A pharmaceutical composition for treating peptic gastric ulcer and its preparation method

Abstract

The present invention relates to a pharmaceutical composition for treating peptic gastric ulcer and a preparation method thereof, belonging to the field of biotechnology. The pharmaceutical composition contains chicken gizzard-membrane peptide, pumpkin seed extract, complex bacteria, white hyacinth bean flower extract and bletilla striata extract; the chicken gizzard-membrane peptide contains the products obtained by successively subjecting chicken gizzard-membrane to fractional enzymolysis with pepsin, chymotrypsin and keratinase; the pumpkin seed extract contains the products obtained by subjecting pumpkin seeds to co-enzymolysis with cellulase and pectinase and then successively to fractional enzymolysis with pepsin, nattokinase and aminopeptidase; the complex bacteria contain the co-fermentation bacteria of Lactobacillus acidophilus, Bifidobacterium and Lactobacillus casei; the white hyacinth bean flower extract is an ethanol percolation extract; the bletilla striata extract contains the products obtained by subjecting bletilla striata tubers to co-enzymolysis with cellulase and pectinase and then successively to fractional enzymolysis with pepsin, bromelain and serrapeptase. The various components are used in combination, having a good therapeutic effect on gastric ulcer, quickly and efficiently repairing gastric mucosa, and being safe and free of side effects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a pharmaceutical composition for treating peptic gastric ulcer and a preparation method thereof. Background Art

[0002] Peptic gastric ulcer is a common digestive system disease. It is mainly caused by the imbalance between the gastric mucosal defense factors and invasive factors, resulting in the digestion and destruction of the gastric mucosa by gastric acid and pepsin, forming ulcers. Its pathogenesis is relatively complex and involves multiple factors, such as Helicobacter pylori (Hp) infection, the use of non-steroidal anti-inflammatory drugs (NSAIDs), abnormal gastric acid secretion, as well as lifestyle and mental stress.

[0003] In terms of drug treatment, antacids such as aluminum hydroxide and sodium bicarbonate were mainly used in the early stage. These drugs can neutralize gastric acid and quickly relieve pain symptoms, but the action duration is short, and long-term use may cause adverse reactions such as constipation or alkalosis, and cannot cure ulcers fundamentally. With the in-depth study of the gastric acid secretion mechanism, cimetidine, ranitidine, omeprazole, lansoprazole, etc. emerged. Cimetidine and ranitidine can promote ulcer healing, with better curative effects than antacids and relatively fewer adverse reactions; however, some patients may experience side effects such as headache, fatigue, and diarrhea after use, and with the prolongation of use time, tolerance problems will occur, reducing the drug efficacy. Omeprazole and lansoprazole can inhibit gastric acid secretion, with strong effects and long duration, can quickly relieve symptoms and promote ulcer healing, but long-term use will affect the absorption of minerals such as calcium and magnesium, increase the risk of fractures, and may also lead to the occurrence of fundic gland polyps, and the treatment effect for some refractory ulcers is still not ideal enough. In addition to acid-suppressing drugs, the treatment of Helicobacter pylori infection is also crucial. Currently, amoxicillin, clarithromycin, etc. are used, but with the widespread use of antibiotics, the problem of Helicobacter pylori drug resistance has gradually emerged, affecting the effect of eradication treatment. It is necessary to continuously explore new treatment regimens and drug combinations, and develop safer and more efficient treatment drugs and regimens to improve the treatment effect of patients. Summary of the Invention

[0004] Aiming at the various side effects, poor safety, and weakened drug efficacy of the existing drugs for treating peptic gastric ulcer. The present invention provides a pharmaceutical composition for treating peptic gastric ulcer and a preparation method thereof. The pharmaceutical composition contains chicken gizzard-membrane peptides prepared by special enzymolysis, pumpkin seed extracts prepared by special enzymolysis, complex bacteriocins prepared by fermenting special substrates with a variety of beneficial bacteria, white hyacinth bean flower extracts extracted by ethanol percolation, and bletilla striata extracts prepared by special enzymolysis; various components are used in combination according to specific proportions, having a good therapeutic effect on gastric ulcers, quickly and efficiently repairing the gastric mucosa, and being safe without side effects. The specific technical solutions are as follows:

[0005] A pharmaceutical composition for treating peptic gastric ulcer, comprising raw materials in the following parts by mass: 1.0 part - 2.0 parts of gallus gallus domesticus gmelin peptide, 1.5 parts - 2.5 parts of pumpkin seed extract, 0.5 part - 1.5 parts of complex bacteria, 2 parts - 5 parts of dolichos lablab flower extract, and 3 parts - 6 parts of bletilla striata extract;

[0006] The gallus gallus domesticus gmelin peptide contains products with a molecular weight below 5 kDa obtained by successively hydrolyzing gallus gallus domesticus gmelin with pepsin, chymotrypsin, and keratinase; the pumpkin seed extract contains products with a molecular weight below 5 kDa obtained by co-hydrolyzing pumpkin seeds with cellulase and pectinase, and then successively hydrolyzing with pepsin, nattokinase, and aminopeptidase; the complex bacteria contains a co-fermentation product of lactobacillus acidophilus, bifidobacterium, and lactobacillus casei; the dolichos lablab flower extract is an ethanol percolation extract of dolichos lablab flower; the bletilla striata extract contains products with a molecular weight below 10 kDa obtained by co-hydrolyzing bletilla striata tubers with cellulase and pectinase, and then successively hydrolyzing with pepsin, bromelain, and serrapeptase.

[0007] In the above pharmaceutical composition, the preparation method of the gallus gallus domesticus gmelin peptide comprises the following steps: taking fresh gallus gallus domesticus gmelin, pulverizing it, adding distilled water with a mass 8 - 12 times that of the gallus gallus domesticus gmelin, adjusting the pH to 1.5 - 2.5, adding pepsin accounting for 0.5% - 1% of the mass of the gallus gallus domesticus gmelin, performing enzymatic hydrolysis at 35°C - 38°C for 60 min - 80 min, heating to inactivate the enzyme, cooling to room temperature, adjusting the pH to 7.5 - 8.5, adding chymotrypsin accounting for 0.8% - 1.5% of the mass of the gallus gallus domesticus gmelin, performing enzymatic hydrolysis at 35°C - 38°C for 50 min - 70 min, heating to inactivate the enzyme, cooling to room temperature, adding keratinase accounting for 1% - 2% of the mass of the gallus gallus domesticus gmelin, performing enzymatic hydrolysis at 40°C - 60°C for 80 min - 100 min, heating to inactivate the enzyme, cooling to room temperature, centrifuging at 4000 r / min - 6000 r / min for 15 min - 20 min, taking the supernatant, performing ultrafiltration with a 5 kDa ultrafiltration membrane, taking the ultrafiltration fraction below 5 kDa, and freeze-drying to obtain a powder, thus obtaining the gallus gallus domesticus gmelin peptide.

[0008] In the above-mentioned pharmaceutical composition, the preparation method of the pumpkin seed extract comprises the following steps: taking the seed flesh of pumpkin seeds, pulverizing it, adding water with a mass 8 to 12 times that of the seed flesh, adjusting the pH to 4.5 - 5.5, adding cellulase with a mass of 0.5% - 2% of the seed flesh and pectinase with a mass of 0.5% - 2% of the seed flesh, carrying out constant-temperature enzymatic hydrolysis at 50°C - 55°C for 2h - 3h, then adjusting the pH to 1.5 - 2.5, adding pepsin with a mass of 0.5% - 1% of the seed flesh, carrying out enzymatic hydrolysis at 35°C - 38°C for 60min - 80min, heating to inactivate the enzyme, cooling to room temperature, adjusting the pH to 6.5 - 7.0, adding nattokinase with a mass of 0.5% - 1.5% of the seed flesh, carrying out enzymatic hydrolysis at 50°C - 55°C for 60min - 90min, heating to inactivate the enzyme, cooling to room temperature, adjusting the pH to 7.0 - 7.5, adding aminopeptidase with a mass of 0.5% - 1.5% of the seed flesh, carrying out enzymatic hydrolysis at 37°C - 45°C for 50min - 70min, heating to inactivate the enzyme, cooling to room temperature, adding ethanol with a mass 0.5 to 2 times that of the seed flesh, mixing and soaking for 1h - 3h, centrifuging at 4000r / min - 6000r / min for 15min - 20min, taking the supernatant, carrying out ultrafiltration with a 5kDa ultrafiltration membrane, taking the ultrafiltration fraction below 5kDa, and freeze-drying to obtain a powder, thus obtaining the pumpkin seed extract.

[0009] In the above-mentioned pharmaceutical composition, the preparation method of the complex bacterin comprises the following steps: steaming Chinese yams and Chinese dates respectively, and grinding them after cooling to make Chinese yam paste and Chinese date paste; preparing a base liquid according to the mass ratio of water: Chinese yam paste: Chinese date paste: glucose: soy peptone: dimethyl hydrogen phosphate: magnesium sulfate = (90 - 100):(5 - 8):(3 - 5):(1 - 3):(0.5 - 1.5):(0.4 - 0.8):(0.05 - 0.2), adjusting the pH to 6.0 - 7.0, then carrying out high-temperature sterilization treatment, and cooling to room temperature to obtain a culture solution; inoculating 1.5%v / v - 3%v / v of Lactobacillus acidophilus, 1.5%v / v - 3%v / v of Bifidobacterium, and 1.5%v / v - 3%v / v of Lactobacillus casei into the culture solution, carrying out anaerobic fermentation at 35°C - 38°C for 60h - 72h to obtain a fermentation broth; centrifuging the fermentation broth at 5000r / min - 8000r / min for 10min - 20min, collecting the supernatant, carrying out ultrasonic treatment at 20kHz - 50kHz for 3min - 10min, carrying out reduced-pressure concentration at 60°C - 80°C, and drying to obtain a powder at 60°C - 80°C, thus obtaining the complex bacterin.

[0010] In the preparation method of the above-mentioned complex bacterin, the Lactobacillus acidophilus is an activated bacterial liquid with a concentration of 100 million CFU / mL - 1 billion CFU / mL; the Bifidobacterium is an activated bacterial liquid with a concentration of 100 million CFU / mL - 1 billion CFU / mL; the Lactobacillus casei is an activated bacterial liquid with a concentration of 100 million CFU / mL - 1 billion CFU / mL.

[0011] In the above preparation method of the complex bacteriocin, the temperature of the high-temperature sterilization is 121°C to 125°C, and the time of the high-temperature sterilization is 15 min to 30 min.

[0012] In the above drug composition, the preparation method of the white hyacinth bean flower extract comprises the following steps: taking white hyacinth bean flowers, pulverizing them, then performing percolation with ethanol 10 to 15 times the mass of the white hyacinth bean flowers to obtain an extraction solution, concentrating under reduced pressure at 60°C to 80°C, and drying to a powder at 60°C to 80°C to obtain the white hyacinth bean flower extract.

[0013] In the above drug composition, the preparation method of the bletilla striata extract comprises the following steps: taking the tubers of fresh bletilla striata, pulverizing them, adding water 8 to 12 times the mass of the tubers, adjusting the pH to 4.5 to 5.5, successively adding cellulase 0.5% to 2% of the mass of the tubers and pectinase 0.5% to 2% of the mass of the tubers, performing enzymatic hydrolysis at a constant temperature of 50°C to 55°C for 2 h to 3 h, then adjusting the pH to 1.5 to 2.5, adding pepsin 0.5% to 1% of the mass of the tubers, performing enzymatic hydrolysis at 35°C to 38°C for 60 min to 80 min, heating to inactivate the enzyme, cooling to room temperature, adjusting the pH to 6.0 to 7.0, adding bromelain 0.5% to 1% of the mass of the tubers, performing enzymatic hydrolysis at 50°C to 60°C for 50 min to 80 min, heating to inactivate the enzyme, cooling to room temperature, adding serrapeptase 0.5% to 1% of the mass of the tubers, performing enzymatic hydrolysis at 45°C to 50°C for 60 min to 90 min, heating to inactivate the enzyme, cooling to room temperature, adding ethanol 1 to 3 times the mass of the tubers, mixing and soaking for 1 h to 3 h, centrifuging at 4000 r / min to 6000 r / min for 15 min to 20 min, taking the supernatant, performing ultrafiltration with a 10 kDa ultrafiltration membrane, taking the ultrafiltration fraction below 10 kDa, and freeze-drying to a powder to obtain the bletilla striata extract.

[0014] In the above preparation method of each raw material, the temperature for heating to inactivate the enzyme is 85°C to 95°C, and the time for heating to inactivate the enzyme is 15 min to 30 min.

[0015] The preparation method of the above drug composition for treating peptic ulcer comprises the following steps: mixing the pumpkin seed extract, complex bacteriocin, white hyacinth bean flower extract and bletilla striata extract evenly by mass parts, and then adding chicken gizzard-membrane peptide and mixing evenly to obtain the drug composition.

[0016] A drug composition for treating peptic ulcer and its preparation method provided by the present invention have the following beneficial effects:

[0017] 1. The pharmaceutical composition of the present invention contains chicken gizzard-membrane peptide, pumpkin seed extract, complex bacteria, white hyacinth bean flower extract and bletilla striata extract. Among them, chicken gizzard-membrane peptide participates in the intracellular metabolic process, provides raw materials for cell proliferation and differentiation, promotes the regeneration of gastric mucosal epithelial cells, and fills the defect at the ulcer site; peptide substances can also regulate the synthesis of extracellular matrix, strengthen the structural integrity of the gastric mucosa, and play a role in repairing and protecting the gastric mucosa; bind to the receptors related to gastric acid secretion, regulate the gastric acid secretion signaling pathway, keep the gastric acid secretion at a reasonable level, and avoid further damage to the gastric mucosa caused by excessive gastric acid secretion. Pumpkin seed extract can enhance the barrier function of the gastric mucosa, prevent the erosion of gastric acid and pepsin on the gastric mucosa; also has anti-inflammatory properties and reduces gastric inflammation; by affecting the neuroendocrine cells in the gastric mucosa, regulates the release of hormones related to gastric acid secretion, thereby indirectly regulating the amount of gastric acid secretion, reducing the stimulation of gastric acid on the gastric mucosa, and promoting the healing of gastric ulcers. Complex bacteria can inhibit the growth of harmful bacteria, prevent the adhesion of harmful bacteria such as Helicobacter pylori on the surfaces of gastric mucosa and intestinal mucosa, and reduce the damage of toxins produced by harmful bacteria to the gastric mucosa; can provide energy for gastric mucosal cells, promote the metabolism and proliferation of gastric mucosal cells; at the same time, the short-chain fatty acids in it can also regulate the pH value of the intestine, improve the microecological environment of the gastrointestinal tract, and are beneficial to the repair of the gastric mucosa and the normalization of gastrointestinal function. During the occurrence of gastric ulcers, oxidative stress reactions can damage gastric mucosal cells, and white hyacinth bean flower extract can scavenge free radicals and reduce the damage of oxidative stress to the gastric mucosa; form a protective film on the surface of the gastric mucosa, similar to the mucus layer of the gastric mucosa itself, and prevent gastric acid and pepsin from directly contacting the gastric mucosa, thereby playing a role in protecting the gastric mucosa. The components in bletilla striata extract can promote the proliferation and migration of gastric mucosal cells, stimulate the release of growth factors of gastric mucosal cells, activate the proliferation signaling pathway in cells, accelerate the migration of gastric mucosal cells to the ulcer site, and fill the ulcer wound surface; can strengthen the connection between cells by promoting the synthesis of tight junction proteins between gastric mucosal cells, prevent the penetration of gastric acid and harmful substances, and protect the integrity of the gastric mucosa.

[0018] 2. In the preparation method of chicken gizzard-membrane peptide, initially, pepsin is used for enzymatic hydrolysis under acidic conditions. Pepsin can specifically decompose the proteins in chicken gizzard-membrane, generating some larger peptide segments. These peptide segments are further decomposed into smaller peptides during the subsequent enzymatic hydrolysis with chymotrypsin and keratinase. The peptides produced in different enzymatic hydrolysis stages have different amino acid sequences and structures, and play roles in different links of gastric mucosa repair respectively. For example, some peptides promote the adhesion of gastric mucosal cells, and some other peptides participate in intercellular signal transduction and promote cell proliferation. Multiple peptides act synergistically to accelerate the repair of the gastric mucosa.

[0019] Chicken gizzard peptides obtained through multiple enzymatic hydrolysis have multiple antibacterial mechanisms. Positively charged peptides can attract negatively charged components on the surface of Helicobacter pylori (such as lipopolysaccharide, etc.), then insert into the bacterial cell membrane, destroy the integrity of the cell membrane, cause the bacterial contents to leak, and thus inhibit bacterial growth. In addition, some peptides bind to key enzymes or proteins in Helicobacter pylori cells, interfering with their normal metabolic processes.

[0020] 3. In the preparation method of pumpkin seed extract, cellulase and pectinase are first used for enzymatic hydrolysis. These two enzymes can decompose the cellulose and pectin components in the pumpkin seed cell wall, rupture the cell wall, and release various components in the cell. These components include protein, fat, carbohydrates and some biologically active substances. Subsequently, pepsin, nattokinase and aminopeptidase are used for enzymatic hydrolysis. Pepsin can decompose protein into peptides and amino acids. Nattokinase and aminopeptidase further decompose peptides into smaller peptides and free amino acids. These small molecules are more easily absorbed by the human body. The amino acids are used to synthesize the proteins needed for gastric mucosal repair. Peptides have the function of regulating the function of gastric mucosal cells, promoting cell proliferation and differentiation, and thus helping to heal gastric ulcers.

[0021] The small molecule peptides produced after enzymatic hydrolysis of pumpkin seed extract have antibacterial activity. These peptides bind to specific targets on the cell membrane of Helicobacter pylori, change the permeability of the cell membrane, cause imbalance of ions and metabolites inside the bacteria, and inhibit bacterial growth. In addition, some organic acids produced during the enzymatic hydrolysis process also inhibit the growth of Helicobacter pylori.

[0022] 4. In the preparation process of compound bacteria, Chinese yam and red dates serve as fermentation substrates to provide rich nutrition for probiotics. The mucin in Chinese yam provides a good adhesion environment for probiotics, and the sugars in red dates provide a carbon source for probiotic fermentation. During the anaerobic fermentation process, probiotics multiply in large numbers and produce a variety of beneficial metabolites, which inhibit the growth of harmful bacteria, reduce the damage of harmful bacteria to the gastric mucosa, and promote the metabolism and repair of gastric mucosal cells. After colonization in the gastrointestinal tract, probiotics can also regulate the body's immune function. They can stimulate intestinal-associated lymphoid tissue, enhance the activity of immune cells, enable the body to better cope with gastric inflammation, and promote the healing of gastric ulcers.

[0023] The bacteriocin produced by fermentation can specifically act on Helicobacter pylori. Bacteriocins bind to receptors on the cell membrane of Helicobacter pylori to form transmembrane channels, which increase the permeability of the bacterial cell membrane and cause leakage of substances in the cell, thereby inhibiting Helicobacter pylori.

[0024] V. The effective components in the flower of Dolichos lablab L. are extracted by ethanol percolation method, which can enrich flavonoids, phenolic substances, etc. Flavonoids enhance the antioxidant capacity of cells and reduce gastric inflammatory response by regulating the activity of antioxidant enzyme systems in cells, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), etc. The phenolic substances in the extract have anti-inflammatory effects, inhibit the infiltration of inflammatory cells and the release of inflammatory mediators, reduce the damage of inflammation to the gastric mucosa, and promote the repair of the gastric mucosa.

[0025] The flavonoids in the extract of the flower of Dolichos lablab L. interact with the phospholipids on the cell membrane of Helicobacter pylori, changing the fluidity and permeability of the cell membrane. At the same time, the flavonoids also interact with the enzyme system in the bacteria, inhibit the activity of the enzymes, interfere with their energy metabolism, and thus inhibit the growth and reproduction of Helicobacter pylori.

[0026] VI. During the preparation of the extract of Bletilla striata, enzymatic hydrolysis with cellulase and pectinase first can decompose the cell wall of the tuber of Bletilla striata, making the effective components in the cells easier to release. Subsequently, pepsin, bromelain and serrapeptase are used for enzymatic hydrolysis. Pepsin decomposes proteins, and bromelain and serrapeptase can further decompose proteins and peptides, producing more small molecule peptides and amino acids. These specific small molecule substances can promote the proliferation and migration of gastric mucosal cells and accelerate the repair of the gastric mucosa. The components produced during the enzymatic hydrolysis process can also enhance the barrier function of the gastric mucosa, promote gastric mucosal cells to secrete mucus, increase the thickness and viscosity of the mucus layer, prevent the penetration of gastric acid and pepsin, and protect the gastric mucosa from damage.

[0027] The small molecule peptides produced after enzymatic hydrolysis of the extract of Bletilla striata also have antibacterial activity. Some peptides bind to the proteins on the cell membrane of Helicobacter pylori, changing the membrane potential, resulting in depolarization of the cell membrane, making the bacteria unable to carry out normal material exchange and energy metabolism, and thus inhibiting its growth and reproduction. Inhibiting the quorum sensing system of bacteria makes Helicobacter pylori unable to coordinate its group behavior normally, such as biofilm formation and virulence factor secretion, etc., thereby reducing its pathogenicity and reproductive ability. Specific Embodiments

[0028] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to these embodiments.

[0029] Example 1: A pharmaceutical composition for treating peptic gastric ulcer, comprising the following raw materials in parts by mass: 1.5 parts of chicken gizzard peptide, 2 parts of pumpkin seed extract, 1 part of complex bacteriocin, 3.5 parts of extract of the flower of Dolichos lablab L. and 4.5 parts of extract of Bletilla striata.

[0030] Among them, the preparation method of chicken gizzard-membrane peptide comprises the following steps: Take fresh chicken gizzard-membrane, crush it, add distilled water with a mass 10 times that of the chicken gizzard-membrane, adjust the pH to 2, add pepsin at 0.8% of the mass of the chicken gizzard-membrane, carry out enzymatic hydrolysis at 37°C for 70 min, raise the temperature to 90°C to inactivate the enzyme for 20 min, cool to room temperature, adjust the pH to 8.0, add chymotrypsin at 1% of the mass of the chicken gizzard-membrane, carry out enzymatic hydrolysis at 37°C for 60 min, raise the temperature to 90°C to inactivate the enzyme for 20 min, cool to room temperature, add keratinase at 1.5% of the mass of the chicken gizzard-membrane, carry out enzymatic hydrolysis at 50°C for 90 min, raise the temperature to 90°C to inactivate the enzyme for 20 min, cool to room temperature, centrifuge at 5000 r / min for 18 min, take the supernatant, carry out ultrafiltration with a 5 kDa ultrafiltration membrane, take the ultrafiltration fraction below 5 kDa, and freeze-dry to powder to obtain chicken gizzard-membrane peptide.

[0031] Among them, the preparation method of pumpkin seed extract comprises the following steps: Take the seed flesh of pumpkin seeds, crush it, add water with a mass 10 times that of the seed flesh, adjust the pH to 5.2, add cellulase at 1.2% of the mass of the seed flesh and pectinase at 1.4% of the mass of the seed flesh, carry out constant-temperature enzymatic hydrolysis at 52°C for 2.5 h, then adjust the pH to 2.1, add pepsin at 0.8% of the mass of the seed flesh, carry out enzymatic hydrolysis at 37°C for 70 min, raise the temperature to 90°C to inactivate the enzyme for 20 min, cool to room temperature, adjust the pH to 6.8, add nattokinase at 1.2% of the mass of the seed flesh, carry out enzymatic hydrolysis at 52°C for 80 min, raise the temperature to 90°C to inactivate the enzyme for 20 min, cool to room temperature, adjust the pH to 7.2, add aminopeptidase at 1% of the mass of the seed flesh, carry out enzymatic hydrolysis at 40°C for 60 min, raise the temperature to 90°C to inactivate the enzyme for 20 min, cool to room temperature, add ethanol with a mass 1 time that of the seed flesh, mix and soak for 2 h, centrifuge at 5000 r / min for 18 min, take the supernatant, carry out ultrafiltration with a 5 kDa ultrafiltration membrane, take the ultrafiltration fraction below 5 kDa, and freeze-dry to powder to obtain pumpkin seed extract.

[0032] Among them, the preparation method of complex bacteria preparation comprises the following steps: Steam the Chinese yam and red dates respectively, and grind them after cooling to make Chinese yam paste and red date paste; Prepare a base solution according to the mass ratio of water: Chinese yam paste: red date paste: glucose: soy peptone: dimethyl hydrogen phosphate: magnesium sulfate = 95:6.5:4:2:1:0.6:0.1, adjust the pH to 6.5, then carry out high-temperature sterilization treatment at 121°C for 20 min, and after cooling to room temperature, obtain a culture solution; Inoculate the culture solution with an activated bacterial solution of Lactobacillus acidophilus with a concentration of 500 million CFU / mL at 2% v / v, an activated bacterial solution of Bifidobacterium with a concentration of 300 million CFU / mL at 2% v / v, and an activated bacterial solution of Lactobacillus casei with a concentration of 800 million CFU / mL at 2% v / v, carry out anaerobic fermentation at 37°C for 65 h to obtain a fermentation broth; Centrifuge the fermentation broth at 6000 r / min for 15 min, collect the supernatant, carry out ultrasonic treatment at 30 kHz for 5 min, carry out reduced-pressure concentration at 70°C, and dry to powder at 70°C to obtain complex bacteria preparation.

[0033] Among them, the preparation method of the white hyacinth bean flower extract comprises the following steps: take white hyacinth bean flowers, crush them, then perform percolation with ethanol 12 times the mass of the white hyacinth bean flowers to obtain an extract, concentrate under reduced pressure at 70°C, and dry at 70°C to obtain a powder, thereby obtaining the white hyacinth bean flower extract.

[0034] Among them, the preparation method of the bletilla striata extract comprises the following steps: take the tubers of fresh bletilla striata, crush them, add water 10 times the mass of the tubers, adjust the pH to 5.2, sequentially add cellulase 1.5% of the mass of the tubers and pectinase 1.5% of the mass of the tubers, perform enzymatic hydrolysis at a constant temperature of 52°C for 2.5 h, then adjust the pH to 2.0, add pepsin 0.8% of the mass of the tubers, perform enzymatic hydrolysis at 37°C for 70 min, raise the temperature to 90°C to inactivate the enzyme for 20 min, cool to room temperature, adjust the pH to 6.5, add bromelain 0.8% of the mass of the tubers, perform enzymatic hydrolysis at 55°C for 60 min, raise the temperature to 90°C to inactivate the enzyme for 20 min, cool to room temperature, add serrapeptase 0.8% of the mass of the tubers, perform enzymatic hydrolysis at 48°C for 80 min, raise the temperature to 90°C to inactivate the enzyme for 20 min, cool to room temperature, add ethanol 2 times the mass of the tubers, mix and soak for 2 h, centrifuge at 5000 r / min for 18 min, take the supernatant, perform ultrafiltration with a 10 kDa ultrafiltration membrane, take the ultrafiltration fraction below 10 kDa, and freeze-dry to obtain a powder, thereby obtaining the bletilla striata extract.

[0035] The preparation method of the above-mentioned drug composition for treating peptic ulcer comprises the following steps: according to parts by mass, mix the pumpkin seed extract, complex bacteriocin, white hyacinth bean flower extract and bletilla striata extract evenly, and then add chicken gizzard-membrane peptide and mix evenly to obtain the drug composition.

[0036] Example 2: A drug composition for treating peptic ulcer, comprising the following raw materials in parts by mass: 1.0 part of chicken gizzard-membrane peptide, 1.5 parts of pumpkin seed extract, 0.5 part of complex bacteriocin, 2 parts of white hyacinth bean flower extract and 3 parts of bletilla striata extract.

[0037] Among them, the preparation method of chicken gizzard-membrane peptide comprises the following steps: Take fresh chicken gizzard-membrane, crush it, add distilled water with a mass 8 times that of the chicken gizzard-membrane, adjust the pH to 1.5, add pepsin with a mass of 0.5% of the chicken gizzard-membrane, carry out enzymatic hydrolysis at 35°C for 60 min, raise the temperature to 85°C to inactivate the enzyme for 15 min, cool to room temperature, adjust the pH to 7.5, add chymotrypsin with a mass of 0.8% of the chicken gizzard-membrane, carry out enzymatic hydrolysis at 35°C for 50 min, raise the temperature to 85°C to inactivate the enzyme for 15 min, cool to room temperature, add keratinase with a mass of 1% of the chicken gizzard-membrane, carry out enzymatic hydrolysis at 40°C for 80 min, raise the temperature to 85°C to inactivate the enzyme for 15 min, cool to room temperature, centrifuge at 4000 r / min for 15 min, take the supernatant, carry out ultrafiltration with a 5 kDa ultrafiltration membrane, take the ultrafiltration fraction below 5 kDa, and freeze-dry to obtain a powder, thus obtaining chicken gizzard-membrane peptide.

[0038] Among them, the preparation method of pumpkin seed extract comprises the following steps: Take the seed kernel of pumpkin seeds, crush it, add water with a mass 8 times that of the seed kernel, adjust the pH to 4.5, add cellulase with a mass of 0.5% of the seed kernel and pectinase with a mass of 0.5% of the seed kernel, carry out enzymatic hydrolysis at a constant temperature of 50°C for 2 h, then adjust the pH to 1.5, add pepsin with a mass of 0.5% of the seed kernel, carry out enzymatic hydrolysis at 35°C for 60 min, raise the temperature to 85°C to inactivate the enzyme for 15 min, cool to room temperature, adjust the pH to 6.5, add nattokinase with a mass of 0.5% of the seed kernel, carry out enzymatic hydrolysis at 50°C for 60 min, raise the temperature to 85°C to inactivate the enzyme for 15 min, cool to room temperature, adjust the pH to 7.0, add aminopeptidase with a mass of 0.5% of the seed kernel, carry out enzymatic hydrolysis at 37°C for 50 min, raise the temperature to 85°C to inactivate the enzyme for 15 min, cool to room temperature, add ethanol with a mass 0.5 times that of the seed kernel, mix and soak for 1 h, centrifuge at 4000 r / min for 15 min, take the supernatant, carry out ultrafiltration with a 5 kDa ultrafiltration membrane, take the ultrafiltration fraction below 5 kDa, and freeze-dry to obtain a powder, thus obtaining pumpkin seed extract.

[0039] Among them, the preparation method of the complex bacteriocin includes the following steps: steaming Chinese yam and red dates respectively, grinding them after cooling to make Chinese yam puree and red date puree; preparing a basal solution according to the mass ratio of water: Chinese yam puree: red date puree: glucose: soy peptone: dimethyl hydrogen phosphate: magnesium sulfate = 90:5:3:1:0.5:0.4:0.05, adjusting the pH to 6.0, then performing high-temperature sterilization treatment at 121 °C for 15 min, and cooling to room temperature to obtain a culture solution; inoculating the culture solution with an activated lactobacillus acidophilus solution with a concentration of 600 million CFU / mL at 1.5% v / v, an activated bifidobacterium solution with a concentration of 200 million CFU / mL at 1.5% v / v, and an activated lactobacillus casei solution with a concentration of 100 million CFU / mL at 1.5% v / v, and performing anaerobic fermentation at 35 °C for 60 h to obtain a fermentation broth; centrifuging the fermentation broth at 5000 r / min for 10 min, collecting the supernatant, performing ultrasonic treatment at 20 kHz for 3 min, concentrating under reduced pressure at 60 °C, and drying to powder at 60 °C to obtain the complex bacteriocin.

[0040] Among them, the preparation method of the white hyacinth bean flower extract includes the following steps: taking white hyacinth bean flowers, crushing them, and then performing percolation with ethanol with a mass 10 times that of the white hyacinth bean flowers to obtain an extraction solution, concentrating under reduced pressure at 60 °C, and drying to powder at 60 °C to obtain the white hyacinth bean flower extract.

[0041] Among them, the preparation method of the bletilla striata extract includes the following steps: taking the tubers of fresh bletilla striata, crushing them, adding water with a mass 8 times that of the tubers, adjusting the pH to 4.5, sequentially adding 0.5% of cellulase based on the mass of the tubers and 0.5% of pectinase based on the mass of the tubers, performing enzymatic hydrolysis at a constant temperature of 50 °C for 2 h, then adjusting the pH to 1.5, adding 0.5% of pepsin based on the mass of the tubers, performing enzymatic hydrolysis at 35 °C for 60 min, raising the temperature to 85 °C to inactivate the enzyme for 15 min, cooling to room temperature, adjusting the pH to 6.0, adding 0.5% of bromelain based on the mass of the tubers, performing enzymatic hydrolysis at 50 °C for 50 min, raising the temperature to 85 °C to inactivate the enzyme for 15 min, cooling to room temperature, adding 0.5% of serrapeptase based on the mass of the tubers, performing enzymatic hydrolysis at 45 °C for 60 min, raising the temperature to 85 °C to inactivate the enzyme for 15 min, cooling to room temperature, adding ethanol with a mass 1 time that of the tubers, mixing and soaking for 1 h, centrifuging at 4000 r / min for 15 min, taking the supernatant, performing ultrafiltration with a 10 kDa ultrafiltration membrane, taking the ultrafiltration fraction below 10 kDa, and freeze-drying to powder to obtain the bletilla striata extract.

[0042] The preparation method of the above-mentioned pharmaceutical composition for treating peptic ulcer includes the following steps: mixing the pumpkin seed extract, complex bacteriocin, white hyacinth bean flower extract and bletilla striata extract evenly according to mass parts, and then adding chicken gizzard peptide and mixing evenly to obtain the pharmaceutical composition.

[0043] Example 3: A pharmaceutical composition for treating peptic gastric ulcer, comprising the following raw materials in parts by mass: 1.0 part of gallus gallus domesticus gizzard peptide, 2.5 parts of pumpkin seed extract, 0.5 part of complex bacterin, 5 parts of white hyacinth bean flower extract, and 3 parts of bletilla striata extract.

[0044] Among them, the preparation method of gallus gallus domesticus gizzard peptide comprises the following steps: Take fresh gallus gallus domesticus gizzard, crush it, add distilled water with a mass 12 times that of the gallus gallus domesticus gizzard, adjust the pH to 1.5, add pepsin accounting for 1% of the mass of the gallus gallus domesticus gizzard, carry out enzymatic hydrolysis at 35 °C for 80 min, raise the temperature to 85 °C to inactivate the enzyme for 30 min, cool to room temperature, adjust the pH to 7.5, add chymotrypsin accounting for 1.5% of the mass of the gallus gallus domesticus gizzard, carry out enzymatic hydrolysis at 35 °C for 70 min, raise the temperature to 85 °C to inactivate the enzyme for 30 min, cool to room temperature, add keratinase accounting for 1% of the mass of the gallus gallus domesticus gizzard, carry out enzymatic hydrolysis at 60 °C for 80 min, raise the temperature to 95 °C to inactivate the enzyme for 15 min, cool to room temperature, centrifuge at 6000 r / min for 15 min, take the supernatant, carry out ultrafiltration with a 5 kDa ultrafiltration membrane, take the ultrafiltration fraction below 5 kDa, and freeze-dry it to powder to obtain gallus gallus domesticus gizzard peptide.

[0045] Among them, the preparation method of pumpkin seed extract comprises the following steps: Take the seed flesh of pumpkin seeds, crush it, add water with a mass 12 times that of the seed flesh, adjust the pH to 4.5, add cellulase accounting for 2% of the mass of the seed flesh and pectinase accounting for 0.5% of the mass of the seed flesh, carry out constant-temperature enzymatic hydrolysis at 55 °C for 2 h, then adjust the pH to 2.5, add pepsin accounting for 0.5% of the mass of the seed flesh, carry out enzymatic hydrolysis at 38 °C for 60 min, raise the temperature to 95 °C to inactivate the enzyme for 15 min, cool to room temperature, adjust the pH to 7.0, add nattokinase accounting for 0.5% of the mass of the seed flesh, carry out enzymatic hydrolysis at 55 °C for 60 min, raise the temperature to 95 °C to inactivate the enzyme for 15 min, cool to room temperature, adjust the pH to 7.5, add aminopeptidase accounting for 0.5% of the mass of the seed flesh, carry out enzymatic hydrolysis at 45 °C for 50 min, raise the temperature to 95 °C to inactivate the enzyme for 15 min, cool to room temperature, add ethanol with a mass 2 times that of the seed flesh, mix and soak for 1 h, centrifuge at 6000 r / min for 15 min, take the supernatant, carry out ultrafiltration with a 5 kDa ultrafiltration membrane, take the ultrafiltration fraction below 5 kDa, and freeze-dry it to powder to obtain pumpkin seed extract.

[0046] Among them, the preparation method of the complex bacteriocin includes the following steps: steaming Chinese yams and red dates respectively, grinding them after cooling to make Chinese yam puree and red date puree; preparing a base solution according to the mass ratio of water: Chinese yam puree: red date puree: glucose: soy peptone: dimethyl hydrogen phosphate: magnesium sulfate = 100:5:5:1:1.5:0.4:0.2, adjusting the pH to 6.0, then performing high-temperature sterilization treatment at 123 °C for 15 min, and after cooling to room temperature, obtaining a culture solution; inoculating the culture solution with 3% v / v of an activated lactobacillus acidophilus solution with a concentration of 100 million CFU / mL, 1.5% v / v of an activated bifidobacterium solution with a concentration of 1 billion CFU / mL, and 3% v / v of an activated lactobacillus casei solution with a concentration of 400 million CFU / mL, performing anaerobic fermentation at 35 °C for 72 h to obtain a fermentation broth; centrifuging the fermentation broth at 5000 r / min for 20 min, collecting the supernatant, performing ultrasonic treatment at 20 kHz for 10 min, performing reduced-pressure concentration at 60 °C, and drying to a powder at 80 °C to obtain the complex bacteriocin.

[0047] Among them, the preparation method of the white hyacinth bean flower extract includes the following steps: taking white hyacinth bean flowers, crushing them, and then performing percolation with ethanol with a mass 10 times that of the white hyacinth bean flowers to obtain an extract, performing reduced-pressure concentration at 80 °C, and drying to a powder at 60 °C to obtain the white hyacinth bean flower extract.

[0048] Among them, the preparation method of the bletilla striata extract includes the following steps: taking the tubers of fresh bletilla striata, crushing them, adding water with a mass 12 times that of the tubers, adjusting the pH to 4.5, successively adding 2% of the tuber mass of cellulase and 0.5% of the tuber mass of pectinase, performing constant-temperature enzymatic hydrolysis at 55 °C for 2 h, then adjusting the pH to 2.5, adding 0.5% of the tuber mass of pepsin, performing enzymatic hydrolysis at 38 °C for 60 min, raising the temperature to 95 °C to inactivate the enzyme for 15 min, cooling to room temperature, adjusting the pH to 7.0, adding 0.5% of the tuber mass of bromelain, performing enzymatic hydrolysis at 60 °C for 50 min, raising the temperature to 95 °C to inactivate the enzyme for 15 min, cooling to room temperature, adding 1% of the tuber mass of serrapeptase, performing enzymatic hydrolysis at 45 °C for 90 min, raising the temperature to 85 °C to inactivate the enzyme for 30 min, cooling to room temperature, adding ethanol with a mass 1 time that of the tubers, mixing and soaking for 3 h, centrifuging at 4000 r / min for 20 min, taking the supernatant, performing ultrafiltration with a 10 kDa ultrafiltration membrane, taking the ultrafiltration fraction below 10 kDa, and freeze-drying to a powder to obtain the bletilla striata extract.

[0049] The preparation method of the above-mentioned pharmaceutical composition for treating peptic gastric ulcer includes the following steps: mixing the pumpkin seed extract, the complex bacteriocin, the white hyacinth bean flower extract, and the bletilla striata extract evenly by mass fraction, and then adding chicken gizzard peptide and mixing evenly to obtain the pharmaceutical composition.

[0050] Example 4: A pharmaceutical composition for treating peptic gastric ulcer, comprising the following raw materials in parts by mass: 2.0 parts of gallus gallus domesticus gizzard peptide, 2.5 parts of pumpkin seed extract, 1.5 parts of complex bacteria, 5 parts of dolichos lablab flower extract, and 6 parts of bletilla striata extract.

[0051] Among them, the preparation method of gallus gallus domesticus gizzard peptide comprises the following steps: Take fresh gallus gallus domesticus gizzard, crush it, add distilled water with a mass 12 times that of the gallus gallus domesticus gizzard, adjust the pH to 2.5, add pepsin accounting for 1% of the mass of the gallus gallus domesticus gizzard, carry out enzymatic hydrolysis at 38 °C for 80 min, raise the temperature to 95 °C to inactivate the enzyme for 15 min, cool to room temperature, adjust the pH to 8.5, add chymotrypsin accounting for 1.5% of the mass of the gallus gallus domesticus gizzard, carry out enzymatic hydrolysis at 38 °C for 70 min, raise the temperature to 95 °C to inactivate the enzyme for 15 min, cool to room temperature, add keratinase accounting for 2% of the mass of the gallus gallus domesticus gizzard, carry out enzymatic hydrolysis at 60 °C for 100 min, raise the temperature to 95 °C to inactivate the enzyme for 15 min, cool to room temperature, centrifuge at 6000 r / min for 20 min, take the supernatant, carry out ultrafiltration with a 5 kDa ultrafiltration membrane, take the ultrafiltration fraction below 5 kDa, and freeze-dry it to powder to obtain gallus gallus domesticus gizzard peptide.

[0052] Among them, the preparation method of pumpkin seed extract comprises the following steps: Take the seed meat of pumpkin seeds, crush it, add water with a mass 12 times that of the seed meat, adjust the pH to 5.5, add cellulase accounting for 2% of the mass of the seed meat and pectinase accounting for 2% of the mass of the seed meat, carry out constant-temperature enzymatic hydrolysis at 55 °C for 3 h, then adjust the pH to 2.5, add pepsin accounting for 1% of the mass of the seed meat, carry out enzymatic hydrolysis at 38 °C for 80 min, raise the temperature to 95 °C to inactivate the enzyme for 15 min, cool to room temperature, adjust the pH to 7.0, add nattokinase accounting for 1.5% of the mass of the seed meat, carry out enzymatic hydrolysis at 55 °C for 90 min, raise the temperature to 95 °C to inactivate the enzyme for 15 min, cool to room temperature, adjust the pH to 7.5, add aminopeptidase accounting for 1.5% of the mass of the seed meat, carry out enzymatic hydrolysis at 45 °C for 70 min, raise the temperature to 95 °C to inactivate the enzyme for 15 min, cool to room temperature, add ethanol with a mass 2 times that of the seed meat, mix and soak for 3 h, centrifuge at 6000 r / min for 20 min, take the supernatant, carry out ultrafiltration with a 5 kDa ultrafiltration membrane, take the ultrafiltration fraction below 5 kDa, and freeze-dry it to powder to obtain pumpkin seed extract.

[0053] Among them, the preparation method of the complex bacteriocin includes the following steps: Steam the Chinese yam and red dates respectively, grind them after cooling to make Chinese yam puree and red date puree; Prepare a base solution according to the mass ratio of water: Chinese yam puree: red date puree: glucose: soy peptone: dimethyl hydrogen phosphate: magnesium sulfate = 100:8:5:3:1.5:0.8:0.2, adjust the pH to 7.0, then perform high-temperature sterilization treatment at 125°C for 30 min, and after cooling to room temperature, obtain a culture solution; Inoculate the culture solution with 3% v / v of an activated Lactobacillus acidophilus bacterial solution with a concentration of 1 billion CFU / mL, 3% v / v of an activated Bifidobacterium bacterial solution with a concentration of 800 million CFU / mL, and 3% v / v of an activated Lactobacillus casei bacterial solution with a concentration of 600 million CFU / mL, and perform anaerobic fermentation at 38°C for 72 h to obtain a fermentation broth; Centrifuge the fermentation broth at 8000 r / min for 20 min, collect the supernatant, perform ultrasonic treatment at 50 kHz for 10 min, concentrate under reduced pressure at 80°C, and dry to powder at 80°C to obtain the complex bacteriocin.

[0054] Among them, the preparation method of the white hyacinth bean flower extract includes the following steps: Take the white hyacinth bean flower, crush it, and then perform percolation with ethanol 15 times the mass of the white hyacinth bean flower to obtain an extraction solution, concentrate under reduced pressure at 80°C, and dry to powder at 80°C to obtain the white hyacinth bean flower extract.

[0055] Among them, the preparation method of the Bletilla striata extract includes the following steps: Take the fresh tubers of Bletilla striata, crush them, add water 12 times the mass of the tubers, adjust the pH to 5.5, sequentially add 2% of the mass of the tubers of cellulase and 2% of the mass of the tubers of pectinase, perform enzymatic hydrolysis at a constant temperature of 55°C for 3 h, then adjust the pH to 2.5, add 1% of the mass of the tubers of pepsin, perform enzymatic hydrolysis at 38°C for 80 min, raise the temperature to 95°C to inactivate the enzyme for 15 min, cool to room temperature, adjust the pH to 7.0, add 1% of the mass of the tubers of bromelain, perform enzymatic hydrolysis at 60°C for 80 min, raise the temperature to 95°C to inactivate the enzyme for 15 min, cool to room temperature, add 1% of the mass of the tubers of serrapeptase, perform enzymatic hydrolysis at 50°C for 90 min, raise the temperature to 95°C to inactivate the enzyme for 15 min, cool to room temperature, add ethanol 3 times the mass of the tubers, mix and soak for 3 h, centrifuge at 6000 r / min for 20 min, take the supernatant, perform ultrafiltration with a 10 kDa ultrafiltration membrane, take the ultrafiltration fraction below 10 kDa, and freeze-dry to powder to obtain the Bletilla striata extract.

[0056] The preparation method of the above-mentioned pharmaceutical composition for treating peptic ulcer includes the following steps: Mix the pumpkin seed extract, complex bacteriocin, white hyacinth bean flower extract, and Bletilla striata extract evenly by mass fraction, and then add chicken gizzard peptide and mix evenly to obtain the pharmaceutical composition.

[0057] Example 5: A pharmaceutical composition for treating peptic gastric ulcer, comprising the following raw materials in parts by mass: 2.0 parts of gallus gallus domesticus gizzard peptides, 1.5 parts of pumpkin seed extract, 1.5 parts of complex bacteria, 2 parts of white hyacinth bean flower extract, and 6 parts of bletilla striata extract.

[0058] Among them, the preparation method of gallus gallus domesticus gizzard peptides comprises the following steps: Take fresh gallus gallus domesticus gizzard, crush it, add distilled water with a mass 8 times that of the gallus gallus domesticus gizzard, adjust the pH to 2.5, add pepsin with a mass of 0.5% of the gallus gallus domesticus gizzard, carry out enzymatic hydrolysis at 38 °C for 60 min, raise the temperature to 95 °C to inactivate the enzyme for 15 min, cool to room temperature, adjust the pH to 8.5, add chymotrypsin with a mass of 0.8% of the gallus gallus domesticus gizzard, carry out enzymatic hydrolysis at 38 °C for 50 min, raise the temperature to 95 °C to inactivate the enzyme for 15 min, cool to room temperature, add keratinase with a mass of 2% of the gallus gallus domesticus gizzard, carry out enzymatic hydrolysis at 40 °C for 100 min, raise the temperature to 85 °C to inactivate the enzyme for 30 min, cool to room temperature, centrifuge at 4000 r / min for 20 min, take the supernatant, carry out ultrafiltration with a 5 kDa ultrafiltration membrane, take the ultrafiltration fraction below 5 kDa, and freeze-dry it to a powder to obtain gallus gallus domesticus gizzard peptides.

[0059] Among them, the preparation method of pumpkin seed extract comprises the following steps: Take the seed flesh of pumpkin seeds, crush it, add water with a mass 8 times that of the seed flesh, adjust the pH to 5.5, add cellulase with a mass of 0.5% of the seed flesh and pectinase with a mass of 2% of the seed flesh, carry out constant-temperature enzymatic hydrolysis at 50 °C for 3 h, then adjust the pH to 1.5, add pepsin with a mass of 1% of the seed flesh, carry out enzymatic hydrolysis at 35 °C for 80 min, raise the temperature to 85 °C to inactivate the enzyme for 30 min, cool to room temperature, adjust the pH to 6.5, add nattokinase with a mass of 1.5% of the seed flesh, carry out enzymatic hydrolysis at 50 °C for 90 min, raise the temperature to 85 °C to inactivate the enzyme for 30 min, cool to room temperature, adjust the pH to 7.0, add aminopeptidase with a mass of 1.5% of the seed flesh, carry out enzymatic hydrolysis at 37 °C for 70 min, raise the temperature to 85 °C to inactivate the enzyme for 30 min, cool to room temperature, add ethanol with a mass 0.5 times that of the seed flesh, mix and soak for 3 h, centrifuge at 4000 r / min for 20 min, take the supernatant, carry out ultrafiltration with a 5 kDa ultrafiltration membrane, take the ultrafiltration fraction below 5 kDa, and freeze-dry it to a powder to obtain pumpkin seed extract.

[0060] Among them, the preparation method of the complex bacteriocin includes the following steps: steaming Chinese yam and red dates respectively, grinding them after cooling to make Chinese yam puree and red date puree; preparing a base solution according to the mass ratio of water: Chinese yam puree: red date puree: glucose: soy peptone: dimethyl hydrogen phosphate: magnesium sulfate = 90:8:3:3:0.5:0.8:0.05, adjusting the pH to 7.0, then performing high-temperature sterilization treatment at 121°C for 30 min, and after cooling to room temperature, obtaining a culture solution; inoculating the culture solution with 1.5% v / v of an activated Lactobacillus acidophilus bacterium solution with a concentration of 700 million CFU / mL, 3% v / v of an activated Bifidobacterium bacterium solution with a concentration of 100 million CFU / mL, and 1.5% v / v of an activated Lactobacillus casei bacterium solution with a concentration of 1 billion CFU / mL, performing anaerobic fermentation at 38°C for 60 h to obtain a fermentation broth; centrifuging the fermentation broth at 8000 r / min for 10 min, collecting the supernatant, performing ultrasonic treatment at 50 kHz for 3 min, performing reduced-pressure concentration at 80°C, and drying at 60°C to a powder to obtain the complex bacteriocin.

[0061] Among them, the preparation method of the white hyacinth bean flower extract includes the following steps: taking white hyacinth bean flowers, crushing them, and then performing percolation with ethanol 15 times the mass of the white hyacinth bean flowers to obtain an extraction solution, performing reduced-pressure concentration at 60°C, and drying at 80°C to a powder to obtain the white hyacinth bean flower extract.

[0062] Among them, the preparation method of the Bletilla striata extract includes the following steps: taking the fresh tubers of Bletilla striata, crushing them, adding water 8 times the mass of the tubers, adjusting the pH to 5.5, sequentially adding 0.5% of the mass of the tubers of cellulase and 2% of the mass of the tubers of pectinase, performing enzymatic hydrolysis at a constant temperature of 50°C for 3 h, then adjusting the pH to 1.5, adding 1% of the mass of the tubers of pepsin, performing enzymatic hydrolysis at 35°C for 80 min, raising the temperature to 85°C to inactivate the enzyme for 30 min, cooling to room temperature, adjusting the pH to 6.0, adding 1% of the mass of the tubers of bromelain, performing enzymatic hydrolysis at 50°C for 80 min, raising the temperature to 85°C to inactivate the enzyme for 30 min, cooling to room temperature, adding 0.5% of the mass of the tubers of serrapeptase, performing enzymatic hydrolysis at 50°C for 60 min, raising the temperature to 95°C to inactivate the enzyme for 15 min, cooling to room temperature, adding ethanol 3 times the mass of the tubers, mixing and soaking for 1 h, centrifuging at 6000 r / min for 15 min, taking the supernatant, performing ultrafiltration with a 10 kDa ultrafiltration membrane, taking the ultrafiltration fraction below 10 kDa, and freeze-drying to a powder to obtain the Bletilla striata extract.

[0063] The preparation method of the above-mentioned pharmaceutical composition for treating peptic ulcer includes the following steps: mixing the pumpkin seed extract, complex bacteriocin, white hyacinth bean flower extract, and Bletilla striata extract evenly by mass, and then adding chicken gizzard peptide and mixing evenly to obtain the pharmaceutical composition.

[0064] In each of the above embodiments, the pH regulator is an aqueous hydrochloric acid solution with a concentration of 0.4 mol / L and an aqueous sodium hydroxide solution with a concentration of 0.4 mol / L.

[0065] Sources of the enzymes and bacteria used in each of the above embodiments: Pepsin is of the aspartic acid type and is sourced from Xi'an Mugo Biotechnology Co., Ltd., with an enzyme activity of 3000 NFU / g; Chymotrypsin is sourced from Hubei Dibo Chemical Co., Ltd., with an enzyme activity of 4000 NFU / g; Keratinase is sourced from Qingdao Haiweisen Biotechnology Co., Ltd., with an enzyme activity of 200,000 NFU / g; Cellulase is sourced from Henan Anrui Biotechnology Co., Ltd., with an enzyme activity of 100,000 NFU / g; Pectinase is sourced from Shandong Zheque Bioengineering Co., Ltd., with an enzyme activity of 100,000 NFU / g; Nattokinase is sourced from Xi'an Wanfang Biotechnology Co., Ltd., with an enzyme activity of 20,000 NFU / g; Aminopeptidase is sourced from Henan Anrui Biotechnology Co., Ltd., with an enzyme activity of 100,000 NFU / g; Bromelain is sourced from Zhongtai Biotechnology (Shanxi) Co., Ltd., with an enzyme activity of 100,000 NFU / g; Serratiopeptidase is sourced from Jinhua Qianxun Biotechnology Co., Ltd., with an enzyme activity of 100,000 NFU / g; Lactobacillus acidophilus is sourced from Shandong Pingo Pharmaceutical Co., Ltd.; Bifidobacterium is sourced from Xi'an Dongchi Biotechnology Co., Ltd.; Lactobacillus casei is sourced from Xi'an Bainenkang Biotechnology Co., Ltd.; Soybean peptone is sourced from Shandong Xinzhuoyuan Biotechnology Co., Ltd.

[0066] Comparative Example 1

[0067] The chicken gizzard membrane peptide is replaced with the freeze-dried powder of sterilized crushed chicken gizzard membrane; other parameters and methods are the same as in Example 1.

[0068] Comparative Example 2

[0069] In the preparation method of the chicken gizzard membrane peptide, chymotrypsin is not used for enzymatic hydrolysis; other parameters and methods are the same as in Example 1.

[0070] Comparative Example 3

[0071] In the preparation method of the chicken gizzard membrane peptide, keratinase is not used for enzymatic hydrolysis; other parameters and methods are the same as in Example 1.

[0072] Comparative Example 4

[0073] In the preparation method of the chicken gizzard membrane peptide, keratinase is used for enzymatic hydrolysis first, and then chymotrypsin is used for enzymatic hydrolysis; other parameters and methods are the same as in Example 1.

[0074] Comparative Example 5

[0075] The pumpkin seed extract is replaced with the crushed dry powder of pumpkin seeds; other parameters and methods are the same as in Example 1.

[0076] Comparative Example 6

[0077] In the preparation method of pumpkin seed extract, nattokinase is not used for enzymatic hydrolysis; other parameters and methods are the same as those in Example 1.

[0078] Comparative Example 7

[0079] In the preparation method of pumpkin seed extract, aminopeptidase is not used for enzymatic hydrolysis; other parameters and methods are the same as those in Example 1.

[0080] Comparative Example 8

[0081] In the preparation method of pumpkin seed extract, aminopeptidase is first used for enzymatic hydrolysis, and then nattokinase is used for enzymatic hydrolysis; other parameters and methods are the same as those in Example 1.

[0082] Comparative Example 9

[0083] In the pharmaceutical composition, amylomyces is used to replace complex bacteriocin; other parameters and methods are the same as those in Example 1.

[0084] Comparative Example 10

[0085] In the pharmaceutical composition, the extract of white hyacinth bean flower is replaced by starch; other parameters and methods are the same as those in Example 1.

[0086] Comparative Example 11

[0087] The extract of Bletilla striata is replaced by the pulverized dry powder of Bletilla striata; other parameters and methods are the same as those in Example 1.

[0088] Comparative Example 12

[0089] In the preparation method of the extract of Bletilla striata, bromelain is not used for enzymatic hydrolysis; other parameters and methods are the same as those in Example 1.

[0090] Comparative Example 13

[0091] In the preparation method of the extract of Bletilla striata, serrapeptase is not used for enzymatic hydrolysis; other parameters and methods are the same as those in Example 1.

[0092] Comparative Example 14

[0093] In the preparation method of the extract of Bletilla striata, serrapeptase is first used for enzymatic hydrolysis, and then bromelain is used for enzymatic hydrolysis; other parameters and methods are the same as those in Example 1.

[0094] The pharmaceutical compositions prepared in the above examples and comparative examples were subjected to safety and efficacy tests.

[0095] I. Safety Test

[0096] (1) Acute toxicity test: 200 healthy adult Kunming mice weighing 18 - 22 g were selected and randomly divided into 20 groups (10 mice in each group, with an equal number of males and females). One group was used as the blank control group and was given normal saline by gavage. The remaining groups were respectively given the drug composition at a dose of 2.5 mg / 20 g body weight to the mice by gavage. The mice were continuously observed for 14 days to observe the poisoning manifestations, including behavior, appearance, and secretions, etc. The results showed that no acute poisoning symptoms occurred in the test mice of each group.

[0097] (2) Long - term toxicity test: 200 healthy adult Kunming mice weighing 18 - 22 g were selected and randomly divided into 20 groups (10 mice in each group, with an equal number of males and females). One group was used as the blank control group and was given normal saline by gavage. The remaining groups were respectively given the drug composition at a dose of 0.5 mg / 20 g body weight to the mice by gavage. The drug was administered continuously for 120 days, once a day. During the administration period, the general conditions of the mice, including behavior, appearance, and secretions, etc., were observed. After the test ended, the mice were sacrificed, and histopathological examinations were performed on the organs of the mice, such as the heart, liver, spleen, lungs, and kidneys. The results showed that the behavior of the mice was normal, and no obvious pathological changes were found in the pathological examinations of each organ, indicating that the safety of each composition was good.

[0098] (3) Allergic reaction test: 60 healthy adult guinea pigs were selected and randomly divided into 20 groups (3 guinea pigs in each group). One group was used as the blank control group, and an active systemic anaphylaxis test for guinea pigs was carried out. On the 0th day, 7th day, and 14th day, 5 mL of the composition samples of each example and comparative example (the composition concentration was 0.05 mg / ml, and the solvent was water) were given to the guinea pigs by gavage. The blank control group was given an equal amount of normal saline by gavage; on the 21st day, 5 mL of the composition sample (the composition concentration was 0.5 mg / ml) was given to the guinea pigs by gavage for challenge. The blank control group was given an equal amount of normal saline by gavage. After 24 hours, 48 hours, and 72 hours of challenge administration, the reactions of the guinea pigs were observed. The results showed that the guinea pigs did not show allergic symptoms such as piloerection, sneezing, dyspnea, convulsions, shock, etc. The composition components were safe and there was no allergic reaction.

[0099] II. Efficacy detection

[0100] Establishment of gastric ulcer animal model: 210 healthy adult SD rats were selected and randomly divided into 21 groups (10 rats in each group). One group was used as the positive control group, and one group was used as the negative control group. After fasting for 24 hours, 0.6 mg / kg of absolute ethanol was injected intraperitoneally.

[0101] Rats in each experimental group and control group were intragastrically administered with 0.35 mg / 20 g body weight of the composition every day. Rats in the negative control group were intragastrically administered with an equal amount of normal saline, and rats in the positive control group were intragastrically administered with 0.35 mg / 20 g body weight of ranitidine, three times a day. After 14 consecutive days of administration, the rats were sacrificed. After removing the stomach: (1) Observe the healing of gastric ulcers, measure the ulcer area, and calculate the ulcer degree. Ulcer degree = ulcer area / total gastric mucosa area × 100%; (2) Detect the pH of gastric juice. The results are shown in Table 1 below.

[0102] III. In vitro antibacterial test against Helicobacter pylori:

[0103] Helicobacter pylori (ATCC43504) was cultured in broth medium at 37 °C for 24 h. After centrifugation, the supernatant was taken. After mixing the supernatant, several 5-mL aliquots of the supernatant were taken and 0.1 g of each drug composition sample was added respectively. Three parallel samples were prepared for each. And 0.1 g of purified water was used as the negative control group (three parallel samples); then it was cultured at 37 °C for 30 min; the number of bacteria in the culture solution was measured by the plate method, and the antibacterial rate was calculated: Antibacterial rate = (number of bacteria in the negative control group - number of bacteria in the test group) / number of bacteria in the negative control group × 100%; The results are shown in Table 1 below.

[0104] Table 1 Results of efficacy detection (average value)

[0105]

[0106] From the above experimental results, it can be seen that the compositions prepared in Examples 1 to 5 are safe in composition, without toxicity and allergic reactions; compared with the negative control group, they can significantly reduce the degree of gastric mucosa ulcer, and the effect is equivalent to that of the positive control group. They can significantly protect the gastric mucosa and improve the efficacy of gastric ulcer, without side effects.

[0107] From the results of Comparative Example 1, it can be seen that the chicken gizzard-membrane peptide was replaced by the pulverized dry powder of chicken gizzard-membrane, and the keratin contained in the pulverized dry powder of chicken gizzard-membrane was not removed. Keratin has strong toughness and poor solubility, and the treatment effect of gastric ulcer is poor; while the chicken gizzard-membrane peptide removes keratin with keratinase, and keratinase can cooperate with other proteases to enzymatically hydrolyze to obtain various specific small peptides, which can play a better role in promoting gastric mucosa repair, regulating gastric acid secretion, and inhibiting the reproduction of Helicobacter pylori.

[0108] From the results of Comparative Examples 2 to 4, it can be seen that in the preparation method of the chicken gizzard-membrane peptide, if chymotrypsin or keratinase is not used for enzymatic hydrolysis, or the use order of the two enzymes is changed, the composition of the extracted product changes, and the effect of the composition in repairing the gastric mucosa and improving gastric ulcer decreases.

[0109] From the results of Comparative Example 5, it can be seen that when the pumpkin seed extract is replaced with crushed dry powder of pumpkin seeds, the crushed dry powder of pumpkin seeds contains macromolecular components and cellulose, etc., which are not conducive to gastrointestinal absorption; while the macromolecular components and cellulose are removed from the pumpkin seed extract, which can reduce adverse reactions, and can obtain more effective specific small molecule peptide components, enhance the overall therapeutic effect of the drug on gastric ulcer, inhibit the reproduction of Helicobacter pylori, synergistically promote the repair of gastric mucosa with other active ingredients, can assist in regulating gastric acid secretion, and enhance the protective effect of gastric mucosa.

[0110] From the results of Comparative Example 6 to Comparative Example 8, it can be seen that in the preparation method of the pumpkin seed extract, nattokinase or aminopeptidase is not used for enzymatic hydrolysis, or the usage order of the two enzymes is changed, and the components of the obtained product are changed, and the effect of the composition in repairing gastric mucosa and improving gastric ulcer decreases.

[0111] From the results of Comparative Example 9, it can be seen that the complex bacterin is made by special fermentation substrates and fermentation of various probiotics. The bacterin can help regulate the normal physiological functions of the gastrointestinal tract, thus indirectly promoting the healing of gastric ulcer and protecting the gastric mucosa. The bacterin can also inhibit the reproduction of Helicobacter pylori; in the pharmaceutical composition, when the complex bacterin is replaced with starch, the therapeutic efficacy of the complex bacterin is lost.

[0112] From the results of Comparative Example 10, it can be seen that the extract of white hyacinth bean flower helps to maintain the normal physiological state of the gastrointestinal tract, has anti-inflammatory and antioxidant effects, can enhance the repair function of other components in the composition on the gastric mucosa, and inhibit the reproduction of Helicobacter pylori; when the extract of white hyacinth bean flower in the pharmaceutical composition is replaced with starch, the therapeutic efficacy of the complex bacterin is lost.

[0113] From the results of Comparative Example 11, it can be seen that when the extract of Bletilla striata is replaced with crushed dry powder of Bletilla striata, the crushed dry powder of Bletilla striata contains macromolecular components, is not easy to absorb, and has a poor effect; the extract of Bletilla striata obtained by specific enzymatic hydrolysis has more efficient components, has better effects of arresting bleeding by astringents and promoting granulation for discharging pus, promotes the repair of gastric mucosa, reduces bleeding, enhances the gastric mucosal barrier function, regulates gastric acid secretion, thereby reducing gastric inflammation and accelerating the healing of gastric ulcer, and the enzymolysis components also have a certain effect of inhibiting the reproduction of Helicobacter pylori.

[0114] From the results of Comparative Example 12 to 14, it can be seen that in the preparation method of the extract of Bletilla striata, bromelain or serrapeptase is not used for enzymatic hydrolysis, or the usage order of the two enzymes is changed, and the components of the obtained product are changed, and the effect of the composition in repairing gastric mucosa and improving gastric ulcer decreases.

Claims

1. A pharmaceutical composition for treating peptic gastric ulcer, characterized in that: The pharmaceutical composition comprises the following raw materials in parts by weight: 1.0 to 2.0 parts of chicken endogenous peptide, 1.5 to 2.5 parts of pumpkin seed extract, 0.5 to 1.5 parts of compound bacteriocin, 2 to 5 parts of white lentil flower extract and 3 to 6 parts of bletilla striata extract; The chicken gizzard peptide contains products below 5 kDa obtained by sequentially enzymolysis of chicken gizzard with pepsin, chymotrypsin and keratinase; the pumpkin seed extract contains products below 5 kDa obtained by sequentially enzymolysis of pumpkin seeds with cellulase and pectinase, and then sequentially enzymolysis of pepsin, nattokinase and aminopeptidase; The composite bacteriocin contains co-fermented bacteriocin of Lactobacillus acidophilus, Bifidobacterium and Lactobacillus casei; the white bean flower extract is an ethanol percolation extract of white bean flower; the bletilla striata extract contains a product below 10 kDa obtained by co-enzymatic hydrolysis of bletilla striata tubers with cellulase and pectinase, followed by graded enzymatic hydrolysis with pepsin, bromelain and serrapeptase.

2. A pharmaceutical composition for treating peptic gastric ulcer according to claim 1, characterized in that: The preparation method of chicken gizzard peptide comprises the following steps: taking fresh chicken gizzard lining and crushing it, adding it into distilled water with the mass of 8 to 12 times of the chicken gizzard lining, adjusting the pH value to 1.5 to 2.5, adding pepsin with the mass of 0.5% to 1% of the chicken gizzard lining, performing enzymolysis at 35 to 38° C. for 60 to 80 minutes, heating up to inactivate the enzyme, cooling down to room temperature, adjusting the pH value to 7.5 to 8.5, adding chymotrypsin with the mass of 0.8% to 1.5% of the chicken gizzard lining, performing enzymolysis at 35 to 38° C. Carry out enzymatic hydrolysis for 50min-70min, heat up to inactivate the enzyme, cool to room temperature, add keratinase (1%-2% by weight of chicken gizzard lining) and carry out enzymatic hydrolysis at 40℃-60℃ for 80min-100min, heat up to inactivate the enzyme, cool to room temperature, centrifuge at 4000r / min-6000r / min for 15min-20min, take the supernatant, perform ultrafiltration using a 5kDa ultrafiltration membrane, take the ultrafiltration component below 5kDa, freeze-dry to powder, and obtain chicken gizzard lining peptide.

3. A pharmaceutical composition for treating peptic gastric ulcer according to claim 1, characterized in that: The preparation method of the pumpkin seed extract comprises the following steps: taking pumpkin seed flesh, crushing it, adding water of 8 to 12 times the mass of the seed flesh, adjusting the pH to 4.5 to 5.5, adding 0.5% to 2% of cellulase by mass of the seed flesh and 0.5% to 2% of pectinase by mass of the seed flesh, performing enzymolysis at a constant temperature of 50° C. to 55° C. for 2 h to 3 h, then adjusting the pH to 1.5 to 2.5, adding 0.5% to 1% of pepsin by mass of the seed flesh, performing enzymolysis at 35° C. to 38° C. for 60 min to 80 min, heating up to inactivate the enzyme, cooling down to room temperature, adjusting the pH to 6.5 to 7.0, and adding 0.5% to 1.5% of nattokinase by mass of the seed flesh. , perform enzymolysis at 50℃~55℃ for 60min~90min, heat up to inactivate the enzyme, cool to room temperature, adjust the pH to 7.0~7.5, add 0.5%~1.5% aminopeptidase by weight of seed meat, perform enzymolysis at 37℃~45℃ for 50min~70min, heat up to inactivate the enzyme, cool to room temperature, add 0.5 times to 2 times the weight of seed meat of ethanol, mix and soak for 1h~3h, centrifuge at 4000r / min~6000r / min for 15min~20min, take the supernatant, ultrafilter with 5kDa ultrafiltration membrane, take the ultrafiltration component below 5kDa, freeze-dry to powder, and obtain pumpkin seed extract.

4. A pharmaceutical composition for treating peptic gastric ulcer according to claim 1, characterized in that: The preparation method of the composite bacteriocin comprises the following steps: steaming yam and red dates respectively, grinding them after cooling, and preparing yam paste and red date paste; preparing a base solution according to the mass ratio of water: yam paste: red date paste: glucose: soy peptone: dimethyl phosphate: magnesium sulfate = (90-100): (5-8): (3-5): (1-3): (0.5-1.5): (0.4-0.8): (0.05-0.2), adjusting the pH to 6.0-7.0, and then performing high-temperature sterilization treatment, cooling to room temperature, and obtaining a culture solution; inoculating 1 .5% v / v ~ 3% v / v of Lactobacillus acidophilus, 1.5% v / v ~ 3% v / v of Bifidobacterium and 1.5% v / v ~ 3% v / v of Lactobacillus casei are anaerobically fermented at 35℃ ~ 38℃ for 60h ~ 72h to obtain a fermentation broth; the fermentation broth is centrifuged at 5000r / min ~ 8000r / min for 10min ~ 20min, the supernatant is collected, ultrasonically treated at 20kHz ~ 50kHz for 3min ~ 10min, concentrated under reduced pressure at 60℃ ~ 80℃, and dried at 60℃ ~ 80℃ to powder to obtain a composite bacteriocin.

5. A pharmaceutical composition for treating peptic gastric ulcer according to claim 4, characterized in that: The Lactobacillus acidophilus is an activated bacterial liquid with a concentration of 100 million CFU / mL to 1 billion CFU / mL; the Bifidobacterium is an activated bacterial liquid with a concentration of 100 million CFU / mL to 1 billion CFU / mL; and the Lactobacillus casei is an activated bacterial liquid with a concentration of 100 million CFU / mL to 1 billion CFU / mL.

6. A pharmaceutical composition for treating peptic gastric ulcer according to claim 4, characterized in that: The temperature of the high temperature sterilization is 121° C. to 125° C., and the time of the high temperature sterilization is 15 min to 30 min.

7. A pharmaceutical composition for treating peptic gastric ulcer according to claim 1, characterized in that: The preparation method of the white lentil flower extract comprises the following steps: taking white lentil flowers, crushing them, then using ethanol with a mass of 10 to 15 times that of the white lentil flowers for percolation to obtain an extract, concentrating under reduced pressure at 60 to 80° C., and drying at 60 to 80° C. to obtain a powder to obtain the white lentil flower extract.

8. A pharmaceutical composition for treating peptic gastric ulcer according to claim 1, characterized in that: The preparation method of the bletilla striata extract comprises the following steps: taking fresh bletilla striata tubers, crushing them, adding water 8 to 12 times the mass of the tubers, adjusting the pH to 4.5 to 5.5, sequentially adding 0.5% to 2% of cellulase by mass of the tubers and 0.5% to 2% of pectinase by mass of the tubers, performing enzymolysis at a constant temperature of 50 to 55° C. for 2 hours to 3 hours, then adjusting the pH to 1.5 to 2.5, adding 0.5% to 1% of pepsin by mass of the tubers, performing enzymolysis at 35 to 38° C. for 60 minutes to 80 minutes, heating up to inactivate the enzyme, cooling down to room temperature, adjusting the pH to 6.0 to 7.0, adding 0.5% to 1% of pectin by mass of the tubers, and performing enzymolysis at a constant temperature of 50 to 55° C. for 2 hours to 3 hours. 1% bromelain is enzymatically hydrolyzed at 50℃-60℃ for 50min-80min, the temperature is raised to inactivate the enzyme, the temperature is lowered to room temperature, 0.5%-1% serrapeptase by weight of the tuber is added, the temperature is raised to inactivate the enzyme, the temperature is lowered to room temperature, 1 to 3 times the weight of the tuber is added with ethanol, the mixture is mixed and soaked for 1h-3h, the mixture is centrifuged at 4000r / min-6000r / min for 15min-20min, the supernatant is taken, ultrafiltration is performed using a 10kDa ultrafiltration membrane, the ultrafiltration fraction below 10kDa is taken, and freeze-dried to powder to obtain a Bletilla striata extract.

9. A pharmaceutical composition for treating peptic gastric ulcer according to claim 2, 3 or 8, characterized in that: The temperature for heating the enzyme to be inactivated is 85°C to 95°C, and the time for heating the enzyme to be inactivated is 15min to 30min.

10. A method for preparing the pharmaceutical composition for treating peptic gastric ulcer according to claim 1, characterized in that: The preparation method comprises the following steps: evenly mixing pumpkin seed extract, compound bacteria, white lentil flower extract and bletilla striata extract according to weight proportions, then adding chicken gizzard peptide and mixing evenly to obtain a pharmaceutical composition.

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