Composition for protecting liver and regulating uric acid level of intestinal tract and application thereof

Abstract

The invention discloses a composition with effects of resisting carbonylation and promoting uric acid excretion of intestinal tracts and application of the composition. The composition comprises ampelopsis grossedentata leaf extract and vitamin C, wherein the weight percentage of the ampelopsis grossedentata leaf extract is 20%-83%, and the weight percentage of the vitamin C is 17%-80%. Compared with a single material, the ampelopsis grossedentata leaf VC composition disclosed by the invention has a more remarkable effect on resisting carbonylation.

Description

Technical Field

[0001] The present invention belongs to the field of functional compositions, and specifically relates to a composition for protecting the liver and regulating intestinal uric acid levels and its application. Background Art

[0002] Hyperuricemia (HUA) is a metabolic disease caused by increased uric acid production and / or decreased uric acid excretion due to purine metabolism disorders in the body. The diagnosis of hyperuricemia is defined internationally as: under a normal purine diet, fasting blood uric acid levels on two different days are >420μmol / L for men and >360μmol / L for women. The latest version of the "Guidelines for the Diagnosis and Treatment of Hyperuricemia and Gout in China (2019)" defines: Regardless of whether it is a man or a woman, if the blood uric acid level exceeds 420μmol / L twice on different days, it is called hyperuricemia.

[0003] When the human blood is oversaturated with uric acid, uric acid anions combine with sodium ions to form monosodium urate (MSU) crystals, which are deposited in the joints and the surrounding joint capsules, bursae, and cartilage, causing blockage and erosion of the joints, and then causing joint edema and inflammation, which is gout. In addition to inducing gout, hyperuricemia is also related to the occurrence and development of many diseases. Such as acute kidney disease, chronic interstitial nephritis, kidney stones (severe cases induce uremia); cause vascular endothelial lesions, leading to atherosclerosis, hypertension and other cardiovascular diseases; cause lipid metabolism disorders, diabetes and obesity, etc.

[0004] The liver is the most important organ for drug metabolism in the human body and an important detoxification organ. Uric acid is mainly produced by the liver metabolism of purine compounds from dietary intake and decomposition in the body, so the liver also plays an important role in the normal physiological metabolism of the human body. There are three main drugs for the clinical treatment of hyperuricemia and gout: allopurinol, febuxostat and benzbromarone. Allopurinol and febuxostat reduce the production of uric acid by inhibiting the activity of xanthine oxidase (XOD), and are uric acid inhibitors; benzbromarone inhibits the reabsorption of uric acid by inhibiting various anion transporters in the kidneys, and is a uricosuric drug. Although these drugs have obvious efficacy, the hypersensitivity syndrome of allopurinol in Asian populations, the cardiovascular risk of febuxostat, and the hepato-renal toxicity of benzbromarone, these limitations limit the scope of application of drug treatment.

[0005] Therefore, it is of great significance to develop a composition that can effectively improve, conveniently and safely reduce uric acid, and at the same time has a protective effect on organs and cells, and apply it to food and health food. Ampelopsis grossedentata leaves are the tender stems and leaves of Ampelopsis grossedentata (Hand-Mazz) WT Wang, which was first recorded in "The Classic of Tea" and is a perennial woody vine of the genus Ampelopsis in the family Vitaceae. Ampelopsis grossedentata leaves can be used as medicine or eaten. According to the "Compilation of Chinese Herbal Medicines" and other documents, Ampelopsis grossedentata leaves can be used as medicine to clear away heat and detoxify, and can treat colds, wind-heat, sore throats, etc. Modern research shows that Ampelopsis grossedentata leaf extract is rich in flavonoid compounds, especially dihydromyricetin, which has the highest content, and has multiple pharmacological activities such as anti-inflammatory, antibacterial, antioxidant, hypoglycemic, hypolipidemic, and antihypertensive.

[0006] Vitamin C is a water-soluble vitamin with the chemical name L-(+)-threose 2,3,4,5,6-pentahydroxy-2-hexenoic acid-4-lactone, also known as L-ascorbic acid. Its molecular formula is C6H8O6 and its molecular weight is 176.12.

[0007] At present, there have been some reports on the study of the uric acid-lowering effect of Agave sphenanthera leaves. For example, the patent disclosures such as CN109275767A, CN109400750B, CN111973704A, and CN112933162A involve the uric acid-lowering effect of Agave sphenanthera leaves, but do not involve the synergistic effect of Agave sphenanthera leaves and their extracts, and vitamin C-related components. No research has been conducted on the uric acid-lowering mechanism of the relevant combination, and the protective effect of Agave sphenanthera leaves and vitamins on organs and cells has not been explored. Summary of the invention

[0008] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0009] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0010] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a composition that can protect the liver and regulate the uric acid level in the intestine.

[0011] In order to solve the above technical problems, the present invention provides the following technical solution: a composition with anti-carbonylation and intestinal excretion promoting uric acid excretion effects, comprising an extract of the leaves of Agave sutchuenensis and a vitamin C-related component; wherein the mass ratio of the extract of the leaves of Agave sutchuenensis to the vitamin C-related component is 1-8:0.5-4.

[0012] As a preferred embodiment of the composition of the present invention, the mass ratio of the Ampelopsis grossedentata leaf extract to the vitamin C-related component is 2-4:0.5-4.

[0013] As a preferred embodiment of the composition of the present invention, the vitamin C-related components are selected from one or more of vitamin C, vitamin C derivatives, and substances rich in vitamin C.

[0014] As a preferred embodiment of the composition of the present invention, the substance rich in vitamin C is selected from roxburghii, acerola, sour cherry and cherry.

[0015] As a preferred embodiment of the composition of the present invention, the content of dihydromyricetin in the Ampelopsis grossedentata leaf extract is greater than or equal to 10%.

[0016] Another object of the present invention is to overcome the deficiencies in the prior art and provide the use of the above composition in the preparation of a product having the ability to reduce carbonylation of liver cell proteins.

[0017] Another object of the present invention is to overcome the deficiencies in the prior art and provide the use of the above composition in preparing a product having the ability to inhibit the secretion of URATI protein.

[0018] Another object of the present invention is to overcome the deficiencies in the prior art and provide the use of the above composition in preparing a product having the function of inhibiting the secretion of GLUT9 protein.

[0019] Another object of the present invention is to overcome the deficiencies in the prior art and provide the use of the above composition in preparing a product having the function of promoting the secretion of ABCG2 protein.

[0020] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of the above composition in the preparation of food / health food.

[0021] Another object of the present invention is to overcome the deficiencies in the prior art and provide the use of the above composition in the preparation of medicines.

[0022] Beneficial effects of the present invention:

[0023] (1) All ingredients in the Agave sphenanthera leaf extract and vitamin C-related components of the present invention are health food / ordinary food raw materials permitted by laws and regulations, have a relatively long history of consumption or are recorded in herbal medicine, and are safer and more secure than existing drugs such as allopurinol, febuxostat and benzbromarone.

[0024] (2) The combination of the extract of Ampelopsis grossedentata leaves and vitamin C-related components in the present invention has a more significant effect on anti-carbonylation than a single material. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0026] Figure 1 This is a graph showing the experimental results of the anti-carbonylation effect of the composition of the Ampelopsis grossedentata leaf extract and vitamin C in the examples of the present invention.

[0027] Figure 2 The figure is a graph showing the effects of different doses of the composition in the examples of the present invention on uric acid in hyperuricemia model mice, wherein ### compared with the control group, P < 0.001, *** compared with the model group, P < 0.001, ** compared with the model group, P < 0.01, * compared with the model group, P < 0.05.

[0028] Figure 3 This is a graph showing the expression level of URAT1 protein in the intestine of hyperuric acid model mice after administration of the drug in an example of the present invention.

[0029] Figure 4 This is a graph showing the expression level of GLUT9 protein in the intestine of hyperuric acid model mice after administration of the drug in an embodiment of the present invention.

[0030] Figure 5 This is a graph showing the intestinal ABCG2 protein expression level in the hyperuric acid model mice after administration in the embodiment of the present invention; wherein, ### compared with the control group, P < 0.001; *** compared with the model group, P < 0.001; ** compared with the model group, P < 0.01; * compared with the model group, P < 0.05. DETAILED DESCRIPTION

[0031] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0032] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0033] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0034] The Ampelopsis grossedentata leaf extract (dihydromyricetin ≥ 10%) and vitamin C in the embodiments of the present invention are both common commercially available products.

[0035] Example 1

[0036] This embodiment provides a composition for protecting the liver and regulating intestinal uric acid levels, the main steps of which are:

[0037] 800 mg of Ampelopsis grossedentata leaf extract and 100 mg of vitamin C were weighed and mixed evenly to prepare an Ampelopsis grossedentata leaf VC composition.

[0038] Example 2

[0039] This embodiment provides a composition for protecting the liver and regulating intestinal uric acid levels, the main steps of which are:

[0040] Weigh 400 mg of Ampelopsis grossedentata leaf extract and 100 mg of vitamin C, mix them evenly, and prepare an Ampelopsis grossedentata leaf VC composition.

[0041] Example 3

[0042] This embodiment provides a composition for protecting the liver and regulating intestinal uric acid levels, the main steps of which are:

[0043] Weigh 200 mg of Ampelopsis grossedentata leaf extract and 100 mg of vitamin C, mix them evenly, and prepare an Ampelopsis grossedentata leaf VC composition.

[0044] Example 4

[0045] This embodiment provides a composition for protecting the liver and regulating intestinal uric acid levels, the main steps of which are:

[0046] Weigh 100 mg of Ampelopsis grossedentata leaf extract and 100 mg of vitamin C, mix them evenly, and prepare an Ampelopsis grossedentata leaf VC composition.

[0047] Example 5

[0048] This embodiment provides a composition for protecting the liver and regulating intestinal uric acid levels, the main steps of which are:

[0049] Weigh 200 mg of Ampelopsis grossedentata leaf extract and 400 mg of vitamin C, mix them evenly, and prepare an Ampelopsis grossedentata leaf VC composition.

[0050] Example 6

[0051] This embodiment provides a composition for protecting the liver and regulating intestinal uric acid levels, the main steps of which are:

[0052] Weigh 200 mg of Ampelopsis grossedentata leaf extract and 50 mg of vitamin C, mix them evenly, and prepare an Ampelopsis grossedentata leaf VC composition.

[0053] Comparative Example 1

[0054] 1600 mg of Ampelopsis grossedentata leaf extract and 100 mg of vitamin C were weighed and mixed evenly to prepare an Ampelopsis grossedentata leaf tea VC composition.

[0055] Comparative Example 2

[0056] Weigh 50 mg of Ampelopsis grossedentata leaf extract and 100 mg of vitamin C, mix them evenly, and prepare an Ampelopsis grossedentata leaf VC composition.

[0057] Comparative Example 3

[0058] Weigh 200 mg of Ampelopsis grossedentata leaf extract and 800 mg of vitamin C, mix them evenly, and prepare an Ampelopsis grossedentata leaf VC composition.

[0059] Comparative Example 4

[0060] Weigh 200 mg of Ampelopsis grossedentata leaf extract and 25 mg of vitamin C, mix them evenly, and prepare an Ampelopsis grossedentata leaf VC composition.

[0061] Set up each experimental sample group according to Table 1 below.

[0062] Table 1

[0063]

[0064]

[0065] Experiment 1: Anti-carbonylation experiment

[0066] (1) Instruments and consumables:

[0067] Incubator, microplate reader, 96-well plate, 12-well plate, different types of pipettes and tips;

[0068] (2) Reagents:

[0069] OxiSelect TM Protein Carbonyl ELISA Kit (cell biolabs) (or other carbonylation kits), BCA protein detection kit, MEM (containing NEAA) basal medium, MEM (containing NEAA) complete medium (MEM (containing NEAA) + 15% serum + 1% double antibody), CCK-8 reagent, ethanol (used with cell biolabs kit), and samples to be tested;

[0070] Source of HepG2 cells: Wuhan Pronocell Life Science Co., Ltd.

[0071] (3) Experimental steps:

[0072] 1) Culture of human hepatocellular carcinoma cells (HepG2)

[0073] Culture the cells in a humidified incubator at 37°C with 5% CO2 using complete culture medium; change the medium 2-3 times per week. When the cell density reaches 80%, subculture at a ratio of 1:2-1:3.

[0074] 2) CCK-8 assay to detect cytotoxicity

[0075] Cells in logarithmic growth phase were collected at 1.5×10 5 Each well was inoculated with 100 μL of the cells / mL into a 96-well plate. The cells were cultured in a 37°C, 5% CO2 incubator.

[0076] When the cell density reaches more than 60%, the cells are randomly divided into a blank group, a control group, and a group treated with experimental samples of different concentrations (the materials are prepared with basal / complete culture medium according to experimental requirements). The blank group is added with 100 μL basal culture medium, and the experimental group is incubated with sample solutions of different concentrations prepared with the culture medium for 24 hours.

[0077] After incubation, the cell viability of each group was detected by CCK-8 method;

[0078] The details are as follows: aspirate the culture medium in the well, add 100 μL of 10% CCK-8 solution, and incubate at 37°C for 1-4 h;

[0079] After incubation, the absorbance of each well was measured at 450 nm.

[0080] The relative cell viability of each group was calculated according to the following formula:

[0081] Relative cell viability % = (OD experimental group - OD blank group) / (OD control group - OD blank group)) × 100%.

[0082] CCK-8 is used to select the most suitable concentration of substances for cell survival, so 62.5 μg / ml was selected.

[0083] 3) Effect of samples on acetaminophen (APAP)-induced carbonylation

[0084] 5×10 cells in logarithmic growth phase were taken 4 / mL was evenly inoculated into a 12-well plate, 1mL per well, and cultured in a 37°C, 5% CO2 incubator for 24h;

[0085] The cells were randomly divided into a control group (only medium was added), a model group (10 mmol / L APAP was added, the modeling concentration (62.5 μg / mL) was obtained by CCK-8 experiment), and a sample group (10 mmol / L APAP and samples of appropriate concentration were added) and incubated for 24 h, where APAP and samples were prepared with medium;

[0086] The well plate was placed at -80°C, and the cells were lysed by freeze-thaw method (limited by the kit, if other Elisa kits are used, other methods can be used to collect proteins), and the solution was collected;

[0087] Centrifuge at 5000 rpm for 10 min at 4°C and collect the supernatant;

[0088] The protein concentration of the collected supernatant was detected using a BCA detection kit and adjusted to 10 μg / mL with PBS. The protein carbonylation level in the sample was detected using an Elisa detection kit (cell biolabs).

[0089] Define the evaluation criteria:

[0090] The higher the protein carbonylation content, the more serious the carbonylation is and the weaker the sample's ability to resist carbonylation is.

[0091] The results are shown in Table 2 and Figure 1 .

[0092] Table 2

[0093]

[0094]

[0095] It can be seen that:

[0096] Single-ingredient Agave sutchuenensis leaf extract and VC have been proven to reduce protein carbonylation levels at the HepG2 cell level (new model) and have anti-cellular carbonylation effects; the combination of Agave sutchuenensis leaf extract and VC at a certain ratio has a significant synergistic effect in reducing protein carbonylation levels.

[0097] Example 7

[0098] Experiment 2: Uric acid lowering experiment

[0099] (1) Reagents

[0100] Acid determination kit (uricase method); physiological saline (sodium chloride injection); yeast powder; oxonicacid potassium salt; RIPA lysis buffer; 20X TBST buffer; xanthine oxidase (XOD) colorimetric test kit;

[0101] Uric acid determination kit (uricase method); One-Step Page Gel Fast Preparation Kit (8%), Vazyme, Lot: 7E751E3; 180kDa Plus Prestained Protein Marker, Vazyme, MP201; 5x loading buffer.

[0102] (2) Specific experimental methods

[0103] Male mice weighing 18-22 g were selected. Before modeling, the mice were randomly divided into groups according to their weight. Except for 13 mice in the blank group who were given normal saline, the other mice were given potassium oxonate combined with yeast for modeling.

[0104] Blood was collected 7 days after modeling, and the subjects were divided into control group, model group, low-dose group, medium-dose group, and high-dose group according to blood uric acid.

[0105] After the adaptive feeding was completed, the experiment began, with modeling from the first to the seventh day of the experiment, and continuous intragastric administration starting from the eighth day and ending after 9 days;

[0106] The blank group was gavaged and intraperitoneally injected with normal saline, and the other experimental animals were modeled using yeast combined with potassium oxonate. After oral administration of yeast, potassium oxonate was immediately injected intraperitoneally. During the medication period, different drugs were given 1 hour after modeling every day to evaluate whether they had the effect of lowering blood uric acid.

[0107] On the 16th day of the experiment, 1 h after administration, the mice were weighed and killed;

[0108] Blood samples were collected and placed in a 4°C refrigerator overnight, then centrifuged at 3500 r / min for 10 min, and the upper serum was collected for the detection of serum uric acid;

[0109] Part of the intestinal tissue of the mice was taken to determine the expression levels of proteins URAT1, GLUT9, and ABCG2.

[0110] In this experiment, yeast combined with potassium oxonate method was used to prepare a mouse hyperuricemia model, and the mice were randomly divided into a blank group, a model group, and an experimental sample treatment group;

[0111] The experimental sample adopts the composition of Example 3, and three dosage concentrations are set, namely, a high-dose group (0.36 g / kg), a medium-dose group (0.18 g / kg), and a low-dose group (0.09 g / kg). The combination of Example 3 is selected to conduct experiments with different concentration settings, see Table 3.

[0112] Table 3

[0113] Group Experimental concentration Blank Group / Model Group / Low dose group 0.09g / kg Medium dose group 0.18g / kg High dose group 0.36g / kg

[0114] Results Figure 2 to Figure 5 , the blood uric acid of mice was significantly lower than that of the model group, indicating that the composition has the effect of lowering uric acid. The levels of URAT1 protein and GLUT9 reabsorption protein in the mouse intestine were significantly lower than those of the model group, and the level of secretory protein ABCG2 was significantly higher than that of the model group, and it was statistically significant, indicating that it has a significant effect of lowering uric acid in the mouse hyperuricemia model replicated by yeast combined with potassium oxonate.

[0115] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A composition for protecting the liver and regulating intestinal uric acid levels, characterized in that: include, Extract of Agave sphenanthera leaf, vitamin C-related components; The mass ratio of the Ampelopsis grossedentata leaf extract to the vitamin C-related component is 1-8:0.5-4.

2. The composition according to claim 1, characterized in that: The vitamin C-related components are selected from one or more of vitamin C, vitamin C derivatives, and substances rich in vitamin C; wherein the substances rich in vitamin C are selected from roxburghii, acerola, sour cherry and cherry.

3. The composition according to claim 1 or 2, characterized in that: The mass ratio of the Ampelopsis grossedentata leaf extract to the vitamin C-related component is 2-4:0.5-4.

4. The composition according to claim 3, characterized in that: The content of dihydromyricetin in the Ampelopsis grossedentata leaf extract is ≥10%.

5. Use of the composition according to any one of claims 1 to 4 in the preparation of a product having the effect of reducing protein carbonylation in liver cells.

6. Use of the composition according to any one of claims 1 to 4 in the preparation of a product capable of inhibiting the secretion of URATI protein.

7. Use of the composition according to any one of claims 1 to 4 in the preparation of a product capable of inhibiting GLUT9 protein secretion.

8. Use of the composition according to any one of claims 1 to 4 in the preparation of a product having the function of promoting the secretion of ABCG2 protein.

9. Use of the composition according to any one of claims 1 to 4 in the preparation of health food, food, and special food.

10. Use of the composition according to any one of claims 1 to 4 in the preparation of medicines.

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