Radish polysaccharide oral liquid with auxiliary protection effect on chemical liver injury

Abstract

The invention provides radish polysaccharide oral liquid with an auxiliary protection effect on chemical liver injury, and belongs to the technical field of prevention and treatment of chemical liver injury. The preparation method of the radish polysaccharide provided by the invention comprises the following steps: drying radishes, and crushing the dried radishes to obtain radish powder; mixing the radish powder with absolute ethyl alcohol, standing at 50 DEG C for 8 hours, and filtering with gauze to obtain filter residues; mixing the filter residues with water to obtain a mixture, mixing the mixture with cellulase, performing enzymolysis for 40-70 minutes at the temperature of 30-55 DEG C, taking filtrate, performing alcohol precipitation, collecting precipitates, adding water, performing redissolution, performing centrifugation, taking supernatant, and performing freeze drying to obtain the radish polysaccharide. According to the method for preparing the radish polysaccharide with the auxiliary protection effect on the chemical liver injury, provided by the invention, the high-content radish polysaccharide with the auxiliary protection effect on the alcoholic liver injury can be obtained from radishes. The radish polysaccharide oral liquid and the green radish polysaccharide oral liquid provided by the invention have a remarkable protection effect on acute liver injury caused by alcohol.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical liver injury prevention and treatment, and specifically relates to a radish polysaccharide oral liquid having an auxiliary protective effect on chemical liver injury. Background Art

[0002] Alcoholic liver damage is a type of chemical liver damage and a liver disease caused by long-term and heavy drinking. Drinking has been shown to disrupt the normal function of the liver, multiple organs of the gastrointestinal system, and the brain. The liver is the main site of alcohol oxidative metabolism. Alcoholic liver damage is a common disease that usually manifests as alcoholic fatty liver in the early stages. Alcoholic fatty liver can be reversed, but if not intervened in time, it will develop into more serious liver diseases such as alcoholic hepatitis, liver fibrosis, cirrhosis, and even deteriorate into liver cancer. At present, the commonly used drugs for the treatment of alcoholic liver damage mainly include metadoxine, glucocorticoids, glycyrrhizic acid preparations, etc. However, most drugs have some adverse side effects, are not suitable for long-term use, and are not specific drugs for alcoholic liver damage.

[0003] White radish is a common vegetable. Since ancient times, there has been a saying that "winter radish is like cold ginseng". Green radish is rich in nutrients needed by the human body, has a high content of amylase, is juicy, sweet and slightly spicy. It is a famous raw food variety and is called "fruit radish". Radish contains fewer calories and more fiber. Eating it regularly helps lose weight. The mustard oil and refined fiber contained in radish can promote gastrointestinal motility and help excrete waste from the body. Polysaccharides are an important component of radish, and have multiple physiological functions such as immune regulation, anti-tumor, anti-oxidation, lowering blood sugar, and lowering blood lipids. So far, there have been no reports on the use of green radish polysaccharides to protect against alcoholic liver damage. Summary of the invention

[0004] In view of this, one of the purposes of the present invention is to provide a radish polysaccharide having an auxiliary protective effect on chemical liver damage and a preparation method thereof.

[0005] The second purpose of the present invention is to provide a green radish polysaccharide oral liquid and a radish polysaccharide oral liquid which have an auxiliary protective effect on chemical liver damage.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a method for preparing radish polysaccharide with auxiliary protective effect on chemical liver damage, comprising the following steps: drying radish and then crushing to obtain radish powder; mixing the radish powder with anhydrous ethanol, standing at 50 DEG C for 8 hours, filtering with gauze to obtain filter residue; mixing the filter residue with water to obtain a mixture, mixing the mixture with cellulase, adjusting the pH value to 7, performing enzymolysis at 30-55 DEG C for 40-70 minutes, taking the filtrate for alcohol precipitation, collecting the precipitate, adding water for redissolution, centrifuging to obtain the supernatant, and freeze-drying to obtain radish polysaccharide.

[0008] Preferably, the material-liquid ratio of the mixture of the filter residue and water is 1-3 g: 10-25 mL.

[0009] Preferably, the amount of cellulase added is 0.2-0.8 g of cellulase per 100 g of the mixture, based on the weight of the mixture.

[0010] Preferably, the weight ratio of radish powder to anhydrous ethanol is 1:5.

[0011] The present invention also provides a green radish polysaccharide having an auxiliary protective effect on chemical liver damage, which is prepared by the above method.

[0012] The present invention also provides a white radish polysaccharide having an auxiliary protective effect on chemical liver damage, which is prepared by the above method.

[0013] The present invention also provides a green radish polysaccharide oral liquid with auxiliary protective effect on chemical liver damage, comprising an oral liquid and the above-mentioned green radish polysaccharide; the weight-to-volume ratio of the green radish polysaccharide to the oral liquid is 0.1-0.3 g:1 mL; the preparation method of the oral liquid comprises the following steps: mixing wolfberry, hawthorn, turmeric, red dates, licorice, and poria with water and soaking for 20 minutes, decocting to a slight boil and maintaining for 30 minutes, taking the filtrate and concentrating it to obtain the oral liquid; the weight-to-volume ratio of the wolfberry, hawthorn, turmeric, red dates, licorice, poria and water is 7.8g:7.8g:2.7g:7.8g:7.8g:7.8g:1000mL.

[0014] The present invention also provides a radish polysaccharide oral liquid having an auxiliary protective effect on chemical liver damage, comprising water, the green radish polysaccharide and the white radish polysaccharide, wherein the volume-to-weight ratio of water, green radish polysaccharide and white radish polysaccharide is 1 mL: 0.05 g: 0.05 g.

[0015] The present invention also provides a green radish polysaccharide oral liquid with auxiliary protective effect on chemical liver damage, comprising the following raw materials in volume proportion: the above-mentioned green radish polysaccharide, 0.1-0.2 weight parts of oats, 0.1-0.2 weight parts of raisins, 2-3 volume parts of pear juice, 2-3 volume parts of tomato juice, and 50-60 volume parts of water; the concentration of green radish polysaccharide in the green radish polysaccharide oral liquid is 0.1g / mL.

[0016] Preferably, the chemical liver injury is alcoholic liver injury.

[0017] Beneficial effects of the present invention:

[0018] The method for preparing radish polysaccharide with auxiliary protective effect on chemical liver damage provided by the invention can obtain high-content radish polysaccharide with auxiliary protective effect on alcoholic liver damage from radish.

[0019] The raw materials used in the radish polysaccharide oral liquid and the green radish polysaccharide oral liquid provided by the present invention are both medicinal and edible raw materials, have no toxic side effects to each other, and the prepared oral liquid can be directly drunk, is easily accepted by patients, and is suitable for promotion and use. The radish used in the present invention is widely available and inexpensive. The radish polysaccharide oral liquid and the green radish polysaccharide oral liquid provided by the present invention can improve liver function and lipid metabolism by inhibiting the ALT, AST activity and TG, TC content in serum; reduce the liver index of mice and the MDA content in liver tissue, and effectively reduce the deposition of fat in the liver; increase the SOD, CAT activity and GSH-Px content in mice, reduce the degree of lipid peroxidation and oxidative stress reaction, and restore the balance of the body's oxidation and antioxidant systems, thereby playing a protective role against acute liver damage caused by alcohol. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of the amount of cellulase added on the polysaccharide content;

[0021] Figure 2 The effect of enzymatic hydrolysis temperature on polysaccharide content;

[0022] Figure 3 The effect of enzymatic hydrolysis time on polysaccharide content;

[0023] Figure 4 The effect of the ratio of filter residue to distilled water on the polysaccharide content;

[0024] Figure 5 The effect of different extraction methods on polysaccharide content, where the letters on any two columns are the same, indicating no significant difference (P ≥ 0.05), and if they are different, indicating a significant difference (P < 0.05);

[0025] Figure 6 The liver index results of different groups, if the letters on any two columns are the same, it means there is no significant difference P ≥ 0.05, if they are different, it means there is a significant difference P < 0.05;

[0026] Figure 7The TC results of different groups, if the letters on any two columns are the same, it means there is no significant difference P ≥ 0.05, if they are different, it means there is a significant difference P < 0.05;

[0027] Figure 8 The TG results of different groups, if the letters on any two columns are the same, it means there is no significant difference P ≥ 0.05, if they are different, it means there is a significant difference P < 0.05;

[0028] Fig. 9 The ALT results of different groups, if the letters on any two columns are the same, it means there is no significant difference P ≥ 0.05, if they are different, it means there is a significant difference P < 0.05;

[0029] Fig.10 The AST results of different groups, if the letters on any two columns are the same, it means there is no significant difference P ≥ 0.05, if they are different, it means there is a significant difference P < 0.05;

[0030] Fig.11 The MDA results of different groups, if the letters on any two columns are the same, it means there is no significant difference P ≥ 0.05, if they are different, it means there is a significant difference P < 0.05;

[0031] Fig.12 The CAT results of different groups, if the letters on any two columns are the same, it means there is no significant difference P ≥ 0.05, if they are different, it means there is a significant difference P < 0.05;

[0032] Fig.13 The SOD results of different groups, if the letters on any two columns are the same, it means there is no significant difference P ≥ 0.05, if they are different, it means there is a significant difference P < 0.05;

[0033] Fig.14 The GSH-PX results of different groups, if the letters on any two columns are the same, it means there is no significant difference P ≥ 0.05, if they are different, it means there is a significant difference P < 0.05;

[0034] Fig.15 The TNF-α results of different groups, if the letters on any two columns are the same, it means there is no significant difference P ≥ 0.05, if they are different, it means there is a significant difference P < 0.05;

[0035] Fig.16 The IL-6 results of different groups, if the letters on any two columns are the same, it means there is no significant difference P ≥ 0.05, if they are different, it means there is a significant difference P < 0.05;

[0036] Fig.17HE staining results of liver tissue. DETAILED DESCRIPTION

[0037] The invention provides a method for preparing radish polysaccharide with auxiliary protective effect on chemical liver damage, comprising the following steps: drying radish and then crushing to obtain radish powder; mixing the radish powder with anhydrous ethanol, standing at 50 DEG C for 8 hours, filtering with gauze to obtain filter residue; mixing the filter residue with water to obtain a mixture, mixing the mixture with cellulase, adjusting the pH value to 7, performing enzymolysis at 30-55 DEG C for 40-70 minutes, taking the filtrate for alcohol precipitation, collecting the precipitate, adding water for redissolution, centrifuging to obtain the supernatant, and freeze-drying to obtain radish polysaccharide.

[0038] In the method of the present invention, it is preferred that the radish be cleaned and then dried and crushed, and the drying temperature is preferably 30°C. The present invention does not specifically limit the specific method of crushing, and preferably passes through a 40-mesh sieve after crushing. In the present invention, when radish powder is mixed with anhydrous ethanol, the weight ratio of radish powder to anhydrous ethanol is preferably 1:5. In the present invention, when the filter residue is mixed with water, the material-liquid ratio of the filter residue to water is preferably 1-3g:10-25mL, more preferably 1g:15-20mL. The amount of the cellulase added is based on the weight of the mixture, preferably 0.2-0.8g of cellulase is added per 100g of the mixture, more preferably 0.4-0.6g of cellulase is added per 100g of the mixture, and the enzymatic activity of the cellulase is preferably 21.067-23.333U / g. In the present invention, the temperature of the enzymolysis is preferably 35-50°C, more preferably 40-45°C, and the time of the enzymolysis is preferably 50-60min, more preferably 52-58min. After the enzymolysis is completed, preferably the enzyme is inactivated, the temperature of the enzyme inactivation is preferably 95°C, the time of the enzyme inactivation is preferably 10min, and after the enzyme inactivation is completed, preferably the filtrate is obtained by suction filtration. In the present invention, the alcohol precipitation is preferably carried out using an ethanol solution with a volume ratio of water to anhydrous ethanol of 3:7. After the alcohol precipitation is completed, the precipitate is preferably collected by centrifugation, the centrifugation condition is preferably 4500rpm centrifugation for 15min, the precipitate is redissolved with water, and the supernatant is taken by centrifugation, and the centrifugation condition is preferably 3500rpm centrifugation for 30min. The present invention is not particularly limited to the specific conditions for freeze drying.

[0039] The present invention also provides green radish polysaccharide and white radish polysaccharide having auxiliary protective effect on chemical liver damage, which are prepared by the above method.

[0040] The present invention also provides a green radish polysaccharide oral liquid with auxiliary protective effect on chemical liver damage, comprising an oral liquid and the above-mentioned green radish polysaccharide; the weight-to-volume ratio of the green radish polysaccharide to the oral liquid is 0.1-0.3 g:1 mL; the preparation method of the oral liquid comprises the following steps: mixing wolfberry, hawthorn, turmeric, red dates, licorice, and poria with water and soaking for 20 minutes, decocting to a slight boil and maintaining for 30 minutes, taking the filtrate and concentrating it to obtain the oral liquid; the weight-to-volume ratio of the wolfberry, hawthorn, turmeric, red dates, licorice, poria and water is 7.8g:7.8g:2.7g:7.8g:7.8g:7.8g:1000mL.

[0041] In the green radish polysaccharide oral liquid of the present invention, the decoction is preferably performed twice, and the second decoction refers to adding 1000 mL of water to the residue after the first decoction and continuing to decoct. The two filtrates are combined, the residue is filtered out, and the filtrate is concentrated. The concentration method is preferably 50° C. rotary evaporation concentration, preferably concentrated to a volume of 3 mL of the concentrate corresponding to each gram of the raw material drug, and the concentrated solution is collected as the oral liquid.

[0042] The present invention also provides a radish polysaccharide oral liquid having an auxiliary protective effect on chemical liver damage, comprising water, the green radish polysaccharide and the white radish polysaccharide, wherein the volume-to-weight ratio of water, green radish polysaccharide and white radish polysaccharide is 1 mL: 0.05 g: 0.05 g.

[0043] The present invention also provides a green radish polysaccharide oral liquid with auxiliary protective effect on chemical liver damage, comprising the following raw materials in volume proportion: the above-mentioned green radish polysaccharide, 0.1-0.2 weight parts of oats, 0.1-0.2 weight parts of raisins, 2-3 volume parts of pear juice, 2-3 volume parts of tomato juice, and 50-60 volume parts of water; the concentration of green radish polysaccharide in the green radish polysaccharide oral liquid is 0.1g / mL.

[0044] In the present invention, the unit of weight is g, the unit of volume is mL, and the chemical liver injury is preferably alcoholic liver injury.

[0045] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0047] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0048] In the following examples, green radish, white radish, hawthorn, wolfberry, red dates, turmeric, Poria, oats, raisins, pears, tomatoes, and 56-proof liquor were all purchased from Jinzhou General Supermarket; triglyceride (TG), total cholesterol (TC), malondialdehyde (MDA), alanine aminotransferase (ALT), catalase (CAT), glutathione peroxidase (GSH-Px), aspartate aminotransferase (AST), superoxide dismutase (SOD) detection kits and total protein quantification kits were all purchased from Nanjing Jiancheng Bioengineering Co., Ltd.; tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) kits were purchased from Shanghai Jingkang Bioengineering Co., Ltd.; acetonitrile and methanol were chromatographically pure, and the rest were analytically pure.

[0049] The animal experiments in the embodiments of the present invention were approved by the Animal Experiment Ethics of Jinzhou Medical University and met the established ethical requirements. The Animal Experiment Certificate Number is SYXK (Liao) 2022-0006, and the Animal Ethics Permit Document Number is 2019051. 60 SPF-grade male Kunming mice, weighing 20±2 g, were provided by Jinzhou Aohong Pharmaceutical Co., Ltd., license number: SCXK (Liao) 2019-0003, quality certificate number: 210772231100004021.

[0050] All statistical analyses of the data in the present invention were performed using SPSS 22.0 and Origin 19.0. The data results were expressed as "mean ± SD", and P < 0.05 was considered significant.

[0051] Example 1

[0052] A method for preparing radish polysaccharide with auxiliary protective effect on chemical liver injury comprises the following steps: washing radish, drying at 30°C to constant weight, crushing, and passing through a 40-mesh sieve to obtain radish powder; accurately weighing 5g of radish powder, adding 25mL of anhydrous ethanol, water bathing at 50°C for 8h, filtering with gauze and discarding the supernatant; mixing the filter residue with distilled water (the solid-liquid ratio of the filter residue to distilled water is 1g:12mL) to obtain a mixture; adding cellulase (the amount of cellulase is 0.3g of cellulase per 100g of the mixture, and the enzyme activity of the cellulase is 23.333U / g), adjusting the pH value to 7.0, enzymolysis at 35°C for 45min, inactivating the enzyme at 95°C for 10min, filtering to obtain a filtrate, using an ethanol solution with a volume ratio of water to anhydrous ethanol of 3:7 for alcohol precipitation, centrifuging at 4500rpm for 15min to collect the precipitate, adding water for redissolution, centrifuging at 3500rpm for 30min to obtain the supernatant, and freeze-drying at -50°C for 72h to obtain radish polysaccharide.

[0053] Example 2

[0054] The difference from Example 1 is that the amount of cellulase used is 0.2g, 0.4g, 0.6g, 0.8g, and 1.0g per 100g of the mixture, respectively, and the rest is the same as Example 1. The polysaccharide content in the radish polysaccharide obtained with different amounts of cellulase is determined by:

[0055] (1) Preparation of standard solution: Accurately weigh 25 mg of glucose standard that has been dried to constant weight, place in a 50 mL volumetric flask, add water to dissolve, make up to volume, and shake well to obtain the solution.

[0056] (2) Drawing of standard curve: Accurately measure 0.5, 1.0, 2.5, 3.0, 4.0, and 5.0 mL of standard solution, place them in 25 mL volumetric flasks, add water to make up, shake well, and obtain a series of standard solutions with concentrations of 0.01, 0.02, 0.05, 0.06, 0.08, and 0.10 mg / mL; respectively take 1.0 mL of the above series of standard solutions and water and place them in 10 mL stoppered test tubes, accurately add 0.3 mL of 6% phenol solution, stir and immediately add 2.5 mL of concentrated sulfuric acid, shake well and place in a boiling water bath for color development for 20 minutes, take out and place in ice water for cooling for 10 minutes, and measure the absorbance at 490 nm. Use the concentration of glucose standard solution as the horizontal axis and the absorbance as the vertical axis to draw a standard curve, and the regression equation is y=12.934x+0.0011; R 2 =0.9943.

[0057] (3) Determination of polysaccharide content in radish polysaccharide: Phenol-sulfuric acid method: Accurately weigh 6.0 g of redistilled phenol, place it in a brown volumetric flask (100 mL), dilute it with distilled water, and prepare a phenol solution for use. Dissolve 100 mg of radish polysaccharide in 10 mL of water, transfer 1.0 mL of the polysaccharide solution to a 25 mL test tube, add 0.3 mL of 6% phenol solution, stir and immediately add 2.5 mL of concentrated sulfuric acid, shake well and color in a boiling water bath for 20 minutes, take out and cool in ice water for 10 minutes, measure the absorbance with a UV-visible spectrophotometer, substitute it into the regression equation obtained in step (2), obtain the concentration value, and then calculate according to the following formula:

[0058]

[0059] Y is the polysaccharide content, mg / g; V is the sample volume, mL; C is the polysaccharide mass concentration, mg / mL; D is the dilution multiple; M is the sample mass, g.

[0060] The results are as follows Figure 1 It can be seen that the amount of cellulase added will directly affect the content of polysaccharides in radish polysaccharides.

[0061] Example 3

[0062] The difference from Example 1 is that the enzymolysis temperatures are 30°C, 40°C, 50°C, 60°C, and 70°C, respectively, and the rest are the same as in Example 1. The polysaccharide content in the radish polysaccharide obtained at different enzymolysis temperatures was determined by the same method as in Example 2. The results are shown in FIG. Figure 2 It can be seen that the enzymatic hydrolysis temperature will directly affect the content of polysaccharides in radish polysaccharides.

[0063] Example 4

[0064] The difference from Example 1 is that the enzymolysis time is 40min, 50min, 60min, 70min, and 80min respectively, and the rest is the same as Example 1. The polysaccharide content in the radish polysaccharide obtained at different enzymolysis times is determined by the same method as Example 2. The results are shown in FIG. Figure 3 It can be seen that the enzymatic hydrolysis time will directly affect the content of polysaccharides in radish polysaccharides.

[0065] Example 5

[0066] The difference from Example 1 is that the material-liquid ratio of the filter residue to distilled water is 1g:10mL, 1g:15mL, 1g:20mL, 1g:25mL, 1g:30mL, and the rest is the same as Example 1. The polysaccharide content in the radish polysaccharide obtained with different material-liquid ratios was determined by the same method as in Example 2. The results are shown in FIG. Figure 4 It can be seen that the solid-liquid ratio of the filter residue to distilled water will directly affect the content of polysaccharides in radish polysaccharides.

[0067] Example 6

[0068] A method for preparing radish polysaccharide with auxiliary protective effect on chemical liver damage, comprising the following steps: washing radish, drying at 30°C to constant weight, crushing, and passing through a 40-mesh sieve to obtain radish powder; accurately weighing 5g of radish powder, adding 25mL of anhydrous ethanol, bathing at 50°C for 8h, filtering with gauze and discarding the supernatant; mixing the filter residue with distilled water (the solid-liquid ratio of the filter residue to distilled water is 1g:15mL) to obtain a mixture; adding cellulase (the amount of cellulase is 0.6g of cellulase per 100g of the mixture), adjusting the pH value to 7.0, enzymolysis at 50°C for 60min, inactivating the enzyme at 95°C for 10min, filtering to obtain a filtrate, ethanol precipitation, centrifuging to collect the precipitate, adding water to re-dissolve, centrifuging to obtain the supernatant, and freeze-drying to obtain radish polysaccharide.

[0069] Example 7

[0070] Radish polysaccharides were prepared by hot water extraction, ultrasonic extraction, cellulase-ultrasonic extraction and cellulase extraction, and the contents of radish polysaccharides prepared by different extraction methods were compared. The specific methods are as follows:

[0071] Hot water extraction: First time: After washing the radish, dry it at 30℃ to constant weight, crush it, pass it through a 40-mesh sieve, accurately weigh 5g of the sample, add 5 times anhydrous ethanol and water bath at 50℃ for 8h to remove pigments, filter with gauze and discard the supernatant, mix the residue with distilled water at a solid-liquid ratio of 1:12, water bath at 60℃ for 4h, filter with gauze to take the supernatant, rotary evaporate to 20% of the original volume, centrifuge to take the supernatant, add 5 times ethanol to precipitate for 24 hours, collect the precipitate by centrifugation at 3500rpm for 30min, re-dissolve it with water, centrifuge at 3500rpm for 15min to collect the supernatant, and freeze-dry at -50℃ to obtain crude polysaccharide.

[0072] The second hot water extraction was as follows: the first extraction was in a 60°C water bath for 4 hours, the residue after filtering the supernatant with gauze was mixed with distilled water again at a solid-liquid ratio of 1:12, in a 60°C water bath for 4 hours, the supernatant was filtered with gauze, and the mixture was evaporated to 20% of the original volume, and the supernatant was obtained by centrifugation. Five times ethanol was added for precipitation for 24 hours, and the precipitate was collected by centrifugation at 3500 rpm for 30 minutes, and water was added for redissolution. The supernatant was collected by centrifugation at 3500 rpm for 15 minutes, and the crude polysaccharide was obtained by freeze-drying at -50°C.

[0073] Ultrasonic extraction: First time: After washing the radish, dry it at 30℃ to constant weight, crush it, pass it through a 40-mesh sieve, accurately weigh 5g of the sample, add 5 times anhydrous ethanol and water bath at 50℃ for 8h to remove pigments, filter it with gauze and discard the supernatant, mix the filter residue with distilled water at a solid-liquid ratio of 1:25, ultrasonically treat it at a power of 225W for 30min, filter it with gauze to take the supernatant, rotary evaporate it to 20% of the original volume, centrifuge it at 4500rpm for 15min to collect the precipitate, add water to re-dissolve it, centrifuge it at 3500rpm for 20min to collect the supernatant, and freeze-dry it at -50℃ to obtain crude polysaccharide.

[0074] The second extraction of ultrasonic extraction is as follows: ultrasonication for 30 minutes, gauze filtration to obtain the supernatant, and the residue is mixed with distilled water again according to a solid-liquid ratio of 1:25, ultrasonication at a power of 225 W for 30 minutes, gauze filtration to obtain the supernatant, rotary evaporation to 20% of the original volume, centrifugation at 4500 rpm for 15 minutes to collect the precipitate, re-dissolve in water, centrifuge at 3500 rpm for 20 minutes to collect the supernatant, and freeze-drying at -50°C to obtain crude polysaccharide.

[0075] Cellulase-ultrasonic extraction: First time: After the radish is cleaned, it is dried at 30℃ to constant weight, crushed, passed through a 40-mesh sieve, 5g of sample is accurately weighed, 5 times of anhydrous ethanol is added to a 50℃ water bath for 8h to remove pigment, the supernatant is discarded by gauze filtering, the filter residue is mixed with distilled water (the solid-liquid ratio of the filter residue to distilled water is 1g:15mL) to obtain a mixture; cellulase is added (the amount of cellulase is 0.6g of cellulase per 100g of the mixture), the pH value is adjusted to 7.0, enzymolysis is performed at 50℃ for 60min, enzyme is inactivated at 95℃ for 10min, ultrasonic power is 350W for 30min, the supernatant is filtered through gauze, rotary evaporated to 20% of the original volume, centrifuged at 4500rpm for 15min to collect the precipitate, re-dissolved with water, centrifuged at 3500rpm for 20min to collect the supernatant, and freeze-dried at -50℃ to obtain crude polysaccharide.

[0076] The second extraction of cellulase-ultrasound is as follows: ultrasonic power is 350W for 30min, the filter residue after the supernatant is filtered through gauze is mixed again with distilled water (the solid-liquid ratio of the filter residue to distilled water is 1g:15mL) to obtain a mixture; cellulase is added (the amount of cellulase is 0.6g of cellulase per 100g of mixture), the pH value is adjusted to 7.0, enzymolysis is performed at 50℃ for 60min, enzyme is inactivated at 95℃ for 10min, ultrasonic power is 350W for 30min, the supernatant is filtered through gauze, rotary evaporated to 20% of the original volume, centrifuged at 4500rpm for 15min, the precipitate is collected, water is added for redissolution, the supernatant is collected by centrifugation at 3500rpm for 20min, and the crude polysaccharide is freeze-dried at -50℃.

[0077] Cellulase extraction: The first extraction was carried out according to the method of Example 6.

[0078] The second extraction of cellulase is to filter the residue after obtaining the filtrate by suction, continue to mix the residue with distilled water (the solid-liquid ratio of the residue to distilled water is 1g:15mL) to obtain a mixture; add cellulase (the dosage of cellulase is 0.6g of cellulase per 100g of mixture), adjust the pH value to 7.0, enzymolysis at 50℃ for 60min, inactivate the enzyme at 95℃ for 10min, filter to obtain the filtrate, precipitate with alcohol, collect the precipitate by centrifugation, re-dissolve with water, take the supernatant by centrifugation, and freeze-dry to obtain radish polysaccharide.

[0079] The polysaccharide content in the radish polysaccharides obtained by different extraction methods and the first and second extractions of each extraction method was determined. The method for determining the polysaccharide content was the same as that in Example 2. The results are as follows Figure 5 It can be seen that the cellulase extraction method provided by the present invention has a higher polysaccharide extraction rate.

[0080] Example 8

[0081] A green radish polysaccharide oral liquid having an auxiliary protective effect on alcoholic liver damage, comprising an oral liquid and green radish polysaccharide prepared by the method described in Example 6; the weight volume ratio of the green radish polysaccharide to the oral liquid is 0.1 g:1 mL; the green radish polysaccharide and the oral liquid are uniformly mixed and allowed to stand, filtered using a membrane filter under the condition of a filter membrane pore size of 3 μm, and then aseptically filled in a specification of 10 mL / bottle, and finally high-temperature instantaneous sterilization is performed at 105°C to obtain the green radish polysaccharide oral liquid.

[0082] The preparation method of the oral liquid comprises the following steps: mixing 7.8g of wolfberry, 7.8g of hawthorn, 2.7g of turmeric, 7.8g of red dates, 7.8g of licorice and 7.8g of poria, and then mixing with 1000mL of water and soaking for 20min, decocting until slightly boiling and maintaining for 30min, decocting twice, combining the filtrate, filtering out the filter residue, and obtaining an extract; concentrating the extract by rotary evaporation at 50°C until the volume of the concentrate corresponding to each gram of the raw material drug is 3mL, collecting the concentrate, and obtaining the oral liquid.

[0083] Example 9

[0084] A radish polysaccharide oral liquid having an auxiliary protective effect on alcoholic liver damage is composed of water, green radish polysaccharide prepared according to the method described in Example 6, and white radish polysaccharide prepared according to the method described in Example 6, wherein the volume weight ratio of water, green radish polysaccharide and white radish polysaccharide is 1 mL:0.05 g:0.05 g. After being evenly mixed, the mixture is allowed to stand, filtered using a membrane filter under the condition that the filter membrane pore size is 3 μm, and then aseptically filled in a specification of 10 mL / bottle, and finally high-temperature instantaneous sterilization is performed under the condition of 105°C to obtain the radish polysaccharide oral liquid.

[0085] Example 10

[0086] A green radish polysaccharide oral liquid with auxiliary protective effect on alcoholic liver damage is prepared by the following steps: 7.8g of oats and 7.8g of raisins are mixed, 234mL of water is added, and 20mL of pear juice (the pear juice is obtained by washing fresh snow pears and squeezing them with a juicer to obtain fresh pear juice) and 20mL of tomato juice (the tomato juice is obtained by washing fresh tomatoes and squeezing them with a juicer to obtain fresh tomato juice) are added, mixed and soaked for 20min, decocted to a slight boil and maintained for 30min, the filter residue is filtered out to obtain an extract, green radish polysaccharide prepared according to the method described in Example 6 is added to make the concentration of green radish polysaccharide in the green radish polysaccharide oral liquid be 0.1g / mL, mixed evenly and then allowed to stand, filtered with a membrane filter under the condition of a filter membrane pore size of 3μm, and then aseptically filled with a specification of 10mL / bottle, and finally high-temperature instantaneous sterilization is performed at 105°C to obtain the green radish polysaccharide oral liquid.

[0087] Embodiment 11

[0088] An oral liquid, the preparation method of the oral liquid comprising: mixing 7.8g of wolfberry, 7.8g of hawthorn, 2.7g of turmeric, 7.8g of red dates, 7.8g of licorice and 7.8g of poria, and then mixing with 1000mL of water and soaking for 20min, decocting until slightly boiling and maintaining for 30min, decocting twice, combining the filtrate, filtering out the filter residue, and obtaining an extract; concentrating the extract by rotary evaporation at 50°C until the volume of the concentrate corresponding to each gram of the raw material drug is 3mL, collecting the concentrate, filtering with a membrane filter under the condition that the filter membrane pore size is 3μm, and then aseptically filling with a specification of 10mL / bottle, and finally high-temperature instantaneous sterilization at 105°C to obtain the oral liquid.

[0089] Example 12

[0090] 1.1 Product sensory and morphological analysis

[0091] The results showed that the green radish polysaccharide oral liquid and radish polysaccharide oral liquid for protecting alcoholic liver damage prepared in Examples 8 to 10 had no suspended matter, no stratification, good clarity, slightly sweet taste, and the sterile microbial limit test met the relevant regulations.

[0092] 1.2 Drug efficacy test methods and results

[0093] The experimental mice were kept under the following conditions: temperature 22±2°C; relative humidity: 55±5%; light exposure 12h per day; free access to food and water.

[0094] After 7 days of adaptive feeding, the experimental mice were randomly divided into a normal group, a model group, a drug (silybin) group, implementation group 1 (oral administration of the oral solution of Example 11), implementation group 2 (oral administration of the green radish polysaccharide oral solution obtained in Example 8), implementation group 3 (oral administration of the radish polysaccharide oral solution of Example 9), and implementation group 4 (oral administration of the green radish polysaccharide oral solution of Example 10), with 12 mice in each group.

[0095] The mice in the normal group and the model group were first gavaged with an equal amount of saline; the groups in implementation 1, implementation 2, implementation 3, and implementation 4 were gavaged with different groups of oral liquid at 400 mg / (kg·d) every day, and the drug group was gavaged with silybin (50 mg / kg / d); 30 minutes later, the normal group continued to be gavaged with an equal amount of saline; the model group and the groups in implementation 1, implementation 2, implementation 3, and implementation 4 were gavaged with 56-degree liquor at 12 mL / (kg·d). The mice were gavaged continuously for 15 days to establish the acute alcoholic liver injury model in mice.

[0096] After the last intragastric administration, the rats were fasted but not watered for 12 hours. The eyeballs were removed and blood was collected and kept at room temperature for 2 hours. The blood was centrifuged at 4°C and 3500r / min for 10 minutes. The upper layer of serum was collected and packaged and stored in a -80°C refrigerator for later use.

[0097] After blood collection, the mice were killed by cervical dislocation, and the liver tissue was taken. The blood stains on the surface of the liver were rinsed with PBS, dried with filter paper, and weighed. The liver tissue of the same part of each mouse was fixed in 4% (V / V) paraformaldehyde for pathological detection; the remaining liver tissue was divided into TG, TC, MDA, ALT, CAT, GSH-PX, AST, SOD, TNF-α and IL-6 detection kits for determination of the required mass, and stored in a -80℃ refrigerator for future use.

[0098] result:

[0099] Alcohol ingested into the body is mainly metabolized in the liver. When metabolism is insufficient, oxidative stress and lipid peroxidation reactions will occur in the body, causing certain toxicity and damage to the liver, increasing the overall mass and volume of the liver. Liver enlargement is a common clinical symptom of alcoholic liver damage.

[0100] In order to study the effect of radish polysaccharide oral liquid on the degree of liver damage, the effect of radish polysaccharide oral liquid on the liver index of mice (the ratio of mouse liver weight to mouse body weight) was investigated. The results are as follows Figure 6 As shown in the figure, the liver index of the model group was significantly higher than that of the normal group by 30.86% (P<0.05), indicating that alcohol caused liver tissue damage and edema, and the model was successfully established. Compared with the model group, the liver index of the drug, implementation 1, implementation 2, implementation 3, and implementation 4 groups was significantly reduced by 19.05%, 20.98%, 20.89%, 24.94%, and 17.86% (P<0.05), indicating that the oral liquid of the drug, implementation 1, implementation 2, implementation 3, and implementation 4 groups can effectively reduce liver edema and acute alcoholic liver injury.

[0101] The presence of alcoholic liver damage is associated with increased levels of serum metabolites and lipid metabolism. TC and TG are important indicators of liver damage. Increased levels of TC and TG can lead to excessive accumulation of liver fat or impaired metabolism. A large number of studies have shown that acute alcohol poisoning can damage the structure and function of organelles and enzymes in liver cells, affect the β-oxidation of fatty acids in mitochondria, cause lipid peroxidation, damage liver cell membranes, and ultimately lead to the accumulation of TG in the liver. There are a large number of ALT and AST in the liver cytoplasm. When alcohol stimulates and damages liver cells, the barrier function of its cell membrane will be affected, resulting in increased permeability. Therefore, ALT and AST will be partially transferred to the blood, and the ALT and AST levels in the blood will be abnormal. Therefore, to a certain extent, the degree of liver damage caused by alcohol can be reflected by detecting the levels of these two transaminases in the blood. Once liver cells are damaged, a large amount of ALT and AST are released into the serum, causing the serum levels to increase.

[0102] In order to study the effect of radish polysaccharide oral liquid on serum metabolites and lipid metabolism in mice with alcoholic liver injury, the effect of radish polysaccharide oral liquid on TC, TG, ALT, and AST in the serum of mice with alcoholic liver injury was investigated. Figure 7-10 As shown; it shows that the radish polysaccharide oral liquid in Experiment 1, Experiment 2, Experiment 3 and Experiment 4 can effectively alleviate acute alcoholic liver injury. Compared with the normal group, the TG and TC levels and ALT and AST levels in the model group were significantly increased by 140.91% and 39.87% and 259.52% and 359.59% (P < 0.05); however, compared with the model group, the TG and TC levels and ALT and AST levels in the drug group were significantly decreased by 55.07%, 29.47% and 59.13%, 68.55%, respectively; compared with the model group, the TG and TC levels and ALT and AST levels in the implementation group 1 were significantly decreased by 42.63%, 24.88%, 46.27%, 73.73%, respectively; compared with the model group, the TG and TC levels and ALT and AST levels in the implementation group 2 were significantly decreased by 42.63%, 24.88%, 46.27%, 73.73%, respectively. Compared with the model group, the levels of TG and TC as well as ALT and AST in implementation group 3 were significantly reduced by 70.09%, 30.69%, 52.81% and 79.06%, respectively; compared with the model group, the levels of TG and TC as well as ALT and AST in implementation group 4 were significantly reduced by 41.09%, 21.94%, 22.99% and 74.57%, respectively (P<0.05), indicating that the drugs, implementation 1, implementation 2, implementation 3 and implementation 4 oral liquid can effectively improve the effects of serum metabolites and lipid metabolism in mice with alcoholic liver injury.

[0103] Alcohol metabolism produces a large number of active free radicals. Oxidative stress is an important mechanism of alcoholic liver damage, and MDA is one of the main products of lipid peroxidation. When alcohol is not metabolized smoothly in the liver, the content of the oxidative damage product MDA will increase, leading to cell damage. CAT is essentially a type of oxidoreductase with strong antioxidant properties. It can enhance the body's inhibition of hydrogen peroxide, causing it to decompose rapidly and reduce the content of harmful substances MDA. Antioxidant enzymes such as SOD and GSH-PX can remove free radicals and reactive oxygen in the body. They are important defense enzymes against acute alcoholic liver damage and can balance the antioxidant system and the oxidative system.

[0104] In order to study the effect of radish polysaccharide oral liquid on the production of a large number of active free radicals in alcohol metabolism in mice with alcoholic liver damage, the effect of radish polysaccharide oral liquid on the MDA, CAT, SOD and GSH-PX indicators in the serum of mice with alcoholic liver damage was investigated. Figure 11-14As shown, it shows that the oral solution of drug, implementation 1, implementation 2, implementation 3 and implementation 4 groups can effectively improve acute alcoholic liver injury. Compared with the normal group, the MDA level in the model group was significantly increased, while the levels of CAT, SOD and GSH-Px were significantly decreased. Compared with the model group, the MDA levels of drug, implementation 1, implementation 2, implementation 3 and implementation 4 groups were significantly reduced by 44.71%, 33.49%, 38.67%, 43.48% and 38.06%, respectively (P<0.05); the CAT levels were significantly increased by 57.86%, 70.80%, 112.04%, 180.91% and 176.32%, and the SOD levels were significantly increased by 61.18%, 110.46%, 13 The levels of GSH-Px in the drug, implementation 1, implementation 2, implementation 3 and implementation 4 groups were significantly increased by 197.33%, 134.05%, 160.37%, 141.85% and 185.46% (P<0.05), respectively. This shows that the drug, implementation 1, implementation 2, implementation 3 and implementation 4 oral solution can effectively improve alcoholic liver injury, and implementation 3 has the best effect.

[0105] Alcoholic liver injury is accompanied by increased levels of inflammatory cytokines. TNF-α can promote neutrophil migration and the production of neutrophil proteolytic enzymes and reactive oxygen species, leading to liver cell damage. IL-6 mainly mediates the inflammatory response.

[0106] In order to study the effect of radish polysaccharide oral liquid on inflammatory cytokines in mice with alcoholic liver injury, the effect of radish polysaccharide oral liquid on the indicators of TNF-α and IL-6 in the liver of mice with alcoholic liver injury was investigated. Figure 15-16 As shown, the oral liquid of radish polysaccharide in drug, experiment 1, experiment 2, experiment 3 and experiment 4 can effectively improve acute alcoholic liver injury. Compared with the normal group, the levels of TNF-α and IL-6 in the model group were significantly increased; compared with the model group, the levels of TNF-α and IL-6 in drug, implementation 1, implementation 2, implementation 3 and implementation 4 were significantly reduced by 3.50%, 5.48%, 7.62%, 7.46%, 27.42% and 10.86%, 5.31%, 12.48%, 23.80%, 22.41% (P<0.05), respectively, indicating that the oral liquid in drug, implementation 1, implementation 2, implementation 3 and implementation 4 can effectively improve alcoholic liver injury, and implementation 4 has the best effect.

[0107] Liver histopathology is the standard for judging the degree of liver damage, and HE-stained pathological sections can directly observe liver damage. Fig.17As shown in the figure, the liver tissue structure of mice in the normal group was normal, the liver lobules and cell nuclei structure were clear, the liver cells were neatly arranged around the central vein in a radial pattern, the liver cells were intact, and there was no significant pathological condition; the liver lobules and cell nuclei structure of mice in the model group were destroyed, the central vein was severely deformed, the arrangement was disordered, and edema occurred, which is a typical pathological feature of alcoholic liver injury. From the results of histopathology, the size of the liver sinusoids of mice in the drug, implementation 1, implementation 2, implementation 3, and implementation 4 groups was normal compared with the model group, and the liver cells were arranged regularly. The experiment proved that alcoholic liver injury was alleviated to a certain extent after gavage with radish polysaccharide oral liquid.

[0108] From the above content, it can be known that the green radish polysaccharide oral liquid and radish polysaccharide oral liquid provided by the present invention can effectively protect the liver cell structure, inhibit liver swelling caused by alcohol, and reduce the liver index; downregulate the activity of liver cell damage-related biological enzymes ALT and AST in serum; increase the activity of liver antioxidant enzymes to inhibit the generation of liver cell oxidation product MDA, and reduce oxidative stress damage to the liver of alcoholic liver mice; at the same time, it can also reduce the levels of TC and TG in the liver and serum, effectively improve lipid metabolism disorders, and reduce lipid accumulation; reduce the release of inflammatory factors such as tumor necrosis factor TNF-α and IL-6, promote the body's inflammation level to return to a balanced state, reduce liver inflammation, and then improve alcohol damage to liver tissue.

[0109] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing radish polysaccharide having auxiliary protective effect on chemical liver damage, characterized in that: The method comprises the following steps: drying and crushing the radish to obtain radish powder; mixing the radish powder with anhydrous ethanol, standing at 50°C for 8 hours, filtering with gauze to obtain filter residue; mixing the filter residue with water to obtain a mixture, mixing the mixture with cellulase, adjusting the pH value to 7, performing enzymolysis at 30-55°C for 40-70 minutes, taking the filtrate for alcohol precipitation, collecting the precipitate, adding water for redissolution, centrifuging to obtain the supernatant, and freeze-drying to obtain radish polysaccharide.

2. The method according to claim 1, characterized in that The material-liquid ratio of the mixture of filter residue and water is 1-3 g: 10-25 mL.

3. The method according to claim 1, characterized in that The amount of the cellulase added is calculated based on the weight of the mixture, and 0.2 to 0.8 g of the cellulase is added per 100 g of the mixture.

4. The method according to claim 1, characterized in that The weight ratio of radish powder to anhydrous ethanol is 1:

5.

5. A green radish polysaccharide having an auxiliary protective effect on chemical liver damage, characterized in that: Prepared by the method according to any one of claims 1 to 4.

6. A white radish polysaccharide having an auxiliary protective effect on chemical liver damage, characterized in that: Prepared by the method according to any one of claims 1 to 4.

7. An oral liquid of green radish polysaccharide having an auxiliary protective effect on chemical liver damage, characterized in that: The invention comprises an oral liquid and the green radish polysaccharide as claimed in claim 5; the weight-to-volume ratio of the green radish polysaccharide to the oral liquid is 0.1-0.3 g:1 mL; the preparation method of the oral liquid comprises the following steps: mixing wolfberry, hawthorn, turmeric, red dates, licorice, and poria with water and soaking them for 20 minutes, decocting them until they are slightly boiled and maintained for 30 minutes, taking the filtrate and concentrating it to obtain the oral liquid; the weight-to-volume ratio of the wolfberry, hawthorn, turmeric, red dates, licorice, poria and water is 7.8 g: 7.8 g: 2.7 g: 7.8 g: 7.8 g: 7.8 g: 1000 mL.

8. A radish polysaccharide oral liquid having an auxiliary protective effect on chemical liver damage, characterized in that: The invention comprises water, the green radish polysaccharide according to claim 5 and the white radish polysaccharide according to claim 6, wherein the volume weight ratio of water, the green radish polysaccharide and the white radish polysaccharide is 1 mL: 0.05 g: 0.05 g.

9. An oral liquid of green radish polysaccharide having an auxiliary protective effect on chemical liver damage, characterized in that: The invention comprises the following raw materials: the green radish polysaccharide as claimed in claim 5, 0.1-0.2 parts by weight of oats, 0.1-0.2 parts by weight of raisins, 2-3 parts by volume of pear juice, 2-3 parts by volume of tomato juice, and 50-60 parts by volume of water; the concentration of the green radish polysaccharide in the green radish polysaccharide oral liquid is 0.1 g / mL.

10. The method according to any one of claims 1 to 4, the green radish polysaccharide according to claim 5, the white radish polysaccharide according to claim 6, the green radish polysaccharide oral liquid according to claim 7, the radish polysaccharide oral liquid according to claim 8 or the green radish polysaccharide oral liquid according to claim 9, characterized in that: The chemical liver injury is alcoholic liver injury.

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