A method for extracting flavonoids and its application in preparing medicine for treating gastric diseases

Abstract

The present invention provides a method for extracting flavonoids and its application in preparing a medicine for treating gastric disease, belonging to the technical field of Chinese medicine extraction, the extraction method comprises the following steps: (1) weighing the following raw materials by weight according to the ratio: 2-7 parts of cassia twig, 1-5 parts of vinegar corydalis, 7-12 parts of calcined oyster, 3-7 parts of fennel, 1-4 parts of amomum, 6-10 parts of galangal, 12-17 parts of liquorice, mixing and crushing to a particle size of less than 100 mesh to obtain a mixed micro powder; (2) preparing a pretreatment material; (3) preparing an enzymolysis solution; (5) preparing a fermentation filtrate; (6) separating and purifying the fermentation filtrate using macroporous resin chromatography and gel chromatography in sequence, freeze drying, and obtaining an extract containing flavonoids. The extract prepared by the extraction method of the present invention has a high flavonoid content, and the flavonoid-containing extract can be used for treating drugs for gastric ulcer and chronic atrophic gastritis.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to a method for extracting flavonoids and application of the method in preparing medicine for treating gastric diseases. Background Art

[0002] Gastric ulcer and gastritis are common clinical diseases. Helicobacter pylori infection, nonsteroidal anti-inflammatory drugs, pepsin, and abnormal gastric acid secretion are common causes; bad daily living habits can also induce them. The occurrence of gastric ulcer and gastritis is due to the imbalance of gastric mucosal damage factors and protective factors. When the mucosal damage factors are greater than the defensive factors, the disease is likely to occur.

[0003] At present, the drugs for treating gastric diseases mainly include Weidele, Ledewei, Lizhudele; synitidine, famotidine and Tagamet; Belladonna tablets, Wei'an; Weibizhi and Weigaining, Cufuan, Prebos (Cisapride), etc. However, most of them are Western medicines, and they have irritating effects on gastric mucosa. Long-term use will produce certain drug resistance. Chinese patent medicines can overcome the disadvantages of Western medicines with large toxic and side effects. In recent years, Chinese patent medicines have been increasingly valued in the treatment of gastric diseases.

[0004] Some studies have shown that flavonoids in Chinese patent medicines play an important role in relieving stomach discomfort and promoting stomach health. At present, the extraction of flavonoids is mainly based on thermal extraction, but high temperature may cause the degradation or denaturation of heat-sensitive components in flavonoids and destroy other beneficial components in the medicinal materials. How to effectively extract flavonoids and other active components from Chinese medicine raw materials is one of the difficulties that need to be overcome.

[0005] Therefore, there is an urgent need for a method for extracting flavonoids and an application thereof in preparing a drug for treating gastric diseases. Summary of the invention

[0006] The purpose of the present invention is to provide a method for extracting flavonoids and application of the method in preparing medicine for treating gastric diseases.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] A method for extracting flavonoids, comprising the following steps:

[0009] (1) Weigh the following raw materials in parts by weight according to the ratio: 2-7 parts of cinnamon twig, 1-5 parts of vinegar corydalis, 7-12 parts of calcined oyster, 3-7 parts of fennel, 1-4 parts of amomum, 6-10 parts of galangal, and 12-17 parts of liquorice, mix and grind to a particle size of less than 100 mesh to obtain a mixed micro powder;

[0010] (2) subjecting the mixed micropowder to microwave pretreatment to obtain a pretreated material;

[0011] (3) mixing the pretreated material and water to obtain a mixed liquid, and immersing the mixed liquid at 10-15° C. for 25-28 hours to obtain an impregnation liquid;

[0012] (4) adding a complex enzyme to the impregnation liquid, performing enzymolysis at 40-45° C. for 4-6 hours, and then inactivating the enzyme to obtain an enzymolysis solution;

[0013] (5) adding Lactobacillus johnsonii to the enzymatic hydrolysate, anaerobic fermentation at 38-40° C. for 24-26 h, sterilizing, then adding Lactobacillus rhamnosus, anaerobic fermentation at 38-40° C. for 35-40 h, sterilizing, then adding Lactobacillus reuteri, anaerobic fermentation at 38-40° C. for 15-20 h, sterilizing, and filtering with a 0.22 μm filter membrane to obtain a fermentation filtrate;

[0014] (6) The fermentation filtrate is separated and purified by macroporous resin chromatography and gel chromatography, and freeze-dried to obtain a flavonoid-containing extract.

[0015] Furthermore, the microwave pretreatment conditions in step (2) are as follows: the power of a single magnetron is 1.6-1.8KW, there are 35-38 magnetrons, and the treatment is carried out at a frequency of 2500-2550MHz for 3-5min.

[0016] Furthermore, in step (3), the weight ratio of the pretreated material to water is 1:(8-10).

[0017] Furthermore, in step (4), the complex enzyme comprises hemicellulase, pectinase and β-glucosidase.

[0018] Furthermore, in the step (4), the amount of hemicellulase used in the soaking liquid is 100-110 U / mL; the amount of pectinase used in the soaking liquid is 70-80 U / mL; and the amount of β-glucosidase used in the soaking liquid is 140-150 U / mL.

[0019] In the prior art, Chinese medicine raw materials are usually crushed and then directly enzymolyzed with a single type of enzyme. This method is not ideal for the extraction of active components such as flavonoids. The present invention can improve the content of flavonoids in the extract by pretreating the raw materials of a specific composition, soaking them for a certain period of time, and then using a specific composite enzyme for enzymolysis. Microwave pretreatment can accelerate the rupture of the cell wall, improve the dissolution rate of the effective ingredients, and allow the subsequent composite enzyme to have more contact area for catalytic reaction, thereby improving the efficiency of enzymolysis; the soaking process can allow water to better penetrate into the medicinal materials, which is conducive to the enzymolysis. The present invention uses a specific enzyme for enzymolysis to optimize the characteristics of flavonoid compounds to optimize the extraction process, improve the selectivity and efficiency of the extraction, and the above three steps work synergistically to effectively improve the extraction rate of flavonoid compounds. At the same time, the flavonoid-containing extract prepared under this condition can significantly reduce the gastric juice secretion of pylorus-ligated rats, and has a significant protective effect on gastric ulcers.

[0020] Furthermore, in the step (5), the amount of Lactobacillus johnsonii used in the enzymatic hydrolysate is 10 6 -10 7 CFU / mL.

[0021] Furthermore, in step (5), the amount of Lactobacillus rhamnosus used in the enzymatic hydrolysate is 10 9 -10 10 CFU / mL.

[0022] Furthermore, in step (5), the amount of Lactobacillus reuteri used in the enzymatic hydrolysate is 10 8 -10 9 CFU / mL.

[0023] The present invention uses three specific strains for gradient fermentation on the basis of enzymolysis, which can increase the content of flavonoids in the extract. At the same time, the flavonoid-containing extract prepared under this condition has a good effect on improving chronic atrophic gastritis. Some intermediate metabolites can be produced during the enzymolysis process of the present invention, and these products can be used as substrates or promoters in the fermentation process, which help to improve the fermentation efficiency and the yield of flavonoid compounds; at the same time, different strains can produce different metabolites, and there may be a synergistic effect between these metabolites, which act on the raw materials together to further promote the release of flavonoid compounds. The content and type of active components in the flavonoid-containing extract are improved, which can improve the activity of SOD and GSH-Px, and has a good effect on improving chronic atrophic gastritis.

[0024] Furthermore, in the step (6), the macroporous resin chromatography method uses a separation column with a diameter-to-height ratio of 1:(18-22); the separation column is filled with a macroporous adsorption resin of model HPD600.

[0025] Furthermore, in step (6), the gel chromatography method uses a separation column with a diameter-to-height ratio of 1:(18-22); the gel used is hydroxypropyl dextran gel.

[0026] The invention also provides an application of a flavonoid extraction method in preparing a medicine for treating gastric disease.

[0027] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0028] 1. The extract prepared by the extraction method of the present invention has a high flavonoid content, and the flavonoid-containing extract can be used as a drug for treating gastric ulcer and chronic atrophic gastritis.

[0029] 2. The present invention pre-treats the raw material, soaks it for a certain period of time, and then uses a specific complex enzyme for enzymolysis, which can improve the content of flavonoids in the product. At the same time, the extract prepared under this condition can significantly reduce the gastric juice secretion of pylorus-ligated rats and has a significant protective effect on gastric ulcers.

[0030] 3. The present invention uses three specific strains for gradient fermentation based on enzymatic hydrolysis to increase the content of flavonoids in the prepared extract. At the same time, the flavonoid-containing extract prepared under this condition has a good effect on improving chronic atrophic gastritis. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The bacteria used in the present invention are commercially available raw materials.

[0033] Lactobacillus johnsonii, number: SHBCC D71369, was purchased from Shanghai Microbiological Collection Center.

[0034] Lactobacillus reuteri, number: SHBCC D12901, was purchased from Shanghai Collection of Microorganisms Center.

[0035] Lactobacillus rhamnosus, number: SHBCC D73158, was purchased from Shanghai Collection of Microorganisms Center.

[0036] Lactobacillus acidophilus, number: CICC 22162, was purchased from China Industrial Microbiological Culture Collection Center.

[0037] Streptococcus thermophilus, number: CCTCCAB 2010206, was purchased from China Center for Type Culture Collection.

[0038] Lactobacillus bulgaricus, number: CCTCC CB 20082295, was purchased from China Center for Type Culture Collection.

[0039] Example 1

[0040] This embodiment provides a method for extracting flavonoids, and the extraction method comprises the following steps:

[0041] (1) Weigh the following raw materials in parts by weight according to the ratio: 4 parts of cinnamon twig, 3 parts of vinegar corydalis, 10 parts of calcined oyster, 5 parts of fennel, 2 parts of amomum, 8 parts of galangal, and 15 parts of liquorice, mix and grind to a particle size of less than 100 mesh to obtain a mixed micro powder;

[0042] (2) pre-treating the mixed micropowder with microwaves, wherein the microwave pre-treatment conditions are: a single magnetron power of 1.7 kW, 36 magnetrons, and a frequency of 2520 MHz for 4 min to obtain a pre-treated material;

[0043] (3) mixing the pretreated material and water in a weight ratio of 1:9 to obtain a mixed liquid, and immersing the mixed liquid at 12° C. for 26 hours to obtain an impregnation liquid;

[0044] (4) Adding a complex enzyme to the impregnation liquid, the complex enzyme includes hemicellulase, pectinase and β-glucosidase. The amount of hemicellulase used in the impregnation liquid is 105 U / mL; the amount of pectinase used in the impregnation liquid is 75 U / mL; and the amount of β-glucosidase used in the impregnation liquid is 145 U / mL; performing enzymolysis at 42° C. for 5 hours, and after inactivating the enzymes, obtaining an enzymolysis liquid;

[0045] (5) adding Lactobacillus johnsonii to the enzymolysis solution, wherein the amount of Lactobacillus johnsonii in the enzymolysis solution is 10 6 CFU / mL, anaerobic fermentation at 39℃ for 25h, after sterilization, continue to add Lactobacillus rhamnosus, the amount of Lactobacillus rhamnosus used in the enzymatic hydrolysate is 10 9 CFU / mL, anaerobic fermentation at 39℃ for 38h, after sterilization, continue to add Lactobacillus reuteri, the amount of Lactobacillus reuteri used in the enzymatic hydrolysate is 10 8 CFU / mL, anaerobic fermentation at 39°C for 17 h, sterilization, and filtration using a 0.22 μm filter membrane to obtain the fermentation filtrate;

[0046] (6) The fermentation filtrate was separated and purified by macroporous resin chromatography and gel chromatography, and freeze-dried to a water content of 2.37% by mass to obtain a flavonoid-containing extract.

[0047] The macroporous resin chromatography method uses a separation column with a diameter-to-height ratio of 1:20; the separation column is filled with a macroporous adsorption resin of the HPD600 model. When the macroporous resin chromatography method is used for separation and purification, the sample concentration is 65 mg / mL, the sample volume is 3 times the column volume, and the sample flow rate is 2 mL / min; the static adsorption time after sample loading is 100 minutes, the distilled water elution volume after sample loading is 6.0 times the column volume, and the elution flow rate is 1.0 mL / min; the eluent is an ethanol aqueous solution with a mass percentage concentration of 55%, the eluent volume is 6.0 times the column volume, and the elution flow rate is 1.0 mL / min, and the eluent is collected to obtain a separated and purified extract.

[0048] The gel chromatography method uses a separation column with a diameter-to-height ratio of 1:20; the gel used is hydroxypropyl dextran gel; when the gel chromatography method is used for separation and purification, the loading concentration of the separated and purified extract is 100 mg / mL, the loading volume is 4.0 times the column volume, and the loading flow rate is 1.0 mL / min; the static adsorption time after loading is 100 minutes, the distilled water elution volume after loading is 4.0 times the column volume, and the elution flow rate is 2.0 mL / min; the eluent is an ethanol aqueous solution with a mass percentage concentration of 70%, the eluent volume is 6.0 times the column volume, and the elution flow rate is 2 mL / min, the eluent is collected, and then subjected to subsequent freeze-drying treatment.

[0049] Example 2

[0050] This embodiment provides a method for extracting flavonoids, and the extraction method comprises the following steps:

[0051] (1) Weigh the following raw materials in parts by weight according to the ratio: 2 parts of cinnamon twig, 5 parts of vinegar corydalis, 7 parts of calcined oyster, 7 parts of fennel, 1 part of amomum, 10 parts of galangal, and 12 parts of liquorice, mix and grind to a particle size of less than 100 mesh to obtain a mixed micro powder;

[0052] (2) pre-treating the mixed micropowder with microwaves, wherein the microwave pre-treatment conditions are: a single magnetron power of 1.8 kW, 35 magnetrons, and a treatment frequency of 2550 MHz for 3 min to obtain a pre-treated material;

[0053] (3) mixing the pretreated material and water in a weight ratio of 1:8 to obtain a mixed liquid, and immersing the mixed liquid at 10° C. for 28 hours to obtain an impregnation liquid;

[0054] (4) Adding a composite enzyme to the impregnation liquid, the composite enzyme includes hemicellulase, pectinase and β-glucosidase. The amount of the hemicellulase used in the impregnation liquid is 110 U / mL; the amount of the pectinase used in the impregnation liquid is 70 U / mL; and the amount of the β-glucosidase used in the impregnation liquid is 150 U / mL; performing enzymolysis at 40° C. for 6 hours, and after inactivating the enzymes, obtaining an enzymolysis liquid;

[0055] (5) adding Lactobacillus johnsonii to the enzymolysis solution, wherein the amount of Lactobacillus johnsonii in the enzymolysis solution is 10 6 CFU / mL, anaerobic fermentation at 38℃ for 26h, after sterilization, continue to add Lactobacillus rhamnosus, the amount of Lactobacillus rhamnosus used in the enzymatic hydrolysate is 10 9 CFU / mL, anaerobic fermentation at 40℃ for 35h, after sterilization, Lactobacillus reuteri was added, and the amount of Lactobacillus reuteri used in the enzymatic hydrolysate was 10 9 CFU / mL, anaerobic fermentation at 38°C for 20 h, sterilization, and filtration using a 0.22 μm filter membrane to obtain the fermentation filtrate;

[0056] (6) The fermentation filtrate was separated and purified by macroporous resin chromatography and gel chromatography, and freeze-dried until the water content by mass was 2.31% to obtain a flavonoid-containing extract.

[0057] The macroporous resin chromatography method uses a separation column with a diameter-to-height ratio of 1:20; the separation column is filled with a macroporous adsorption resin of the HPD600 model. When the macroporous resin chromatography method is used for separation and purification, the sample concentration is 65 mg / mL, the sample volume is 3 times the column volume, and the sample flow rate is 2 mL / min; the static adsorption time after sample loading is 100 minutes, the distilled water elution volume after sample loading is 6.0 times the column volume, and the elution flow rate is 1.0 mL / min; the eluent is an ethanol aqueous solution with a mass percentage concentration of 55%, the eluent volume is 6.0 times the column volume, and the elution flow rate is 1.0 mL / min, and the eluent is collected to obtain a separated and purified extract.

[0058] The gel chromatography method uses a separation column with a diameter-to-height ratio of 1:20; the gel used is hydroxypropyl dextran gel; when the gel chromatography method is used for separation and purification, the loading concentration of the separated and purified extract is 100 mg / mL, the loading volume is 4.0 times the column volume, and the loading flow rate is 1.0 mL / min; the static adsorption time after loading is 100 minutes, the distilled water elution volume after loading is 4.0 times the column volume, and the elution flow rate is 2.0 mL / min; the eluent is an ethanol aqueous solution with a mass percentage concentration of 70%, the eluent volume is 6.0 times the column volume, and the elution flow rate is 2 mL / min, the eluent is collected, and then subsequent freeze-drying is performed.

[0059] Comparative Example 1

[0060] The difference between this comparative example and Example 1 is that no pretreatment and immersion are performed, and direct enzymolysis is performed.

[0061] The mixed micro powder and water are mixed in a weight ratio of 1:9 to obtain a mixed solution, and a composite enzyme is added for enzymolysis. The composite enzyme includes hemicellulase, pectinase and β-glucosidase. The amount of hemicellulase used in the mixed solution is 105U / mL; the amount of pectinase used in the mixed solution is 75U / mL and the amount of β-glucosidase used in the mixed solution is 145U / mL; enzymolysis is performed at 42°C for 5h, and after the enzyme is inactivated, an enzymolysis solution is obtained.

[0062] Comparative Example 2

[0063] The difference between this comparative example and Example 1 is that the usage amount of each enzyme is different.

[0064] The complex enzyme includes hemicellulase, pectinase and beta-glucosidase. The hemicellulase is used in an amount of 145 U / mL in the impregnation liquid; the pectinase is used in an amount of 105 U / mL in the impregnation liquid; and the beta-glucosidase is used in an amount of 75 U / mL in the impregnation liquid.

[0065] Comparative Example 3

[0066] The difference between this comparative example and Example 1 is that the types of enzymes are different.

[0067] The complex enzyme includes neutral protease, cellulase and amylase. The neutral protease is used in an amount of 105 U / mL in the impregnation liquid; the cellulase is used in an amount of 75 U / mL in the impregnation liquid; and the amylase is used in an amount of 145 U / mL in the impregnation liquid.

[0068] Comparative Example 4

[0069] The difference between this comparative example and Example 1 is that the amount of each bacteria added is different.

[0070] Add Lactobacillus johnsonii to the enzymatic hydrolysate. The amount of Lactobacillus johnsonii used in the enzymatic hydrolysate is 10 8 CFU / mL, anaerobic fermentation at 39℃ for 25h, after sterilization, continue to add Lactobacillus rhamnosus, the amount of Lactobacillus rhamnosus used in the enzymatic hydrolysate is 10 6 CFU / mL, anaerobic fermentation at 39℃ for 38h, after sterilization, continue to add Lactobacillus reuteri, the amount of Lactobacillus reuteri used in the enzymatic hydrolysate is 10 10 CFU / mL, anaerobic fermentation at 39°C for 17 h, sterilization, and filtration using a 0.22-μm filter membrane to obtain the fermentation filtrate.

[0071] Comparative Example 5

[0072] The difference between this comparative example and Example 1 is that the types of bacteria are different.

[0073] Add Lactobacillus acidophilus to the enzymatic hydrolysate. The amount of Lactobacillus acidophilus used in the enzymatic hydrolysate is 10 6CFU / mL, anaerobic fermentation at 39℃ for 25h, after sterilization, thermophilic streptococcus was added, and the amount of thermophilic streptococcus used in the enzymatic hydrolysate was 10 9 CFU / mL, anaerobic fermentation at 39℃ for 38h, after sterilization, continue to add Lactobacillus bulgaricus, the amount of Lactobacillus bulgaricus used in the enzymatic hydrolysate is 10 8 CFU / mL, anaerobic fermentation at 39°C for 17 h, sterilization, and filtration using a 0.22-μm filter membrane to obtain the fermentation filtrate.

[0074] Comparative Example 6

[0075] The difference between this comparative example and Example 1 is that no gradient fermentation is performed.

[0076] Lactobacillus johnsonii, Lactobacillus rhamnosus and Lactobacillus reuteri were added to the enzymatic hydrolysate at the same time. The amount of Lactobacillus johnsonii in the enzymatic hydrolysate was 10 6 CFU / mL, the amount of Lactobacillus rhamnosus used in the enzymatic hydrolysate is 10 9 CFU / mL, the amount of Lactobacillus reuteri used in the enzymatic hydrolysate is 10 8 CFU / mL, anaerobic fermentation at 39°C for 80 h, sterilization, and filtration using a 0.22-μm filter membrane to obtain the fermentation filtrate.

[0077] Comparative Example 7

[0078] The difference between this comparative example and Example 1 is that the raw material ratio is different.

[0079] The following raw materials were weighed in parts by weight according to the ratio: 10 parts of cinnamon twigs, 10 parts of vinegar-corydalis, 4 parts of calcined oyster shells, 10 parts of fennel, 8 parts of amomum villosum, 2 parts of galangal, and 3 parts of liquorice; mixed and crushed to a particle size of less than 100 mesh to obtain a mixed micropowder.

[0080] Performance Testing

[0081] The performance tests were performed on the flavonoid-containing extracts prepared in Examples 1-2 and Comparative Examples 1-7.

[0082] 1. Determine the percentage of total flavonoids in the extract according to the method specified in the 2015 edition of the Chinese Pharmacopoeia.

[0083] The percentage of total flavonoids / %=100%×the mass of total flavonoids / the mass of the extract containing flavonoids. The test results are shown in Table 1.

[0084] Table 1 Extraction percentage results of total flavonoids

[0085]

[0086] The results show that the total flavonoid content in the extracts of Examples 1-2 is high, and the changes in the extraction methods in Comparative Examples 1-6 will lead to a decrease in the total flavonoid content. In Comparative Example 7, the raw material ratio was changed, and the total flavonoid content decreased. The analysis is that the flavonoid content in different raw materials is different. At the same time, the raw materials are used as nutrients for subsequent enzymatic hydrolysis and fermentation, and their ratio will also affect the activity of enzymes and bacteria.

[0087] 2. Protective effect of flavonoid extract on gastric ulcer in rats caused by pyloric ligation 110 SPF SD rats, all male, were randomly divided into a model group, a famotidine group (dosage of 0.01 g / kg rat) and a flavonoid extract group of Examples 1-2 and Comparative Examples 1-7 (dosage of 0.5 g / kg rat), with 10 rats in each group. Each group was gavaged with 10 mL of drug solution / kg rat, once a day for 10 consecutive days, and the model group was given an equal volume of pure water; wherein, the drug solution is a solution after the specific drug is diluted with pure water. After the last administration, all animals were fasted but not watered for 24 hours, and the rats were anesthetized with 3.5% chloral hydrate, the abdominal wall was cut open, the pylorus was ligated, and the drug was administered again through the duodenum. The incision was sutured, and water supply and diet were stopped. After 24 hours, the animals were killed, the cardia was ligated, and the whole stomach was removed. Collect gastric juice and centrifuge at 3000r / min for 15min, and take the supernatant for later use. Wash the whole stomach, inject 6mL of 1% formaldehyde solution into the stomach, and immerse the whole stomach in 1% formaldehyde solution for 10min. Then cut the stomach open along the greater curvature of the stomach, and calculate the ulcer index under a microscope. Ulcer index: measure the maximum long diameter of the ulcer surface and the maximum wide diameter perpendicular to the maximum long diameter, and take the product of the two as the ulcer index. The test results are shown in Table 2.

[0088] Table 2 Protective effect on gastric ulcer in rats induced by pyloric ligation

[0089]

[0090]

[0091] The results in Table 2 show that compared with the model group, the gastric ulcer index of Example 1-2 was significantly reduced, and the effect was close to that of famotidine, while Comparative Example 1-7 changed the preparation steps, the types and amounts of complex enzymes and bacterial agents, and the composition of raw materials, all of which led to a decrease in the protective effect against gastric ulcers in rats caused by pyloric ligation.

[0092] 3. The therapeutic effect of flavonoid extracts on chronic atrophic gastritis

[0093] Take 120 healthy clean SD rats, weighing 180-200g, both male and female. The concentration is 180mg·L -1The model rats were fed with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) aqueous solution instead of drinking water for 8 weeks. All rats were fed with ordinary feed.

[0094] Rats were randomly divided into 12 groups, 10 rats in each group, namely normal control group, model group, omeprazole positive drug group, experimental group of Example 1-2 and Comparative Example 1-7. Starting from the 9th week of modeling, drinking MNNG solution was stopped, and rats in each group began to be gavaged once a day, 10 mL / kg rat each time. Examples 1-2 and Comparative Example 1-7 of the present invention were administered at a dosage of 5 g / kg rat, omeprazole positive drug was administered at a dosage of 1.6 g / kg rat per day, and rats in the normal control group and model group were gavaged with physiological saline, once a day, 10 mL / kg rat each time, for 90 consecutive days.

[0095] After 90 days of administration, rats were fasted for 16 hours, anesthetized with urethane, and the rat stomach was dissected, the stomach contents were washed, rinsed with distilled water twice, and the water was absorbed by filter paper. Carefully scrape 1 / 3 of the whole gastric mucosa, weigh it, add an appropriate amount of distilled water to homogenize it, make a gastric mucosal homogenate, and test the GSH-Px and SOD content. Among them, the determination of GSH-Px and SOD was carried out by superoxide dismutase (SOD) and glutathione (GSH-Px) kits (purchased from Nanjing Jiancheng Bioengineering Institute). The results are shown in Table 3.

[0096] Table 3 GSH-Px and SOD contents in rat gastric mucosal tissue.

[0097] <![CDATA[GSH-Px(mg·g·prot -1 )]]> SOD(U / mL) Example 1 52.64±4.30 342.73±9.21 Example 2 51.27±4.21 341.69±10.52 Comparative Example 1 44.32±5.17 314.76±8.65 Comparative Example 2 48.53±5.03 325.11±7.98 Comparative Example 3 47.75±5.18 321.02±9.11 Comparative Example 4 48.48±5.26 327.13±10.28 Comparative Example 5 46.11±4.69 317.94±11.47 Comparative Example 6 45.29±5.87 319.83±8.74 Comparative Example 7 47.86±4.95 320.08±11.05 Normal control group 55.65±5.89 371.27±11.12 Model Group 31.23±4.21 259.53±8.24 Positive drug group 44.89±5.82 337.21±1.53

[0098] The active components in the flavonoid-containing extract prepared in Examples 1-2 of the present invention can increase the activity of SOD and GSH-Px, thereby reducing lipid peroxidation and playing a role in protecting gastric mucosa and repairing damaged gastric mucosa. Comparative Examples 1-7 change the preparation steps, the types and amounts of compound enzymes and bacterial agents, and the composition of raw materials, all of which will lead to a decrease in the effect of protecting gastric mucosa and repairing damaged gastric mucosa.

[0099] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting flavonoids, characterized in that: The extraction method comprises the following steps: (1) Weigh the following raw materials in parts by weight according to the ratio: 2-7 parts of cinnamon twig, 1-5 parts of vinegar corydalis, 7-12 parts of calcined oyster shell, 3-7 parts of fennel, 1-4 parts of amomum villosum, 6-10 parts of galangal, and 12-17 parts of liquorice, mix and grind to a particle size of less than 100 mesh to obtain a mixed micro powder; (2) pre-treating the mixed micro powder by microwave to obtain a pre-treated material; (3) Mixing the pretreated material and water to obtain a mixed liquid, and soaking the mixed liquid at 10-15° C. for 25-28 hours to obtain an impregnated liquid; (4) adding a complex enzyme to the impregnation liquid, performing enzymolysis at 40-45°C for 4-6 hours, and then inactivating the enzyme to obtain an enzymolysis solution; (5) adding Lactobacillus johnsonii to the enzymatic hydrolysate, and fermenting anaerobically at 38-40° C. for 24-26 hours. After sterilization, adding Lactobacillus rhamnosus, and fermenting anaerobically at 38-40° C. for 35-40 hours. After sterilization, adding Lactobacillus reuteri, and fermenting anaerobically at 38-40° C. for 15-20 hours. After sterilization, filtering with a filter membrane to obtain a fermentation filtrate; (6) Separating and purifying the fermentation filtrate by macroporous resin chromatography and gel chromatography, and freeze-drying to obtain a flavonoid extract; In step (4), the complex enzyme is hemicellulase, pectinase and β-glucosidase; the amount of hemicellulase used in the impregnation liquid is 100-110 U / mL; the amount of pectinase used in the impregnation liquid is 70-80 U / mL; the amount of β-glucosidase used in the impregnation liquid is 140-150 U / mL; In the step (5), the amount of Lactobacillus johnsonii used in the enzymatic hydrolysate is 10 6 -10 7 CFU / mL, the amount of Lactobacillus rhamnosus used in the enzymatic hydrolysate is 10 9 -10 10 CFU / mL, the amount of Lactobacillus reuteri used in the enzymatic hydrolysate is 10 8 -10 9 CFU / mL.

2. The method for extracting flavonoids according to claim 1, characterized in that: The microwave pretreatment conditions in step (2) are: the power of a single magnetron group is 1.6-1.8KW, 35-38 magnetron groups, and the treatment is carried out at a frequency of 2500-2550MHz for 3-5min.

3. The method for extracting flavonoids according to claim 1, characterized in that: The weight ratio of the pretreated material to water in step (3) is 1:(8-10).

4. The method for extracting flavonoids according to claim 1, characterized in that: In the step (6), the macroporous resin chromatography method uses a separation column with a diameter-to-height ratio of 1:(18-22); the separation column is filled with a macroporous adsorption resin of model HPD600; the gel chromatography method uses a separation column with a diameter-to-height ratio of 1:(18-22); the gel used is hydroxypropyl dextran gel.

5. Use of a flavonoid extract obtained by the flavonoid extraction method according to any one of claims 1 to 4 in preparing a drug for treating gastric ulcer.

6. Use of a flavonoid extract obtained by the flavonoid extraction method according to any one of claims 1 to 4 in the preparation of a drug for treating chronic atrophic gastritis.

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