A traditional Chinese medicine extraction polypeptide for inhibiting staphylococcus aureus and application thereof in treating eczema in medicated bath

Abstract

This invention relates to the field of pharmaceutical technology, specifically disclosing a polypeptide extracted from the traditional Chinese medicine Artemisia argyi, the amino acid sequence of which is shown in SEQ ID No. 1. This polypeptide can be prepared from Artemisia argyi through steps such as ultrasonic disruption, enzymatic hydrolysis, and purification. Experiments show that the Artemisia argyi polypeptide at a concentration of 2 μg / mL inhibits Staphylococcus aureus by 92±4.72%. This invention also provides a medicated bath component containing the Artemisia argyi polypeptide, which can significantly alleviate itching, lesion area, and morphology in patients with chronic eczema. After 30 days of treatment, the experimental group's score decreased from 7.8 to 3.1 (the control group showed no significant change). The polypeptide of this invention is naturally derived and highly safe, providing a new strategy for the treatment of eczema.

Description

Technical Field

[0001] This invention relates to the field of biopeptide technology, and more specifically, to a traditional Chinese medicine extract polypeptide that inhibits Staphylococcus aureus and its application in the treatment of eczema in medicated baths. Background Technology

[0002] Eczema is a common inflammatory skin disease characterized by itching, erythema, papules, and infiltrative lesions, severely impacting patients' quality of life. Chronic eczema has a long course and is prone to recurrence. Current treatments mostly rely on corticosteroids, immunosuppressants, or antibiotics, but long-term use may lead to skin atrophy, drug resistance, or systemic side effects. Recent studies have indicated that Staphylococcus aureus plays a crucial role in the pathogenesis of eczema. This bacterium can stimulate immune responses and exacerbate skin inflammation by secreting toxins. Therefore, inhibiting Staphylococcus aureus has become a new target for eczema treatment. Existing eczema medicated bath products, such as the Sophora flavescens-Phellodendron amurense compound bath, mainly relieve symptoms through anti-inflammatory and moisturizing effects, but their inhibition rate against Staphylococcus aureus is only 40-60%, and they lack specific targeting components, resulting in limited efficacy. This highlights the targeted advantages of the present invention.

[0003] Traditional Chinese medicine extracts have garnered significant attention due to their natural origin and multiple therapeutic effects. Artemisia argyi is traditionally used for its effects in dispelling dampness and relieving itching, warming the meridians and promoting blood circulation, antibacterial and anti-inflammatory properties, regulating immunity, calming the fetus and stopping bleeding, and repelling insects and foul odors. However, the antifungal activity of its polypeptide components has not been fully explored. Current technologies for polypeptide extraction mostly employ chemical synthesis or microbial fermentation, which are costly and may introduce impurities. Furthermore, the process for extracting polypeptides from traditional Chinese medicine lacks standardization, resulting in insufficient activity evaluation. In addition, medicated baths, as an adjunct therapy for eczema, have the advantages of gentle systemic effects and high patient compliance; however, existing medicated bath products mostly focus on moisturizing or anti-inflammatory effects, lacking specific components targeting Staphylococcus aureus.

[0004] Therefore, there is an urgent need in this field for a polypeptide component derived from traditional Chinese medicine that is highly effective in inhibiting Staphylococcus aureus and suitable for medicated bath applications, in order to solve the problems of significant side effects and insufficient targeting in existing treatments. This invention, based on the development of a novel polypeptide from Artemisia argyi, fills a gap in this field. Summary of the Invention

[0005] This invention first provides a traditional Chinese medicine extract polypeptide that inhibits Staphylococcus aureus, the amino acid sequence of which is shown in SEQ ID No.1.

[0006] In some embodiments, the polypeptide is extracted from the traditional Chinese medicine Artemisia argyi.

[0007] The present invention also provides a method for preparing the above-mentioned polypeptide, comprising the following steps: crushing Artemisia argyi powder by ultrasonication, centrifuging to obtain the supernatant, adding trypsin, alkaline protease and papain in sequence for enzymatic hydrolysis, precipitating with trichloroacetic acid, concentrating by ultrafiltration and purifying by gel chromatography to obtain the polypeptide.

[0008] In some embodiments, the gel chromatography uses a Sephadex G-15 column, and the absorbance is detected by UV after elution to collect the main peak component.

[0009] The present invention also provides a pharmaceutical composition comprising the above-described polypeptide and pharmaceutically acceptable excipients.

[0010] In some embodiments, the excipients include at least one selected from β-cyclodextrin, chitosan, fumaric acid, sodium hydroxide, and microcrystalline cellulose.

[0011] In some embodiments, the composition is in the form of a medicated bath, with an appropriate water volume of 200L, wherein the polypeptide content is 100-300mg.

[0012] The present invention also provides the use of the above-mentioned polypeptide in the preparation of a drug for inhibiting Staphylococcus aureus.

[0013] The present invention also provides the use of the above-described pharmaceutical composition in the preparation of medicated bath products for treating eczema.

[0014] The present invention also provides a medicated bath method for treating eczema, comprising using the above-mentioned medicated bath composition, soaking in water at a temperature of 38-42°C for 15-20 minutes, once a day.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects:

[0016] (1) Highly effective antibacterial: The inhibition rate of mupirocin at a concentration of 0.1 μg / mL was 90±8.35%, and the inhibition rate of Artemisia argyi polypeptide at a concentration of 2 μg / mL was 92±4.72%. Although the concentration of Artemisia argyi polypeptide is slightly higher, as a natural extract, it does not have the risk of skin irritation or drug resistance associated with mupirocin. Moreover, it can be administered to the whole body through a medicated bath, making it safer and more suitable overall. In addition, it is a natural extract, which reduces the side effects of chemical drugs.

[0017] (2) Significant therapeutic effect: Clinical trials have shown that medicated baths containing Artemisia argyi polypeptide can reduce eczema scores (from 7.8 to 3.1 after 30 days) and improve itching and skin lesions.

[0018] (3) Stable process: The extraction method is highly reproducible, and gel chromatography ensures the purity of peptides.

[0019] (4) Good compatibility with medicated baths: The combination of excipients enhances the stability of polypeptides and makes it convenient for patients to use at home. Attached Figure Description

[0020] Figure 1 This is a gel chromatography result of Artemisia argyi polypeptide.

[0021] Figure 2 This is an LC-MS / MS image of Artemisia argyi polypeptide.

[0022] Figure 3 The scores of eczema in the subjects after the medicated bath, where * indicates P<0.05 and ** indicates P<0.01. Detailed Implementation

[0023] The present invention will be further described in detail and completely below with reference to the embodiments. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0024] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are all commercially available.

[0025] Example 1: Extraction and Identification of Artemisia argyi Polypeptides

[0026] Take 500g of commercially available dried mugwort leaves and perform preliminary pulverization using a pulverizer. The pulverizer parameters were set to 10000rpm, operating temperature 20℃, single cycle time 30s, and total operating time 15min. Pass the pre-pulverized mugwort powder through a 40-mesh sieve and collect the qualified powder as raw material for peptide extraction. Place the mugwort powder in 5L of ultrapure water for ultrasonic disruption. The ultrasonic disruptor parameters were set as follows: power 150W, single cycle time 30s, single cycle time 2min, total operating time 1h, and maximum temperature 20℃. Centrifuge the ultrasonically treated suspension at 4℃ and 8000rpm for 30min, and collect the supernatant. This supernatant contains soluble proteins. Extract the precipitate once more with a small amount of ultrapure water, and combine the two supernatants for later use. Add 0.2% (w / v) trypsin and 0.1% (w / v) alkaline protease to the supernatant, mix thoroughly, adjust the pH to 8.0-8.2, and incubate at 55℃ with stirring for 4 hours. Add 0.1% (w / v) papain to the hydrolysate, adjust the pH to 7.0, and incubate at 55℃ with stirring for 3 hours. After hydrolysis, boil the solution for 10 minutes or adjust the pH to 7.0 to inactivate the enzyme and terminate the hydrolysis. Add TCA (trichloroacetic acid) to the hydrolysate to a final concentration of 4%, and incubate at 4℃ for 30 minutes. Centrifuge at 10,000 rpm for 20 minutes at 4℃, collect the supernatant (i.e., the preliminarily purified peptide solution), and adjust the pH to 7.5. Concentrate the preliminarily purified peptide solution to 1 / 100 of its original volume using a 3.5 kDa ultrafiltration tube, and replace the solution with ultrapure water. The preliminarily purified polypeptide solution was freeze-dried using a vacuum freeze dryer to obtain Artemisia argyi pre-purified polypeptide powder.

[0027] The initial purified Artemisia argyi polypeptide powder was prepared into a 5 mg / mL solution using ultrapure water and filtered through a 0.45 μm aqueous filter membrane. The polypeptides were separated using a Sephadex G-15 (1.6 cm × 60 cm) gel column. The sample loading volume was 5 mL, the flow rate was 0.5 mL / min, and one tube was collected every 2 min. Elution was performed with ultrapure water, and the absorbance was measured using a UV spectrophotometer. Eluent fractions with similar absorbance values ​​were combined. Four fractions with similar peaks were obtained and named F1, F2, F3, and F4. The gel chromatography purification results of the Artemisia argyi polypeptides are shown below. Figure 1 As shown.

[0028] The eluent from the main peak F3 was concentrated and desalted using a 5 kDa ultrafiltration tube. The specific steps were as follows: The eluent was added to the ultrafiltration tube and centrifuged at 12000g for 15 min at 4°C. The filtrate was discarded, and the protein precipitate was resuspended in 50 mM NH4HCO3 solution. This process was repeated three times. The protein concentration was adjusted to 0.5 mg / mL using 50 mM NH4HCO3 solution. DTT was added to a final concentration of 10 mM, and the mixture was incubated at 37°C for 60 min. After the sample cooled to room temperature, IAA was added to a final concentration of 20 mM, and the mixture was incubated at room temperature in the dark for 30 min to block the reduced thiol groups and prevent disulfide bond renaturation. Trypsin working solution was added at a protein:enzyme ratio of 100:1. After vortexing, the mixture was incubated at 4°C for 30 min to allow for complete enzyme-protein binding. After incubating overnight at 37°C, 10% TFA was added to a final concentration of 0.1%, vortexed, and incubated at room temperature for 10 min to terminate the enzymatic digestion reaction. The C18 solid-phase extraction column was activated by washing it sequentially with 3 mL of ACN and 3 mL of 0.1% TFA. The enzymatically digested peptide solution was then slowly passed through the column at a flow rate of 1 drop / 2 s. The C18 column was washed with 3 mL of 0.1% TFA to remove salt ions and water-soluble impurities. Elution was performed with 1 mL of 60% ACN (containing 0.1% TFA), and the eluent was collected, freeze-dried under vacuum to a dry powder, and stored at -20℃ for later use. The amino acid sequence of the collected main peak F3 fraction was determined by LC-MS / MS, and the amino acid sequence of the Artemisia argyi peptide was identified as NARGIITAG (SEQ ID No: 1).

[0029] Example 2: Evaluation of the inhibitory activity of Artemisia argyi polypeptide against Staphylococcus aureus

[0030] Activate frozen Staphylococcus aureus (ATCC 6538) by adding 200 μL of the thawed bacterial suspension to a TSA agar plate and spreading it evenly with a sterile spreader. Invert the plate and incubate at 37°C for 24 hours until cream-colored or pale yellow colonies with a raised center and neat edges appear (1-2 mm). Use a sterile inoculation loop to pick a single colony from the solid medium and inoculate it into an Erlenmeyer flask containing TSB liquid medium. Place the flask in a shaker at 37°C and incubate with shaking at 180 rpm. After the bacterial suspension becomes noticeably turbid, subculture three times to enhance bacterial viability. Dilute the bacterial suspension to 1×10⁻⁶ with PBS. 8 CFU / mL, then serially diluted 10-fold to obtain 1×10⁻⁶ CFU / mL. 4 CFU / mL bacterial suspension. Add 1 mL of each concentration of bacterial suspension to a sterile 1.5 mL EP tube. Add the test drug diluted with PBS to the tube according to the groupings in Table 1. Mix thoroughly, then spread 100 μL onto a Petri dish containing solid culture medium. Perform three replicates for each group. Incubate the Petri dishes at 37°C for 48 hours, then remove and count the colonies.

[0031] Table 1. Staphylococcus aureus inhibition experiment groupings

[0032]

[0033] The inhibition rate of Staphylococcus aureus was calculated as follows: T = (BA) / B * 100%, where T is the inhibition rate, B is the number of colonies in the negative control group, and A is the number of colonies in the positive control group or experimental group. The results are shown in Table 2. The Artemisia argyi polypeptide in the experimental group showed a slightly better inhibitory effect on Staphylococcus aureus than mupirocin, demonstrating good potential and value for pharmaceutical development.

[0034] Table 2. Results of Staphylococcus aureus inhibition rate

[0035]

[0036] Example 3: Medicated Bath Components and Their Preparation Method

[0037] In this embodiment, the experimental group bath components, by weight, consist of the following raw materials: 300mg Artemisia argyi polypeptide (prepared in Example 1), 6g β-cyclodextrin, 5g chitosan (8kDa), 14g fumaric acid, 27g sodium hydroxide, and 8g microcrystalline cellulose.

[0038] This formula is suitable for 200L of standard adult medicated bath water. Use 20L of warm water at approximately 40℃ as the stock solution preparation water, and the remaining 180L of warm water for subsequent dilution. Accurately weigh each ingredient according to the formula. Slowly add 14g of fumaric acid to 5L of 40℃ warm water, stirring at 200 rpm until completely dissolved, forming an acidic stock solution. Then, add 27g of sodium hydroxide in 6 small portions to the above acidic stock solution, stirring after each addition until no obvious particles are visible. Monitor the pH during the process; the final system pH ≈ 7.0. Add 300mg of Artemisia argyi polypeptide and mix well. Take 5g of chitosan, add it to 2L of 40℃ warm water, and stir at 300 rpm for 30 minutes until the chitosan is completely dissolved, forming a transparent viscous liquid. Set aside. Add 6g of β-cyclodextrin to 3L of 40℃ warm water and stir for 15 minutes until uniformly suspended. Pour the entire prepared solution into the remaining 9L of warm water and stir at 200 rpm for 30 minutes to ensure uniform mixing of all components and the formation of a stable composite excipient mother liquor. Slowly pour the prepared 20L mother liquor into 180L of bath water at 38-42℃ and stir for 15 minutes before use for medicated baths.

[0039] 100mg is suitable for mild eczema (score 6-8), 200mg is suitable for moderate eczema (score 9-12), and 300mg is suitable for severe eczema (score ≥13).

[0040] The control group bath composition, by weight, consisted of the following ingredients: 6g β-cyclodextrin, 5g chitosan (8kDa), 14g fumaric acid, 27g sodium hydroxide, and 8g microcrystalline cellulose. The preparation method was the same as the experimental group.

[0041] Example 4: Evaluation of the therapeutic effect of Artemisia argyi polypeptide on eczema

[0042] Sixty patients with chronic eczema, all over 18 years of age, were randomly divided into two groups of 15 males and 15 females in each group. All patients met the diagnostic criteria for chronic eczema in the "Chinese Guidelines for the Diagnosis and Treatment of Eczema (2024 Edition)". Patients received a medicated bath once daily using the bath components described in Example 3, with a water temperature of 38-42℃ and a duration of 15 minutes, for 30 days. The severity of itching, area of ​​skin lesions, and morphology were recorded before treatment and after 7, 15, and 30 days of treatment. Scores were assigned and recorded; the specific scoring criteria are shown in Table 3.

[0043] Table 3 Eczema Symptom Scoring Table

[0044]

[0045] The statistical results of patient scores are shown in Table 4. The sum of all symptom scores was the patient's total score for that day, and the t-test was used for statistical analysis. There was no significant difference between the experimental group and the control group before the medicated bath. After 7, 15, and 30 days of medicated bath treatment, the scores in the experimental group were significantly lower than those in the control group (P < 0.01 or P < 0.05), indicating that the medicated bath components containing Artemisia argyi polypeptide can effectively relieve the symptoms of patients with chronic eczema and reduce patient scores.

[0046] Table 4 Patient Score Statistics

[0047]

[0048] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polypeptide extracted from a traditional Chinese medicine that inhibits Staphylococcus aureus, characterized in that, Its amino acid sequence is shown in SEQ ID No.

1.

2. The polypeptide according to claim 1, characterized in that, The polypeptide was extracted from the traditional Chinese medicine Artemisia argyi.

3. A method for preparing the polypeptide according to claim 1, characterized in that, Includes the following steps: Artemisia argyi powder was ultrasonically crushed, and the supernatant was collected by centrifugation. Trypsin, alkaline protease and papain were added sequentially for enzymatic hydrolysis. After precipitation with trichloroacetic acid, ultrafiltration concentration and gel chromatography purification, Artemisia argyi polypeptide was obtained.

4. The method as described in claim 3, characterized in that, The gel chromatography was performed using a Sephadex G-15 column. After elution, the absorbance was detected by UV light, and the main peak component was collected.

5. A pharmaceutical composition, characterized in that, It comprises the Artemisia argyi polypeptide of claim 1 and pharmaceutically acceptable excipients.

6. The pharmaceutical composition according to claim 5, characterized in that, The excipients include at least one of β-cyclodextrin, chitosan, fumaric acid, sodium hydroxide, and microcrystalline cellulose.

7. The pharmaceutical composition according to claim 5 or 6, characterized in that, The composition is in the form of a medicinal bath, with a suitable water volume of 200L, and contains 300mg of Artemisia argyi polypeptide.

8. The use of the polypeptide of claim 1 in the preparation of a drug for inhibiting Staphylococcus aureus.

9. The use of the pharmaceutical composition of claim 5 in the preparation of a medicated bath product for treating eczema.

10. A medicated bath method for treating eczema, characterized in that, This includes using the medicated bath composition according to any one of claims 5-7, with a water temperature of 38-42°C, soaking for 15 minutes, once daily.

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