Polypeptides for the treatment of rhinitis, methods of making and uses

Abstract

The present application relates to a kind of polypeptide for treating rhinitis, preparation method and use, belong to biotechnology field.The present application extracts natural active small molecule polypeptide from white tea plant, the polypeptide is safe and nontoxic to cell, and has significant bacteriostatic and bactericidal effect to rhinitis pathogenic bacteria such as staphylococcus aureus, pseudomonas aeruginosa, candida albicans and aspergillus niger;In addition, in guinea pig in-vivo test, the symptoms and IgE, IL6 and IFN-γ etc. of allergic rhinitis can be significantly improved inflammatory factors, and treatment effect is better than positive control group or comparable with positive control group.The white tea polypeptide of the present application has significant effect of treating rhinitis, provides a new type, safe, efficient treatment choice for rhinitis patient, has important scientific value and clinical application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a polypeptide for treating rhinitis, a preparation method and use. BACKGROUND

[0002] Rhinitis is inflammation of the nasal cavity caused by viruses, bacteria, allergens, various physical and chemical factors, and certain systemic diseases. The main pathological changes are congestion, swelling, exudation, hyperplasia, atrophy, or necrosis of the nasal mucosa. Clinically, it can be divided into acute rhinitis and chronic rhinitis according to the speed of onset, and chronic rhinitis can be further divided into allergic rhinitis (allergic rhinitis) and non-allergic rhinitis. The main symptoms of rhinitis patients are runny nose, nasal congestion, sneezing, and nasal itching. Chronic rhinitis often recurs and can cause certain difficulties in the patient's life, such as difficulty falling asleep for patients with severe nasal congestion. Some patients may develop acute sinusitis, acute otitis media, asthma, and other complications. Some patients may also experience psychological effects due to the long-term presence of rhinitis symptoms.

[0003] The most common method of drug treatment is nasal administration, and the treatment of rhinitis mainly relies on western medicine (such as antihistamines, glucocorticoids, decongestants, etc.) and traditional Chinese medicine (such as extracts of Xinyi, Cang'erzi, Huangqi, etc.). Western medicine has a quick effect, but long-term use can lead to drug resistance, drug-induced rhinitis, and systemic side effects; while traditional Chinese medicine has fewer side effects, but has a slow onset, complex components, and insufficient quality control and mechanism research. In addition, the physiological structure of the nasal mucosa is complex, and is covered with a mucus layer and a cilia system, which pose additional challenges to drug penetration and absorption. Traditional nasal administration preparations often fail to overcome these barriers, resulting in uneven distribution of drugs in the nasal cavity and difficulty in achieving the desired therapeutic effect. Therefore, finding a new rhinitis treatment drug with high efficiency, safety, and stability has become a research hotspot.

[0004] In recent years, the application of natural active ingredients in drug research and development has attracted much attention. Plant polypeptides show great potential due to their low toxicity, high biological activity, and good tissue penetration. White tea polypeptide is a small molecule active peptide extracted from white tea, which has significant anti-inflammatory, antioxidant, and immunomodulatory effects. However, compared with green tea polypeptide or other plant active ingredients, the research of white tea polypeptide in the medical field is still in its infancy. Existing studies have shown that white tea polypeptide can inhibit histamine release, reduce mucosal inflammatory response, and alleviate allergic rhinitis symptoms by regulating Th1 / Th2 immune balance. However, its specific target, pharmacodynamic mechanism, and clinical applicability still need further verification. Compared with traditional Western medicine and traditional Chinese medicine, white tea polypeptide has unique advantages: on the one hand, its small molecular weight and clear structure facilitate quality control and meet the requirements of modern precision medicine; on the other hand, its natural source characteristics reduce the risk of side effects and are suitable for long-term use. However, there is little research on the pharmacology of white tea polypeptide, and its potential in the treatment of rhinitis has not been fully explored. Therefore, developing rhinitis treatment drugs based on white tea polypeptide not only fills the research gap in this field but also provides a new, safe, and efficient treatment option for rhinitis patients, which has important scientific value and clinical application prospects. SUMMARY

[0005] To solve the above technical problems, the present application provides a polypeptide for treating rhinitis, a preparation method and use. The white tea polypeptide prepared has high safety, and shows significant anti-inflammatory and antibacterial effects in vitro against rhinitis-related pathogenic bacteria and animal model experiments, and can be widely used for treating rhinitis and has good therapeutic effect.

[0006] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0007] The present application provides a white tea polypeptide, characterized in that the amino acid sequence of the white tea polypeptide is GGYITMS (SEQ ID NO: 1), ILGDDKYA (SEQ ID NO: 2), TDKMSTGW (SEQ ID NO: 3), and KEPDGAEW (SEQ ID NO: 4).

[0008] Further, the white tea polypeptide represented by SEQ ID NO: 1-4 is synthesized by GenScript Biotech (Shanghai) Co., Ltd. and stored for future use.

[0009] The present application also provides a preparation method of white tea polypeptide, characterized by comprising the following steps:

[0010] (1) Extraction of white tea protein: 20 g of Fuding Da Bai tea was weighed, crushed through a 60-mesh sieve to obtain white tea powder, and then 40 mL of protein extraction solution was added, which contained 0.7 M sucrose, 0.1 M NaCl, 0.5 M Tris-HCl with a pH of 7.5, 50 mM EDTA-2Na, 0.2% DTT and 0.1% benzylsulfonyl fluoride, and homogenate grinding was performed;

[0011] (2) Layering of Tris equilibrated phenol saturated solution: The protein extraction solution was supplemented to 100 mL and mixed, 100 mL of Tris equilibrated phenol saturated solution with a pH of 8.0 was added, 4°C mixing was performed for 30 min, and mixing was performed multiple times; 4°C centrifugation was performed at 10,000 g for 15 min, the upper liquid was collected, 5 times the volume of pre-cooled 0.1 M ammonium acetate-methanol solution was added to precipitate the protein in the tea leaves, and overnight precipitation was performed at -20°C;

[0012] (3) Desalination: 4°C centrifugation was performed at 12,000 g for 10 min, the precipitate was collected, water was added to the precipitate and mixed, the dialysis bag was injected, and dialysis was performed in pure water for 24-48 h, during which pure water was replaced 4-6 times until the water outside the dialysis bag was colorless and the conductivity was close to that of pure water, and then 12,000 g centrifugation was performed for 10 min to obtain the desalted white tea protein precipitate;

[0013] (4) Methanol-acetone washing of the protein precipitate: 5 times the volume of pre-cooled methanol was added to wash the white tea protein precipitate, and slight mixing was performed; acetone was used to replace methanol, and the above step was repeated twice to completely remove the methanol; 4°C centrifugation was performed at 12,000 g for 10 min, and the precipitate was collected; the precipitate was freeze-dried, the dried powder was dissolved in 100 mL of sterile double-distilled water, vortex washing was performed, and then 12,000 g centrifugation was performed at room temperature for 10 min, and the supernatant was taken;

[0014] (5) Ultrafiltration and freeze-drying: the supernatant was transferred to a 10 KD ultrafiltration centrifuge tube, 4°C centrifugation was performed at 12,000 g for 15 min, and the filter membrane retained protein liquid and the filtrate were collected, respectively. The filtrate was transferred to a 1 KD ultrafiltration centrifuge tube, 4°C centrifugation was performed at 12,000 g for 15 min, and the filter membrane retained protein liquid and the filtrate were collected, respectively, the filtrate was the prepared white tea polypeptide crude extract, freeze-drying was performed, the finished product white tea polypeptide was obtained, and the product was stored at -20°C for standby use.

[0015] (6) White tea polypeptide sequencing: for high-throughput sequencing of the prepared white tea polypeptide, the peptide sequences with high sequencing result score, high reliability and high frequency of occurrence were screened, and the white tea polypeptide with SEQ ID NO: 1-4 sequences was obtained.

[0016] The application further provides a pharmaceutical composition, characterized in that the pharmaceutical composition contains the white tea polypeptide.

[0017] Further, the weight ratio of the polypeptide in the pharmaceutical composition is 0.1%-99.9%.

[0018] Further, it also contains pharmaceutically acceptable diluents, excipients or carriers.

[0019] Further, the pharmaceutical composition can be prepared into any pharmaceutically acceptable dosage form.

[0020] Further, the dosage form of the pharmaceutical composition includes oral preparations.

[0021] Further, the oral preparation is a solid preparation, a semi-solid preparation or a liquid preparation.

[0022] Further, the oral preparation is selected from any one of a bolus, a powder, a granule, an oral liquid, a decoction, a tablet.

[0023] The present application also provides the use of the white tea polypeptide or the pharmaceutical composition in the preparation of a medicament for treating rhinitis.

[0024] Further, the rhinitis includes acute rhinitis, chronic rhinitis, sinusitis, allergic rhinitis, atrophic rhinitis or vasomotor rhinitis.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] The present application extracts a natural active small molecule polypeptide from a white tea plant. The polypeptide is safe and non-toxic to cells, has a significant bacteriostatic and bactericidal effect on rhinitis pathogenic bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger, and can significantly improve the symptoms of allergic rhinitis and inflammatory factors such as IgE, IL6 and IFN-γ in animal body tests. The treatment effect is better than or comparable to the positive control group. The white tea polypeptide of the present application has a significant effect on treating rhinitis, provides a new, safe and efficient treatment option for rhinitis patients, and has important scientific value and clinical application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiment or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0028] Figure 1 The figure is a three-dimensional structure prediction diagram of the white tea polypeptide of the present application;

[0029] Figure 2 The figure is a cell toxicity test result of the white tea polypeptide of the present application; DETAILED DESCRIPTION

[0030] The starting materials, reagents or apparatuses used in the following examples, if not specifically stated, are available from commercial suppliers or can be obtained by known methods.

[0031] "comprise," "comprising," "containing", "including", "have", or any other variation thereof, are intended to cover a non-exclusive inclusion, and are not intended to exclude other steps, components, or constituents. The terms "comprise" and "comprising" are to be construed as "including" and "including without limitation." The compositions and methods / processes of the present application can comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein.

[0032] Example 1 Preparation of white tea polypeptide

[0033] The preparation of white tea polypeptide comprises the following steps:

[0034] (1) Take 20g of fresh Fuding Da Bai tea, cut it into pieces, and crush it through a 60-mesh sieve to obtain white tea powder. Add 40mL of plant total protein extraction solution (P1258, purchased from Beijing Puli Lei Gene Technology Co., Ltd., containing 0.7M sucrose, 0.1M NaCl, 0.5M Tris-HCl pH 7.5, 50mM EDTA-2Na, 0.2% DTT, and 0.1% benzylsulfonyl fluoride) and grind it into a homogenate until no large chunks or pieces of tissue are visible, ensuring that the sample is completely ground and broken up;

[0035] (2) Tris equilibrated phenol saturated solution layering: supplement the protein extraction solution to 100mL and mix well. Add 100mL of Tris equilibrated phenol saturated solution at pH 8.0, mix at 4°C for 30min, and mix multiple times. Centrifuge at 4°C at 10000g for 15min, collect the upper liquid, and add 5 volumes of pre-cooled 0.1M ammonium acetate-methanol solution to precipitate the proteins in the tea leaves. Precipitate at -20°C overnight;

[0036] (3) Desalination: centrifuge at 4°C at 12000g for 10min, collect the precipitate, mix with water in the precipitate, inject into a dialysis bag, and dialyze in pure water for 24-48h, changing the pure water 4-6 times during the period until the water outside the dialysis bag is colorless and the conductivity is close to that of pure water. Then centrifuge at 12000g for 10min to obtain the desalted white tea protein precipitate;

[0037] (4) Methanol-acetone washing protein precipitate: add 5 times the volume of pre-cooled methanol to wash the above white tea protein precipitate, mix slightly; replace methanol with acetone and repeat the above steps twice to completely remove methanol; centrifuge at 4°C, 12000g for 10 min, collect the precipitate; freeze-dry the precipitate, dissolve the dried powder in 100 mL of sterile double distilled water, vortex wash, then centrifuge at 12000g for 10 min at room temperature, and take the supernatant;

[0038] (5) Ultrafiltration and freeze-drying: transfer the supernatant to a 10KD ultrafiltration centrifuge tube, centrifuge at 4°C, 12000g for 15 min, and collect the filter membrane retained protein solution and the filtrate, respectively. Transfer the filtrate to a 1KD ultrafiltration centrifuge tube, centrifuge at 4°C, 12000g for 15 min, and collect the filter membrane retained protein solution and the filtrate, respectively. The filtrate is the prepared white tea polypeptide crude extract, which is freeze-dried to obtain the finished white tea polypeptide, which is stored at -20°C for standby.

[0039] (6) High-throughput sequencing of the prepared white tea polypeptide, screening of peptide sequences with high sequencing result score, high reliability and high frequency of occurrence: GGYITMS (SEQ ID NO: 1), ILGDDKYA (SEQ ID NO: 2), TDKMSTGW (SEQ ID NO: 3), KEPDGAEW (SEQ ID NO: 4), entrusted to Shengong Biological Engineering (Shanghai) Co., Ltd. for synthesis and storage for standby.

[0040] The polypeptides of SEQ ID NO: 1-4 were predicted using the bioinformatics prediction analysis tool Expasy-ProtParam (https: / / web.expasy.org / protparam / ), and the results are shown in Table 1.

[0041] Table 1 Sequence and physicochemical properties of white tea polypeptides

[0042]

[0043] The polypeptides of SEQ ID NO: 1-4 were predicted using the tertiary structure prediction tool PEP-FOLD (https: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD3 / ), and the prediction results are shown in Table 2. Figure 1

[0044] Example 2 Safety evaluation of white tea polypeptide

[0045] Take HeLa cells growing to 70%-80% of the culture bottle bottom area, 0.25% trypsin digestion, and prepare a cell density of 1×10 5 ​The 100 μL cell suspension was inoculated in a 96-well culture plate and placed in a 37°C, 5% CO2 saturated humidity incubator for culture. After 24 h of cell culture, the complete culture solution was aspirated. In the experimental group, the polypeptide solution of SEQ ID NO: 1-4 prepared in Example 1 was added in a concentration of 0.2 mg / ml diluted with high-sugar DMEM medium, and the control group was the cells cultured in DMEM medium, and the blank group was DMEM medium without cells. The culture was continued in a 37°C, 5% CO2 saturated humidity incubator for 24 h. CCK-8 reagent 10 μL was added to each group, and the cells were incubated in the cell incubator for 1-4 h. The absorbance value (OD value) of each well was measured using an enzyme-linked immunoassay instrument at a wavelength of 450 nm. The cell survival rate was calculated according to the average absorbance value of each group according to the following formula:

[0046]

[0047] The experimental results are shown in Table 1. Figure 2 The cell survival rates of TP-1, TP-3 and TP-4 of the four polypeptides of the present application and the control group are all above 100%, and the cell survival rate of TP-2 is only 68.46%, indicating that TP-2 has a certain toxicity to cells, while the other three polypeptides TP-1, TP-3 and TP-4 have a certain effect of promoting cell growth and proliferation, and are non-toxic and safe to cells.

[0048] Example 3: In vitro antibacterial test evaluation of white tea polypeptides

[0049] Test materials: Staphylococcus aureus (No. bio-52471), Pseudomonas aeruginosa (No. bio-52545), Candida albicans (No. bio-00033) and Aspergillus niger (No. bio-00016) were purchased from Beijing Baoerbo Biotechnology Co., Ltd.

[0050] Test sample: The three white tea polypeptides TP-1, TP-3 and TP-4 were diluted to concentrations of 2000 μg / mL, 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL and 15.625 μg / mL by the half-cup dilution method. After co-culturing with microorganisms, the last clear well in the co-cultured plate was observed under a daylight lamp with the naked eye, and the MIC (minimum inhibitory concentration) was determined. After detection of the MIC, the co-cultured material was detected by the plate method to determine the MBC (minimum bactericidal concentration) of the test sample.

[0051] The test results are shown in Table 2.

[0052] Table 2 The antibacterial test results of the white tea polypeptide of the present application (μg / mL)

[0053]

[0054] From the results of Table 2, it can be seen that the polypeptide of the present application has obvious bacteriostatic and bactericidal effects on the pathogenic bacteria related to rhinitis.

[0055] Example 4 Animal in vivo allergic rhinitis test evaluation

[0056] Test animals and grouping: 36 SPF guinea pigs, half male and half female, 250-300g, purchased from Guangdong Weitong Lihua Experimental Animal Technology Co., Ltd., experimental animal production license number: SCXK (Yue) 2022-0063, quarantine for 4 days. Randomly divided into 6 groups, 6 in each group, namely blank group, model control group (0.9% sodium chloride injection), positive drug control group (desloratadine dry suspension (Fubiting), Hainan Puli Pharmaceutical Co., Ltd., national drug code H20041111), test group (TP-1, TP-3, TP-4).

[0057] Establishment of sensitized animal model: On the 1st, 3rd, 5th, 7th, 9th, 11th, and 13th days, 0.2 ml of sensitizing solution (10 mg / ml OVA-Al(OH)3 adjuvant solution) was injected intraperitoneally, once every other day, for a total of 7 times. Challenge: starting on the 15th day, 10% OVA solution was administered bilaterally by nasal drops, 50 μl per side, once a day, for 7 consecutive days. The normal group was challenged with normal saline by bilateral nasal drops. After the challenge, the guinea pigs were observed for 15 minutes, and the number of sneezes, the number of nose scratches, and the amount of nasal discharge were recorded to score the symptoms of allergic rhinitis. The scoring criteria are shown in Table 3. If the total symptom score of the guinea pig is ≥5, the modeling is considered successful.

[0058] Table 3 Scoring criteria

[0059]

[0060] Dosing method: After the guinea pigs were successfully modeled, the drug was administered according to the human-mouse conversion. The positive control group of mice was given desloratadine dry suspension 10 mg / kg by gavage, and the test group of mice was given each test polypeptide 50 mg / kg by gavage. The blank control group and the model control group were given the same volume of normal saline by gavage, with a dose of 1 mL / 100g, twice a day, for 2 consecutive weeks.

[0061] Statistical analysis: The data were analyzed using SPSS 16.0 statistical general linear model software, and the data were represented as mean ± standard deviation. Duncan's test was used for comparison between groups, with p<0.05 or p<0.01 as the significant difference standard.

[0062] Detection index:

[0063] (1) Behavioral observation: 60 min after the last administration, the number of scratching nose, sneezing and running nose was recorded according to the scoring criteria in Table 3, and the score was given;

[0064] (2) Serum inflammatory factor detection: 2 days after the last administration, the guinea pigs in each group were anesthetized with ether and fixed on the experimental table, the heart was exposed by opening the chest with scissors, and about 5 mL of blood was collected from the heart. The collected blood was allowed to stand at room temperature for 2 h, then centrifuged at 1500 g for 15 min, and the serum was collected in a clean EB tube and stored for later use. ELISA kit (purchased from Wuhan Fenn Biological Technology Co., Ltd.) was used to detect the levels of IgE (item number: EM2035), IL6 (item number: ER0042) and IFN-γ (item number: EM0093) in the serum of guinea pigs in each group.

[0065] (3) Nasal mucosa pathological section detection: for the blank group, test substance 1 and test substance 3 after administration, remove the skin on the surface of the maxilla, mandible and brain, cut the animal head from the cervical vertebrae, take the whole head and put it into 10% formalin solution for fixation, paraffin section, HE staining and pathological histological observation.

[0066] Test results:

[0067] (1) Behavioral observation results

[0068] Table 4 Comparison of behavioral scores of guinea pigs before and after treatment with test substances

[0069]

[0070] Note: compared with the blank group, # P<0.05, ## P<0.01; compared with the model control group *P<0.05, **P<0.01.

[0071] From the results in Table 4, the total score of behavior of the model control group, the positive control group and the test group before treatment was >5, indicating that the model was successfully constructed. After treatment, the behavioral scores of the positive control group and the test group were significantly lower than those of the model control group, indicating that the white tea polypeptide of the application had obvious effect on improving rhinitis, and its effect was comparable to that of the positive control group.

[0072] (2) Serum inflammatory factor detection results

[0073] Table 5 Serum inflammatory factor detection results

[0074]

[0075] Note: compared with the blank group, # P<0.05, ##P<0.01; compared with the model control group *P<0.05, **P<0.01.

[0076] According to the results of Table 5, the model control group is extremely significantly different from the blank group (P<0.01), indicating that the modeling is successful. In addition, the polypeptides TP-1, TP-3 and TP-4 can significantly improve the symptoms of allergic rhinitis and inflammatory factors such as IgE, IL6 and IFN-γ in the behavior score and serum inflammatory factor detection, and the treatment effect is better than or comparable to the positive control group, indicating that the white tea polypeptide of the application has a significant effect on treating rhinitis.

[0077] (3) Nasal mucosa pathological section detection results

[0078] After continuous oral administration for 7 days, the HE staining results show that the overall structure of the nasal mucosa is basically normal, the tissue mucosa layer is covered with a repair layer of ciliated columnar epithelium, the tissue mucosa epithelial layer is clear, the arrangement is relatively regular, there is no obvious exfoliation, edema, necrosis and other degeneration of the mucosa epithelium, and there is no obvious inflammatory cell infiltration in the tissue, indicating that the prescription of the application is non-toxic and non-irritating.

[0079] The above describes the basic principles, main features and advantages of the application. Those skilled in the art should understand that the application is not limited by the above examples, and the above examples and descriptions in the specification are only the principles of the application. Without departing from the spirit and scope of the application, various changes and improvements can be made to the application, and these changes and improvements all fall within the scope of the claimed application. The scope of protection claimed by the application is defined by the appended claims and their equivalents.

Claims

1. A white tea polypeptide, characterized in that, The amino acid sequence of the white tea polypeptide is shown in any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:

4.

2. A pharmaceutical composition, characterized by, The pharmaceutical composition contains any one of the white tea polypeptides shown in claim 1.

3. The pharmaceutical composition of claim 2, wherein, It also contains a pharmaceutically acceptable diluent, excipient or carrier.

4. The pharmaceutical composition of claim 3, wherein, The pharmaceutical composition can be prepared into any pharmaceutically acceptable dosage form, and the dosage form of the pharmaceutical composition is oral preparation.

5. The pharmaceutical composition of claim 4, wherein, The oral preparation is solid preparation, semi-solid preparation or liquid preparation.

6. The pharmaceutical composition of claim 4, wherein, The oral preparation is selected from any one of pill, powder, granule, oral liquid, decoction, tablet.

7. Use of any one of the white tea polypeptides of claim 1 or the pharmaceutical composition of any one of claims 2-6 in the preparation of a medicament for treating allergic rhinitis.

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