Use of black currant (Ribes nigrum) polysaccharides in the preparation of a product for preventing and treating radiation-induced liver injury

Abstract

The application discloses an application of a blackcurrant ribes polysaccharide in preparation of a product for preventing and treating radioactive liver injury, and belongs to the technical field of biological medicine preparation. The application provides an application of a polysaccharide in preparation of a product for preventing and treating liver injury, in particular, a new application of the blackcurrant ribes polysaccharide in preparation of a medicine, health care product, food or feed for preventing and treating liver injury caused by ionizing radiation. The polysaccharide can relieve liver injury by increasing the content of superoxide dismutase in liver tissue and reducing the content of glutamic-pyruvic transaminase, and the polysaccharide has superiority and no toxic side effect in the medicine, health care product and the like for preventing and treating liver injury.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine preparation, and in particular relates to the application of Aronia melanocarpa polysaccharide in the preparation of a product for preventing and treating radiation-induced liver damage. Background Art

[0002] Radiation-induced liver injury (RALI) refers to a pathological condition in which liver function is impaired after prolonged or short-term exposure to high doses of radioactive substances. This damage can occur through various pathways, including oral ingestion or inhalation of radioactive substances, radiation-induced oxygen free radicals and oxidative stress, inflammation and liver fibrosis, and an increased risk of liver cancer. Typical symptoms of RALI include abnormal liver function indicators, enlarged liver volume, yellowing of the skin and eyes, and liver cell damage. To prevent RALI, the most effective way is to avoid all exposure to radioactive substances or, if exposure is unavoidable, to take effective protective measures to reduce the harm caused by radioactive substances.

[0003] At present, the treatment of radiation-induced liver injury mainly includes 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, although antioxidants can reduce oxidative stress to a certain extent, their repair capacity is limited for liver cells that have been severely damaged. Moreover, 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 nigra 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 valuable component naturally found in Aronia nigra fruit. Research has shown that this polysaccharide not only possesses excellent antioxidant properties but also effectively inhibits cell damage and inflammatory responses. Antioxidant activity plays a crucial role in the treatment of liver diseases. The liver is a crucial metabolic organ in the body, and its detoxification, metabolic, and immune functions require adequate antioxidant support. Aronia polysaccharide is a powerful antioxidant that scavenges excess free radicals, mitigates oxidative stress damage to the liver, and thus protects normal liver function. Aronia polysaccharide also exhibits significant anti-inflammatory effects. Inflammation often plays a role in the development of liver diseases. Aronia polysaccharide can inhibit the production and release of inflammatory factors, reducing liver inflammation and thus improving liver pathology. Notably, Aronia polysaccharide also has a protective effect against liver cell damage. When the liver is stimulated by external factors such as drugs and alcohol, liver cells are susceptible to damage and even death. Aronia melanocarpa 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 melanocarpa polysaccharide also has multiple effects such as improving liver metabolic function and improving liver function. These effects make Aronia melanocarpa polysaccharide have broad application prospects in preventing and treating liver damage. However, the current research on Aronia melanocarpa polysaccharide in preventing and treating 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 melanocarpa polysaccharide disclosed in this application has a significant effect on the prevention and treatment of liver damage caused by ionizing radioactivity, and has very important application prospects.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0007] Application of Aronia nigra polysaccharide in the preparation of drugs for resisting radioactive liver injury.

[0008] The invention discloses an application of Aronia nigra polysaccharide in the preparation of a health product or feed having an auxiliary protective effect on chemical liver damage.

[0009] The invention discloses an application of Aronia nigra polysaccharide in the preparation of health products or feeds having auxiliary protective effects against ionizing radiation hazards.

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

[0011] (1) Dissolve the finished Aronia nigra fruit powder in water, stir in a water bath at 40-50°C for 1 hour, then adjust the pH to 5.0 with hydrochloric acid, add the enzyme mixture, and perform enzymolysis at a constant temperature of 40-50°C for 3 hours, then heat to boiling for 20 minutes and cool 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 rotary centrifugation. Anhydrous ethanol is then added for alcohol precipitation, and the solution is redissolved after alcohol precipitation. After deproteinization, the solution is freeze-dried to obtain the black fruit glandular costus polysaccharide.

[0013] In one embodiment, during the water dissolution process in 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 in the form of one or more of a liquid agent, a powder, and a granule.

[0019] In one embodiment, the Aronia melanocarpa polysaccharide alleviates liver damage by increasing superoxide dismutase (SOD) in liver tissue and reducing the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and pro-inflammatory factors IL-6 and TNF-α in liver tissue. Therefore, when Aronia melanocarpa polysaccharide is used in the preparation of medicines, health products, foods, or feeds for preventing and treating liver damage, it has greater advantages and 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 melanocarpa polysaccharide in the preparation of medicines, health products, foods or feeds for preventing and treating liver damage caused by ionizing radiation.

[0022] 2. The drug, health product, food, or feed prepared from the Aronia nigra polysaccharide for preventing and treating liver damage can reduce the levels of alanine aminotransferase and aspartate aminotransferase in the liver, enhance the antioxidant capacity of liver tissue, and reduce liver inflammatory responses. Thus, the drug can prevent and treat liver damage 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 These are H&E images of the livers of mice in each group in Example 1 of the present invention;

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

[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 The SOD content in the tissues of the mice in each group in Example 1 of the present invention. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below with reference to experimental examples. These experimental examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following experiments are conventional methods unless otherwise specified. The materials and reagents used are commercially available 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, shake, 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 a 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 the 1 mg / mL glucose standard solution into eight 10 mL volumetric flasks, then bring to volume with deionized water. Take 2 mL of each solution and add 1 mL of 5% phenol. Mix thoroughly, then quickly add 5 mL of concentrated sulfuric acid. Shake well, place in a boiling water bath for 15 minutes, and cool to room temperature (20°C). Measure the absorbance at 490 nm. Plot a standard curve with glucose concentration (mg / mL) as the horizontal axis to 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] Place 1g of finished Aronia nigra fruit powder in 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 aqueous extract in a high-speed centrifuge (4000r / min, 10min), retain the supernatant and discard the precipitate, and use a rotary evaporator to reduce the supernatant to 400 About 1 mL (this step reduces the sample volume) was centrifuged at 8000 rpm for 20 minutes, the precipitate was discarded, and the above extraction method was repeated twice. The sample was concentrated to one-tenth of the original volume by rotary evaporation at 50°C, and 2 volumes of anhydrous ethanol were added. After alcohol precipitation, the sample was reconstituted with a small amount of distilled water. The sample was then repeatedly deproteinized using the Sevega method. After dialysis with running water for 72 hours using a dialysis bag (molecular weight cutoff 3500 Da), the sample was freeze-dried to obtain a purple-black flocculent Aronia polysaccharide. The polysaccharide content of the Aronia polysaccharide prepared above was determined using the phenol-sulfuric acid method, with a polysaccharide content of 8.48%. The protein content was determined using a BCA kit, with a value of 1.4%.

[0039] Example 2 Effect of Aronia nigra 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, and they were given free access to food and water to maintain a circadian rhythm.

[0041] Grouping: Mice were randomly divided into a normal control group (NC group), a radiation model group (IR group), a low-dose Aronia melanocarpa polysaccharide intervention group (AMP-L group), and a high-dose Aronia melanocarpa polysaccharide intervention group (AMP-H group), with 5 mice in each group. All animal experiments were approved by the Animal Ethics Committee of Dalian Polytechnic University and 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.

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

[0046] Following the above protocol, prior to the first oral gavage, the radioactive model group (IR group), the low-dose intervention group (AMP-L group), and the high-dose group (AMP-H group) were uniformly irradiated with timed, quantitative abdominal X-rays on the first day to induce an ionizing radiation-induced liver injury model. After the final oral gavage, mice were fasted for 12 hours and allowed to drink water. The mice were then sacrificed by dislocation. Blood samples were collected and centrifuged (3000 rpm / 10 min). Serum was separated and stored at -20°C for ELISA analysis of ALT, AST, TNF-α, and IL-6 levels. Following sacrifice, liver tissue was removed and washed with pre-chilled saline to remove blood. The liver was weighed and the liver index was calculated. Liver tissue was fixed and stained with H&E. Liver tissue was excised, added to PBS, homogenized, and centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was aspirated and superoxide dismutase (SOD) content was determined using a kit from the Nanjing Jiancheng Bioengineering Institute. Total protein content was determined by BCA assay and corrected.

[0047] Result measurement and 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 significantly depressed state, with a significant decrease in activity level, and some mice even remained still for a long 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 hepatocyte nuclear morphology is normal, and the nuclear membrane is clearly visible.

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

[0051] In the low-dose Aronia melanocarpa polysaccharide-treated group, mice receiving 200 mg / mL of Aronia melanocarpa polysaccharide for two consecutive weeks showed reduced inflammatory cell infiltration in the liver tissue compared to the model group, and hepatic granulomatous lesions were no longer significant. However, significantly enlarged hepatocytes were still visible in the lower right side of the portal area, suggesting the presence of cellular edema. This suggests that Aronia melanocarpa polysaccharide has a certain repair effect on liver ionizing radiation damage.

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

[0053] 2. Effect of Mouse Liver Index

[0054] The liver index, defined as the ratio of a mouse's liver weight to its total body weight, is an important indicator of liver health. Under normal physiological conditions, the liver-to-body weight ratio remains within a relatively stable range. However, when an animal experiences some degree of damage, the corresponding organ weight may fluctuate significantly.

[0055] like Figure 3 As shown in the figure, the liver index of the blank group mice remained stable within the normal range, which fully demonstrated that these mice were in good health and their livers had not suffered obvious damage. 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] Notably, the liver indices of the two groups of mice treated with Aronia melanocarpa polysaccharide significantly improved compared to the control group. This is primarily due to the antioxidant properties of Aronia melanocarpa polysaccharide, which effectively alleviates liver damage caused by oxidative stress. This finding clearly demonstrates that Aronia melanocarpa polysaccharide exhibits a significant protective effect against radiation-induced liver damage in mice.

[0057] 3. Effects of transaminase levels in mice

[0058] Transaminases primarily catalyze the transamination reaction between amino acids and keto acids. They are widely present in animal tissues, particularly in the myocardium, brain, liver, and kidneys. Found primarily in hepatocytes, transaminases are essential catalysts for the liver's normal functioning and a key indicator of liver health.

[0059] like Figure 4 、 Figure 5 As shown, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice in the blank group remained stable within the normal range, demonstrating that in healthy mice with normal liver function, ALT and AST perform their normal functions. However, the ALT and AST levels in irradiated mice were significantly higher than those in the other groups. This is due to damage to the liver cells in irradiated mice, which results in the release of transaminases into the serum. In the presence of Aralia nigra polysaccharide, the levels of both transaminases decreased significantly compared to the control group, demonstrating the role of 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 damage 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 the black fruit glandular costa polysaccharide has an intervention effect on the content of TNF-α inflammatory factors in mice. Figure 7 As shown in the results, compared with the blank group, the IL-6 content in the serum of the control group mice was significantly increased (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 and high-dose groups mice was significantly increased. However, after the intervention of Aronia nigra polysaccharide, the content was reduced 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 (SOD) in liver tissue was significantly increased (P < 0.0001), and the levels of pro-inflammatory factors (TNF-α, IL-1β) in serum were significantly reduced (P < 0.05).

[0062] In the above examples, the use of Aronia melanocarpa polysaccharide in preparing a product for preventing and treating liver damage was verified only in mice, particularly for preventing and treating acute liver damage induced by radiation. However, the use of Aronia melanocarpa polysaccharide in preparing a product for preventing and treating liver damage is also applicable to other animals or humans, including but not limited to pigs, cattle, sheep, and humans.

[0063] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A use of Aronia nigra polysaccharide in the preparation of a drug for preventing radiation-induced liver injury, 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, and adding an enzyme mixture for enzymolysis to obtain a water extract; (2) The aqueous extract obtained in step (1) is centrifuged, the supernatant is collected, and the supernatant is concentrated by rotary centrifugation. Anhydrous ethanol is then added for alcohol precipitation. After alcohol precipitation, the extract is redissolved, deproteinized, and freeze-dried to obtain the black fruit arbutus polysaccharide.

2. The use according to claim 1, characterized in that In step (1), during the water dissolution process, the liquid-to-material ratio of water to Aronia nigra fruit powder is 25:1, mL:g.

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

4. The use according to claim 3, characterized in that The mass ratio of cellulase, pectinase and papain is 2.66:2.66:

1.

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

6. The use according to claim 1, characterized in that In step (2), deproteinization is performed by repeatedly deproteinizing the reconstituted solution using the Sevega method.

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