Application of aronia melanocarpa polysaccharide in preparation of product for preventing and treating radioactive liver injury

Abstract

The invention discloses application of aronia melanocarpa polysaccharide in preparation of a product for preventing and treating radiation liver injury, and belongs to the technical field of biological medicine preparation. The invention provides application of polysaccharide in preparation of products for preventing and treating liver injury, and particularly relates to novel application of aronia melanocarpa polysaccharide in preparation of drugs, health care products, food or feed for preventing and treating liver injury caused by ionizing radiation. The polysaccharide can relieve liver injury by increasing superoxide dismutase of liver tissues and reducing the content of glutamic-pyruvic transaminase and the like, has superiority when being used for medicines, health care products and the like for preventing and treating liver injury, and has no toxic or side effect.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine preparation, and particularly relates to the application of Aronia nigra polysaccharide in the preparation of products for preventing and treating radioactive liver damage. Background Art

[0002] Radiation liver injury refers to a pathological state in which liver function is damaged after people are exposed to high doses of radioactive substances for a long period of time or in a short period of time. This damage may occur in many ways, such as oral ingestion of radioactive substances or inhalation through the respiratory tract, radiation-induced oxygen free radicals and oxidative stress reactions, inflammatory reactions and liver fibrosis, and increased risk of liver cancer. Typical symptoms of radiation liver injury include abnormal liver function indicators, enlarged liver volume, yellowing of the skin and eyes, and liver cell damage. In order to prevent the occurrence of radiation liver injury, the most effective way is to avoid contact with any radioactive substances, or, if contact is unavoidable, take effective protective measures to reduce the harm of radioactive substances to the body.

[0003] At present, the treatment methods for radiation-induced liver injury mainly include symptomatic treatment and supportive treatment, such as taking measures to protect liver function and reduce inflammatory response. Among them, drug treatment has many deficiencies and shortcomings in dealing with radiation-induced liver injury. First of all, although antioxidants can reduce oxidative stress to a certain extent, their repair ability is limited for liver cells that have been severely damaged. And long-term and large-scale use of antioxidants may disrupt the original antioxidant-oxidation balance in the body and cause other health problems. Secondly, long-term use of anti-inflammatory drugs such as glucocorticoids can bring serious side effects, such as increased blood sugar, blood pressure fluctuations, osteoporosis, decreased immunity, etc., which increase the risk of patients contracting other diseases. Summary of the invention

[0004] The purpose of the present invention is to provide an application of Aronia nigromaculata polysaccharide in the preparation of a product for preventing and treating radiation-induced liver injury, so as to provide a new substance, method and approach for preparing a product for preventing and treating radiation-induced liver injury.

[0005] Aronia polysaccharide is a precious ingredient naturally present in Aronia fruit. Studies have shown that this polysaccharide not only has excellent antioxidant properties, but also can effectively inhibit cell damage and inflammatory response. Antioxidation is an important link in the treatment of liver diseases. The liver is an important metabolic organ in the body, and its detoxification, metabolism and immunity functions require sufficient antioxidant support. Aronia polysaccharide is such a powerful antioxidant. It can remove excessive free radicals in the body, reduce the damage of oxidative stress to the liver, and thus protect the normal function of the liver. In addition, Aronia polysaccharide also has a significant anti-inflammatory effect. In the onset of liver disease, inflammatory response often plays a role in fueling the flames. Aronia polysaccharide can inhibit the production and release of inflammatory factors, reduce the inflammatory response of the liver, and thus improve the pathological state of the liver. It is worth mentioning that Aronia polysaccharide also has a certain protective effect on liver cell damage. When the liver is stimulated by external factors such as drugs and alcohol, liver cells are easily damaged or even die. Aronia nigra polysaccharide can promote the repair and regeneration of liver cells and reduce the degree of liver cell damage by regulating the signal pathways in the cells. In addition to the above-mentioned effects, Aronia nigra polysaccharide also has multiple effects such as improving liver metabolic function and improving liver function. These effects make Aronia nigra polysaccharide have broad application prospects in the prevention and treatment of liver damage. However, the current research on Aronia nigra polysaccharide in the prevention and treatment of liver damage is not in-depth enough, and more research is needed to explore its mechanism of action, optimal dosage, and interaction with other drugs. At the same time, more clinical trials are also needed to verify its efficacy and safety. The Aronia nigra polysaccharide disclosed in this application has a significant effect on the prevention and treatment of liver damage caused by ionizing radioactivity, and has a very important application prospect.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] Application of Aralia nigromaculata polysaccharide in the preparation of drugs for preventing radiation-induced liver injury.

[0008] Application of Aralia nigra polysaccharide in the preparation of health products or feeds with auxiliary protective effects on chemical liver damage.

[0009] The invention discloses an application of Aronia nigra polysaccharide in preparing a health product or feed having an auxiliary protective effect against the hazards of ionizing radiation.

[0010] In one embodiment, the method for preparing the Aronia melanocarpa polysaccharide comprises the following steps:

[0011] (1) dissolving the finished Aronia nigra fruit powder in water, stirring in a water bath at 40-50° C. for 1 hour, then adjusting the pH to 5.0 with hydrochloric acid, adding an enzyme mixture, performing enzymolysis at 40-50° C. for 3 hours, then heating to boiling for 20 minutes and cooling for 8-12 hours to obtain a water extract;

[0012] (2) The aqueous extract solution obtained in step (1) is centrifuged, the supernatant is collected, and the solution is concentrated by rotation, and then anhydrous ethanol is added for alcohol precipitation. After the alcohol precipitation, the solution is redissolved, deproteinized, and freeze-dried to obtain the Aronia nigra polysaccharide.

[0013] In one embodiment, during the water dissolution process of step (1), the liquid-to-solid ratio of water to Aronia melanocarpa fruit powder is 25:1, mL:g.

[0014] In one embodiment, in step (1), the enzyme mixture refers to a mixture of cellulase, pectinase and papain, and the mass ratio of cellulase, pectinase and papain is 2.66:2.66:1.

[0015] In one embodiment, the centrifugation condition in step (2) is: centrifugation at a speed of 3000-5000 r / min for 5-15 min.

[0016] In one embodiment, the rotary concentration in step (2) is to concentrate the reaction solution to one tenth of its original volume.

[0017] In one embodiment, the deproteinization in step (2) is to repeatedly deproteinize the reconstituted solution by the Sevega method.

[0018] In one embodiment, the drug is one or more of a liquid agent, a powder, and a granule.

[0019] In one embodiment, the Aronia nigra polysaccharide relieves liver damage by increasing superoxide dismutase (SOD) in liver tissue, reducing alanine aminotransferase (ALT), aspartate aminotransferase (AST) in liver tissue, and proinflammatory factors IL-6 and TNF-α. Therefore, when Aronia nigra polysaccharide is used to prepare medicines, health products, foods or feeds for preventing and treating liver damage, it has more advantages and also provides a new safe and effective means for preventing and treating liver damage.

[0020] Beneficial effects of the present invention:

[0021] 1. The present invention provides an application of a polysaccharide in the preparation of a product for preventing and treating liver damage, specifically a new application of Aronia nigra polysaccharide in the preparation of medicines, health products, foods or feeds for preventing and treating liver damage caused by ionizing radioactivity.

[0022] 2. The medicine, health product, food or feed for preventing and treating liver damage made from the polysaccharide of Aronia nigra can reduce the levels of alanine aminotransferase and aspartate aminotransferase in the liver, enhance the antioxidant capacity of liver tissue, and reduce the inflammatory response of the liver. Thus, the effect of preventing and treating liver damage can be achieved from various angles.

[0023] 3. The medicine, health product, food or feed for preventing and treating liver damage prepared from the Aronia nigra polysaccharide has no toxic or side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 H&E images of the liver of each group of mice in Example 1 of the present invention;

[0025] Figure 2 The weight change images of each group of mice in Example 1 of the present invention;

[0026] Figure 3 The liver index images of each group of mice in Example 1 of the present invention;

[0027] Figure 4 is the serum ALT content of each group of mice in Example 1 of the present invention;

[0028] Figure 5 is the serum AST content of each group of mice in Example 1 of the present invention;

[0029] Figure 6 is the serum TNF-α content of each group of mice in Example 1 of the present invention;

[0030] Figure 7 is the serum IL-6 content of each group of mice in Example 1 of the present invention;

[0031] Figure 8 It is the tissue SOD content of each group of mice in Example 1 of the present invention. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with experiments, which are only used to explain the present invention and are not used to limit the scope of the present invention. The experimental methods used in the following experiments are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0033] 1. Cellulase, pectinase and papain: cellulase (Macklin9012-54-8 10000U / g); pectinase (Macklin9032-75-1 30000U / g); papain (Macklin9001-73-4>200U / mg);

[0034] 2. Sevega method: Mix the extract with Sevage reagent [chloroform: n-butanol = 5:1 (V / V)] in a ratio of 5:1, oscillate, and centrifuge. The denatured protein will be located at the interface between the extract and the Sevage reagent.

[0035] 3. Determination of polysaccharide content:

[0036] Phenol-sulfuric acid method: accurately prepare 1 mg / mL glucose standard solution. Accurately pipette 0.00, 1.00, 2.00, 3.00, 4.00, 5.00, 6.00, and 7.00 mL of 1 mg / mL glucose standard solution into 8 10 mL volumetric flasks, and dilute to volume with deionized water. Take 2 mL of each solution, add 1 mL of 5% phenol, mix well, and quickly add 5 mL of concentrated sulfuric acid. Shake well and place in a boiling water bath for 15 minutes. Cool to room temperature (20°C), measure the absorbance at 490 nm, draw a standard curve with glucose concentration (mg / mL) as the horizontal axis, and construct a quantitative model; then calculate the polysaccharide content in the sample based on the absorbance of the measured sample.

[0037] Example 1 Preparation of Aronia nigra polysaccharide

[0038] Put 1g of the finished product of Aronia nigra fruit powder into a beaker and add 25mL of distilled water. Stir in a 50℃ water bath (400 rpm) for 1 hour, then adjust the pH to 5.0 with hydrochloric acid, add an enzyme mixture (cellulase 0.1064g, pectinase 0.1064g, papain 0.04g), and perform enzymolysis at 50℃ for 3h, then heat to boiling for 20min and leave overnight; centrifuge the obtained water extract in a high-speed centrifuge (4000r / min, 10min), retain the supernatant and discard the precipitate, and use a rotary evaporator to reduce the pressure and concentrate the supernatant to 400 mL (this step reduces the sample volume) and centrifuge at 8000r / min for 20min, discard the upper precipitate, repeat the above extraction method twice, 50°C rotary evaporation to concentrate to one tenth of the original volume, add 2 times the volume of anhydrous ethanol, remelt with a small amount of distilled water after alcohol precipitation, and then repeatedly deproteinize by Sevega method, dialyze with flowing water for 72h in a dialysis bag (molecular weight cutoff 3500Da), and freeze-dry to obtain purple-black flocculent Aronia polysaccharide. The prepared Aronia polysaccharide was determined for polysaccharide content by phenol sulfuric acid method, wherein the polysaccharide content was 8.48%; the protein content was determined by BCA kit, which was 1.4%.

[0039] Example 2 Effect of Aralia nigromaculata polysaccharide on the treatment of radiation-induced liver injury

[0040] Twenty 6-week-old Balb / C male SPF mice weighing 20-22 g were purchased from Liaoning Changsheng Biotechnology Co., Ltd. There were 5 mice in each group, with free access to food and water to maintain circadian rhythm.

[0041] Grouping experiment: The mice were randomly divided into normal control group (NC group), radiation model group (IR group), low-dose Aronia nigra polysaccharide intervention group (AMP-L group), and high-dose Aronia nigra polysaccharide intervention group (AMP-H group), with 5 mice in each group for experiment. All animal experiments were approved by the Animal Ethics Committee of Dalian Polytechnic University and were conducted in accordance with the guidelines of the National Institute of Animal Experimentation.

[0042] NC group: 200 μL PBS, once a day, for a total of 14 times.

[0043] IR group: 200 μL PBS, once a day, for a total of 14 times.

[0044] AMP-L group: 200 mg / mL Aronia nigra polysaccharide, once a day, for a total of 14 times of gavage.

[0045] AMP-H group: 400 mg / mL Aronia nigra polysaccharide, once a day, for a total of 14 times of gavage.

[0046] According to the above scheme, before the first intragastric administration, the radioactive model group (IR group), the low-dose intervention group (AMP-L group) and the high-dose group (AMP-H group) were uniformly given regular and quantitative abdominal X-ray irradiation on the first day to induce the ionizing radiation liver injury model. After the last intragastric administration, the mice were fasted for 12 hours and allowed to drink water, then the mice were killed by dislocation, blood samples were collected, centrifuged (3000r / 10min), serum was separated, and stored at -20℃ for ELISA determination of ALT, AST, TNF-α, and IL-6 levels. After the mice were killed, the liver tissue was removed and washed with pre-cooled saline to remove blood stains, the liver weight was weighed, and the liver index was calculated. The liver tissue was selected for fixation and H&E staining. The liver tissue was cut, PBS solution was added, the tissue was homogenized, centrifuged at 4℃, 10000×g for 10min, the supernatant was aspirated, and the superoxide dismutase (SOD) content was determined using the kit of Nanjing Jiancheng Bioengineering Institute. The total protein content was determined by BCA method for correction.

[0047] Results analysis

[0048] 1. Degree of liver tissue damage in mice

[0049] On the first day of the experiment, after experiencing quantitative and timed ionizing radiation from the CT machine, the mice showed a markedly depressed state, with a significant decrease in activity, and some mice even remained still for long periods of time. Figure 1These are H&E staining images of mouse liver tissue after the experiment. The liver tissue structure of mice in the blank group is clear, inflammatory cell infiltration is rare, the nuclear morphology of hepatocytes is normal, and the nuclear membrane is clearly visible.

[0050] After exposure to ionizing radiation, the liver tissue of the mice in the control group showed obvious inflammatory response, with lymphocytes and monocytes forming focal infiltration in the portal area and accumulating to form hepatic granuloma-like lesions, accompanied by punctate necrosis of hepatocytes and the appearance of small vacuoles. This result shows that the radiation liver injury model was successfully established.

[0051] For mice in the low-dose Aronia nigra polysaccharide treatment group, after taking 200 mg / mL Aronia nigra polysaccharide for two consecutive weeks, the inflammatory cell infiltration in the liver tissue was reduced compared with the model group, and the hepatic granuloma-like lesions were no longer significant. However, significantly enlarged hepatocytes were still visible on the lower right side of the portal area, indicating the presence of cell edema. This shows that Aronia nigra polysaccharide has a certain repair effect on liver ionization radiation damage.

[0052] The mice in the high-dose Aronia nigra polysaccharide treatment group showed significant improvement in liver tissue performance after taking 400 mg / mL Aronia nigra polysaccharide for two consecutive weeks, and there was almost no significant inflammatory cell infiltration compared with the control group. This shows that the liver tissue damage of the mice has been significantly improved, and its overall condition is closer to that of the blank group. This result further verifies that there is a close relationship between the repair effect of Aronia nigra polysaccharide on liver damage and the dose.

[0053] 2. Effect of mouse liver index

[0054] The liver index, which is the ratio of the mouse liver weight to its total body weight, is an important indicator of liver health. Under normal physiological conditions, the ratio of liver to body weight will remain within a relatively stable range. However, when the animal body is damaged to a certain extent, the corresponding organ weight may change significantly.

[0055] like Figure 3 As shown in the figure, the liver index of the blank group mice was stable within the normal range, which fully demonstrated that these mice were in good health and their livers were not significantly damaged. In contrast, the liver indexes of the other three groups of mice were significantly lower than those of the blank group, which clearly revealed that the livers of these mice had suffered radiation damage.

[0056] It is worth noting that the liver index of the two groups of mice given aronia polysaccharide was significantly improved compared with the control group. This is mainly due to the antioxidant properties of aronia polysaccharide, which can effectively alleviate the symptoms of liver damage caused by oxidation. This finding clearly proves that aronia polysaccharide has a significant protective effect in intervening in radiation-induced liver damage in mice.

[0057] 3. Effects of transaminase levels in mice

[0058] The main function of transaminase is to catalyze the amino transfer reaction between amino acids and keto acids. It is widely present in animal tissues, especially in the myocardium, brain, liver, kidney, etc. Transaminase mainly exists in liver cells. It is an indispensable "catalyst" for the normal functioning of the liver and an important indicator of liver health.

[0059] like Figure 4 , Figure 5 As shown in the figure, the levels of alanine aminotransferase and aspartate aminotransferase in the blank group mice were stable within the normal range, proving that in healthy mice, when the liver function was normal, alanine aminotransferase and aspartate aminotransferase played their normal roles. However, the levels of alanine aminotransferase and aspartate aminotransferase in irradiated mice were significantly higher than those in other groups. This was because the liver cells of irradiated mice were damaged, causing the transaminases to be released into the serum. Under the intervention of Aralia nigromaculata polysaccharide, the levels of the two transaminases decreased significantly compared with those in the control group, which played a role in repairing damaged liver cells.

[0060] 4. Effects on other physiological indicators of mice

[0061] like Figure 6 As shown in the figure, compared with the blank group, the TNF-α content of the control group mice increased significantly, indicating that the degree of oxidative stress and inflammation in the body was high, and the radiation injury mouse model was successfully established. In the two groups of mice given polysaccharides, the TNF-α content in the mouse serum was slightly higher than that in the blank group, and lower than that in the control group. This shows that Aralia nigromaculata polysaccharide has an intervention effect on the content of TNF-α inflammatory factors in mice. Figure 7 As shown in the figure, compared with the blank group, the IL-6 content in the serum of the control group mice increased significantly (p < 0.001), indicating that the radiation injury mouse model was successfully established. Compared with the blank group, the IL-6 content in the low-dose group and the high-dose group mice increased significantly, but under the intervention of Aronia nigra polysaccharide, the content decreased compared with the control group mice, indicating that Aronia nigra polysaccharide has an intervention effect on the IL-6 content in the serum of radiation-injured mice. The antioxidant level of liver tissue (SOD was significantly increased (P < 0.0001), and the levels of pro-inflammatory factors in serum (TNF-α, IL-1β) were significantly reduced (P < 0.05).

[0062] In the above examples, the application of Aronia nigra polysaccharide in the preparation of products for preventing and treating liver damage was verified only on mice, especially in the prevention and treatment of acute liver damage induced by radioactive irradiation. However, the application of Aronia nigra polysaccharide in the preparation of products for preventing and treating liver damage is also applicable to other animals or humans, including but not limited to pigs, cattle, sheep, humans, etc.

[0063] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of Aralia nigromaculata polysaccharide in the preparation of drugs for preventing radiation-induced liver injury.

2. Use of Aronia nigra polysaccharide in the preparation of health products or feeds with auxiliary protective effects on chemical liver damage.

3. Use of Aronia nigra polysaccharide in the preparation of health products or feeds with auxiliary protective effects against ionizing radiation hazards.

4. The use according to any one of claims 1 to 3, characterized in that: The preparation method of Aronia nigra polysaccharide comprises the following steps: (1) dissolving the Aronia nigra fruit powder in water, adjusting the pH to 5.0 with hydrochloric acid, adding an enzyme mixture for enzymolysis, and obtaining a water extract; (2) The aqueous extract obtained in step (1) is centrifuged, the supernatant is collected, and the extract is concentrated by rotation, and then anhydrous ethanol is added for alcohol precipitation. After alcohol precipitation, the extract is redissolved, deproteinized, and freeze-dried to obtain Aronia nigra polysaccharide.

5. The use according to claim 4, characterized in that: In step (1), during the process of adding water to dissolve, the liquid-to-solid ratio of water to the aronia melanocarpa fruit powder is 25:1, mL:g.

6. The use according to claim 4, characterized in that: In step (1), the enzyme mixture refers to a mixture of cellulase, pectinase and papain.

7. The use according to claim 6, characterized in that: The mass ratio of cellulase, pectinase and papain is 2.66:2.66:

1.

8. The use according to claim 4, characterized in that: In step (1), the enzymatic hydrolysis condition is 40-50° C. for 3 h.

9. The use according to claim 4, characterized in that: In step (2), deproteinization is performed by repeatedly deproteinizing the reconstituted solution using the Sevega method.

10. The use according to claim 1, characterized in that: The medicine is one or more of liquid reagent, powder, and granule.

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