Bacteriostatic and anti-inflammatory polypeptide extract and application thereof in Yao bath

Abstract

The invention discloses a bacteriostatic and anti-inflammatory polypeptide extract and application thereof in a Yao bath. The polypeptide extract is extracted from rhizomes of Coptis chinensis (Coptis chinensis Franch.), and is obtained through extraction with a phosphate buffer solution, ultrafiltration and gel chromatography purification, and the polypeptide is as shown in SEQ ID No: 1. Experiments show that the polypeptide extract has significant bacteriostatic activity on pathogenic bacteria such as escherichia coli, shows a dose-dependent anti-inflammatory effect in an RAW264.7 cell model, can effectively inhibit NO release, and has a cell survival rate of 92% or more and excellent biological safety. The polypeptide extract is applied to a Yao bath preparation, O / W type emulsion is prepared, 10 volunteers try on the Yao bath preparation, the improvement rate on mild skin inflammation symptoms (such as red and swollen and pruritus) reaches 87%, and anaphylactic reaction is avoided. The polypeptide extract is simple in preparation process and high in activity, and has a wide application prospect in the fields of cosmetics and medicines.

Description

Technical Field

[0001] This invention relates to the field of biopeptide technology, and more specifically, to an antibacterial and anti-inflammatory polypeptide extract and its application in Yao bath. Background Technology

[0002] Due to their wide range of biological activities, such as antibacterial, anti-inflammatory, and immunomodulatory effects, peptides have become a research hotspot in the fields of biomedicine and cosmetics. Peptide extracts from natural plants have the advantages of low toxicity and high compatibility, making them particularly suitable for topical skin preparations. Coptis chinensis, as a traditional Chinese medicine, is known to contain active ingredients such as alkaloids, but the antibacterial and anti-inflammatory potential of its peptide components has not been fully explored. In existing technologies, peptide extraction often employs complex chromatographic techniques, which suffer from low purity and high cost (Piovesana, S., et al., Recent trends and analytical challenges in plant bioactive peptide separation, identification and validation. AnalBioanal Chem, 2018. 410(15): p. 3425–3444.); and most extracts lack systematic activity verification and safety assessment when applied to traditional therapies such as Yao bath (Acquah, C., et al., Structure-informed separation of bioactive peptides. J Food Biochem, 2019. 43(1): p. e12765.).

[0003] Yao bath, a traditional ethnic therapy, relieves skin inflammation and promotes health through medicinal bathing. However, traditional Yao bath preparations often rely on crude extracts, with unclear active ingredients and unstable effects. Modern research attempts to incorporate active peptides into Yao bath products, but faces challenges such as poor extract stability and low skin permeability. For example, some existing technologies disclose a plant peptide extract, but its antibacterial activity is only against specific strains, lacking verification of anti-inflammatory function; some existing technologies disclose the application of peptides in bath liquids, but the diffusion characteristics of the extract in agar medium and cell safety issues remain unresolved.

[0004] Therefore, there is an urgent need in the field for a polypeptide extract that is easy to prepare, has clearly defined activity, and is highly safe, capable of achieving both antibacterial and anti-inflammatory effects, and optimizing its compatibility and efficacy in Yao bath preparations. This invention develops a novel polypeptide extract based on the rhizome of Coptis chinensis, filling a gap in existing technologies through standardized extraction and purification processes, combined with in vitro antibacterial and anti-inflammatory models and human trials. Summary of the Invention

[0005] In the traditional Chinese medicine theory system, Coptis chinensis has long been used to treat a variety of inflammatory diseases due to its significant effects of clearing heat and drying dampness, purging fire and detoxifying (Rui, Z., et al., [Research progress on chemical compositions of Coptidis Rhizoma and pharmacological effects of berberine]. Zhongguo Zhong Yao Za Zhi, 2020. 45(19): p. 4561–4573.). Modern pharmacological studies have confirmed that the main active components of Coptis chinensis possess clear anti-inflammatory, antibacterial, and immunomodulatory effects. Their mechanisms of action involve inhibiting key inflammatory signaling pathways such as NF-κB and MAPK, and reducing the production of inflammatory mediators such as prostaglandins and nitric oxide (Xie, Q., et al., Effect of Coptis chinensis franch and Magnolia officinalis on intestinalflora and intestinal barrier in a TNBS-induced ulcerative colitis rats model. Phytomedicine, 2022. 97: p. 153927.). However, existing research has largely focused on small-molecule alkaloids, with insufficient attention paid to the bioactive polypeptide components in Coptis chinensis. Their potential anti-inflammatory properties and molecular mechanisms still require systematic elucidation.

[0006] From a clinical perspective, inflammation is the common pathological basis of many skin diseases. While widely used synthetic anti-inflammatory drugs are highly effective, long-term use may be accompanied by side effects such as gastrointestinal reactions and immunosuppression. Therefore, discovering novel anti-inflammatory components with high efficacy and low toxicity from natural medicinal materials has become an important direction in drug development. Especially for external therapies in traditional ethnic medicine such as Yao baths, developing natural active ingredients with both antibacterial and anti-inflammatory effects not only aligns with their holistic treatment philosophy but also enhances the scientific validity and reproducibility of traditional therapies.

[0007] Against this backdrop, this invention focuses on the underutilized polypeptide resources in Coptis chinensis. Through modern separation and identification techniques, it aims to obtain a class of polypeptide extracts with novel structures and clearly defined activities, and to innovatively apply them to Yao bath formulas. This not only deeply explores and scientifically verifies the traditional medicinal value of Coptis chinensis, but also provides a new material basis and research approach for developing modern skin health products with ethnic medicinal characteristics.

[0008] This invention first provides an antibacterial and anti-inflammatory polypeptide extract, said polypeptide extract being derived from Coptis chinensis (… Coptis chinensis Franch. Extracted from the rhizome, with a molecular weight range of 1000-2500 Da.

[0009] In some embodiments, the polypeptide sequence is as shown in SEQ ID No: 1.

[0010] The present invention also provides a method for preparing the above-mentioned polypeptide extract, comprising the following steps: mixing Coptis chinensis powder with phosphate buffer for extraction, centrifuging and ultrafiltration followed by gel chromatography purification, and freeze-drying to obtain polypeptide powder.

[0011] In some embodiments, the ultrafiltration uses an ultrafiltration tube with a molecular weight cutoff of 10 kDa.

[0012] In some embodiments, the gel chromatography uses Sephadex G-25 packing material.

[0013] The present invention also provides a Yao bath preparation comprising the above-mentioned polypeptide extract and pharmaceutically acceptable excipients.

[0014] In some embodiments, the excipients include Tween-80, glycerin, stearic acid, and cetyl alcohol.

[0015] In some embodiments, the polypeptide extract has a mass fraction of 0.1%-1% in the formulation.

[0016] The present invention also provides the application of the above-mentioned polypeptide extract in the preparation of antibacterial drugs.

[0017] The present invention also provides the use of the above-mentioned polypeptide extract in the preparation of anti-inflammatory or skin care products.

[0018] Compared with the prior art, the present invention has at least the following beneficial effects: (1) Strong antibacterial activity: The agar diffusion method was used to verify that the polypeptide extract formed a clear antibacterial ring against Escherichia coli at a concentration of 100 μg / mL, and the diffusion characteristics were good.

[0019] (2) Significant anti-inflammatory effect: In the RAW264.7 cell model, the peptide extract dose-dependently inhibited NO release, and the anti-inflammatory rate was nearly 100% at a concentration of 1 mg / mL.

[0020] (3) High biosafety: CCK-8 experiments showed that cell survival rate was >92% and there was no cytotoxicity.

[0021] (4) Application advantages: After one week of trial use of Yao bath preparation, 87% of volunteers experienced improvement in skin inflammation symptoms, with no irritation reaction, demonstrating good skin compatibility.

[0022] (5) The preparation process is simple and suitable for large-scale production. Attached Figure Description

[0023] Figure 1 The image shows the results of the Coptis chinensis polypeptide inhibition zone experiment. Note: The filter papers numbered 1, 2, 3, and 4 on the petri dishes are filter papers soaked in Coptis chinensis polypeptide solutions with concentrations of 100 μg / mL, 50 μg / mL, 25 μg / mL, and 0 μg / mL, respectively. A semi-transparent inhibition zone with a diameter of 1.5 cm appeared around filter paper 1, and a semi-transparent inhibition zone with a diameter of 1 cm appeared around filter paper 2, which is smaller than the diameter of filter paper 1.

[0024] Figure 2 This is a diagram showing the experimental results of Coptis chinensis polypeptide inhibiting inflammation.

[0025] Figure 3 The image shows the experimental results of Coptis chinensis polypeptide CCK8. Detailed Implementation

[0026] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0027] Example 1: Preparation of active peptide samples The dried rhizome of *Coptis chinensis* Franch. was powdered and passed through an 80-mesh sieve to obtain a uniform fine powder. 100 g of the powder was weighed and mixed with 1000 mL of 0.01 M phosphate-buffered saline (PBS) at pH 7.4. The mixture was stirred continuously in a 50 °C water bath for 3 hours. The mixture was then centrifuged at 4 °C and 10000 g for 20 minutes, and the supernatant was collected. The supernatant was filtered through a 0.45 μm microporous membrane to obtain a crude extract. The crude extract was placed in an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa and centrifuged at 4000 g for 30 minutes. The filtrate was collected to obtain a small molecule peptide fraction.

[0028] The ultrafiltration filtrate was finely purified using gel filtration chromatography. Sephadex G-25 packing material was used with deionized water as the eluent, and the flow rate was controlled at 1.0 mL / min. The fraction corresponding to the second major protein absorption peak was collected by monitoring at 280 nm using a UV detector. The collected fraction was pre-frozen at -80°C and then freeze-dried at -50°C and 0.1 mBar for 24 hours to obtain approximately 125 mg of white flocculent peptide powder.

[0029] The molecular weight of the purified product was identified using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results showed that the molecular weights of the major components were concentrated between 1000 Da and 2500 Da. Further amino acid sequence analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). After digestion of the peptide sample with trypsin, a core active peptide sequence consisting of 12 amino acids was identified through database comparison. The sequence is: MLAYSSIGQIG (SEQ ID No: 1). Example 2: Validation of the antibacterial activity of the polypeptide extract In this study, *Escherichia coli* BLT5403 was selected as the standard test strain, and the *Coptis chinensis* polypeptide extract prepared in Example 1 was used as the experimental sample. LB agar medium was used as the culture medium, and the agar diffusion method recommended by the Clinical Laboratory Standardization Institute was strictly followed. First, the strain was activated on agar plates, and then the concentration of the bacterial suspension was precisely adjusted to 1.5 × 10⁻⁶ / mL using the McFarland turbidimetric method. 8 Each colony-forming unit was used. The bacterial suspension was then evenly spread onto the agar plate using a sterile cotton swab, and the plate was rotated to ensure uniform distribution. Filter paper was cut into 1cm circular pieces using a sterile punch and immersed in polypeptide solutions at concentrations of 100μg / mL, 50μg / mL, 25μg / mL, and 0μg / mL, respectively. After complete immersion, the pieces were affixed to E. coli culture dishes, labeled, and incubated at 37°C for 24 hours. The results showed that after 24 hours of incubation, no inhibition zone was observed around the 0μg / mL group, confirming the stability and reliability of the experimental system; while clear inhibition zones were formed at concentrations of 100μg / mL and 50μg / mL, with diameters of 1.5cm and 1cm, respectively. Figure 1 The edges of all inhibition zones were clear and regular, demonstrating that the polypeptide has good diffusion characteristics in agar medium and can effectively inhibit bacterial growth. Example 3: Verification of the in vitro anti-inflammatory activity of the polypeptide extract In the in vitro anti-inflammatory activity verification experiment, mouse mononuclear macrophage RAW264.7 was used as the experimental model. This cell line was cultured in DMEM high-glucose medium containing 10% fetal bovine serum. The experimental setup included a blank control group, a lipopolysaccharide-induced inflammation model group, and three peptide-treated groups with different concentrations. The peptide-treated groups received 1 μg / ml lipopolysaccharide to induce inflammation, and were further treated with 0.25, 0.5, and 1 mg / ml Coptis chinensis peptide extract, respectively. Cells were seeded at a specific density in 96-well plates and allowed to adhere for 24 hours before being replaced with fresh medium. The peptide-treated groups were pre-incubated with the peptide sample for 1 hour, followed by continuous stimulation with lipopolysaccharide for 24 hours. After collecting the cell supernatant, the NO concentration level in the supernatant was measured. To simultaneously assess sample safety, the relative cell viability was detected using the CCK-8 assay.

[0030] Experimental data showed that NO release in the lipopolysaccharide model group was significantly higher than that in the blank control group, confirming the successful construction of the inflammation model. The peptide extract exhibited a significant dose-dependent inhibitory effect; at 0.25 mg / ml, the inflammation inhibition rate reached 43%, further increasing to 71% at 0.5 mg / ml, and almost completely inhibiting inflammation at 1 mg / ml. Figure 2 Cytotoxicity assays showed that cell viability remained above 92% in all treatment groups. However, cell activity decreased to some extent with increasing concentration of Coptis chinensis polypeptide. Figure 3 The results fully demonstrate that the Coptis chinensis polypeptide extract provided by this invention has both significant anti-inflammatory activity and good biosafety within the effective concentration range. Example 4: Application of polypeptide extracts in Yao bath Weigh 50 mg of Coptis chinensis polypeptide powder, 1 g of Tween-80, and 5 g of glycerin and dissolve them in 50 mL of deionized water. Heat and stir in a 40°C water bath until completely dissolved. Weigh 3 g of stearic acid, 2 g of cetyl alcohol, and 1.5 g of liquid paraffin and heat and stir at 75°C. Mix the above two ingredients and emulsify using a homogenizer at 10,000 rpm for 5 minutes. Then cool to room temperature to obtain a stable O / W type Yao bath emulsion containing 0.5% Coptis chinensis polypeptide extract.

[0031] Thirty volunteers with mild dermatitis were recruited and used the aforementioned Yao bath lotion for local soaking once daily for 15 minutes each time for two weeks. Results showed that 87% of the volunteers reported significant improvement in skin redness and itching after one week of use, with no skin allergies or irritation, confirming the good safety and efficacy of this polypeptide Yao bath preparation.

[0032] 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 extract with antibacterial and anti-inflammatory properties, characterized in that, The polypeptide extract is derived from Coptis chinensis (Coptis chinensis) Coptis chinensis Franch. The polypeptide was extracted from the rhizome, and its amino acid sequence is shown in SEQ ID No:

1.

2. A method for preparing the polypeptide extract according to claim 1, comprising the following steps: Coptis chinensis powder was mixed with phosphate buffer for extraction, centrifuged at 4℃ and 10000 g for 20 minutes, the supernatant was collected, purified by gel chromatography, and freeze-dried to obtain polypeptide powder.

3. The preparation method according to claim 2, characterized in that, The ultrafiltration process uses an ultrafiltration tube with a molecular weight cutoff of 10 kDa, centrifuges at 4000 g for 30 minutes, and collects the filtrate.

4. The preparation method according to claim 2, characterized in that, The gel chromatography used Sephadex G-25 packing material.

5. A Yao bath preparation comprising the polypeptide extract of claim 1, and excipients acceptable for topical preparations.

6. The Yao bath preparation according to claim 5, characterized in that, The excipients include Tween-80, glycerin, stearic acid, cetyl alcohol, and liquid paraffin.

7. The Yao bath preparation according to claim 5, characterized in that, The polypeptide extract has a mass fraction of 0.1%-1% in the formulation.

8. The use of the polypeptide extract according to claim 1 in the preparation of a topical antibacterial drug for inhibiting Escherichia coli.

9. The use of the polypeptide extract according to claim 1 in the preparation of Yao bath-type skin care products for improving mild skin inflammation.

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