Composition with auxiliary protection effect on chemical liver injury

Abstract

The invention provides a composition. The composition is prepared from spleen aminopeptide, phycocyanin, folic acid substances and health-care food or medicine acceptable auxiliary materials. The composition disclosed by the invention has the advantages that the composition has a synergistic effect, can effectively reduce liver enzyme, improve degeneration and necrosis of liver cells and relieve chemical liver injury, has an immune regulation effect and can inhibit liver inflammatory response, so that the composition can be used for assisting in preventing and treating the chemical liver injury. In addition, the multi-component compound form of the product can obviously improve the taking compliance of users, and the total use cost is saved.

Description

Technical Field

[0001] The present invention relates to a composition containing spleen aminopeptide, phycocyanin and folic acid substances, which is used for assisting in preventing and treating chemical liver damage. The present invention belongs to the fields of pharmacy and health food. Background Art

[0002] The liver is the most important metabolic organ in the human body, responsible for important physiological functions such as metabolism, oxidation, and reduction. Liver disease has become one of the major diseases that endanger human health. Liver damage is a pathological condition common to all types of liver disease. Common liver damage factors can be divided into physical liver damage, chemical liver damage, and viral liver damage, among which chemical liver damage is the most common in clinical practice. Chemical liver damage includes environmental toxicant liver damage, alcoholic liver damage, and drug-induced liver damage. The degree of damage is proportional to the dose and time of exposure to these substances. Continuous damage can lead to liver fibrosis, and then evolve into cirrhosis and liver cancer. In recent years, due to environmental pollution, drug abuse, or changes in lifestyle, the incidence of chemical liver damage has tended to increase. Early detection and timely intervention are the key to preventing and treating liver damage and preventing it from developing into severe liver disease.

[0003] Currently, there are very limited drugs for treating liver damage, and some drugs have significant side effects, with a relatively low benefit / risk ratio. For example, in order to suppress the excessive inflammatory response or immune response caused by liver damage, a large number of steroid drugs (glucocorticoid drugs) are often used clinically. However, these corticosteroid drugs have a strong immunosuppressive effect. Although they can reduce liver cell inflammation and necrosis, long-term use can lead to secondary bacterial or fungal infections, resulting in serious consequences. Therefore, how liver protection treatment products can be both effective and safe still needs further exploration.

[0004] Spleen aminopeptide is an immunomodulator. Its main ingredients are peptides and nucleotides extracted from the spleens of fresh pigs, cows, and other animals. It can trigger and enhance the body's cellular immune function and promote immune balance, increase T cell activity, promote interferon release, and enhance overall immunity. Phycocyanin is a dark blue powder isolated from spirulina, primarily found in cyanobacteria, red algae, and cryptoalgae. Phycocyanin helps regulate and synthesize several important enzymes required for human metabolism and plays an important role in promoting elastin synthesis and even cell regeneration. Folic acid, a B vitamin essential for human growth and development, plays an important role in promoting nervous system development and nutrition. Pregnant women require four times more folic acid than the average person. Folic acid deficiency in pregnant women may lead to adverse consequences such as neural tube defects, placental abruption, megaloblastic anemia, and fetal growth retardation.

[0005] In exploratory experiments, we combined the above substances in various forms and proportions and applied them to animals with liver damage. The results showed that the combined use of animal spleen extract, phycocyanin, and folic acid substances in a certain ratio can effectively reduce the release of liver function-related enzymes (ALT, AST) in the serum of experimental animals, reduce the content of lipid peroxide MDA, alleviate liver steatosis, and improve the pathological state of liver damage. Based on this, the present invention provides a synergistic multi-component composition through optimization, which can protect the liver and detoxify, can be used to prevent or treat chemical liver damage, and has an immunomodulatory effect. Summary of the Invention

[0006] In view of the shortcomings of current products for preventing and treating chemical liver damage, the present invention provides a safe and effective nutritional composition.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A composition comprising:

[0009] a) animal spleen extract; b) phycocyanin; c) folic acid substances; d) acceptable excipients or carriers or a mixture thereof.

[0010] In the present invention, the animal spleen extract is spleen aminopeptide.

[0011] In the present invention, the mass ratio of spleen aminopeptide:phycocyanin:folic acid substances is 1-20:400-5000:0.2-5.

[0012] In the present invention, as a preferred embodiment, the mass ratio of spleen aminopeptide:phycocyanin:folic acid substances is 2-10:1000-2000:0.4-1.2.

[0013] In the present invention, the spleen aminopeptide is 1 to 20 parts, the phycocyanin is 400 to 5000 parts, and the folic acid substance is 0.2 to 5 parts.

[0014] In the present invention, as a preferred embodiment, the spleen aminopeptide is 1 to 10 parts, the phycocyanin is 400 to 2000 parts, and the folic acid substance is 0.2 to 1.2 parts.

[0015] In the present invention, as a more preferred embodiment, the spleen aminopeptide is 2 to 10 parts, the phycocyanin is 1000 to 2000 parts, and the folic acid substance is 0.4 to 1.2 parts.

[0016] In the present invention, a portion can be micrograms, milligrams, grams, microliters, milliliters, deciliters, etc.

[0017] In the present invention, the spleen aminopeptide is selected from one or both of porcine spleen peptide and bovine spleen peptide.

[0018] In the present invention, the main components of spleen aminopeptide are polypeptides and / or nucleotide substances extracted from pig or bovine spleen.

[0019] In the present invention, spleen aminopeptide can be formulated into liquid and powder forms.

[0020] In the present invention, the spleen aminopeptide can be a spleen aminopeptide solution or a spleen aminopeptide oral lyophilized powder, and the spleen aminopeptide solution can be a spleen aminopeptide oral liquid or a spleen aminopeptide injection.

[0021] In the present invention, the folic acid substance is selected from one of 5-methyltetrahydrofolate, formyltetrahydrofolate, active metabolites of folic acid or folate, and substances that can release / generate folic acid in the body.

[0022] In the present invention, acceptable excipients or carriers or mixtures thereof include one or more of sugars or functional sweeteners for medicines or foods, fillers, wetting agents, binders and lubricants.

[0023] In the present invention, the dosage form of the composition is a health food or a food-acceptable dosage form, including but not limited to freeze-dried powder, oral liquid, injection, ordinary tablets, double-layer tablets, multi-layer tablets, granules, capsules, pills, compressed candies, solid beverages, syrups, liquid preparations, powders and the like.

[0024] In the present invention, the composition can be further prepared into fluid dairy products, dairy products, fermented beverages, milk powder, soy milk, juice, sports drinks, desserts, jelly, candy, milk, soft drinks, yogurt, cheese, bread, biscuits, soda crackers, energy bars, pizza crust, formulated milk powder, liquid food, food for special dietary needs, food for patients, nutritional food, frozen food, processed food, other commercially available food and the like, baby food, health food, animal feed, herbal composition or dietary supplement.

[0025] In the present invention, the composition can be used in combination with a traditionally known pharmaceutical product or food having a protective effect against liver damage.

[0026] In the present invention, the composition can be used in combination with traditionally known pharmaceutical products or foods having the effect of enhancing immunity.

[0027] In the present invention, the composition is selected from the administrable forms of pharmaceuticals, nutritional formulations, tube feeding formulations, dietary supplements, functional foods and beverage products.

[0028] In the present invention, the composition is used in preparing a product for preventing or assisting in treating chemical liver damage.

[0029] In the present invention, chemical liver injury includes alcoholic liver injury, drug-induced liver injury, and immune liver injury.

[0030] In the present invention, the composition is used in preparing a product for enhancing immunity.

[0031] In the present invention, the composition can be in the form of medicine, health food, functional food or food for special medical purposes.

[0032] The beneficial effects of the present invention are as follows: the present invention provides a composition with auxiliary protective effect on chemical liver damage, and the combined supplementation of the various components produces an unexpected synergistic effect, which has the functions of auxiliary prevention and treatment of chemical liver damage (reducing the content of oxidation products, reducing the content of serum alanine aminotransferase and aspartate aminotransferase, protecting liver cells, and alleviating liver pathological damage), enhancing the body's immunity (regulating CD8 + The role of the immune system in regulating the proportion of T lymphocytes, NK cells and other immune cells) provides an effective solution for the clinical prevention and treatment of alcoholic liver injury, drug-induced liver injury and immune liver injury. DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to specific embodiments, which is not intended to limit the present invention. Any equivalent replacements in the art made according to the contents of the present invention shall fall within the scope of protection of the present invention.

[0034] In order to verify the scientific nature of the pharmaceutical composition provided by the present invention and to demonstrate that the three components of the pharmaceutical composition are reasonably formulated and can be combined to exert a synergistic effect rather than simply superimposing the pharmacological effects, the Jin Zhengjun Q value method is introduced for analysis. The Jin Zhengjun Q value method, also known as the probability addition method, calculates the pharmacological effects of the two drugs in combination and the pharmacological effects of the two drugs alone within the dose-effect curve using the following calculation formula: Q = E A+B / (E A +E B -E A *E B ), where the numerator represents the "measured combined effect" and the denominator represents the "expected combined effect". (In order to analyze the relationship between the pharmacological effects of components and compositions, their pharmacological effects are converted into effects that can intuitively reflect the strength of the pharmacological effects. The calculation formula is: E i =1-P i / P 模型组 , P i is the pharmacological index of each component, P 模型组 = is the pharmacological index of the model group), Q is the ratio of the two: when Q is less than 0.85, the two drugs are considered to have an antagonistic effect; when it is less than 1.15 and greater than 0.85, it is considered to have an additive effect; when it is greater than or equal to 1.15, it is considered to have a synergistic effect. Based on the calculation formula for the combined use of two drugs, the pharmacological effects of the three drugs in combination and the pharmacological effects of the three drugs alone are calculated using the following formula: Q = E A+B+C / (E A+E B +E C -E A *E B -E A *E C -E B *E C -E A *E B *E C ).

[0035] Example 1: Protective effect of the composition of the present invention on rats with carbon tetrachloride-induced liver damage

[0036] 1. Methods

[0037] Experimental Animals and Grouping: Sprague-Dawley rats, half male and half female, weighing 200 ± 20 g, were purchased from the Guangdong Medical Laboratory Animal Center. They were housed at a room temperature of 18–28°C and a relative humidity of 40%–70% with free access to water. Following a standard diet for 1 week, they were randomly assigned to the groups shown in Table 2 below, with 10 rats per group.

[0038] Model Construction and Dosing: Each group received daily oral administration at the dosages listed in Table 2 below. The blank control group and the model control group received an equal volume of normal saline once daily for 30 consecutive days. On the 30th day, the rats were fasted overnight for 16 hours. All groups except the blank control group were given a single oral administration of 2% carbon tetrachloride (diluted in peanut oil) at a volume of 5 mL / kg body weight to establish an acute chemical liver injury model. The blank control group received a single dose of peanut oil. Five hours later, each group underwent a final oral administration. Twenty-four hours later, blood was collected from the femoral artery, allowed to stand at room temperature for 2 hours, and then centrifuged at 3500 rpm for 10 minutes. The supernatant serum was obtained for later use.

[0039] Detection indicators: (1) Scoring of liver tissue lesion severity: Rats were quickly killed by cervical dislocation, and liver tissue of the same size and location in the left lateral lobe of the rat liver was obtained. The liver tissue was fixed with formaldehyde, embedded in paraffin, and stained with HE. The pathological damage of the liver was observed under a light microscope. During microscopic examination, the pathological changes of cells were recorded starting from one end of the liver field of view, and the entire tissue section was continuously observed with a 100x objective lens. The main types of lesions included ballooning of hepatocytes, fatty degeneration, cytoplasmic aggregation, hydropic degeneration, and cell necrosis. The area occupied by various lesions in each field of view was recorded separately, and the total score of the lesions in the observed field of view was accumulated. The scores of various pathological changes were added together, and the score of hepatocyte necrosis was twice included in the total score. The scoring indicators and methods are shown in Table 1. (2) Determination of malondialdehyde (MDA) and reduced glutathione (GSH) in liver tissue: A small piece of liver tissue was cut and homogenized in ice water to make a 10% homogenate. MDA was determined by TBA method, and GSH was determined by colorimetry. The specific steps were operated according to the instructions of the kit.

[0040] Table 1 Scoring method for liver tissue lesion degree

[0041]

[0042] 2. Results

[0043] As shown in Tables 2 and 3, the liver tissue lesion severity score of rats in the blank control group was 0, and the liver tissue cell structure was clear and intact, with no inflammatory cell infiltration. Compared with the blank control group, the liver MDA of rats in the model control group was significantly increased, and GSH was significantly decreased, indicating peroxidative liver damage. The liver tissue lesion severity score was significantly increased (P < 0.01), and inflammatory cell infiltration, cytoplasmic aggregation, and cell necrosis were observed in the hepatocytes, indicating that the chemical liver injury model was successfully established. Compared with the model control group, there were no significant changes in the folic acid group and the porcine spleen peptide group. On the basis of the phycocyanin group, the liver MDA of rats in the porcine spleen peptide + phycocyanin + folic acid group was further significantly decreased, GSH was further significantly increased, and the liver tissue lesion severity score was significantly reduced. Microscopic examination showed that obvious inflammatory cell infiltration, partial cell cytoplasm aggregation, and cell necrosis were still observed in the phycocyanin group; while in the porcine spleen peptide + phycocyanin + folic acid group, most of the liver cells were intact and arranged neatly, and the hydropic degeneration, ballooning degeneration and inflammatory cell infiltration of the liver cells were significantly reduced, indicating that the above-mentioned combination has a significant effect on reducing the content of the oxidative product MDA, protecting liver cells, and reducing liver pathological damage in rats.

[0044] At the same time, the Q values ​​of the three indicators of liver MDA, liver tissue lesion score and GSH content in the porcine spleen peptide + phycocyanin + folic acid group and the corresponding porcine spleen peptide group, phycocyanin group and folic acid group were 1.53, 1.45 and 1.36 respectively (Q values ​​were all >1.15), indicating that the triple combination group had an unexpected synergistic effect compared with the three single-drug groups in inhibiting lipid peroxidation in the liver, preventing oxidative damage to liver cells, and reducing pathological inflammation and necrosis of liver tissue, so it can be used to prevent or treat chemical liver damage.

[0045] Table 2 Effect score of the composition of the present invention on rat liver tissue lesions ( n=9~10)

[0046]

[0047] Note: Compared with the blank control group, a P<0.05, aa P<0.01; compared with the model control group, b P<0.05, bb P<0.01.

[0048] Table 3 Effects of the composition of the present invention on MDA and GSH in rat liver ( n=9~10)

[0049]

[0050]

[0051] Note: Compared with the blank control group, a P<0.05, aa P<0.01; compared with the model control group, b P<0.05, bb P<0.01.

[0052] Example 2: Study on the improvement effect and immunomodulatory effect of the composition of the present invention on rats with alcoholic liver damage

[0053] 1. Methods

[0054] Experimental Animals and Grouping: SPF Wistar rats, half male and half female, weighing 200 ± 20 g, were purchased from the Guangdong Medical Laboratory Animal Center. They were housed at a room temperature of 18–28°C and a relative humidity of 40%–70% with free access to water. They were fed a standard diet for 1 week.

[0055] Modeling and drug administration: The blank control group was gavaged with normal saline, and the model control group was gavaged with liquor (6 mL / (kg·d) in the first week, 8 mL / (kg·d) in the second week, 10 mL / (kg·d) in the third week, and 11 mL / (kg·d) in weeks 4 to 10). The other experimental groups were gavaged with the test substances at the doses listed in Table 3 below, and then gavaged with liquor 5 hours later. The alcohol dose was the same as that in the model control group. The experiment lasted for 10 weeks.

[0056] Index measurement: After the last oral gavage, the rats were fasted but not watered for 12 hours. The rats were anesthetized with 3% sodium pentobarbital. Blood was collected from the abdominal aorta. A portion of the whole blood was used for flow cytometry to measure the proportion of immune cells. The remaining blood was centrifuged to separate the serum for biochemical index detection. (1) Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) liver function index measurement: Serum ALT and AST were measured using an automatic biochemical analyzer. (2) T lymphocyte subsets and natural killer (NK) cell detection: Whole blood (anticoagulated) was collected from the abdominal aorta of each rat and divided into two test tubes. 100 μL of blood was collected from each test tube. 300 μL of red blood cell lysis buffer was added to each test tube. The tubes were vortexed and placed on ice for 15 minutes. The cells were centrifuged at 450×g for 10 minutes at 4°C to separate the leukocytes and the supernatant was discarded. 200 μL of red blood cell lysis buffer was added to the leukocyte pellet and the above steps were repeated once. Add 100 μL of pH 7.2 PBS to each test tube, and add two antibody combinations: anti-CD3-FITC 1 μL / anti-CD8a-APC 1 μL, anti-TCRαβ-FITC 2 μL / anti-CD161a-PE 3 μL (divide the blank control group into five single-staining controls, and add only one of the antibodies to each control at the same dosage as above); gently shake to mix, incubate at room temperature in the dark for 30 minutes, add 2 mL of PBS, centrifuge at 400 × g for 5 minutes, discard the supernatant, add 1 mL of PBS, and detect by flow cytometry.

[0057] 2. Results

[0058] There is immune dysfunction in the development and progression of alcoholic liver damage. Long-term drinking will inhibit the body's natural immune cells, reduce the number and activity of lymphocyte subsets and NK cells, impair the ability of NK cells to degranulate, significantly inhibit their immune function, cause direct damage to liver cells, and enhance the susceptibility of long-term alcoholics to develop hepatitis, cirrhosis or liver cancer.

[0059] As shown in Table 4 and Table 5, compared with the blank control group, the serum ALT and AST activities of the rats in the model control group were significantly increased, and the peripheral blood CD3 + CD8 + T lymphocyte ratio and TCRαβ + CD161a +The proportion of NK cells was significantly reduced (P < 0.01), indicating that the model was successfully constructed. Compared with the model control group, the serum ALT and AST activities of rats in the high-dose phycocyanin group, low-dose bovine spleen peptide + 5-MTHF + phycocyanin group, and high-dose bovine spleen peptide + 5-MTHF + phycocyanin group were significantly reduced, and the proportion of peripheral blood T lymphocyte subsets and NK cells was significantly increased (P < 0.05 or P < 0.01), while there was no significant change in the other groups. This shows that high-dose phycocyanin and different doses of the combination have the effect of alleviating alcohol-induced liver dysfunction and regulating CD8 + The role of the ratio of immune cells such as T lymphocytes and NK cells.

[0060] At the same time, the Q values ​​of the low-dose bovine spleen peptide + 5-MTHF + phycocyanin group in reducing the activity of rat serum ALT and AST and increasing the proportion of peripheral blood T lymphocyte subsets and NK cells were 1.29, 1.22, 1.26 and 1.41, respectively. The Q values ​​of the high-dose bovine spleen peptide + 5-MTHF + phycocyanin group were 1.19, 1.19, 1.23 and 1.38, respectively. The Q values ​​were all >1.15, indicating that the components of the combination have a synergistic effect in improving liver function and enhancing the body's immune regulation.

[0061] Table 4 Effects of the composition on the activity of ALT and AST in rat serum ( n=9~10)

[0062]

[0063]

[0064] Note: Compared with the blank control group, a P<0.05, aa P<0.01; compared with the model control group, b P<0.05, bb P<0.01.

[0065] Table 5 Effects of the composition on the ratio of T lymphocyte subsets and NK cells in rat peripheral blood ( n=9~10)

[0066]

[0067] Note: Compared with the blank control group, a P<0.05, aa P<0.01; compared with the model control group, b P<0.05, bb P<0.01.

Claims

1. A composition comprising: a) Animal spleen extract; b) phycocyanin; c) folic acid substances; d) an acceptable excipient or carrier or a mixture thereof.

2. The composition according to claim 1, characterized in that The animal spleen extract is spleen aminopeptide.

3. The composition according to claim 2, characterized in that The mass ratio of the spleen aminopeptide:phycocyanin:folic acid substances is 1-20:400-5000:0.2-5.

4. The composition according to claim 3, characterized in that The spleen aminopeptide is 1 to 20 parts, the phycocyanin is 400 to 5000 parts, and the folic acid substance is 0.2 to 5 parts.

5. The composition according to claim 4, characterized in that The spleen aminopeptide is 1 to 10 parts, the phycocyanin is 400 to 2000 parts, and the folic acid substance is 0.2 to 1.2 parts; as a preferred embodiment, the spleen aminopeptide is 2 to 10 parts, the phycocyanin is 1000 to 2000 parts, and the folic acid substance is 0.4 to 1.2 parts.

6. The composition according to claim 2, characterized in that The spleen aminopeptide is selected from one or both of porcine spleen peptide and bovine spleen peptide, and its main components are polypeptides and / or nucleotide substances extracted from the spleen of porcine or bovine.

7. The composition according to claim 1, characterized in that The folic acid substance is selected from one of 5-methyltetrahydrofolate, formyltetrahydrofolate, active metabolites of folic acid or folate, and substances that can release / generate folic acid in the body.

8. Use of the composition according to any one of claims 1 to 7 in the preparation of a product for preventing or assisting in the treatment of chemical liver damage.

9. The use according to claim 8, characterized in that The chemical liver injury includes alcoholic liver injury, drug-induced liver injury, and immune liver injury.

10. Use of the composition according to any one of claims 1 to 7 in the preparation of a product for enhancing immunity.

Images