Traditional Chinese medicine composition for resisting gastric mucosal lesion as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention provides a traditional Chinese medicine composition for resisting gastric mucosal lesion as well as a preparation method and application thereof. The preparation method comprises the following steps: S1, performing vacuum drying on the red-knees herb and the daphniphyllum calycinum, crushing to obtain mixed powder, and collecting dry condensate water for later use; s2, dry condensate water and compound enzyme are mixed to prepare enzymatic hydrolysate, the enzymatic hydrolysate is evenly sprayed to the mixed powder, then enzymolysis is conducted, and an enzymolysis mixture is obtained; s3, adding the enzymolysis mixture into an aqueous two-phase extraction solvent, performing ultrasonic extraction, filtering, centrifuging, taking supernate, performing vacuum concentration, eluting with macroporous adsorption resin, collecting eluent, performing vacuum concentration, and performing low-temperature vacuum drying to obtain the traditional Chinese medicine composition. According to the method, the red-knees herb and the daphniphyllum calycinum are subjected to ultrasonic extraction through the combination of proper enzymolysis and an aqueous two-phase extraction solvent, so that the dissolution of total flavonoid substances can be enhanced, the total flavonoid substances can be prevented from being damaged, the extraction yield of the total flavonoid substances is increased, and the extraction time is effectively shortened. The prepared traditional Chinese medicine composition can also obviously relieve gastric mucosal lesion and is good in acting effect.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine compositions for resisting gastric mucosal damage, and in particular to a traditional Chinese medicine composition for resisting gastric mucosal damage, and a preparation method and application thereof. Background Art

[0002] The occurrence of gastric mucosal injury is closely related to the damage of gastric mucosal epithelial cells caused by various stimuli. HP infection, NAISDS, ethanol, stress and other harmful damaging factors can cause damage to gastric mucosal cells, leading to gastric mucosal lesions with the main characteristics of gastric mucosal congestion, bleeding, erosion or superficial ulcers, which is the main pathological link of acute and chronic gastritis, gastric ulcer, gastric cancer and other diseases. In recent years, with the accelerated pace of life, increased work pressure, and irregular diet and work and rest time, the incidence of gastritis and gastric ulcer has continued to increase, becoming one of the most common clinical diseases. Therefore, the prevention and treatment of gastric mucosal injury has become a public health issue of urgent concern to the current society.

[0003] The formation of gastritis and gastric ulcer usually develops on the basis of gastric mucosal damage. Oxidative stress and inflammatory response play a key role in the occurrence, development, evolution and prognosis of gastric mucosal damage. Therefore, anti-oxidation and anti-inflammatory treatment strategies are of great significance for reducing gastric mucosal damage. Ethnic medicine has rich experience in preventing and treating gastric mucosal damage, and has relatively few side effects. Its effects are characterized by multiple pathways, multiple links and multiple targets, and it has good application prospects.

[0004] Polygonum hydropiper and Rhizoma Acerolae are two traditional Chinese medicines used to treat gastric diseases. They have the effects of regulating qi and strengthening the stomach, removing dampness and removing stagnation. They are used for acute gastroenteritis caused by poor circulation, qi stagnation and dampness, and the resulting indigestion symptoms such as abdominal distension, abdominal pain and diarrhea. When using the above two traditional Chinese medicines, the effective ingredients contained in them must be extracted first, so as to prepare corresponding pharmaceutical preparations. At present, due to unreasonable extraction methods, the extraction effect of Polygonum hydropiper and Rhizoma Acerolae is not high, resulting in a low content of total flavonoids in the medicines and poor effects. Summary of the invention

[0005] In view of this, the present invention provides a traditional Chinese medicine composition for resisting gastric mucosal damage, and a preparation method and application thereof.

[0006] The technical solution of the present invention is achieved in this way:

[0007] A method for preparing a Chinese medicine composition for resisting gastric mucosal damage comprises the following steps:

[0008] S1, placing Polygonum hydropiper and Rhizoma Atractylodis Macrocephalae in a vacuum drying oven for vacuum drying, and then crushing to obtain a mixed powder, and collecting and drying condensed water for later use;

[0009] S2, mixing the dried condensed water with the composite enzyme to obtain an enzymatic solution, spraying the enzymatic solution evenly into the mixed powder, and then performing enzymatic hydrolysis for 5-6 hours at a temperature of 30-50° C. and a relative humidity of 70%-100% to obtain an enzymatic mixture;

[0010] S3. Add the enzymatic mixture into the aqueous two-phase extraction solvent and shake it evenly, then perform ultrasonic extraction, filter, centrifuge the filtered extract, take the supernatant and distill it under reduced pressure to obtain a crude extract, apply the crude extract to a macroporous adsorption resin, elute with ethanol at a volume concentration of 50%-90%, collect the eluate, concentrate it under reduced pressure, and dry it in a vacuum at low temperature to obtain a Chinese medicine composition.

[0011] Furthermore, the particle size of the mixed powder is 40-60 mesh.

[0012] Furthermore, the mass ratio of the dried condensed water to the complex enzyme is 30-40:1; the complex enzyme includes cellulase, pectinase, and β-glucanase in a mass ratio of 3-5:2-3:0.5-1.4.

[0013] Furthermore, the two-phase aqueous extraction solvent includes choline chloride, betaine, ethylene glycol and glycerol in a mass ratio of 1.2-1.8:1.5-2.0:0.5-0.8:0.5-0.8.

[0014] Furthermore, the preparation method of the aqueous two-phase extraction solvent is as follows: under a stirring state with a rotation speed of 150-200 r / min, ethylene glycol is added to glycerol, stirred and mixed, and then choline chloride and betaine are added and stirred and mixed to obtain a mixed solvent, and then 20-50% of water by mass of the mixed solvent is added and mixed again, and then heated in a water bath at a temperature of 75-80°C and a rotation speed of 60-80 r / min, and stirred until a stable, uniform transparent liquid is formed, so as to obtain the aqueous two-phase extraction solvent.

[0015] Furthermore, the ultrasonic extraction process is: firstly, ultrasonic treatment is performed for 50-60 minutes at a temperature of 30-50°C, a power of 100-150W, and a frequency of 20-25kHz, and then standing for 2-3 minutes, and then the power is adjusted to 400-500W and the frequency is adjusted to 35-40kHz for ultrasonic treatment for 20-30 minutes.

[0016] Furthermore, the centrifugal speed is 4000-6000r / min and the centrifugal time is 10-12min.

[0017] Furthermore, the macroporous adsorption resin is AB-8 type macroporous adsorption resin.

[0018] The Chinese medicinal composition for resisting gastric mucosal injury prepared by the preparation method of the invention comprises Polygonum hydropiper and Rhizoma Atractylodis Macrocephalae in a mass ratio of 1:1.8-2.2.

[0019] The invention discloses an application of the Chinese medicine composition in preparing a drug for resisting gastric mucosal injury.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The invention obtains mixed powder by vacuum drying Polygonum hydropiper and Rhizoma Atractylodis Macrocephalae, and then moistens the dried mixed powder with collected dry condensed water and performs enzymolysis under suitable conditions, so that the cell walls of Polygonum hydropiper and Rhizoma Atractylodis Macrocephalae can be effectively broken to make the effective components of the Polygonum hydropiper and Rhizoma Atractylodis Macrocephalae be better dissolved, and the loss of flavonoid active substances in the Polygonum hydropiper and Rhizoma Atractylodis Macrocephalae after enzymolysis in an aqueous solution environment can be avoided, and further, a biphasic aqueous extraction solvent composed of choline chloride, betaine, ethylene glycol and glycerol is appropriately mixed and extracted under suitable ultrasonic conditions, so that the dissolution of total flavonoids in the Polygonum hydropiper and Rhizoma Atractylodis Macrocephalae can be enhanced, and the destruction of the flavonoids can be avoided, so that the extraction yield of the total flavonoids can be improved, and the extraction time can be effectively shortened.

[0022] Through the specific extraction method of the present invention, the total flavonoids in the obtained traditional Chinese medicine composition are significantly increased, the proliferation activity of cells is significantly increased after administration, the anti-oxidation and anti-inflammatory abilities of cells are significantly enhanced, and gastric mucosal damage is significantly reduced. Pharmacodynamic studies have shown that the traditional Chinese medicine composition has a significant improvement effect on gastric mucosal damage and enhances anti-inflammatory and antioxidant abilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The content of flavonoids in the traditional Chinese medicine compositions obtained by different preparation methods.

[0024] Figure 2 This is a comparison chart of the effects of the Chinese medicine composition prepared in Example 3 of the present invention on macroscopic observation of gastric mucosal lesions in mice.

[0025] Figure 3 This is a comparison chart of the effect of the Chinese medicine composition prepared in Example 3 of the present invention on HE staining of gastric mucosal lesions in mice.

[0026] Figure 4 This is a comparison chart of the effects of the Chinese medicine composition prepared in Example 3 of the present invention on normal cell proliferation; Cellviability refers to cell survival rate, and SMSO refers to dimethyl sulfoxide.

[0027] Figure 5 This is a fluorescence image comparison diagram of the effect of the traditional Chinese medicine composition prepared in Example 3 of the present invention on intracellular ROS, Control refers to the normal group, and Model refers to the model group.

[0028] Figure 6 for Figure 5 Corresponding reactive oxygen species fluorescence intensity diagram; ROS fluorescence intensity refers to the reactive oxygen species fluorescence intensity, Control refers to the normal group, and Model refers to the model group.

[0029] Figure 7 This is a comparison chart of the antioxidant effects of the traditional Chinese medicine composition prepared in Example 3 of the present invention. Relavite expression of Gpx-1 mRNA refers to the relative expression of glutathione peroxidase 1 messenger RNA, Relavite expression of SOD-1 mRNA refers to the relative expression of superoxide dismutase 1 messenger RNA, Control refers to the normal group, and Model refers to the model group.

[0030] Figure 8 This is a comparison chart of the anti-inflammatory effects of the traditional Chinese medicine composition prepared in Example 3 of the present invention. Relaviteexpression of TNF-a mRNA refers to the relative expression of tumor necrosis factor α messenger RNA, Relaviteexpression of IL-1βmRNA refers to the relative expression of recombinant human interleukin-1β messenger RNA, Control refers to the normal group, and Model refers to the model group. DETAILED DESCRIPTION

[0031] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0032] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0033] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0034] The cellulase of the present invention was purchased from Guangdong Mingtong Biotechnology Co., Ltd., and the enzyme activity was 100,000 U / g.

[0035] The pectinase of the present invention was purchased from Guangdong Mingtong Biotechnology Co., Ltd., and the enzyme activity was 30,000 U / g.

[0036] The β-glucanase of the present invention was purchased from Guangdong Ousman Biotechnology Co., Ltd., and the enzyme activity was 20,000 U / g.

[0037] The materials used in the experimental process of the present invention are as follows:

[0038] (1) Reagents: CCK-8 kit, cDNA detection kit, qPCR kit, ROS kit.

[0039] (2) Instruments: electronic analytical balance (Sartorius, Germany), tissue slicer, carbon dioxide incubator, electron microscope, low-temperature centrifuge (Eppendorf, Germany), vortex mixer (Crystal, USA), microplate reader (USA), flow cytometer.

[0040] Example 1

[0041] The Chinese medicine composition of this embodiment comprises Polygonum hydropiper and Rhizoma Atractylodis Macrocephalae in a mass ratio of 1:2.

[0042] The aqueous two-phase extraction solvent of this embodiment includes choline chloride, betaine, ethylene glycol, and glycerol in a mass ratio of 1.6:2.0:0.8:0.6.

[0043] The preparation method of the two-phase aqueous extraction solvent comprises the following steps: adding ethylene glycol to propylene glycol and stirring and mixing them evenly under a stirring state at a rotation speed of 200 r / min, then adding choline chloride and betaine and stirring and mixing them evenly to obtain a mixed solvent, then adding 30% of water by mass of the mixed solvent and mixing them evenly again, and then heating in a water bath under the conditions of a temperature of 80°C and a rotation speed of 80 r / min, and stirring until a stable, uniform and transparent liquid is formed, so as to obtain the two-phase aqueous extraction solvent.

[0044] The preparation method of the Chinese medicine composition for resisting gastric mucosal injury of this embodiment comprises the following steps:

[0045] S1, placing Polygonum hydropiper and Acer truncatum in a vacuum drying oven for vacuum drying, and then crushing to obtain a mixed powder with a particle size of 60 mesh, and collecting and drying condensed water for later use;

[0046] S2, the dried condensed water and the composite enzyme are mixed to obtain an enzymatic solution, the enzymatic solution is evenly sprayed into the mixed powder so that the mixed powder is completely and evenly soaked, and then enzymatic hydrolysis is performed for 5 hours at a temperature of 30° C. and a relative humidity of 70% to obtain an enzymatic hydrolysis mixture; the mass ratio of the dried condensed water to the composite enzyme is 30:1; the composite enzyme includes cellulase, pectinase, and β-glucanase in a mass ratio of 4:2.5:0.5;

[0047] S3. Prepare a two-phase aqueous extraction solvent, add the enzymatic hydrolysis mixture into the two-phase aqueous extraction solvent and shake it evenly, then perform ultrasonic treatment for 60 minutes at a temperature of 50°C, a power of 150W, and a frequency of 20kHz, let it stand for 2 minutes, then adjust the power to 450W and the frequency to 40kHz for ultrasonic treatment for 20 minutes, filter, centrifuge the filtered extract at a speed of 4000r / min for 12 minutes, take the supernatant and distill it under reduced pressure to obtain a crude extract, apply AB-8 type macroporous adsorption resin to the crude extract, and elute it with ethanol with volume concentrations of 90%, 70%, and 60% in sequence, collect the eluate with a volume concentration of 60%, concentrate under reduced pressure, and dry it in vacuum at low temperature, concentrate under reduced pressure, and dry it in vacuum at 40°C to obtain a Chinese medicine composition.

[0048] Example 2

[0049] The Chinese medicine composition of this embodiment comprises Polygonum hydropiper and Rhizoma Atractylodis Macrocephalae in a mass ratio of 1:2.

[0050] The aqueous two-phase extraction solvent of this embodiment includes choline chloride, betaine, ethylene glycol, and glycerol in a mass ratio of 1.2:1.5:0.7:0.5.

[0051] The preparation method of the two-phase aqueous extraction solvent comprises the following steps: adding ethylene glycol to propylene glycol and stirring and mixing at a rotation speed of 150 r / min, then adding choline chloride and betaine and stirring and mixing to obtain a mixed solvent, then adding 50% of water by mass of the mixed solvent and mixing again, and then heating in a water bath at a temperature of 75°C and a rotation speed of 60 r / min, stirring until a stable, uniform and transparent liquid is formed, so as to obtain the two-phase aqueous extraction solvent.

[0052] The preparation method of the Chinese medicine composition for resisting gastric mucosal injury of this embodiment comprises the following steps:

[0053] S1, placing Polygonum hydropiper and Acer truncatum in a vacuum drying oven for vacuum drying, and then crushing to obtain a mixed powder with a particle size of 40 mesh, and collecting and drying condensed water for later use;

[0054] S2, mixing the dried condensed water with the composite enzyme to obtain an enzymatic solution, spraying the enzymatic solution evenly into the mixed powder so that the mixed powder is completely and evenly soaked, and then performing enzymolysis for 6 hours at a temperature of 50° C. and a relative humidity of 90% to obtain an enzymatic mixture; the mass ratio of the dried condensed water to the composite enzyme is 40:1; the composite enzyme includes cellulase, pectinase, and β-glucanase in a mass ratio of 5:3:1.4;

[0055] S3. Prepare an aqueous two-phase extraction solvent, add the enzymatic mixture into the aqueous two-phase extraction solvent and shake evenly, then perform ultrasonic treatment for 55 minutes at a temperature of 45°C, a power of 100 W, and a frequency of 25 kHz, let it stand for 3 minutes, then adjust the power to 400 W and the frequency to 35 kHz for ultrasonic treatment for 30 minutes, filter, centrifuge the filtered extract at a speed of 5000 r / min for 10 minutes, take the supernatant and distill it under reduced pressure to obtain a crude extract, apply AB-8 type macroporous adsorption resin to the crude extract, and elute with ethanol with volume concentrations of 90%, 70%, and 60% in sequence, collect the eluate with a volume concentration of 60%, concentrate under reduced pressure, and dry in vacuum at low temperature, concentrate under reduced pressure, and dry in vacuum at a temperature of 40°C to obtain a Chinese medicine composition.

[0056] Example 3

[0057] The Chinese medicine composition of this embodiment comprises Polygonum hydropiper and Rhizoma Atractylodis Macrocephalae in a mass ratio of 1:2.

[0058] The aqueous two-phase extraction solvent of this embodiment includes choline chloride, betaine, ethylene glycol, and glycerol in a mass ratio of 1.8:1.6:0.5:0.8.

[0059] The preparation method of the two-phase aqueous extraction solvent comprises the following steps: adding ethylene glycol to propylene glycol and stirring and mixing at a rotation speed of 180 r / min, then adding choline chloride and betaine and stirring and mixing to obtain a mixed solvent, then adding 20% ​​of water by mass of the mixed solvent and mixing again, and then heating in a water bath at a temperature of 80°C and a rotation speed of 70 r / min, and stirring until a stable, uniform and transparent liquid is formed, so as to obtain the two-phase aqueous extraction solvent.

[0060] The preparation method of the Chinese medicine composition for resisting gastric mucosal injury of this embodiment comprises the following steps:

[0061] S1, placing Polygonum hydropiper and Acer truncatum in a vacuum drying oven for vacuum drying, and then crushing to obtain a mixed powder with a particle size of 50 mesh, and collecting and drying condensed water for later use;

[0062] S2, mixing the dried condensed water with the composite enzyme to obtain an enzymatic solution, spraying the enzymatic solution evenly into the mixed powder so that the mixed powder is completely and evenly soaked, and then performing enzymolysis for 5 hours at a temperature of 40° C. and a relative humidity of 100% to obtain an enzymatic mixture; the mass ratio of the dried condensed water to the composite enzyme is 35:1; the composite enzyme includes cellulase, pectinase, and β-glucanase in a mass ratio of 3:2:1.2;

[0063] S3. Prepare a two-phase aqueous extraction solvent, add the enzymolysis mixture into the two-phase aqueous extraction solvent and shake it evenly, then perform ultrasonic treatment for 50 minutes at a temperature of 40°C, a power of 120W, and a frequency of 20kHz, let it stand for 3 minutes, then adjust the power to 500W and the frequency to 35kHz for ultrasonic treatment for 25 minutes, filter, centrifuge the filtered extract at a speed of 6000r / min for 11 minutes, take the supernatant and distill it under reduced pressure to obtain a crude extract, apply AB-8 type macroporous adsorption resin to the crude extract, and elute it with ethanol with volume concentrations of 90%, 70%, and 60% in sequence, collect the eluate with a volume concentration of 60%, concentrate under reduced pressure, and dry it in vacuum at low temperature, concentrate under reduced pressure, and dry it in vacuum at 40°C to obtain a Chinese medicine composition.

[0064] Comparative Example 1

[0065] The difference between this comparative example and Example 3 is that the enzymatic hydrolysis step is changed, and the rest is the same as Example 3. The preparation method of the Chinese medicine composition for resisting gastric mucosal injury in this comparative example comprises the following steps:

[0066] S1, placing Polygonum hydropiper and Acer truncatum in a vacuum drying oven for vacuum drying and then crushing to obtain a mixed powder with a particle size of 50 mesh;

[0067] S2, mixing deionized water and complex enzyme to obtain enzymolysis solution, adding mixed powder to the enzymolysis solution, stirring evenly, and then enzymolyzing for 5 hours at a temperature of 40°C to obtain an enzymolysis mixture; the mass ratio of deionized water to complex enzyme is 35:1; the complex enzyme includes cellulase, pectinase, and β-glucanase in a mass ratio of 3:2:1.2; the mass ratio of the enzymolysis solution to the mixed powder is 50:1;

[0068] S3. Prepare a two-phase aqueous extraction solvent, add the enzymolysis mixture into the two-phase aqueous extraction solvent and shake it evenly, then perform ultrasonic treatment for 50 minutes at a temperature of 40°C, a power of 120W, and a frequency of 20kHz, let it stand for 3 minutes, then adjust the power to 500W and the frequency to 35kHz for ultrasonic treatment for 25 minutes, filter, centrifuge the filtered extract at a speed of 6000r / min for 11 minutes, take the supernatant and distill it under reduced pressure to obtain a crude extract, apply AB-8 type macroporous adsorption resin to the crude extract, and elute it with ethanol with volume concentrations of 90%, 70%, and 60% in sequence, collect the eluate with a volume concentration of 60%, concentrate under reduced pressure, and dry it in vacuum at low temperature, concentrate under reduced pressure, and dry it in vacuum at 40°C to obtain a Chinese medicine composition.

[0069] Comparative Example 2

[0070] The difference between this comparative example and Example 3 is that the composition and ratio of the two-phase aqueous extraction solvent are changed, and the rest is the same as Example 3.

[0071] The aqueous two-phase extraction solvent of this embodiment includes choline chloride and glycerol in a mass ratio of 3.4:1.3.

[0072] The preparation method of the two-phase aqueous extraction solvent is as follows: under a stirring state with a rotation speed of 180 r / min, choline chloride is added to glycerol and stirred to obtain a mixed solvent, then 20% of water by mass of the mixed solvent is added and mixed again, and then heated in a water bath under the conditions of a temperature of 80°C and a rotation speed of 70 r / min, and stirred until a stable, uniform and transparent liquid is formed, so as to obtain the two-phase aqueous extraction solvent.

[0073] Comparative Example 3

[0074] The difference between this comparative example and Example 3 is that the conditions of ultrasonic extraction are changed, and the rest are the same as Example 3.

[0075] The preparation method of the Chinese medicine composition for resisting gastric mucosal injury of this comparative example comprises the following steps:

[0076] S1, placing Polygonum hydropiper and Acer truncatum in a vacuum drying oven for vacuum drying, and then crushing to obtain a mixed powder with a particle size of 50 mesh, and collecting and drying condensed water for later use;

[0077] S2, mixing the dried condensed water with the composite enzyme to obtain an enzymatic solution, spraying the enzymatic solution evenly into the mixed powder so that the mixed powder is completely and evenly soaked, and then performing enzymolysis for 5 hours at a temperature of 40° C. and a relative humidity of 100% to obtain an enzymatic mixture; the mass ratio of the dried condensed water to the composite enzyme is 35:1; the composite enzyme includes cellulase, pectinase, and β-glucanase in a mass ratio of 3:2:1.2;

[0078] S3. Prepare an aqueous two-phase extraction solvent, add the enzymatic hydrolysis mixture into the aqueous two-phase extraction solvent and shake it evenly, then perform ultrasonic treatment for 75 minutes at a temperature of 40°C, a power of 200 W, and a frequency of 60 kHz, filter, centrifuge the filtered extract at a speed of 6000 r / min for 11 minutes, take the supernatant and distill it under reduced pressure to obtain a crude extract, apply AB-8 type macroporous adsorption resin to the crude extract, and elute it with ethanol with volume concentrations of 90%, 70%, and 60%, collect the eluate with a volume concentration of 60%, concentrate it under reduced pressure, and dry it in vacuum at low temperature, concentrate it under reduced pressure, and dry it in vacuum at 40°C to obtain a Chinese medicine composition.

[0079] Test situation

[0080] 1. Determination of the content of total flavonoids

[0081] Using NaNO 2 -Al(NO 3 )3 -NaOH colorimetric method and UV spectrophotometry were used to determine the contents of total flavonoids in the traditional Chinese medicine compositions prepared in the above Examples 1-3 and Comparative Examples 1-3.

[0082] Preparation of standard curve: Accurately measure 1.0, 2.0, 3.0, 4.0, and 5.0 mL of the above rutin reference solution and place them in 10 mL volumetric flasks, add water to 5.0 mL, and add 5% NaNO 2 0.3 mL of solution was shaken and allowed to stand for 6 min, and then 10% A1 (NO 3 ) 3 Solution 0.3mL, shake well, let stand for 6min, add NaOH test solution 4mL, dilute to the scale with water, shake well, let stand for 15min, use the corresponding reagent as blank, measure the absorbance at a wavelength of 500nm, and draw a standard curve of total flavonoids with absorbance as the ordinate and concentration as the abscissa.

[0083] Determination of Chinese medicine composition: 1.0 mL of the sample solution of the Chinese medicine composition prepared in Examples 1-3 and Comparative Examples 1-3 was accurately pipetted into a 10 mL volumetric flask, water was added to 5.0 mL, and 5% NaNO 2 0.3 mL of solution was shaken and allowed to stand for 6 min, and then 10% A1 (NO 3 ) 3 0.3mL of solution, shake well, let stand for 6 minutes, add 4mL of NaOH test solution, dilute to scale with water, shake well, let stand for 15 minutes, measure absorbance at 500nm wavelength, and use the corresponding reagent without adding sample solution as blank. The test results are as follows Figure 1 shown.

[0084] Depend on Figure 1 The results show that the Chinese medicinal composition composed of Polygonum hydropiper and Acer truncatum obtained by the appropriate extraction method of Examples 1-3 of the present invention has a high content of flavonoids. Compared with Example 3, Comparative Example 1 adopts the method of placing the mixed powder in an aqueous solution for enzymatic hydrolysis, which causes the loss of flavonoid active ingredients in Polygonum hydropiper and Acer truncatum, resulting in a decrease in the extraction yield. Compared with Example 3, Comparative Example 2 changes the raw material composition and ratio of the aqueous two-phase extraction solvent, and Comparative Example 3 changes the conditions of ultrasonic treatment, but both fail to fully extract flavonoids, and the loss is large, resulting in a decrease in the final extraction yield.

[0085] 2. Experimental results of the application of Chinese medicine combination on gastric mucosal injury

[0086] The Chinese medicine combination prepared in Example 3 of the present invention was used in a mouse gastric mucosal injury experiment. The gastric mucosal injury in the mouse experiment of the present invention was induced by ethanol.

[0087] SPF grade, half male and half female Kunming mice weighing 20±2g were randomly divided into 3 groups: normal group, model group, and the Chinese medicine composition group prepared in Example 3 as the example group, with 10 mice in each group. The normal group and the model group were given distilled water, and the example group was given once a day, and both were given at 0.2ml / 10g for 6 consecutive days; before modeling, fasting but not water for 24h, 2h after the last administration, the mice except the normal group were gavaged with 0.1ml / 10g of anhydrous ethanol, and were killed 2h later, and the stomach was removed for macroscopic observation and pathological analysis. Pathological analysis process:

[0088] Sample collection - dehydration - embedding - sectioning - dewaxing to water - hematoxylin staining of cell nuclei, eosin staining of cytoplasm - dehydration and sealing - microscopic examination, image acquisition and analysis. The results are as follows Figure 2-3 shown.

[0089] The macroscopic observation results are as follows Figure 2 As shown in the figure, the gastric mucosa of the normal group has normal morphology and color, and no abnormal phenomena such as bleeding and ulcer were observed. A large number of bleeding bands appeared in the gastric mucosa of the mice in the model group, accompanied by obvious erosion, indicating that the modeling of acute gastric mucosal injury caused by ethanol in mice was successful. Compared with the model group, the lesion area and degree of lesion of the gastric mucosa of the mice in the embodiment group of the present invention were significantly reduced, indicating that the Chinese medicine composition extracted by the method of the present invention can reduce ethanol-induced gastric mucosal injury.

[0090] See also Figure 3 The HE results showed that the normal group had normal mucosal epithelial morphology, regular and tight arrangement, no shedding, necrosis and inflammatory cell infiltration, and intact glandular structure, flat and smooth. The model group mice had severe damage to the tissue mucosal layer, obvious congestion, bleeding, shedding of mucosal epithelial cells, etc. After pretreatment in the embodiment group of the present invention, the damage to the gastric mucosa of the mice was significantly alleviated, the gastric glandular structure was relatively intact, and only a small amount of local mucosal congestion was observed, indicating that the Chinese medicine composition extracted by the method of the present invention has a good protective effect on ethanol-induced gastric mucosal damage.

[0091] 3. Cell experiment of Chinese medicine combination

[0092] The Chinese medicine combination prepared in Example 3 of the present invention was used as an example group in a cell experiment to determine its effect on RAW264.7 cells. The specific process is as follows.

[0093] 1. Effect of Chinese medicine combination on cell viability

[0094] Take cells in the logarithmic growth phase, add 1 mL of 0.25% trypsin, digest for 1 min at room temperature, add 1640 culture medium to terminate the digestion, and prepare a 1×10 6The single cell suspension of 100 μL / well of cell solution was inoculated on a 96-well plate. After the cells adhered to the wall, different concentrations (unit of concentration was ug / mL) of the Chinese medicine composition of Example 3 were added as the example group. Each group of samples had 6 parallel wells and was incubated at 37°C and 5% CO. 2 After 24 hours of culture, the stock solution was discarded, and 10 μL of CC-K8 reagent and 90 μL of culture medium were added to each well. After 3 hours of culture, the absorbance was measured at a wavelength of 450 nm using an enzyme marker. The cell survival rate was determined by the CCK-8 method.

[0095] See also Figure 4 The Chinese medicine composition prepared in Example 3 of the present invention can significantly improve cell viability at a concentration of 40-125 ug / mL, and shows an obvious dose-dependent relationship.

[0096] 2. Effect of Chinese herbal medicine combination on ROS (reactive oxygen species) levels in LPS (lipopolysaccharide)-induced cells

[0097] Take cells with good growth status and adjust the cell density to 2.5×10 5 / mL, inoculated in a 6-well plate, divided into a normal group (Control), a model group (Model), a low, medium and high concentration group of a Chinese medicine composition, and after 24h, low, medium and high concentrations of a Chinese medicine composition were added to treat the cells and cultured for 24h. Then, except for the normal group, the remaining groups were added with a culture medium containing LPS (lipopolysaccharide) and continued to incubate for 12h. After the culture was completed, the culture fluid was discarded, and the cells were gently rinsed with PBS (phosphate buffered saline) twice. Then the Chinese medicine composition of Example 3 was diluted with serum-free culture medium to a concentration of 10mmol / L, 1mL was added to each well, placed in a 37°C incubator and incubated in dark for 20min, and the cells were washed twice with serum-free cell culture fluid. Finally, the images were observed and collected under an inverted fluorescence microscope.

[0098] See also Figure 5-6 In the normal control group, almost no ROS fluorescence signal was detected, and the ROS level was extremely low. The model group treated with LPS produced a large amount of ROS, and the ROS level was significantly increased compared with the normal group (P < 0.001). Compared with the model group, the green fluorescence of the medium and high doses was significantly weakened, indicating that the ROS level was significantly reduced in a dose-dependent manner, and the difference was statistically significant (P < 0.05 or 0.01).

[0099] 3. Effects of Chinese medicine combination on mRNA expression of inflammatory cytokines and antioxidant enzymes

[0100] Take cells with good growth status, inoculate them in 6-well plates, and group and treat them in the same way as LPS-induced cells in the previous 2. After the culture, discard the culture medium and gently rinse the cells twice with PBS. Use TRNzol reagent to extract total RNA, and detect the quality and concentration of RNA in each group. Then reverse transcribe RNA into cDNA and amplify it using fluorescent quantitative PCR instrument. The amplification conditions are: 95℃-2min; 95℃-10s, 60℃-30s, and 40 cycles. β-actin is used as the internal reference gene, and 2 -ΔΔCt The relative expression levels of genes were determined by the primer sequences shown in Table 1.

[0101] Table 1 Primer sequences

[0102]

[0103] See also Figure 7 After 12 hours of LPS stimulation, the expression levels of Gpx-1 and SOD-1 mRNA in the model group cells were significantly increased compared with the normal group; compared with the model group, the Gpx-1 and SOD-1 mRNA levels were significantly reduced after 24 hours of pretreatment with the Chinese medicine composition prepared in Example 3 of the present invention (p<0.05).

[0104] See also Figure 8 The expression of IL-1β and TNF-α mRNA in the model group cells was significantly higher than that in the normal group, indicating that LPS stimulation caused obvious inflammatory response in the cells; compared with the model group, after pretreatment with the Chinese medicine composition prepared in Example 3 of the present invention for 24 hours, the mRNA expression of IL-1β and TNF-α could be significantly reduced (P<0.05), and the inflammatory response of the cells was improved.

[0105] In summary, the Chinese medicine composition prepared by the method of the present invention can obtain flavonoids with higher activity, and at the same time, the viability of cells is significantly improved after administration, and the anti-inflammatory and antioxidant abilities of cells are significantly enhanced. Therefore, the Chinese medicine composition prepared by the present invention has a good application effect in the preparation of anti-gastric mucosal injury drugs.

[0106] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a Chinese medicine composition for resisting gastric mucosal damage, characterized in that: The following steps are involved: S1, placing Polygonum hydropiper and Rhizoma Atractylodis Macrocephalae in a vacuum drying oven for vacuum drying, and then crushing to obtain a mixed powder, and collecting and drying condensed water for later use; S2, mixing the dried condensed water with the composite enzyme to obtain an enzymatic solution, spraying the enzymatic solution evenly into the mixed powder, and then performing enzymatic hydrolysis for 5-6 hours at a temperature of 30-50° C. and a relative humidity of 70%-100% to obtain an enzymatic mixture; S3. Add the enzymatic mixture into the aqueous two-phase extraction solvent and shake it evenly, then perform ultrasonic extraction, filter, centrifuge the filtered extract, take the supernatant and distill it under reduced pressure to obtain a crude extract, apply the crude extract to a macroporous adsorption resin, elute with ethanol at a volume concentration of 50%-90%, collect the eluate, concentrate it under reduced pressure, and dry it in a vacuum at low temperature to obtain a Chinese medicine composition.

2. The method for preparing the Chinese medicine composition for resisting gastric mucosal damage according to claim 1, characterized in that: The particle size of the mixed powder is 40-60 meshes.

3. The method for preparing the Chinese medicine composition for resisting gastric mucosal injury according to claim 1, characterized in that: The mass ratio of the dried condensed water to the complex enzyme is 30-40:1; the complex enzyme comprises cellulase, pectinase and beta-glucanase in a mass ratio of 3-5:2-3:0.5-1.

4.

4. The method for preparing the Chinese medicine composition for resisting gastric mucosal injury according to claim 1, characterized in that: The two-phase aqueous extraction solvent comprises choline chloride, betaine, ethylene glycol and glycerol in a mass ratio of 1.2-1.8:1.5-2.0:0.5-0.8:0.5-0.

8.

5. The method for preparing the Chinese medicine composition for resisting gastric mucosal injury according to claim 4, characterized in that: The preparation method of the two-phase aqueous extraction solvent is as follows: under a stirring state of a rotation speed of 150-200 r / min, ethylene glycol is added to glycerol, stirred and mixed, and then choline chloride and betaine are added and stirred and mixed to obtain a mixed solvent, and then 20-50% of water by mass of the mixed solvent is added and mixed again, and then heated in a water bath under the conditions of a temperature of 75-80°C and a rotation speed of 60-80 r / min, and stirred until a stable, uniform and transparent liquid is formed, so as to obtain the two-phase aqueous extraction solvent.

6. The method for preparing the Chinese medicine composition for resisting gastric mucosal injury according to claim 1, characterized in that: The ultrasonic extraction process is: firstly, ultrasonic treatment is performed for 50-60 minutes at a temperature of 30-50°C, a power of 100-150W, and a frequency of 20-25kHz, and then the ultrasonic treatment is performed for 20-30 minutes by adjusting the power to 400-500W and the frequency to 35-40kHz.

7. The method for preparing the Chinese medicine composition for resisting gastric mucosal injury according to claim 1, characterized in that: The centrifugal speed is 4000-6000r / min and the centrifugal time is 10-12min.

8. The method for preparing the Chinese medicine composition for resisting gastric mucosal injury according to claim 1, characterized in that: The macroporous adsorption resin is AB-8 type macroporous adsorption resin.

9. A Chinese medicinal composition for preventing gastric mucosal injury prepared by the preparation method according to any one of claims 1 to 8, characterized in that: The traditional Chinese medicine composition comprises polygonum hydropiper and rhizoma aconitifolia in a mass ratio of 1:1.8-2.

2.

10. The Chinese medicine composition according to claim 9, characterized in that: The application of the traditional Chinese medicine composition in preparing drugs for resisting gastric mucosal damage.

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