A kind of earthworm peptide and its medical use

Abstract

The present invention belongs to the field of biomedicine technology, and provides an earthworm peptide and its medical use. The earthworm peptide with an amino acid sequence as shown in SEQ ID NO: 1 can be used alone or in combination with the earthworm peptide shown in SEQ ID NO: 2 to prepare a drug for treating cough and asthma. The earthworm peptide provided by the present invention is a high-content, high-activity small molecule bioactive peptide component obtained by refining and purifying the earthworm biomimetic enzymatic hydrolysis small molecule peptides and analyzing them; animal experiments have confirmed its improvement effect on cough and asthma, providing new drugs and development directions for the future treatment of cough and asthma, and providing a reference for the research of animal traditional Chinese medicine polypeptide components.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to an earthworm peptide and its medical use. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be taken as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] Asthma is a chronic inflammatory disease of the airways, which is caused by the involvement of airway inflammatory cells and structural cells (such as eosinophils, mast cells, T lymphocytes, neutrophils, smooth muscle cells, airway epithelial cells, etc.) and cellular elements. Ancient physicians believed that the cause of the disease was the internal accumulation of phlegm and fluid, and the obstruction of qi by phlegm, which led to abnormal lung qi diffusion and descent. At present, inhaled corticosteroids (ICS) are routinely used in Western clinical practice to treat asthma, but long-term use of inhaled corticosteroids may cause severe bone loss in children and adults. Therefore, the development of new, safe and effective antiasthmatic drugs is the key to solving this problem. Earthworm is a commonly used medicine for the treatment of asthma in traditional Chinese medicine. Many prescriptions for the treatment of asthma, such as Wuwei Dilong Decoction and Dilong Xixin Decoction, contain earthworm ingredients, which have been proven to have a good antiasthmatic effect. In clinical practice, earthworm injection is used to treat bronchial asthma and asthmatic tracheitis with an effective rate of 78%, and has a certain inhibitory effect on inhibiting asthmatic airway remodeling; compound earthworm oral liquid has the functions of unblocking meridians, activating blood circulation, clearing heat, calming the nerves, relieving asthma, diuresis, lowering blood pressure, and lowering blood lipids; earthworm capsules can also effectively treat asthma; the compound of earthworm and other Chinese medicines such as white mustard and saposhnikovia divaricata, when applied on acupoints, has a good therapeutic effect on bronchial asthma.

[0004] Pheretima is rich in nutrients such as protein, polypeptides and unsaturated fatty acids, and has multiple functions such as preventing cardiovascular and cerebrovascular diseases, anti-inflammatory, anti-tumor and regulating blood lipids. For example, patent CN101584853A discloses a worm protein polypeptide preparation and its preparation method and application. The preparation obtains hydrolyzed protein polypeptides by enzymatic hydrolysis of worm homogenate crude extract, or the worm homogenate crude extract is first hydrolyzed by enzyme to obtain hydrolyzed protein polypeptides, and the hydrolyzate is then separated by ultrafiltration membranes with different pore sizes to obtain protein polypeptide components of different molecular weights. Experiments show that the worm protein polypeptide preparation provided by the present invention has a better inhibitory effect on angiotensin converting enzyme after enzymatic hydrolysis, and can effectively prevent and treat hypertension. Patent CN117384256A discloses a worm polypeptide, its preparation method and application. The sequence of the worm polypeptide is NSGPFPRPPHQK, which has anti-inflammatory activity. Patent CN115216508A discloses a preparation method and application of worm polypeptides. The preparation method comprises the following steps: (1) induction treatment: take earthworm, add water with pH 7.8-8.5, use 5-10V voltage electric shock, crush to obtain homogenate, centrifuge, and collect supernatant; (2) composite enzymatic hydrolysis: obtain crude protein from the supernatant obtained in step (1); perform composite enzymatic hydrolysis on the crude protein to obtain a crude enzymatic sample; (3) dextran gel purification: purify the crude enzymatic sample of step (2) using dextran gel; (4) high performance liquid chromatography purification: take the sample obtained in step (3) and purify it by high performance liquid chromatography to obtain earthworm peptide, which has an inhibitory effect on Shigella dysenteriae, hemolytic streptococcus, Candida albicans, and saccharomyces cerevisiae. Patent CN114262727A discloses a fibrinolytic active polypeptide HEPLPEP and EYPLPEP in earthworm, which have good thrombolytic activity. Patent CN113845565A discloses a small peptide derived from earthworm and having antioxidant biological activity, and the amino acid sequence of the small peptide is PGAGAVY and KDLY. Patent CN110935006A discloses the use of earthworm protein peptides in the preparation of drugs for preventing and / or treating thrombotic diseases, and the earthworm protein peptides are selected from one or more of Leu-Val-Thr-Leu-Gly-Asn-Glu, Leu-Leu-Ala-Pro-Pro, Leu-Leu-Pro-Ala-Pro and Thr-Val-Ala-Pro-Phe. The above earthworm protein peptides can significantly inhibit thrombin activity, thereby achieving the effect of anticoagulation; in vivo, earthworm protein peptides have a significant inhibitory effect on carrageenan-induced rat tail thrombosis; in vitro and in vivo in thrombotic rats, the earthworm protein peptides show good anticoagulant effects. The earthworm protein peptide has the effects of inhibiting thrombosis, thrombolysis, anticoagulation and promoting fibrinolysis, and can effectively prevent and treat thrombotic diseases. However, the types of specific active ingredients for relieving cough and asthma have not been studied or reported yet. Summary of the invention

[0005] In order to solve the problems in the prior art and expand the medical application of earthworm peptide, the present invention provides an application of earthworm peptide in the preparation of a drug for treating or alleviating cough and asthma.

[0006] To achieve the above purpose, the present invention adopts the following technical solution.

[0007] A use of an earthworm peptide having an amino acid sequence as shown in SEQ ID NO: 1 in preparing a medicine for treating cough and asthma.

[0008] The above earthworm peptides can be obtained by enzymatically hydrolyzing the earthworm ( Pheretima aspergillum ) or prepared by total synthesis.

[0009] The medicine also contains an earthworm peptide with an amino acid sequence as shown in SEQ ID NO: 2.

[0010] The cough and asthma include its complications, which refer to systemic and local diseases that occur directly or indirectly at the same time as the cough and asthma occur, specifically severe coughing, asthma attacks, and inflammation of the lungs and respiratory tract.

[0011] The medicine containing the above-mentioned earthworm peptide can be prepared into various dosage forms, such as granules, capsules, tablets, pills, etc.

[0012] The present invention has the following advantages:

[0013] The earthworm peptide provided by the present invention is a high-content, high-activity small molecule bioactive peptide component obtained by refining and purifying earthworm biomimetic enzymatic hydrolysis small molecule peptides and analyzing them; animal experiments have confirmed its improvement effect on cough and asthma, providing new drugs and development directions for the future treatment of cough and asthma, and providing a reference for the research of animal traditional Chinese medicine polypeptide components. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The nano-liquid chromatography-mass spectrometry diagram of the enzymatic hydrolysis of the earthworm peptide in Pheretima lumbricoides in Example 1;

[0015] Figure 2 The effects of E1, E2, E3, and E4 earthworm peptide treatment groups on the viability of RAW264.7 cells;

[0016] Figure 3 The effects of E1, E2, E3, and E4 earthworm peptide treatment groups on NO production in RAW264.7 cells;

[0017] Figure 4 The effects of different treatment groups on rats with citric acid-induced cough model, where a is the cough latency period and b is the number of coughs;

[0018] Figure 5The effects of different treatment groups on the latency period of cough in rats with histamine-induced asthma model. DETAILED DESCRIPTION

[0019] A groundworm peptide consisting of at least one of AVFPSIVGR and FEEILPIYK.

[0020] Among them, AVFPSIVGR, referred to as AR-9, has a molecular weight of 944.54 Da and a structural formula as follows:

[0021]

[0022] That is, alanyl-valyl-phenylalanyl-prolyl-seryl-isoleucine-valyl-glycyl-arginine (Ala-Val-Phe-Pro-Ser-Ile-Val-Gly-Arg), which has good water solubility.

[0023] Among them, FEEILPIYK, referred to as FK-9, has a molecular weight of 1150.63Da and a structural formula as follows:

[0024]

[0025] That is, phenylalanyl-glutamyl-glutamyl-isoleucyl-leucyl-prolyl-isoleucyl-tyrosyl-lysine (Phe-Glu-Glu‑Ile-Leu-Pro-Ile-Tyr-Lys), with a molecular weight of 1150.63Da and good water solubility.

[0026] The above-mentioned earthworm peptide can be completely composed of the AR-9 peptide or the AR-9 peptide and the FK-9 peptide, under the condition that cough or asthma is manifested, and can also be prepared together with pharmaceutically acceptable carriers and / or additives.

[0027] The above-mentioned earthworm peptide with antitussive and antiasthmatic activity can be prepared by enzymatic degradation or hydrolysis of a polypeptide or protein containing the above amino acid sequence, such as enzymatic hydrolysis of Guang earthworm powder by pepsin and trypsin in sequence, and further, the enzymatic hydrolyzate can be ultrafiltered and nanofiltered to increase the content of the target peptide segment; the above-mentioned earthworm peptide can also be prepared by total synthesis. In order to improve the purity, eliminate the immunogenic reaction caused by taking foreign proteins, etc., and reduce the incidence of adverse reactions, it is prepared by solid phase synthesis.

[0028] In some embodiments, the method for preparing the above-mentioned earthworm peptide comprises the following steps:

[0029] (1) Soak the dried Dilong in water;

[0030] (2) Adjust the pH value to 1.5-2.0, add pepsin with a substrate mass ratio of 3%, and perform enzymatic hydrolysis at an appropriate temperature;

[0031] (3) Adjust the pH value to 7.5-8.5, add trypsin with a mass ratio of 3% to substrate, and perform enzymatic hydrolysis at an appropriate temperature;

[0032] (4) Adjust the pH value to neutral, inactivate the enzyme at high temperature, and obtain the enzymatic hydrolyzate after solid-liquid separation.

[0033] In order to speed up the infiltration, Guangdilong is powdered, and the fineness can be passed through a No. 3 sieve or above; the amount of water added is 6-10 times the mass of the earthworm. Heating can also be used, such as 80℃-100℃. The temperature for inactivating enzymes is 80℃-100℃. The optimum temperature for enzymatic hydrolysis is 25℃-45℃.

[0034] In order to improve the yield, the solid can be washed with water 1-2 times, and the washing liquid and the enzymatic hydrolyzate are combined to obtain a liquid containing earthworm peptide. In order to increase the content of earthworm peptide in the above-mentioned earthworm peptide-containing liquid, it can also be filtered and concentrated by ultrafiltration with a retention pore size of 1000 Da-3000 Da. In order to facilitate storage and processing, the earthworm peptide-containing liquid can also be dried to obtain a powder. For example, freeze-drying or spray drying.

[0035] The above-mentioned earthworm peptide can be used to prepare antitussive and antiasthmatic drugs; the antitussive and antiasthmatic drugs containing the earthworm peptide can be made into various dosage forms, such as granules, capsules, pills, tablets, etc.

[0036] In some embodiments, a method for preparing earthworm peptide capsules and tablets using enzymatic hydrolyzed lyophilized powder of P. lucidum as raw material is provided. Since the main component of lyophilized powder is polypeptide, its fluidity is poor and it is highly hygroscopic, the moisture absorption rate is reduced and the fluidity is improved by adding auxiliary materials. Since earthworm peptide is easily oxidized, the effect of oxygen and moisture isolation can be achieved by spraying zeinol solution on the surface of the particles, making the preparation more stable.

[0037] More specifically, the preparation process of the capsule is as follows: after the freeze-dried powder and starch are evenly mixed, 80% ethanol is used as a wetting agent to make a soft material and then granulate. After the wet granules are dried, corn protein is evenly sprayed on the surface to achieve the effect of oxygen and moisture isolation to obtain wet granules; after drying, the granules are sized and filled into capsules. Wet granules are obtained according to the above method, and then rolled into small pellets, which can be used as pills after drying; wet granules can be directly dried and sized to obtain granules; disintegrants and lubricants can also be added to the granules to make tablets.

[0038] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0039] Example 1 Preparation of earthworm peptide

[0040] The dried Guangdilong was crushed and passed through a No. 5 sieve to obtain earthworm powder, 10 times the amount of distilled water was added, and the mixture was placed in a constant temperature water bath at 85°C for 15 min. After cooling to room temperature, hydrochloric acid was added to adjust the pH value of the solution to 1.5, pepsin with an enzyme substrate ratio of 3% was added, and the mixture was placed in a constant temperature water bath at 45°C for 1 h. NaOH solution was added to adjust the pH value of the solution to 8.5, trypsin with an enzyme substrate ratio of 3% was added, and the mixture was placed in a constant temperature water bath at 45°C for 3 h. The pH value of the solution was adjusted to 7.0, and the mixture was placed in a constant temperature water bath at 85°C for 15 min. After cooling to room temperature, the mixture was centrifuged at 5000 r / min for 15 min to obtain a supernatant. The precipitate was washed twice with distilled water and centrifuged again (5000 r / min) for 15 min. The supernatants were combined, frozen at -20°C for 24 h, and freeze-dried to obtain freeze-dried powder.

[0041] The obtained freeze-dried powder was subjected to nano-liquid chromatography-mass spectrometry to analyze the peptide sequence. All the peptide segments obtained by the analysis were used as ligands for molecular docking using SYBYL-X 2.0 software: the ligand compounds were dehydrated, hydrogenated, charged, and mechanically optimized.

[0042] Table 1 Peptide sequence information and relative quantitative information

[0043]

[0044] The relative quantitative information of the four peptides with the highest scores obtained by molecular docking in lyophilized powder is shown in Table 1. Among them, the nonapeptide AVFPSIVGR ( Figure 1 a) Nonapeptide FEEILPIYK ( Figure 1 b), the octapeptide AIYQQIGR (SEQ ID NO: 3) and the nonapeptide FRVPTPNVS (SEQ ID NO: 4) had higher simulation activities, and their activities were further verified.

[0045] Example 2 Solid Phase Synthesis of Pterozoite Peptide

[0046] (1) Resin treatment: Fmoc-Leu-Wang resin (molar substitution coefficient of 0.6 mmol / g) was selected as the starting resin, placed in a 3 L reaction column, soaked in dichloromethane (DCM), and drained to complete the swelling of the resin; then, a 20% piperidine solution in DMF was added, and nitrogen was passed through and stirred for 30 min. The solvent was filtered off, and the resin was washed with N,N-dimethylformamide (DMF) for 6 times in a cycle, and filtered to complete the deprotection of the resin.

[0047] (2) Amino acid coupling reaction: Weigh the corresponding amount of O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) and protected amino acid into a beaker and add DMF to dissolve; then add the reaction solution to the resin, add N,N-diisopropylethylamine (DIEA), and blow nitrogen for 90 min. Detection is performed by ninhydrin reaction;

[0048] After the reaction, the solvent was removed, and the resin was washed with DMF for 3 times. A 20% piperidine DMF solution was added to the resin, and nitrogen was introduced for 30 min. Then the solvent was removed, and the resin was washed with DMF for 6 times. The coupling of the amino acid was completed.

[0049] Repeat the above reaction procedure in the order of Ala-Val-Phe-Pro-Ser-Ile-Val-Gly-Arg or Phe-Glu-Glu‑Ile-Leu-Pro-Ile-Tyr-Lys until the condensation reaction of all protected amino acids is completed, couple to the last amino acid, drain and put into a centrifuge tube, add FITC, HATU, DIEA, DMF and DCM, put on a shaker and shake for 8-12 hours, protect from light, and centrifuge. After centrifugation, shrink the polypeptide, wash it three times with DMF / DCM / MEOH in sequence, drain and weigh it.

[0050] (3) Trifluoroacetic acid cleavage: Add the resin to the prepared cleavage solution (86% TFA / 5% EDT / 5% thioanisole / 3% phenol / 2% pure water), stir for 150 min, then extract the resin and cleavage solution, add ether to fully precipitate the peptide, filter, and wash with ether for 6 times to obtain solid phase synthesized nonapeptide AVFPSIVGR (AR-9 peptide), nonapeptide FEEILPIYK (FK-9 peptide), octapeptide AIYQQIGR (AR-8 peptide), and nonapeptide FRVPTPNVS (FS-9 peptide).

[0051] Example 3 Effects of earthworm peptide on macrophages

[0052] 1. Cytotoxicity

[0053] RAW264.7 cells were prepared at 5 × 10 5100 μL of cell suspension was inoculated into 96-well plates at 37°C and 5% CO2 for 24 h. The cells were divided into blank group, control group, drug E1 (AR-9 peptide group), drug E2 (FK-9 peptide group), drug E3 (AR-8 peptide) group, and drug E4 (FS-9 peptide) group. The blank group did not contain cells and only added 200 μL of complete medium; the control group added cell suspension and complete medium; each drug group added 200 μL of drug-containing medium with a concentration gradient of 0.5, 1, 2, 4, 8, 16, 32, and 64 mg / mL, and each group was set up with 6 replicates and placed in a 37°C, 5% CO2 cell culture incubator for 24 h.

[0054] After the cells were administered and cultured for 24 h, 10 μL of CCK8 working solution was added to each well, and the cells were placed in a 37°C, 5% CO2 cell culture incubator for further culturing for 1 h. The cell supernatant was aspirated into a 96-well plate, and the absorbance at 450 nm was measured by an ELISA reader. The cell viability was calculated as follows:

[0055] Cell viability (%) = [(OD 加药 -OD 空白 ) / (OD 对照 -OD 空白 )]×100%.

[0056] The results are as follows Figure 2 As shown (compared with the normal group, P <0.0001); CCK-8 method was used to detect the effects of different earthworm peptides on the activity of RAW264.7 cells. The results showed that when the drug concentration was greater than 8 mg / mL, the cell viability was significantly reduced compared with the normal group ( P <0.0001), when the drug concentration was greater than 8 mg / mL, there was no statistical difference in cell viability compared with the normal group ( P >0.05). The results showed that 0.5, 1, 2, 4, and 8 mg / mL of earthworm peptide solution did not affect the viability of RAW264.7 cells and had no cytotoxicity. Therefore, in subsequent cell experiments, the drug concentration used should not exceed 8 mg / mL.

[0057] 2. NO content (Griess method)

[0058] RAW264.7 cells were prepared at 5 × 10 5100 μL of cell suspension was added to each well of a 96-well plate and cultured at 37°C and 5% CO2 for 24 h. The cells were divided into a blank group, an LPS group, a drug E1 (AR-9 peptide) group, a drug E2 (FK-9 peptide) group, a drug E3 (AR-8 peptide) group, and a drug E4 (FS-9 peptide) group. After adding 100 μL of cell suspension, the blank group was added with only 100 μL of complete medium for 24 h; the LPS group was added with 200 μL of complete medium for 12 h, the cell supernatant was aspirated, and 200 μL LPS (1 μg / mL) was added for 12 h; each drug group was added with 200 μL of medium containing the corresponding drug with a concentration gradient of 2, 4, and 8 mg / mL for 12 h, the cell supernatant was aspirated, and 200 μL LPS (1 μg / mL) was added for 12 h.

[0059] After 24 h of cell modeling and drug culture in each group, the 96-well plate was taken out of the cell culture incubator, 50 μL of supernatant culture medium was aspirated from each well into a new 96-well plate, and 50 μL of Griess Reagent I and Griess Reagent II were added to each well in a dark environment according to the instructions of the NO detection kit. The absorbance at 540 nm was measured with an enzyme marker, and the NO content in the supernatant of the cell culture fluid of each group was calculated based on the NaNO2 content standard curve.

[0060] like Figure 3 As shown (compared with the model group, P <0.0001, P <0.001, P <0.01); compared with the normal group, the NO content in the cell supernatant of the LPS group increased significantly, indicating that LPS induced RAW264.7 cell polarization successfully, and the NO production levels in the four drug groups decreased in a dose-dependent manner. In the E1 group (AR-9 peptide group), the low-dose group significantly reduced the NO production level compared with the LPS group ( P <0.05), the middle and high dose groups significantly reduced the NO production level ( P <0.0001); in the E2 group (FK-9 peptide group), compared with the LPS group, the low, medium and high dose groups all significantly reduced the NO production level ( P <0.0001); in the E3 group (AR-8 peptide), there was no significant difference in the low-dose group compared with the LPS group ( P >0.05), while the medium and high dose groups significantly reduced the NO production level ( P<0.0001); in the E4 group (FS-9 peptide), compared with the LPS group, the low, medium and high dose groups all significantly reduced the NO production level ( P <0.0001). The results showed that E1, E2, E3, and E4 all had an inhibitory effect on the production of NO in RAW264.7 cells induced by LPS and had anti-inflammatory activity, and E2 had the strongest activity.

[0061] Example 4 Effects of earthworm peptide on cough and asthma model animals

[0062] 1. Citrate-induced cough

[0063] Healthy SD rats weighing 200±20 g were selected, half male and half female, and placed in a transparent sealed container. A 17.5% citric acid solution was placed in the nebulizer cup of the ultrasonic nebulizer nebulizer chamber, and the citric acid solution nebulizer gas was sprayed into the transparent sealed container at a constant speed for 1 min. The number of coughs of each rat within 5 min was recorded. The rats with loud coughs and audible coughs were counted as one cough, and rats with less than 10 coughs were discarded.

[0064] Sixty pre-selected rats were randomly divided into model group, compound methoxyphenamine (28.6 mg / kg) group, E1 (AR-9 peptide, 171.4 mg / kg) group, E2 (FK-9 peptide, 171.4 mg / kg) group, E3 (AR-8 peptide, 171.4 mg / kg) group, and E4 (FS-9 peptide, 171.4 mg / kg) group, with 10 rats in each group. Each group was given the corresponding drug by oral gavage for 3 consecutive days. One hour after the drug administration on the third day, the rats were placed in a sealed transparent container, and the atomized gas of citric acid solution was sprayed into the container at a constant speed for 1 min. The latency period from the start of spraying to the onset of coughing and the number of coughs within 5 min after the spraying stopped were observed. The quantitative data were expressed as mean ± standard deviation (x ± s) and analyzed using SPSS 20 statistical software.

[0065] Table 2 Effects of different earthworm peptides on the rat citrate-induced cough model (n=10)

[0066]

[0067] According to Table 2 and Figure 4 It can be seen that compared with the model group, the compound methamphetamine group can significantly prolong the cough latency of rats ( P <0.0001), inhibiting the coughing behavior of rats stimulated by citric acid ( P <0.0001); E1 can significantly prolong the cough latency of rats ( P <0.001), significantly reduced the number of rat coughs ( P<0.0001); E2 can significantly prolong the cough latency of rats ( P <0.0001), and reduced the number of coughing in rats ( P <0.0001); E3 can significantly prolong the cough latency of rats ( P <0.01), reduce the number of rat coughing ( P <0.05); E4 can only prolong the cough latency of rats ( P <0.05), and could not effectively reduce the number of rat coughs ( P >0.05). The results showed that E1 and E2 had obvious antitussive activity, E3 had only weak antitussive activity, and E4 had almost no antitussive activity.

[0068] 2. Histamine-induced cough

[0069] Healthy SD rats were selected, both male and female. The rats were placed in a sealed transparent container, and an equal amount of a mixture of 2% acetylcholine chloride and 0.1% histamine phosphate was sprayed at constant pressure using an ultrasonic nebulizer. Each rat was sprayed at a constant pressure and speed for 15 s, and its asthma latency was measured. The asthma latency was less than 150 s and was qualified. 60 rats were randomly divided into 4 groups: model group, aminophylline (114.3 mg / kg) group, E1 (AR-9 peptide, 171.4 mg / kg) group, E2 (FK-9 peptide, 171.4 mg / kg) group, E3 (AR-8 peptide, 171.4 mg / kg) group, and E4 (FS-9 peptide, 171.4 mg / kg), with 10 rats in each group.

[0070] The drug was administered orally for 3 consecutive days. One hour after administration on the third day, the rats were placed in a sealed transparent container and sprayed with an equal amount of a mixture of 2% acetylcholine chloride and 0.1% histamine phosphate at constant pressure for 15 seconds using an ultrasonic nebulizer. The asthma induction latency was measured, that is, the time from the start of spraying to asthma attack, dyspnea, or even convulsion and fall. The quantitative data were expressed as mean ± standard deviation (x ± s) and analyzed using SPSS 20 statistical software.

[0071] Table 3 Effects of different earthworm peptides on histamine-induced asthma model in rats (n=10)

[0072]

[0073] According to Table 3 and Figure 5 It can be found that after giving rats equal amounts of E1 and E2, the results showed that both E1 and E2 had strong antiasthmatic activity. According to the dosage of Guangdilong prescribed in the pharmacopoeia, according to the extraction rate of E1 and E2 parts, the dosage concentration of E1 was 198.65-397.31 mg / kg, and that of E2 was 28.93-57.86 mg / kg.

[0074] In summary, AR-9 peptide, FK-9 peptide, and AR-9 and FK-9 combined peptide can inhibit the production of NO in RAW264.7 cells induced by LPS and have anti-inflammatory activity; both have obvious antitussive and antiasthmatic activities.

[0075] Example 5 Preparation of medicine containing earthworm peptide

[0076] 1. Preparation of capsules

[0077] (1) The raw material powder and corn starch are mixed evenly in a ratio of 2:1, and 80% ethanol is used as a wetting agent to make a soft material, which is then granulated using a 20-mesh sieve. After the wet granules are dried, 10% corn protein 80% ethanol solution is evenly sprayed on the surface, and finally the granules are weighted by 1% to obtain wet granules;

[0078] (2) Dry the wet granules and fill them into size 0 capsules to obtain earthworm peptide capsules.

[0079] 2. Tablet Preparation

[0080] (1) The raw material powder and corn starch are mixed evenly in a ratio of 2:1, and 80% ethanol is used as a wetting agent to make a soft material, which is then granulated using a 20-mesh sieve. After the wet granules are dried, 10% corn protein 80% ethanol solution is evenly sprayed on the surface, and finally the granules are weighted by 1% to obtain wet granules;

[0081] (2) After the wet granules are dried, a disintegrant and a lubricant are added and the granules are compressed into tablets using a tablet press.

[0082] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A use of earthworm peptide in preparing a drug for treating cough and asthma, characterized in that: The amino acid sequence of the earthworm peptide is shown in SEQ ID NO:

1.

2. A composition for treating cough and asthma, characterized in that: The invention comprises the earthworm peptides with amino acid sequences as shown in SEQ ID NO: 1 and SEQ ID NO:

2.

3. Use of the composition as claimed in claim 2 in preparing a medicine for treating cough and asthma.

4. The use according to claim 1 or 3, characterized in that: The cough and asthma also include complications of cough and asthma; the complications include asthma, lung and / or respiratory tract inflammation.

5. The use according to claim 1 or 3, characterized in that: The dosage form of the drug is selected from granules, capsules, tablets or pills.

6. The use according to claim 5, characterized in that The preparation method of the drug comprises the following steps: S1. Mixing the earthworm peptide or the combination with starch, using 80% ethanol as a wetting agent, making a soft material and then granulating, after the wet granules are dried, spraying the corn protein solution evenly on the surface to obtain wet granules; S2a, drying the wet granules in S1 and then granulating them to obtain granules; S2b, filling the granules in S2a into capsules to obtain capsules; S2c, adding a disintegrant and a lubricant to the granules in S2a, and compressing the granules to obtain tablets; S2d. Roll the wet granules in S1 into small pellets and obtain pellets after drying.

Images