic conditions: ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm); mobile phase A was 0.1% (v / v) TFA aqueous solution, and mobile phase B was 0.1% (v / v) TFA acetonitrile solution; elution gradient: 0-10 min, 5% B; 10-70 min, 5%-60% B; 70-75 min, 60%-95% B; 75-80 min, 95% B; flow rate 1.0 mL / min; detection wavelength 220 nm. Based on the chromatogram ( Figure 1 Collect the fractions corresponding to the main absorption peaks and freeze-dry them separately.

[0031] 1.4 Peptide Identification and Nomenclature: The lyophilized main peak sample was dissolved in 0.1% formic acid aqueous solution and analyzed by matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry (MALDI-TOF MS) to obtain a single peak. The sample was subjected to Edman degradation N-terminal sequencing to obtain its amino acid sequence as: VLKCKRFFKVCKLR (SEQ ID NO:1). Figure 2 The image shows the extracted ion chromatogram, primary mass spectrum, secondary mass spectrum, and structural diagram of this polypeptide. This polypeptide is named Xa-AMP1.

[0032] 1.5 Determination of antimicrobial peptide activity and verification of synergistic effect

[0033] Minimum inhibitory concentration (MIC) gradient test: Prepared at a concentration of 5.0 × 10⁻⁶ 5 CFU / mL ~ 4.5×10 6CFU / mL suspensions of Staphylococcus aureus (ATCC 6538), Escherichia coli (8099), and Candida albicans (ATCC 10231) were used. The MIC of Xa-AMP1 was determined using the microbroth dilution method. Xa-AMP1 was serially diluted 2-fold in 96-well plates with MH broth (bacteria) or RPMI-1640 medium (fungi), ranging from 128 μg / mL to 0.25 μg / mL. An equal volume of bacterial suspension was added to each well to achieve a final inoculum of approximately 5 × 10^5 CFU / mL. A negative control for the culture medium and a positive control for bacterial growth were also included. After incubating the bacterial plates at 36℃ ± 1℃ for 24 h and the fungal plates at 35℃ for 48 h, the lowest drug concentration at which no microbial growth was observed visually was defined as the MIC. The results are shown in Table 1.

[0034] Table 1: Results of MIC gradient assay for the antimicrobial peptide Xa-AMP1

[0035]

[0036] Example 2: Preparation of olive leaf extract matrix

[0037] 2.1 Preparation method

[0038] Weigh 1 kg of dried, pulverized pharmaceutical-grade olive leaves and place them in an extraction vessel. Add 10 times (10 L) of 70% ethanol solution, heat to 80°C, and reflux for extraction, 2 hours each time, for a total of two extractions. Combine the two extracts, filter through a 200-mesh filter cloth, and concentrate the filtrate under reduced pressure at 60°C to approximately 1 L (without alcohol odor). Slowly load the concentrate onto an AB-8 macroporous adsorption resin column (2 L resin volume) pre-equilibrated with deionized water. Elute sequentially with 5 column volumes of deionized water, 3 column volumes of 30% ethanol, and 5 column volumes of 50% ethanol. Collect the 50% ethanol eluent, concentrate under reduced pressure to a thick paste, transfer to a spray drying tower, inlet air temperature 180°C, outlet air temperature 85°C, and spray dry to obtain approximately 125 g of brownish-yellow powdered olive leaf extract matrix.

[0039] 2.2 Verification of synergistic effect with olive leaf substrate (checkerboard method)

[0040] Olive leaf extract matrix and Xa-AMP1 were prepared in series of dilutions. In 96-well plates, a checkerboard pattern was used: different concentrations of matrix solution (0×MIC, 0.25×MIC, 0.5×MIC, 1×MIC, 2×MIC) were added to each row, and different concentrations of Xa-AMP1 solution were added to each column. Then, an equal volume of Candida albicans suspension (final concentration 5×10⁻⁶) was added to each well. 4(CFU / mL). After incubation, the MIC of each combination was measured. The fractional inhibitory concentration index (FICI) was calculated as follows: (MIC of the substrate in the combination / MIC of the substrate alone) + (MIC of Xa-AMP1 in the combination / MIC of Xa-AMP1 alone). A synergistic effect was defined as FICI ≤ 0.5. The experimental results showed that the FICI of Xa-AMP1 combined with olive leaf substrate against Candida albicans was 0.31, confirming a strong synergistic effect between the two. Therefore, Xa-AMP1 was identified as the synergistic antimicrobial peptide of this invention.

[0041] Example 3: Preparation of an antibacterial care solution containing synergistic antibacterial peptides from olive leaf matrix

[0042] 3.1 Formulation composition and accurate weighing (unit: g / 100g finished product)

[0043] Core antibacterial synergistic component: 0.40 g of olive leaf extract matrix prepared in Example 2,

[0044] Example 1: 0.05 g of the antimicrobial peptide Xa-AMP1 screened.

[0045] pH adjuster: Approximately 0.10-0.15 g of pharmaceutical-grade lactic acid (for pH adjustment)

[0046] Solvent: Add purified water to a final volume of 100.00 g.

[0047] 3.2 Preparation process

[0048] a. Weighing: Weigh approximately 95.00 g of purified water into a beaker.

[0049] b. Dissolving the matrix: Add 0.40 g of olive leaf extract matrix to a beaker and stir at room temperature until completely dissolved to obtain a homogeneous solution.

[0050] c. Adjust pH: Use a dropper to draw 10% lactic acid solution, add it slowly while stirring continuously, and monitor it in real time with a precision pH meter to accurately adjust the pH value of the solution to 4.2 ± 0.1.

[0051] d. Add peptide: Accurately weigh 0.05 g of the synergistic antimicrobial peptide Xa-AMP1, add it to the above solution, and gently stir for 30 minutes to ensure that the peptide is completely dissolved.

[0052] e. Volume adjustment: Transfer the entire solution from the beaker to a 100 mL volumetric flask. Wash the beaker several times with a small amount of purified water, adding the washings to the volumetric flask. Finally, bring the volume to the 100.00 g mark with purified water, stopper the flask, and invert it repeatedly to mix thoroughly.

[0053] f. Filtration and Filling: Filter the diluted solution through a 0.45 μm microporous membrane, and collect the filtrate in a clean reagent bottle. In a sterile operating table or clean work area, dispense the filtrate into sterilized 10 mL or 20 mL HDPE plastic bottles or brown glass bottles, seal the bottles, and label them to obtain 100 grams of the finished product, the olive leaf matrix antibacterial care solution containing synergistic antimicrobial peptides.

[0054] Example 4: Evaluation of the efficacy of antibacterial care solution

[0055] To comprehensively, scientifically, and rigorously verify the inhibitory effect of the product of this invention on microorganisms, this invention strictly follows the multiple standard tests specified in "WS / T 650—2019 Evaluation Method for Antibacterial and Bacteriostatic Effects" to systematically evaluate the antibacterial care solution prepared in Example 2.

[0056] 4.1 Test Samples and Test Strains

[0057] The test samples were divided into an experimental group and a control group. The experimental group consisted of the olive leaf matrix antibacterial care solution containing the synergistic antimicrobial peptide Xa-AMP1 prepared in Example 3, which was tested using the undiluted solution. The control group consisted of the same care solution undiluted solution as the experimental group, except that 0.05 g (0.05%) of the synergistic antimicrobial peptide Xa-AMP1 was not added.

[0058] Test strains: Staphylococcus aureus ATCC 6538, Escherichia coli 8099, Candida albicans ATCC 10231.

[0059] General reagents and equipment: Diluent: 0.03 mol / L phosphate buffer (PBS, pH 7.2-7.4), prepared strictly according to standard.

[0060] Culture media: Nutrient agar medium, nutrient broth (for bacteria); Sabouraud agar medium, Sabouraud liquid medium (for Candida albicans).

[0061] Neutralizing agent: Based on preliminary testing and identification of the olive leaf extract, traditional Chinese medicine ingredients and antimicrobial peptides contained in this product, a PBS solution containing 3% Tween 80 and 0.3% lecithin was determined to be the neutralizing agent, and its effectiveness and non-toxicity to microorganisms were confirmed.

[0062] Main equipment: constant temperature water bath (temperature control 20℃±1℃ and 36℃±1℃), timer, Class II biosafety cabinet, vernier calipers (accuracy 0.02mm), vortex shaker, electronic balance (d=0.01g), sterile test tubes, petri dishes, micropipette, 10mm×10mm sterile degreased white plain cloth, etc.

[0063] 2. Experimental Methods and Results

[0064] 2.1 Quantitative Antibacterial Test of Suspension

[0065] Preparation of bacterial suspension: Take fresh 24-hour slant culture of each test bacterium, wash with PBS, and dilute to a concentration of 5.0 × 10⁻⁶. 5 CFU / mL ~ 4.5 × 10 6 Prepare a bacterial suspension of CFU / mL for later use.

[0066] Sample addition and procedure: Take a sterile test tube, add 5.0 mL of the test sample, and incubate in a water bath at 20℃±1℃ for 5 min to equilibrate. Then add 0.1 mL of the above bacterial suspension, mix quickly, and start timing immediately.

[0067] Neutralization and Incubation: After the test bacteria have interacted with the sample for the set 2 and 5 minutes, take 1.0 mL of the mixture and add it to 9.0 mL of PBS (to terminate the reaction by a 10-fold dilution), and mix thoroughly. Perform serial 10-fold dilutions with PBS as needed. Select an appropriate dilution, take 1.0 mL of the sample solution, inoculate it into two sterile Petri dishes, and immediately pour in approximately 15 mL of the corresponding agar medium (nutrient agar for bacteria, Sabouraud agar for Candida albicans) cooled to 45-50°C, and shake well.

[0068] Simultaneously, PBS was used to replace the samples in completely parallel experiments as a positive control. A separate batch of PBS and culture medium was mixed and cultured as a negative control. All petri dishes were incubated at 36℃±1℃. After culturing bacterial vegetative cells for 48 hours and Candida albicans for 72 hours, the number of colonies on each petri dish was counted. The above experiments were independently repeated three times.

[0069] Antibacterial rate calculation and judgment:

[0070] The inhibition rate (X) was calculated strictly according to the standard formula (1): X (%) = [(A0 - A1) / A0] × 100. Where A0 is the average number of recovered colonies (CFU / mL) in the positive control group, and A1 is the average number of recovered colonies (CFU / mL) in the experimental or control group. An inhibition rate ≥50%~90% is considered to have an inhibitory effect; an inhibition rate ≥90% is considered to have a strong inhibitory effect.

[0071] The results are shown in Table 2. At 2 and 5 minutes, the experimental group showed an inhibition rate of ≥90% against the three test bacteria, meeting the standard of "strong antibacterial effect", and the effect was significantly better than that of the control group.

[0072] Table 2: Results of Quantitative Antibacterial Test of Suspension (%)

[0073]

[0074] 4.2 Quantitative Antibacterial Test by Carrier Immersion

[0075] Take a fresh 24-hour slant culture of the test bacteria, wash it with PBS and dilute it to a bacterial suspension of approximately 5.0 × 10^6 CFU / mL to 5.0 × 10^7 CFU / mL. Use a micropipette to drop 10 μL of the bacterial suspension into the center of a sterile carrier (10 mm × 10 mm defatted white cloth), and dry it in an oven at 36℃ ± 1℃ for 30 minutes before use.

[0076] Weigh 5.0 g of sample into a sterile Petri dish using an electronic balance and equilibrate in a water bath at 20℃±1℃ for 5 min. Use sterile forceps to pick up a piece of contaminated carrier and completely immerse it in the sample, then start timing immediately. Incubate for 10 minutes and 20 minutes respectively.

[0077] After the specified time, remove the carrier with tweezers and place it in a test tube containing 5.0 mL of PBS. Shake vigorously for 1 minute to wash off the bacteria. Then, repeat the dilution, pour culture, and counting steps as in 2.1. Use a matrix without antibacterial components instead of the sample, performing the same procedure in parallel as a positive control.

[0078] Antibacterial rate calculation and judgment: Calculated according to standard formula (2): X (%) = [(A0 - A1) / A0] × 100. A0 is the average number of recovered colonies in the control sample (CFU / tablet), and A1 is the average number of recovered colonies in the test sample (CFU / tablet). The judgment criteria are the same as 2.1.

[0079] The results are shown in Table 3. Under the carrier immersion conditions, the experimental group still showed a rapid and strong antibacterial effect.

[0080] Table 3: Results of Quantitative Antibacterial Test of Carrier Immersion (Average Antibacterial Rate, %)

[0081]

[0082] 4.3 Inhibition ring test

[0083] The 24-hour culture of Staphylococcus aureus (ATCC 6538) was washed down with PBS and diluted to 5.0 × 10^5 CFU / mL to 5.0 × 10^6 CFU / mL. A sterile cotton swab was used to collect the bacterial suspension, which was then spread evenly three times on a nutrient agar plate. The plate was then covered and allowed to dry at room temperature for 5 minutes.

[0084] Prepare 5 mm diameter discs from double-layered qualitative filter paper using a sterile punch, then sterilize and dry. Add 20 μL of the test or control group stock solution to each disc, wetting it before attaching it to the surface of the aforementioned bacterial agar plate. Gently press with sterile forceps to ensure firm adhesion. Place two test discs and one negative control disc (with PBS added) on each plate, ensuring a center-to-center distance of more than 25 mm between the discs. Incubate the plates at 36℃±1℃ for 16–18 h. Measure the diameter of the inhibition zone with calipers, accurate to 0.1 mm. Repeat the experiment three times.

[0085] Result determination: The negative control sample should have no inhibition zone, and the test sample with an inhibition zone diameter > 7 mm is judged to have antibacterial effect.

[0086] Figure 3 The results showed that the diameter of the inhibition zone against Staphylococcus aureus in the experimental group was 12.5 ± 0.8 mm (mean ± standard deviation of three replicates), which was much larger than 7 mm, indicating an inhibitory effect. The diameter of the inhibition zone in the control group was 8.2 ± 0.5 mm. This indicates that the addition of the synergistic antimicrobial peptide Xa-AMP1 significantly enhanced the diffusion ability and / or intensity of the lysing antimicrobial substance in the experimental group.

[0087] 4.4 Simulated Retention Antibacterial Effect Test

[0088] Soak sterilized cotton cloths in the original solution of the experimental or control group for 5 minutes, remove them, squeeze out excess liquid, and air dry at room temperature.

[0089] Add 0.1 mL of Candida albicans suspension (approximately 1 × 10^5 CFU) to the center of each dried sample and spread evenly. Place the sample in a sterile Petri dish and incubate at 36℃±1℃ and relative humidity>90%.

[0090] Immediately after inoculation (0 h) and after 6 hours of incubation, samples were transferred to glass Erlenmeyer flasks containing 10 mL of pre-identified neutralizing agent and vigorously shaken for 5 minutes to elute bacteria. The eluent was appropriately diluted and poured onto Sabouraud dextrose agar plates, incubated for 72 hours, and then counted. Sterile cotton cloths were used as positive controls (0 h and 6 h) for direct inoculation of bacterial suspension without sample treatment.

[0091] Antibacterial rate calculation and judgment: The antibacterial rate after 6 hours of culture is calculated according to the standard formula (3). If the antibacterial rate is ≥50%, the sample can be judged to have antibacterial effect.

[0092] After 6 hours of incubation, the sample treated with this product showed an inhibition rate of 87.3% against Candida albicans, indicating that the surface treated with this product could still effectively inhibit the growth of Candida albicans within 6 hours, demonstrating good sustained antibacterial potential. The inhibition rate of the control group sample was 62.1%.

[0093] 4.5 Quantitative sterilization test of suspension

[0094] Take a sterile test tube, add 5.0 mL of sample (test group stock solution), and incubate in a water bath at 20℃±1℃ for 5 min. Add 0.1 mL of bacterial suspension (concentration as in 2.1), mix well, and start timing. At 5 minutes and 10 minutes, respectively, take 0.5 mL of the sample-bacterial suspension mixture and immediately add it to a 4.5 mL neutralizing agent test tube, mix well, and incubate for 10 minutes to ensure complete neutralization. Subsequent dilution, pouring, incubation, and counting procedures are the same as in 2.1.

[0095] Control setup: Use PBS instead of the sample, and perform the operation in parallel as a positive control.

[0096] Sterilization rate calculation and judgment: Sterilization rate (X) is calculated according to standard formula (5): X (%) = [(A - B) / A] ×100. A is the amount of bacteria recovered in the positive control group (CFU / mL), and B is the amount of bacteria recovered in the experimental group (CFU / mL).

[0097] Judgment: If the sterilization rate is ≥90%, it is judged to have antibacterial effect; if the sterilization rate is ≥99%, it is judged to have strong antibacterial effect.

[0098] The results are shown in Table 4. Within a 10-minute contact time, the experimental group achieved a bactericidal rate of over 99.9% against all three test bacteria, demonstrating strong antibacterial activity.

[0099] Table 4: Results of Quantitative Sterilization Test of Suspension (%)

[0100]

[0101] Quantitative suspension antibacterial tests showed that the product of this invention (containing Xa-AMP1) achieved a "strong antibacterial effect" against Candida albicans, Staphylococcus aureus, and Escherichia coli within 2 minutes, demonstrating extremely rapid onset of action. The product completely killed these three standard pathogenic microorganisms within 10 minutes, exhibiting a "strong antibacterial effect," indicating its potential to thoroughly eliminate pathogens. Carrier immersion tests proved that even when the product adhered to a carrier surface, its active ingredients could still effectively dissolve and exert their effects, achieving a strong antibacterial effect within 10-20 minutes, simulating actual usage conditions. The inhibition zone test visually confirmed that the product contains soluble and effectively diffused antibacterial substances, and the inhibition zone of the experimental group was significantly larger than that of the control group, indicating that the synergistic peptides enhanced the overall activity. Simulated retention tests showed that the surface treated with the product maintained a significant antibacterial effect for 6 hours, suggesting that it may provide longer-lasting protection and help reduce the risk of recurrence. In all parallel controlled trials, the experimental group showed comprehensive and significant superiority over the control group in all antibacterial and bactericidal efficacy indicators. Most importantly, in the quantitative antibacterial test of Candida albicans suspension, the inhibition rate at 2 minutes increased from 68.5% in the control group to 96.1% in the experimental group. This improvement proves that the synergistic antibacterial peptide Xa-AMP1 obtained in Example 1, when combined with olive leaf extract matrix and classic Chinese herbal medicines such as Sophora flavescens and Phellodendron chinense, produced a significant synergistic antibacterial effect of "1 + 1 > 2" in the final product.

[0102] This invention not only fully meets the clinical needs of "rapid effectiveness and low recurrence rate" mentioned in the background art, but its comprehensive antibacterial efficacy also surpasses that of traditional Chinese medicine compound formulas. The product's pH value is adjusted to 4.0-4.5, consistent with the acidic environment of a healthy vagina, aiming to "regulate and restore the vagina's own internal environment and pH," thereby achieving the invention's objective of treating both the symptoms and the root cause.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is SEQ ID NO:

1.

2. The polypeptide according to claim 1, characterized in that, The polypeptide was prepared from the African clawed frog.

3. The use of the polypeptide of claim 1 in the preparation of products that inhibit pathogenic microorganisms.

4. The application as described in claim 3, characterized in that, The pathogenic microorganisms mentioned are pathogenic microorganisms in the female reproductive system.

5. The application as described in claim 4, characterized in that, The pathogenic microorganisms mentioned are Staphylococcus aureus and Candida albicans.

6. An antibacterial product, characterized in that, The active component of the antibacterial product contains the polypeptide described in claim 1.

7. The antibacterial product as described in claim 6, characterized in that, The antibacterial product also contains olive leaf matrix extract.

8. The antibacterial product according to claim 7, characterized in that, The olive leaf matrix extract is prepared by adding olive leaves to an ethanol solution for reflux extraction, filtering the extract, concentrating the filtrate, adsorbing the concentrate onto a macroporous adsorption resin column, and eluting sequentially with deionized water, 30% ethanol, and 50% ethanol; collecting the 50% ethanol eluent, concentrating under reduced pressure, and spray drying.

9. The antibacterial product as described in claim 6, characterized in that, The antibacterial product also contains other antibacterial components.

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