Preparation method of paste porridge extract and application of paste porridge extract in stomach injury repair

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a preparation method of a paste porridge extract and application of the paste porridge extract in stomach injury repair. The method is realized by the following steps: (1) respectively adding water into millet and soybeans, soaking, pulping and filtering to obtain rice milk and soybean milk; (2) preheating an iron pot, firstly adding 1 / 5 of the volume of soybean milk, boiling, then adding all the remaining soybean milk, then adding the rice milk, and boiling to obtain pasty porridge; (3) adding 5 times of distilled water into the paste porridge, performing ultrasonic extraction, centrifuging, taking supernatant, performing suction filtration, performing rotary evaporation concentration, and freezing to obtain a paste porridge water extract; the millet and the soybeans in a specific proportion are boiled, so that the prepared paste porridge water extract has relatively good oxidation resistance; compared with soybean milk, rice paste aqueous extract and paste porridge extract, the reducing power of the paste porridge extract is improved, and the paste porridge extract has scavenging capacity on hydroxyl free radicals; the paste porridge aqueous extract extracted by the method has no obvious cytotoxicity to normal GES-1 cells, and can promote cell proliferation. And the cell proliferation promoting activity of the paste porridge aqueous extract is superior to that of a rice paste aqueous extract and a soybean milk aqueous extract. The paste porridge water extract can reduce ROS in cells, improve the SOD level in the cells, reduce the MDA level and improve the GSH level. The aqueous extract has the effect of protecting gastric mucosa.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a preparation method of a porridge extract and an application of the porridge extract in repairing gastric damage. Background Art

[0002] Huzhou, also known as white porridge, is one of the many types of porridge. It is one of the oldest morning foods in the southern part of Shandong Province, and has been around for more than two thousand years. "Lunan porridge" refers to porridge made by grinding and cooking a mixture of yellow millet and soybeans. It is different from the rice soup made by boiling rice for a long time in the southern region. Making Huzhou requires more complicated steps. First, select the best soybeans and yellow millet, soak them in a specific amount, grind them into a slurry, and then slowly pour them into the cooker according to the specified time. It is cooked over a long slow fire. Because the special flavor - the taste of paste - is produced during the cooking process in an iron pot, it is called Huzhou.

[0003] Currently, the commonly used drugs for the treatment of gastric mucosal injury in clinical practice include prostaglandins, gastric mucosal protectants and H2 receptor blockers. Although these drugs are effective overall, they still have disadvantages, may cause some side effects, and often need to be used in combination with other drugs to improve the therapeutic effect. In addition, in the treatment of gastric mucosal injury, although existing drugs can provide a certain degree of relief and treatment, there are still some limitations. In recent years, diet therapy has been increasingly valued in the medical and nutritional communities as an auxiliary treatment method. Diet therapy, with its lower side effects and potential repair effects, provides a new perspective for the treatment of gastric mucosal injury.

[0004] There is a relative lack of research reports on the nutritional value and health benefits of porridge at home and abroad, which is still a blank in academic research. This phenomenon shows that as a traditional specialty food, the potential health benefits of porridge have not been fully studied and recognized. Therefore, it is of great significance to carry out research on the nutritional value and efficacy of porridge, which can promote the modernization and scientific development of traditional porridge foods and enhance their competitiveness in the domestic and foreign markets. Summary of the invention

[0005] In view of the research and development gap in the prior art, the present invention provides a method for preparing a porridge extract.

[0006] Another object of the present invention is to provide the use of the porridge extract prepared by the above preparation method in the preparation of a drug for repairing alcoholic gastric damage in GES-1 cells.

[0007] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: The present invention provides a method for preparing a porridge extract, comprising the following steps: (1) Soaking millet and soybeans in water respectively, then beating and filtering to obtain rice milk and soybean milk; (2) After preheating the iron pot, first add 1 / 5 of the volume of soy milk. After boiling, add all the remaining soy milk, and then add rice milk and cook to obtain porridge; (3) Add distilled water to the porridge, perform ultrasonic extraction, centrifuge, collect the supernatant, filter, concentrate by rotary evaporation, and freeze to obtain the porridge water extract.

[0008] Preferably, in step (1), the mass ratio of millet to soybean is 75-125:50-100; and the ratio of millet or soybean to water is 1 g:1-8 mL.

[0009] Preferably, in step (1), the mass ratio of millet to soybean is 103:75; and the liquid-to-solid ratio is 4.8:1.

[0010] Preferably, in step (2), the preheating is heating at 160-180° C. for 2-4 minutes, until the bottom of the iron pot turns red; the boiling time is 35-55 minutes; preferably, the boiling time is 44 minutes.

[0011] Preferably, in step (3), the volume ratio of the porridge to distilled water is 1:5.

[0012] Preferably, in step (3), the ultrasonic extraction is performed by intermittent ultrasonic extraction (100w) at 50°C for 2h.

[0013] Preferably, in step (3), the volume ratio of the porridge to distilled water is 1:5; and the centrifugation is performed at 10,000 r / min for 15 min.

[0014] The present invention also provides an application of the porridge extract prepared by the above preparation method in preparing a medicine for repairing gastric damage.

[0015] Preferably, the method is used in the preparation of a drug for repairing alcoholic gastric damage in GES-1 cells.

[0016] The beneficial effects of the present invention are: (1) The porridge water extract prepared by boiling millet and soybeans in a specific ratio has good antioxidant capacity; compared with soybean milk and rice paste water extracts, the porridge extract has improved reducing power and has the ability to scavenge hydroxyl free radicals; (2) The porridge water extract extracted by the present invention has no obvious cytotoxicity to normal GES-1 cells and can promote cell proliferation. The cell proliferation promoting activity of the porridge water extract is better than that of rice paste water extract and soy milk water extract. The porridge water extract can reduce intracellular ROS, increase intracellular SOD level, reduce MDA level, and increase GSH level. The water extract has the effect of protecting gastric mucosa.

[0017] (3) The present invention evaluates the effect of porridge on acute alcoholic gastric injury in mice. The porridge can reduce the damage caused by ethanol to mice by reducing the activity levels of MDA and MPO enzymes, enhancing SOD and increasing the activity level of GSH enzymes, thereby achieving a protective effect on acute alcoholic gastric mucosal injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are the sample pictures of porridge water extract, rice paste water extract and soy milk water extract; Figure 2 The effects of porridge water extract (A), rice paste water extract (B), soy milk water extract (C) and Vc (D) on the scavenging rate of DPPH free radicals, compared with low concentrations* P < 0.05, ** P < 0.01,*** P < 0.001; Figure 3 shows the determination of reducing power in A, B, C, and D, respectively, of porridge water extract, rice paste water extract, soy milk water extract, and Vc. Compared with low concentrations* p < 0.05, ** p < 0.01,*** p < 0.001; Figure 4 Determination of hydroxyl radical scavenging rate in porridge water extract (A), rice paste water extract (B), soy milk water extract (C), and Vc (D), compared with low concentration* P < 0.05, ** P < 0.01,*** P < 0.001; Figure 5 Effects of different concentrations of porridge water extract CWE, rice paste water extract RPWE and soy milk water extract SMWE on GES-1 cell activity, compared with the normal group, * p < 0.05, ** p < 0.01, *** p < 0.001; Figure 6 Effects of porridge water extract CWE, rice paste water extract RPWE and soy milk water extract SMWE on GES-1 cell activity. Compared with the normal group,### p < 0.001; compared with the model group* p < 0.05, ** p < 0.01,*** p <0.001; Fig. 7 The morphological effects of porridge water extract CWE, rice paste water extract RPWE and soy milk water extract SMWE on ethanol-induced GES-1 cell damage; Figure 8 Changes of ROS in GES-1 cells after 24 h of treatment with porridge water extract CWE, rice paste water extract RPWE and soy milk water extract SMWE. Compared with the normal group, ### p < 0.001; compared with the model group* p < 0.05, ** p < 0.01,*** p <0.001; Fig. 9 Effects of porridge water extract CWE, rice paste water extract RPWE and soybean milk water extract SMWE on the activity level of SOD in GES-1 cells after ethanol injury. ### p < 0.001; compared with the model group* p <0.05,** p < 0.01,*** p < 0.001; Figure 10 Effects of porridge water extract CWE, rice paste water extract RPWE and soy milk water extract SMWE on the MDA activity level in GES-1 cells after ethanol injury. Compared with the normal group, ### p < 0.001; compared with the model group* p <0.05,** p < 0.01,*** p < 0.001; Figure 11 Effects of porridge water extract CWE, rice paste water extract RPWE and soy milk water extract SMWE on the GSH activity level in GES-1 cells after ethanol injury. Compared with the normal group, ### p < 0.001; compared with the model group* p <0.05,** p < 0.01,*** p < 0.001; Fig.12A is the effect of porridge on the weight change of mice with acute alcoholic gastric injury, control: ranitidine; L-PCW: low-dose porridge group; M-PCW: medium-dose porridge group; H-PCW: high-dose porridge group; B is the effect of porridge on gastric mucosal injury in mice; C is the effect of porridge on the tissue morphological changes in mice; Fig.13 A and B show the effects of porridge on serum MDA and GSH in mice. C, D, and E show the effects of porridge on GSH, MPO, and SOD in gastric tissue of mice. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0020] The millet (Longshan millet) used in the present invention was purchased from Shandong Provincial Supply and Marketing Cooperative Agricultural Products Co., Ltd., and the soybean was purchased from Wuchang Caiqiao Rice Industry Co., Ltd.

[0021] The human gastric mucosal epithelial cells (GES-1 cells) used in the present invention were purchased from Shanghai Cell Bank.

[0022] In some embodiments, the filtering step is performed using cotton gauze.

[0023] In some embodiments, the iron pan is preheated until the bottom of the iron pan turns red, and heated at 160-200° C. for 2-4 minutes.

[0024] Example 1 (1) Select 103 g of millet and 75 g of soybeans of good quality, add water to soak the millet and soybeans respectively for 6 hours; (2) Use a pulper to add water to the soaked millet and soybeans (1 g: 8 mL of water, respectively) for at least two times to ensure that there is no granularity, and then filter with cotton gauze; (3) Preheat the iron pot until the bottom of the pot is red, add 1 / 5 of the soy milk and boil it, then add the remaining soy milk to the pot, add rice milk, and boil for 44 minutes. To prevent the pot from sticking, stir it constantly during the boiling process to make a mushy porridge; (3) Dilute with 5 times distilled water, extract with intermittent ultrasound (100 W) at 50°C for 2 h, and then centrifuge at 10,000 r / min for 15 min. Collect the supernatant, filter, concentrate by rotary evaporation (concentrate to 1 / 5 of the original volume), and freeze to obtain Congee water extract (CWE).

[0025] Effect Example The experiments involved in the present invention were conducted in parallel for at least 3 times, and the data were expressed as mean ± standard deviation. Origin 2021, GraphPad, and SPSS software were used for data analysis and drawing charts. P <0.05 indicated significant differences.

[0026] (I) The porridge prepared in Example 1 was tested for protein, fat and starch contents according to GB 5009.5-2016; the dietary fiber content was tested according to GB 5009.88-2014, the polyphenol content was determined by Folin phenol colorimetry, and the polysaccharide content was determined by absorption photometry. The specific results are shown in Table 1.

[0027] Table 1 (ii) The porridge prepared in Example 1 was subjected to headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) method to determine the flavor substances therein: Headspace solid phase microextraction-gas chromatography-mass spectrometry conditions: about 20.0 mL of sample was added to a sample bottle prepared in advance, the sample bottle prepared in advance was placed in a 60°C water bath for equilibration for 10 min, an aged (aging time: 5 min) extraction needle was placed in the sample bottle, the quartz fiber head was exposed to the headspace gas in the sample bottle, constant temperature extraction was performed for 30 min, the fiber head was pushed back into the needle head and pulled out, inserted into the injector of GC-MS and analyzed at 250°C for 1 min, and the instrument was started to collect data at the same time.

[0028] A total of 80 volatile components were detected, including 19 alkanes, 15 esters, 10 oxygen-containing heterocyclics, 8 alcohols, 7 rares, 7 aromatics, 4 aldehydes, 4 ketones, 3 nitrogen-containing heterocyclics, and 3 ethers. Alkanes accounted for 23.75%, esters accounted for 18.75%, oxygen-containing heterocyclics accounted for 12.5%, alcohols accounted for 10%, rares accounted for 8.75%, aromatics accounted for 8.75%, aldehydes accounted for 5%, ketones accounted for 5%, nitrogen-containing heterocyclics accounted for 3.75%, and ethers accounted for 3.75%. Among them, alkanes and esters accounted for the largest proportion. Alkane compounds have a large threshold value and contribute less to flavor. Esters are mainly produced by fatty acid hydrolysis and microbial metabolism, and have a great effect on flavor. Overall, the characteristic aroma of porridge is rich and strong, especially the aroma of porridge.

[0029] (III) Effect of porridge water extract on the repair of alcoholic gastric injury in gastric mucosal GES-1 cells A control group was set up: rice or soybean milk was soaked in water for 6 hours, and then water was added to pulp according to the same proportion as in Example 1, and filtered to obtain rice paste or soybean milk; then diluted with 5 times distilled water, respectively, and intermittently ultrasonically (100 W) water extracted for 2 hours at 50°C, and then centrifuged at 10000 r / min for 15 minutes. After centrifugation, the supernatant was taken, filtered, concentrated by rotary evaporation (concentrated to 1 / 5 of the original volume), and frozen. Rice paste water extract (RPWE) and soybean milk water extract (SMWE) were obtained respectively.

[0030] Congee water extract (CWE), rice paste water extract (RPWE), and soy milk water extract (SMWE) samples were prepared (Figure 1). The extraction rates of congee, soy milk, and rice paste were 2.27% ± 0.23, 3.7% ± 0.261, and 1.4% ± 0.181, respectively.

[0031] The contents of starch, dietary fiber, protein, fat, polysaccharide, polyphenol and amino acid in the porridge water extract are shown in Table 2. The highest content is starch at 2.7 ± 0.020 g / 100g, polysaccharide at 2.020 ± 0.020g / 100g, and protein at 1.650 ± 0.010g / 100g. A total of 16 amino acids were detected, of which glutamic acid with the highest content was 0.309 ± 0.001g / 100g.

[0032] Table 2 Contents of dietary fiber, starch, protein, fat, polyphenols, polysaccharides and amino acids in porridge water extract 1. Determination of DPPH free radical scavenging rate (1) Congee water extract, soy milk water extract, and rice paste water extract were prepared with concentrations of 5, 10, 15, 20, and 25 mg / mL, respectively; the concentrations of Vc were 0, 0.25, 0.5, 0.75, 1.00, and 1.25 mg / mL.

[0033] (2) Add 1 mL of sample of each concentration into a fixed container, add 1 mL of DPPH ethanol solution (0.1 mol / L) and react in the dark for 30 min. Measure the absorbance at 517 nm, then use anhydrous ethanol as a blank control and Vc as a positive control.

[0034] (3) Calculate its clearance rate and IC according to formula (4.1) 50 value.

[0035] (4.1) Where: A i is the absorbance value measured by sample solution + DPPH solution; A j A is the absorbance value measured by sample solution + anhydrous ethanol; C It is the absorbance value measured for anhydrous ethanol + DPPH solution.

[0036] (4) The results of DPPH free radical scavenging ability of porridge, soy milk, and rice paste water extracts are shown in Figure 2. The DPPH free radical scavenging ability increases with the increase of concentration. When the mass concentration is 25 mg / mL, the DPPH free radical scavenging rates of porridge water extract, rice paste water extract, and soy milk water extract are 87.52%, 92.98%, and 84.14%, respectively. The scavenging rate of high concentrations is significantly increased compared with the lowest concentration ( P <0.05), the concentration scavenging ability of porridge water extract was between those of soy milk and rice paste water extract. SPSS analysis showed that the half-inhibitory mass concentrations of porridge, rice paste, soy milk water extract and Vc on DPPH free radicals were 8.64 mg / mL, 13.93 mg / mL, 7.67 mg / mL and 2.18 μg / mL, respectively, indicating that porridge water extract had a better antioxidant capacity between those of soy milk and rice paste water extract.

[0037] 2. Determination of reducing power (1) Congee water extract, soy milk water extract, and rice paste water extract were prepared with concentrations of 5, 10, 15, 20, and 25 mg / mL, respectively; the concentrations of Vc were 0, 0.25, 0.5, 0.75, 1.00, and 1.25 mg / mL.

[0038] (2) 2 mL of sample (concentrations of 5, 10, 15, 20, and 25 mg / mL), 1 mL of phosphate buffer and 1 mL of 1% potassium ferrocyanide, incubate the mixture at 50 °C for 20 min, and then cool rapidly; (3) Add 0.5 mL of 10% trichloroacetic acid to the mixture to stop the reaction. After centrifuging at 5000 r / min for 10 minutes, take 1 mL of the supernatant, add 1 mL of double distilled water and 200 μL of 1% ferric chloride, and measure the absorbance at 700 nm. An increase in absorbance indicates that the reducing power of the reaction mixture has increased.

[0039] (4) As shown in Figure 3, the reducing power of porridge water extract, rice paste water extract, soy milk water extract and Vc increased with the increase of concentration ( P<0.05). This indicates that the porridge water extract, rice paste water extract, soybean milk water extract and Vc all have reducing ability, among which the reducing power of porridge water extract is higher than that of soybean milk and rice paste water extract, but much lower than that of Vc.

[0040] 3. Hydroxyl radical scavenging rate (o-phenanthroline - Fe 2+ Determination of (1) Prepare porridge water extract, soy milk water extract, and rice paste water extract with concentrations of 5, 10, 15, 20, and 25 mg / mL, respectively; the concentration of Vc is 0, 0.25, 0.5, 0.75, 1.00, and 1.25 mg / mL. ; (2) Add 1 mL of o-phenanthroline solution (0.75 mmol / L) and 1 mL of PBS solution (0.1 mol / L, pH 7.4), and then add 1 mL of sample solutions of different concentrations; (3) After mixing evenly, add 1 mL FeSO4 (0.75 mmol / L), mix evenly, then add 1 mL H2O2 (0.01%) in a water bath (37°C) for 1 hour, and finally measure the absorbance at 536 nm. Calculate the clearance and IC according to formula (4.2): 50 value.

[0041] (4.2) Where: As is the absorbance value measured after adding the sample; A B A is the absorbance value measured without adding sample and using distilled water instead of H2O2; P The absorbance value is measured without adding sample but with H2O2 added.

[0042] Table 3 Hydroxyl radical scavenging rate Sampling method for scavenging rate determination (4) As shown in Figure 4, Vc, porridge water extract, rice paste water extract and soy milk water extract all have a certain ability to scavenge hydroxyl free radicals. Compared with the lowest concentration, the scavenging ability of high concentration is significantly increased ( P <0.05), but the clearance ability of different samples was different, and the IC 50The values ​​are: Vc is 0.394 mg / mL, CWE is 0.614 mg / mL, RPWE is 1.340 mg / mL, and SMWE is 2.088 mg / mL. It can also be seen that within the measured concentration range, the scavenging rates of porridge water extract, rice paste water extract, soybean milk water extract and Vc on hydroxyl free radicals increase with the increase of concentration. Among them, the scavenging rate of porridge water extract on hydroxyl free radicals can reach up to 93.242%. Although the effect is not as significant as that of Vc, it also has a certain scavenging ability.

[0043] 4. Analysis of GES-1 cell proliferation activity (1) Cell recovery First, add 5 mL of complete high-glucose culture medium into a 6 cm culture dish to restore to room temperature, turn on the water bath to adjust to 37°C, then take out the stored GES-1 cells from liquid nitrogen, thaw the cells in a water bath, put the thawed cells into the prepared culture dish, and culture them in a cell culture incubator for 6 hours, observe the cell adhesion, change the cell medium, and finally continue to culture in the incubator.

[0044] (2) Cell passaging Use a suction pump to remove the original culture medium in the cell culture dish, add 3 mL PBS buffer to wash, add trypsin to digest for 2 minutes, and add 4 mL cell culture medium to terminate digestion. Then gently beat the cells and mix them evenly, place them in a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 3 mL cell culture medium, gently beat the cells, add them to the prepared culture dish, and culture them in a cell culture incubator.

[0045] (3) Cell cryopreservation First, prepare the freezing solution (serum + DMSO, the ratio is 9:1), take out the cells from the incubator, use a suction pump to remove the original culture medium in the culture dish, wash with PBS buffer, add 2 mL of trypsin to digest for 2 minutes, add the prepared cell freezing solution, gently blow the cells, and finally put them into 1 mL cryovials, freeze them in a -80℃ refrigerator for one day, and then transfer them to liquid nitrogen for storage.

[0046] (4) If Figure 5 As shown in the results, the porridge water extract, rice paste water extract and soybean milk water extract all have good effects on promoting the growth of GES-1 cells, among which the porridge water extract > soybean milk water extract > rice paste water extract. The three sample water extracts acted on the proliferation activity of normal GES-1 cells in the concentration range of 1.25-20 mg / mL. After 24 hours of culture in the cell culture incubator, the cells in the sample group showed significant changes compared with the blank group ( P<0.05), when the concentration was 20 mg / mL, the cell activity was inhibited. Therefore, the concentrations of 2.5, 5, and 10 mg / mL were selected as the low, medium, and high dose groups of the three sample water extracts, respectively. Studies have shown that the three sample water extracts can increase the permeability of the cell membrane to a certain extent by adsorbing on the cell surface, thereby promoting the water extract to enter the cell and make it work better, but the porridge water extract has the best effect.

[0047] 5. Establishment of GES-1 ethanol injury model (1) GES-1 cells with good growth were inoculated into 96-well cell culture plates at a density of 1×10 5 cells / well, 100 μL per well, set up blank group and model group, and set up 5 replicates in each group. After culturing in the incubator for 24 hours, the adhesion was observed under an optical inverted microscope. After waiting for the cells to adhere, the blank group was replaced with DMEM high-glucose incomplete medium, and the model group was replaced with DMEM incomplete high-glucose medium containing different concentrations of ethanol (0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%) to continue culturing cells. The modeling time was 2, 3, 4, 5, and 6 h, respectively. The degree of damage to GES-1 cells was observed under an optical inverted microscope to screen out the optimal concentration and action time of ethanol modeling. The CCK-8 method was used to detect cell activity, the absorbance at 450 nm was measured, and the inhibition rate was calculated. The inhibition rate was 40%, which was the best modeling condition.

[0048] (4.4) A: Absorbance value of experimental group B: Absorbance value of control group C: Blank absorbance value (2) Cell grouping and drug administration GES-1 cells were divided into blank group, model group and positive control group (ranitidine 160 μg / mL); porridge water extract (CWE), rice paste water extract (RPWE) and soy milk water extract (SMWE) were divided into low-dose group (2.5 mg / mL), medium-dose group (5 mg / mL) and high-dose group (10 mg / mL).

[0049] (3) CCK-8 method to detect cell survival rate The cell density of GES-1 cells was 1×10 5 / mL was inoculated in a 96-well plate, 100 μL per well, and 5 replicates were set up at the same time. After culturing for 24 h until the cells adhered to the wall, complete culture medium was added to the normal group GES-1 cells, and the other experimental groups were added to screen the optimal modeling concentration and time. After culturing for a certain period of time, the supernatant was aspirated and washed with PBS. After modeling, 100 μL of culture medium was added to the normal group and model group, 100 μL of culture medium with a concentration of 160 μg / mL ranitidine was added to the group, and 100 μL of low, medium, and high doses of culture medium containing porridge water extract, soy milk water extract, and rice paste water extract were added to the drug administration group, respectively, and cultured for 24 hours. The activity of cells in each group was detected by CCK-8 method, the absorbance at 450 nm was measured by microplate reader, and the cell proliferation rate was calculated. The above experiment was repeated 3 times.

[0050] (4.5) (4) Observation under an inverted microscope revealed that in the normal group, GES-1 cells showed tight cell-to-cell connections, the nucleus was round and located in the center of the cell, and the cells adhered to the surface of the culture dish to form irregular shapes such as polygons or rhombuses. This morphology indicated that the cells had normal growth and adhesion characteristics, good cell-to-cell interactions, and intact nuclear structures; however, after being damaged by ethanol, the GES-1 cells in the model group showed significant morphological changes. A large number of cells fell off the surface of the culture container and floated in the culture medium, which was a manifestation of the loss of cell adhesion ability. The number of remaining attached cells decreased, and the connections between cells were significantly reduced, and the gaps became larger, which may be due to the destruction of cell-to-cell adhesion molecules and cytoskeleton structures by ethanol. Compared with the model group, the cell growth morphology of the ranitidine group and the high, medium, and low dose groups of porridge water extract, rice paste water extract, and soy milk water extract improved, and the cell connections became tighter ( Figure 7 The effects of the aqueous extracts of the three samples on the survival of GES-1 cells damaged by ethanol are shown in Figure 2. Figure 6 As shown in Figure 2, the survival rate of GES-1 cells in the model group was significantly decreased compared with that in the blank group ( P <0.05), after 24 hours of exposure to three samples of different concentrations, the cells damaged by ethanol were improved compared with the model group, among which the effects of the samples in the medium-dose group and the high-dose group were the most obvious. Among them, the effect of the porridge water extract on the proliferation of GES-1 was higher than that of the rice paste water extract and the soy milk water extract.

[0051] 6. Detection of Reactive Oxygen Species (ROS) in GES-1 Cells (1) Inoculate well-growing GES-1 cells into a 12-well cell culture plate, carefully remove the cell culture medium, trypsinize the cells, discard the trypsin, and pipette them into a single cell suspension with 4 mL of DMEM medium and count them. 5 GES-1 cells were inoculated into 12-well plates at a density of 2 mL per well. After culturing for 24 h, the culture medium was discarded. After modeling with 5% anhydrous ethanol for 3 h, the cells were cultured for another 24 h with DMEM high-glucose medium containing 2.5, 5, and 10 mg / mL porridge water extract (CWE), rice paste water extract (RPWE), and soy milk water extract (SMWE). The DCFA-DA probe was diluted with serum-free DMEM high-glucose medium at a ratio of 1:1000, and 0.5 mL of the diluted DCFA-DA probe was added to each well and incubated for another half an hour. The cells were washed three times with serum-free DMEM high-glucose medium to fully remove the DCFA-DA probe that did not enter the cells. Then 1 mL PBS was added to the cells, stored in the dark, and measured with a fluorescence spectrophotometer.

[0052] (2) Oxidative stress refers to the imbalance between the production and removal of reactive oxygen species (ROS) in cells. This imbalance usually leads to a large accumulation of ROS. ROS plays a dual role in cells. On the one hand, it can activate a variety of signaling pathways that are sensitive to redox status. When the level of ROS increases moderately, they can promote the adaptive response of cells and enhance the antioxidant defense ability of cells. On the other hand, excessive ROS can cause oxidative damage in cells, thereby triggering inflammatory responses, leading to the production and release of inflammatory factors, and inducing cellular inflammatory responses. Therefore, the level of ROS in cells can directly reflect the degree of cell damage. As shown in Figure 8, as the drug concentration increases, the fluorescence intensity of the green fluorescence of the fluorescence microscope decreases, indicating that ROS is significantly reduced. The porridge water extract reduces the content of intracellular ROS, and the effect is better than that of soy milk and rice paste water extracts.

[0053] 7. Detection of biochemical indicators (1) GES-1 cells were seeded in 6-well plates at 1.5 × 10 cells per well. 52 mL per well. Refer to 4.2.9 for group administration. After the cells are completely attached to the wall, the old culture medium is removed and replaced with culture medium. Except for the blank control group, all other groups are replaced with 2 mL / well of 5% ethanol solution. After incubation for 3 hours, the supernatant is discarded. 2 mL / well of DMEM culture medium is added to the blank control group and the model group, and 2 mL / well of 2.5 mg / mL, 5 mg / mL, and 10 mg / mL samples are added to the experimental group, respectively, and incubated for 24 hours. 1 mL of PBS is added to each well for washing, and then 1 mL of trypsin is added for digestion for 2 minutes. The wells are taken out and placed in sterile EP tubes. Ultrasonic disruption is performed (add a certain amount (10 6 Cells of the same order of magnitude are generally added with 0.3-0.5 mL of PBS buffer), centrifuged to obtain the supernatant, and the cell protein concentration is detected using a BCA kit and stored at -80°C. The cell supernatant is detected using SOD, MDA, and GSH kits, respectively. For specific test methods and operating steps, refer to the kit instructions.

[0054] (2) Analysis of SOD enzyme activity in ethanol-damaged GES-1 cells Under oxidative stress, ROS is overproduced, especially in ethanol-induced gastric mucosal damage. The accumulation of ROS can lead to damage to cell structure and function. SOD is an important antioxidant enzyme that converts ROS into more stable hydrogen peroxide by catalyzing the reaction of superoxide anions and hydrogen peroxide. This process helps to reduce the toxic effects of ROS and protect cells from oxidative damage. As shown in Figure 9, the SOD activity level of the blank control group was 7.09 U / mgprot, and the SOD activity level of the model group was 4.193 U / mgprot. The SOD activity level of the model group was lower ( P <0.01). One of the mechanisms of ethanol-induced cell damage may involve the inhibition of SOD enzyme activity. The SOD activity of the medium-dose and high-dose groups of the three experimental groups of water extracts was higher than that of the model group, and the activity level of the high-dose group was equivalent to that of the positive drug ranitidine group. The SOD enzyme activity value of the porridge water extract was higher than that of the rice paste water extract and soy milk water extract groups, that is, the porridge water extract has a better ability to enhance the SOD enzyme activity level.

[0055] (3) Analysis of MDA enzyme activity in ethanol-damaged GES-1 cells Malondialdehyde (MDA) is a commonly used biomarker of lipid peroxidation. It is the metabolic end product of cell membrane lipid peroxidation. The level of MDA is usually used as an indicator of the degree of lipid peroxidation, reflecting the severity of oxidative stress in cells or tissues. In the study of gastric mucosal injury, the increase in MDA levels is usually associated with the severity of gastric mucosal inflammation and injury. Therefore, ROS-mediated lipid peroxidation and increased MDA levels play a key role in the pathogenesis of gastric mucosal injury. Fig.10 The MDA level in the model group was higher than that in the blank control group, and there was a large difference between the two groups ( P <0.001), indicating that ethanol can increase the level of lipid peroxidation in cells, thus leading to cell damage. The MDA of the experimental group was lower than that of the model group. Among them, the MDA value of the high-dose porridge water extract group was lower than that of the high-dose rice paste water extract and soy milk water extract groups. The results showed that the high-dose porridge water extract group had a better ability to reduce the level of lipid peroxidation than the rice paste water extract and soy milk water extract, and was lower than the positive drug ranitidine group.

[0056] (4) Analysis of GSH enzyme activity in ethanol-damaged GES-1 cells In the gastric mucosa, the level of GSH is essential for maintaining the integrity and function of cells. The lack of GSH may lead to the accumulation of free radicals, aggravate the oxidative stress response, and thus deepen the damage of the gastric mucosa. Therefore, GSH and its related enzyme system play an important protective role in defending against ethanol-induced gastric tissue oxidative stress. As shown in Figure 11, the GSH level of the model group was 107.456 μmol / gprot, which was lower than the GSH level of the blank control group, indicating that the GSH level in GES-1 cells after ethanol induction was reduced, causing cell damage. In addition to the low-dose group of soy milk water extract, the GSH levels of the other three experimental groups were higher than the model group, and there was a significant difference in the high-dose group (P < 0.05), indicating that the porridge water extract can increase the activity of GSH in cells.

[0057] 4. Animal testing (1) Experimental animal grouping and drug administration Sixty male Kunming mice were randomly divided into blank group, model group, positive group, low-dose porridge group, medium-dose porridge group, and high-dose porridge group, with a total of 6 groups, 10 mice in each group. Mice in the blank group and model group were gavaged with 0.5% sodium carboxymethylcellulose (CMC-Na) solution; the positive group was given ranitidine at a dose of 50 mg / kg; the international standard recommends that the intake of grains is 40 g / d for each healthy adult. According to the experimental regulations, it was converted into the daily gavage amount of the medium-dose group of experimental mice, and the calculated results were that the gavage amounts of the high, medium, and low-dose groups of porridge were 6, 3, and 1.5 g / (kg.BW), respectively. All drugs were dissolved in 0.5% CMC-Na solution. The drug was gavaged once a day for 15 consecutive days. After the 14th day of administration, the mice were fasted for 24 hours, but water was not allowed. On the 15th day, except for the blank group, the drugs were still administered. 2 hours after administration, except for the normal group, the other groups were gavaged with 75% ethanol 10mL / kg, and the mice were killed 1 hour later. Blood was collected through the eyeballs, and then the mice were killed by cervical dislocation. The gastric tissue of the mice was removed and cut along the greater curvature of the stomach. Then the gastric tissue was placed in ice-cold PBS and thoroughly rinsed to remove the chyme. A part of the gastric tissue was taken from the same part and placed in 4% paraformaldehyde solution for pathological tissue sectioning (He staining). The rest of the gastric tissue was frozen at -80℃ for subsequent experiments.

[0058] (2) Treatment of experimental mice and sample collection After gavage, the mice were gently placed in cages, one mouse per cage, and the behavioral state of each mouse was observed. One hour after gavage, blood was collected after anesthesia, and the mice were killed by cervical dislocation and dissection. The abdominal cavity was cut open, the stomach was removed, and it was cut along the maximum curvature. The stomach contents were rinsed clean with saline, placed on tin foil, weighed and recorded, and then fixed on a flat surface for photography and archiving. At the same time, the stomach damage was observed and evaluated. The evaluated stomach tissue was cut open with surgical scissors, one part was placed in paraformaldehyde for histological evaluation, and one part was placed in liquid ammonia for quick freezing for the determination of indicators of oxidative stress and inflammatory factors.

[0059] Ⅰ. Determination of serum GSH and MDA. Plasma was separated by centrifugation at 4000r / min for 10 min. The test kit (Nanjing Jiancheng Bioengineering Institute) was used for detection.

[0060] Ⅱ. The gastric tissues to be observed were fixed in 10% formalin solution for 48 hours, dehydrated, cleared, waxed, embedded, sectioned, and stained with hematoxylin and eosin (H&E). The morphological changes of the tissues were observed using an optical microscope.

[0061] III. Determination of SOD, MPO, and GSH in gastric tissue. The gastric tissue was refrigerated in liquid nitrogen, taken out before the experiment, and ground in a mortar at low temperature. Accurately weigh the ground tissue, add physiological saline according to weight (g): volume (mL) = 1:9, and continue to stir evenly. After stirring, centrifuge at 3000 r / min, 4℃ for 10 min, and take the supernatant. Use the kit (Nanjing Jiancheng Bioengineering Institute) for detection.

[0062] (3) Analysis of the preventive effect of porridge on acute alcoholic gastric injury in mice Ⅰ. Changes in mouse body weight After 6-week-old male KM mice were adapted to the experimental environment for seven days, they were given medication according to the experimental groups, and their body weights were recorded every day. Figure 4 As shown, 0-5 days were the adaptation period, during which the weight of mice slowly increased; 6-15 days were the medication period, during which the weight of mice slowly decreased and then tended to stabilize; the weight of mice in the medication group had the same trend as that in the normal group, and the mice were in good condition, indicating that the porridge had no significant effect on the weight of mice.

[0063] Ⅱ. Observation of the Morphology of Mouse Gastric Mucosa The whole stomach of the mouse was cut open along the greater curvature of the stomach, and the contents were rinsed with cold saline. After the stomach wall was unfolded, the internal gastric mucosa was shown in Figure 12. The gastric mucosa of the mice in the normal group was shiny and ruddy, intact and without damage. After ethanol gavage, the surface of the gastric mucosa of the mice in the model group had obvious bleeding, damage and ulceration. In the positive group and the low, medium and high dose groups of porridge, there was partial bleeding in the gastric mucosa, but the degree of damage was reduced.

[0064] like Fig.12 As shown in the figure, the structures of the gastric mucosa of mice in the normal group were complete and clear, the cell morphology was intact, the glands were arranged regularly, and no abnormal lesions such as tissue defects, bleeding, and inflammation were observed. In the model group, diffuse necrosis and shedding of mucosal cells in the mice were serious, and a large number of red blood cells were seen in the mucosal layer. The cells were swollen and loosely arranged, and there was inflammatory cell infiltration. In the positive group and the low, medium, and high dose groups of porridge, there was partial bleeding in the gastric mucosa, but the degree of damage was reduced.

[0065] III. Determination of MDA and GSH in mouse serum Fig.13-A shows that increased MDA levels can lead to gastric mucosal damage. It is widely used to evaluate lipid peroxidation. A decrease in MDA levels can prove that oxidative reactions are being inhibited in the body. As shown in 13-B, the MDA level in the model group increased compared with the normal group. Compared with the model group, the MDA levels in the low, medium and high dose groups of porridge were significantly improved. Glutathione (GSH) is an important antioxidant in the body. GSH can maintain the morphology of red blood cells, prevent them from being oxidized by ROS, and maintain the transport of oxygen in the body. Therefore, an increase in GSH levels can indicate that oxidative reactions are being inhibited in the body. As shown in 13, the GSH level in the model group was significantly reduced compared with the normal group. Compared with the model group, the GSH level in the low, medium and high dose groups of porridge was significantly improved.

[0066] IV. Determination of SOD, GSH and MPO in mouse gastric tissue As shown in Figure 13-C, the main physiological function of SOD enzyme is to remove free radicals in the body, which can protect the gastric mucosa from the effects of ethanol-induced oxidative stress. The increase in SOD enzyme activity can be considered as an inhibition of oxidative reactions in the tissue. As shown in Figure 13, compared with the normal group, the SOD activity of the model group was significantly reduced. Compared with the model group, the SOD levels of the low, medium and high dose groups of porridge were significantly improved. Fig.13 -D shows that MPO is a peroxidase in the body. MPO plays a decisive role in the development of gastric mucosal damage. The higher the MPO activity, the more severe the gastric damage. As shown in 13, compared with the normal group, the MPO activity of the model group was significantly increased. Compared with the model group, the MPO levels of the low, medium and high dose groups of the porridge were significantly improved. Fig.13 -E After intragastric administration of alcohol, the antioxidant enzyme activity in the gastric tissue of mice in the model group decreased, the peroxide content increased, and the antioxidant capacity decreased. Intragastric administration of porridge improved the oxidative stress level in the gastric tissue of mice. The GSH content in the gastric tissue of mice in the model group decreased significantly. The GSH content in the gastric tissue of mice in the porridge treatment group increased.

[0067] Paste porridge reduces the damage caused by ethanol by lowering the activity levels of MDA and MPO enzymes and increasing the activity levels of SOD and GSH enzymes, thereby achieving a protective effect against acute alcoholic gastric mucosal injury.

[0068] The present invention uses water extraction for porridge, soybean milk and rice paste, and measures the main components of the porridge water extract, respectively, by measuring the DPPH free radical scavenging rate, reducing power and hydroxyl free radical scavenging rate. The invention provides a scientific basis for studying the antioxidant capacity of porridge in vivo and its influence on human health. The GES-1 cell injury model is established by ethanol induction, and the repair effect of the porridge water extract on ethanol-damaged GES-1 cells is studied. The rice paste water extract, soybean milk water extract and positive drug ranitidine are used as comparisons to explore the influence of the porridge water extract on its protective effect on gastric mucosa and the related mechanism.

[0069] The following conclusions were drawn: (1) The main components of the porridge water extract were determined. The most abundant components were starch (2.7 ± 0.020 g / 100 g), polysaccharide (2.020 ± 0.020 g / 100 g), and protein (1.650 ± 0.010 g / 100 g). A total of 16 amino acids were detected, of which glutamic acid (0.309 ± 0.001 g / 100 g) was the most abundant. Moreover, the DPPH radical scavenging ability of porridge, soy milk and rice paste water extracts increased with the increase of concentration, indicating that porridge water extract has better antioxidant capacity between soy milk and rice paste water extracts; the absorbance of porridge water extract, rice paste water extract and soy milk water extract is increasing, and the increase in absorbance indicates that the reducing power of the three sample water extracts is improved, among which the reducing power of porridge water extract is higher than that of soy milk and rice paste water extracts; Vc, porridge water extract, rice paste water extract and soy milk water extract have certain scavenging ability for hydroxyl free radicals, but at the same concentration, the four samples show different scavenging abilities for hydroxyl free radicals, all of which are positive controls Vc>CWE>RPWE>SMWE.

[0070] (2) The porridge water extract had no obvious cytotoxicity to normal GES-1 cells and could promote cell proliferation. The cell proliferation promoting activity of the porridge water extract was better than that of the rice paste water extract and soy milk water extract. The results of the determination of reactive oxygen species (ROS) showed that the ability of the porridge water extract to reduce intracellular ROS was better than that of soy milk and rice paste water extract. The porridge water extract could increase the intracellular SOD level. The SOD levels of the medium-dose group and the high-dose group of the experimental group were higher than those of the model group. The activity level of the high-dose group was equivalent to that of the positive drug ranitidine group. The SOD enzyme activity value of the porridge water extract was higher than that of the rice paste water extract and soy milk water extract groups. It could reduce the MDA level. The MDA value of the experimental group was lower than that of the model group. Among them, the MDA value of the high-dose group of the porridge water extract was lower than that of the high-dose group of the rice paste water extract and soy milk water extract. It could increase the GSH level. The GSH levels of the three experimental groups were higher than those of the model group except for the low-dose group of the soy milk water extract. There was a significant difference in the high-dose group. This indicates that the porridge water extract can protect GSH in cells and may have the effect of protecting the gastric mucosa.

[0071] The present invention studies the protective effect of porridge on alcoholic gastric mucosal damage, indicating that the porridge water extract may achieve the effect of gastric mucosal protection through antioxidant capacity, in order to provide theoretical support for the rational development of porridge as a food.

Claims

1. A method for preparing a porridge extract, characterized in that: The following steps are involved: (1) Soaking millet and soybeans in water respectively, then beating and filtering to obtain rice milk and soybean milk; (2) After preheating the iron pot, first add 1 / 5 of the volume of soy milk. After boiling, add all the remaining soy milk, and then add rice milk and cook to obtain porridge; (3) Add distilled water to the porridge, perform ultrasonic extraction, centrifuge, collect the supernatant, filter, concentrate by rotary evaporation, and freeze to obtain the porridge water extract.

2. The preparation method according to claim 1, characterized in that: In step (1), the mass ratio of millet to soybean is 75-125:50-100; the ratio of millet or soybean to water is 1 g:1-8 mL.

3. The preparation method according to claim 2, characterized in that: In step (1), the mass ratio of millet to soybean is 103:75; and the liquid-to-solid ratio is 4.8:

1.

4. The preparation method according to any one of claims 1 to 3, characterized in that: In step (2), the preheating is heating at 160-180°C for 2-4 minutes, until the bottom of the iron pot turns red; the boiling time is 35-55 minutes; preferably, the boiling time is 44 minutes.

5. The preparation method according to any one of claims 1 to 4, characterized in that: In step (3), the volume ratio of the porridge to distilled water is 1:

5.

6. The preparation method according to claim 1 or 5, characterized in that: In step (3), the ultrasonic extraction is performed by intermittent ultrasonic extraction (100w) at 50°C for 2h.

7. The preparation method according to claim 6, characterized in that: In step (3), the volume ratio of the porridge to distilled water is 1:5; and the centrifugation is performed at 10000 r / min for 15 min.

8. Use of the porridge extract prepared by the preparation method according to any one of claims 1 to 7 in preparing a drug for repairing gastric damage.

9. The use according to claim 8, characterized in that: Application in the preparation of drugs for repairing alcoholic gastric damage in GES-1 cells.

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