Application of dihydromyricetin as regulator for reducing content of vitamin K
By regulating the expression of the NQO1 gene and protein through dihydromyricetin, vitamin K is converted into a precursor, solving the problem that existing technologies cannot reduce vitamin K in the blood and achieving safe and effective regulation of vitamin K in the blood.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-03
- Publication Date
- 2026-03-10
AI Technical Summary
Current technology lacks drugs that directly target vitamin K, making it impossible to effectively reduce the level of vitamin K in the blood, leading to adverse reactions such as thrombosis and allergic dermatitis.
Dihydromyricetin was used to regulate the expression of the NQO1 gene and protein, converting vitamin K into a vitamin K precursor, and then the vitamin K content in the blood was reduced by oral administration.
It effectively reduces the level of vitamin K in the blood, avoids adverse reactions such as thrombosis and allergic dermatitis, and dihydromyricetin is well absorbed in the intestine, with significant effects when administered orally.
Smart Images
Figure CN121622647A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedicine, and in particular to the application of dihydromyricetin as a regulator for reducing vitamin K levels. Background Technology
[0002] Vitamin K, a fat-soluble vitamin, is essential for maintaining bone health, regulating vascular calcification, and blood clotting. It plays a vital role in preventing neonatal hemorrhage, internal bleeding and hemorrhoids, reducing heavy menstrual bleeding, and promoting normal blood clotting. However, excessively high blood levels can accelerate blood clotting, promote thrombus formation, potentially leading to cerebral thrombosis. Furthermore, it can induce heart disease and cause adverse reactions such as allergic dermatitis. Currently, vitamin K regulators include warfarin, acetonitrile coumarin, dicumarin, benzylacetone coumarin, and phenylindanedione. These drugs inhibit vitamin K synthesis by interfering with the synthesis of vitamin K-dependent factors, thereby suppressing the clotting process. However, there are currently no drugs that directly target vitamin K, breaking it down into vitamin K precursor compounds to lower blood vitamin K levels.
[0003] Dihydromyricetin (DMY) is a natural product extracted from vine tea. It belongs to the flavonoid family and possesses various protective effects, including anti-inflammatory, antioxidant, antitumor, antimicrobial, blood glucose and lipid regulation, and alcohol detoxification and liver protection. Current research indicates that DMY can treat hyperuricemia, inhibit hepatocyte aging and promote liver regeneration, and lower fasting and postprandial blood glucose levels in hyperglycemic and diabetic patients. It can also be used as a plasma-type kallikrein inhibitor and in the preparation of coagulation factor X (FXa) inhibitors. While numerous studies suggest that DMY can treat circulatory-related diseases, its effect on blood vitamin K levels and its mechanism of action remain unreported. Summary of the Invention
[0004] This invention provides an application of dihydromyricetin as a regulator for reducing vitamin K levels. The invention's research found that dihydromyricetin can downregulate the expression of the NQO1 gene and protein, converting vitamin K into a vitamin K precursor, thereby reducing its level in the blood. This is specifically achieved through the following techniques.
[0005] The first aspect of this invention provides the use of dihydromyricetin in the preparation of products that reduce the vitamin K content in the blood.
[0006] Furthermore, the product is a health food, a pharmaceutical, or a laboratory preparation.
[0007] A second aspect of the present invention provides: a product for reducing the vitamin K content in the blood, wherein the raw material of the product includes dihydromyricetin.
[0008] Furthermore, the dosage form of the product includes any one of tablets, capsules, intravenous injections, intraperitoneal injections, inhalers, nebulizers, lyophilized agents, patches, gels, sprays, or suppositories.
[0009] A third aspect of the present invention provides: a method for reducing the level of vitamin K in the blood without the purpose of disease diagnosis and treatment: administering dihydromyricetin, or the above-mentioned products including dihydromyricetin, to the subject.
[0010] Furthermore, the dosage of the dihydromyricetin was 0.1-10 mg / kg based on the subject's body weight.
[0011] Compared with the prior art, the advantages of the present invention are:
[0012] This invention demonstrates through cell and animal models that dihydromyricetin can upregulate the expression of the NQO1 gene and protein, convert vitamin K into vitamin K precursors, and reduce its content in the blood. Dihydromyricetin is well absorbed in the intestine and can be administered orally, producing its effects at relatively low doses. Attached Figure Description
[0013] Figure 1 A diagram showing the vitamin K cycle in the body;
[0014] Figure 2 This is a graph showing the effect of dihydromyricetin on reducing the blood vitamin K content after a high dose of vitamin K intake in Experiment Example 1.
[0015] Figure 3 This is a graph showing the effect of dihydromyricetin on reducing blood vitamin K levels after a long-term high-fat diet in Experiment Example 2.
[0016] Figure 4 (A) is a graph showing the changes in NQO1 gene expression after administration of different concentrations of dihydromyricetin; Figure 4 (B) is a graph showing the changes in NQO1 protein expression after administration of different concentrations of dihydromyricetin; Figure 4 (C) is a Western blot image of NQO1 protein after administration of different concentrations of dihydromyricetin;
[0017] Figure 5 The time-content curve of dihydromyricetin excreted in feces in Experiment Example 4. Detailed Implementation
[0018] The application of the dihydromyricetin of the present invention in reducing vitamin K in the blood will be clearly and completely described below through specific embodiments.
[0019] There are two main types of vitamin K in the body: vitamin K1 and vitamin K2. The human body has a vitamin K cycle, in which vitamin K relies on γ-carboxylase and cyclooxygenase to convert vitamin K precursors into vitamin K, while NADPH dehydrogenase (a quinone) can convert vitamin K into vitamin K precursors, thereby regulating the vitamin K content in the blood.
[0020] The circulation diagram of vitamin K in the body is as follows: Figure 1 As shown. Figure 1 In this formula, Menaquinone is vitamin K2, Phylloquinone is vitamin K1, and the enzyme in 1.6.5.2 is NADPH dehydrogenase (quinone), i.e., NQO1.
[0021] Experimental Example 1: Dihydromyricetin reduces blood vitamin K levels after high-dose vitamin K intake.
[0022] Six-week-old SD rats were randomly divided into cages of eight and housed in individually ventilated, isolated IVC cages. Throughout the modeling period, water and food were changed every two days; bedding was changed twice a week to ensure cage cleanliness. The indoor temperature was maintained at 20-25℃, and humidity at 50-60%. Experiments were conducted on rats after one week of acclimatization, when they reached approximately 200g in weight.
[0023] The groups are as follows:
[0024] Blank group: No processing is performed.
[0025] Model group: Rats were given vitamin K by gavage at a dose of 3.4 mg / kg.
[0026] Low-dose group: Rats were given dihydromyricetin by gavage 1 hour in advance at a dose of 0.63 mg / kg (equivalent to 0.1 mg / kg in humans), followed by vitamin K by gavage at a dose of 3.4 mg / kg.
[0027] High-dose group: Rats were given dihydromyricetin by gavage 1 hour in advance at a dose of 63 mg / kg (equivalent to 10 mg / kg in humans), and then vitamin K was given by gavage at a dose of 3.4 mg / kg.
[0028] Blood samples were collected 1 hour after oral administration of vitamin K in the model group, low-dose group, and high-dose group. Plasma samples were processed using anhydrous ethanol-ether (volume ratio 1:3) extraction. Blood concentrations of vitamin K were determined by reversed-phase high-performance liquid chromatography (RP-HPLC) according to the detection method for vitamin K in the 2020 edition of the Chinese Pharmacopoeia.
[0029] Experimental results are as follows Figure 2 As shown. By Figure 2It was found that after injecting a large dose of vitamin K into rats, the vitamin K content in the rat blood was about 0.8 mg / L. However, when dihydromyricetin was administered 1 hour in advance followed by a large dose of vitamin K, the vitamin K content in the rat blood was significantly lower than that in the model group, proving that dihydromyricetin has the effect of reducing the vitamin K content in the blood.
[0030] Experimental Example 2: Dihydromyricetin reduces blood vitamin K levels after a long-term high-fat diet.
[0031] Six-week-old SD rats were divided into cages of eight and housed in individually ventilated, isolated IVC cages. Throughout the modeling period, water and food were changed every two days; bedding was changed twice a week to ensure cage cleanliness. The indoor temperature was maintained at 20-25℃, and humidity at 50-60%. Modeling was initiated after one week of acclimatization feeding.
[0032] The groups are as follows:
[0033] Blank group: No processing is performed.
[0034] Model group: Rats were fed a high-fat diet (manufacturer: Jiangsu Xiehe Pharmaceutical Biotechnology Co., Ltd.) daily for 3 months, and then blood was collected to determine the vitamin K content in the blood by HPLC-MS / MS.
[0035] In the treatment group, rats were fed a high-fat diet daily and were also given dihydromyricetin by gavage at a dose of 63 mg / kg (equivalent to 10 mg / kg in humans) daily for 3 months. Blood samples were then collected and the vitamin K content in the blood was determined by HPLC-MS / MS.
[0036] Experimental results are as follows Figure 3 As shown, Figure 3 In the diagram, A represents the blank group, B represents the model group, and C represents the group treated with dihydromyricetin. Figure 3 It can be seen that after a long-term high-fat diet, rats will have a significant increase in blood vitamin K levels. However, when dihydromyricetin is administered by gavage while on a long-term high-fat diet, blood vitamin K levels will decrease significantly.
[0037] Experimental Example 3: Dihydromyricetin can upregulate the expression of NQO1 gene and protein.
[0038] RAW264.7 cells were incubated in a specialized culture medium containing 10% fetal bovine serum and 1% penicillin-drug antibodies. Cells were seeded into appropriate culture plates at a density of 5000 cells / well. After 24 hours of culture, drugs could be administered or models established. For model establishment, cells were treated with 50 μg / ml oxidized low-density lipoprotein (ox-LDL). Different concentrations of dihydromyricetin were added 2 hours before the addition of ox-LDL. After 24 hours, cells were collected, lysed with Trizol, and subjected to q-PCR. Cells were also lysed with RIPA and subjected to Western blotting to detect NQO1 gene expression and protein expression.
[0039] Experimental results are as follows Figure 4 As shown, Figure 4 (A) is a graph showing the changes in NQO1 gene expression after administration of different concentrations of dihydromyricetin; Figure 4 (B) is a graph showing the changes in NQO1 protein expression after administration of different concentrations of dihydromyricetin; Figure 4 (C) Western blot images of NQO1 protein after administration of different concentrations of dihydromyricetin. Figure 4 It was found that after treatment of cells with oxidized low-density lipoprotein, the NQO1 gene was downregulated and the protein level decreased. This result is consistent with that of Experiment 2, where a long-term high-fat diet suppressed the NQO1 gene, preventing vitamin K from being properly circulated and converted into its precursor, thus increasing blood vitamin K levels. Cellular experiments confirmed that administration of dihydromyricetin promoted NQO1 gene expression and upregulated NQO1 protein expression.
[0040] Experimental Example 4: Dihydromyricetin Absorption Experiment
[0041] 4.1 Fecal kinetics experiment of dihydromyricetin
[0042] Nine KM mice were randomly selected and divided into groups 1, 2, and 3. Each mouse was administered 0.4 mL of a 50 mg / mL dihydromyricetin (DMY) solution by gavage and placed in a metabolic cage. All fecal samples from each group were collected at this time and recorded as the 0-hour sample, labeled 0-X. Subsequently, fecal samples were collected from each group at 2, 4, 6, 8, 12, 16, 20, 24, 30, 36, 48, and 60 hours, labeled 1-12. After collection at the corresponding time points, the fecal samples were weighed and recorded. Methanol (5 times the weight of the fecal sample) was added to dissolve the fecal sample. The mixture was shaken well to ensure the methanol completely covered the fecal sample, and the sample was sonicated for 20 minutes. Then, 1 mL of the solution was added to a chromatographic sample vial, and the dihydromyricetin content was detected by HPLC. After all samples were collected, liquid chromatography experiments were performed, and the data were recorded, processed, and analyzed.
[0043] The experimental results show the trend of DMY content in feces collected from the three parallel experiments over time, as shown in the following graph. Figure 5 As shown. By Figure 5 It was found that the peak excretion of unabsorbed dihydromyricetin occurred around 6 hours. Calculations based on the total amount of unabsorbed DMY revealed that mice have a strong ability to absorb DMY, with approximately 99.3% of the DMY being absorbed and converted.
[0044] 4.2 Study on the absorption of dihydromyricetin by in vivo intestinal perfusion experiment.
[0045] This experiment employed the Doluisio closed-loop intestinal perfusion model. SD rats were anesthetized, and a cyclic intestinal perfusion experiment was conducted using dihydromyricetin saline solution as the perfusion fluid. Samples were taken from the perfusion fluid vial every 30 minutes to measure drug concentration. Sample analysis was used to assess intestinal absorption in the rats.
[0046] Accurately weigh 80 mg of dihydromyricetin salt and add 8 mL of RO water to prepare a 10 mg / mL dihydromyricetin salt solution. After anesthetizing rats with sodium pentobarbital via intraperitoneal injection, establish a circulatory pathway. After 3 minutes of connection, use a pipette to take 1 mL of liquid from the solution bottle as initial sample 0. Then, time the sampling for 90 minutes, taking samples every 30 minutes to complete the sampling of samples 1-3. Note that the solution should be gently shaken before each sampling. A total of 4 samples should be taken from each rat. A total of 6 rats were used. The retention time, peak area, and other data of dihydromyricetin in the samples were detected by liquid chromatography, recorded, and analyzed. The intestinal permeation rate of dihydromyricetin was calculated using the following formula:
[0047]
[0048] The results are shown in Table 1 below.
[0049] Table 1. Intestinal absorption rate and apparent absorption coefficient in rats
[0050]
[0051] As shown in Table 1, the Ka of dihydromyricetin across the intestine is 1.16 × 10⁻⁶. -2 min -1 The Kapp value is 1.70 × 10⁻⁶. -3 cm -1 ·min -1 Dihydromyricetin has a good rate of intestinal permeation and absorption, and can be administered orally to reduce the level of vitamin K in the blood.
[0052] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.
Claims
1. Use of dihydromyricetin in the preparation of a product for reducing the vitamin K content in blood.
2. Use of dihydromyricetin according to claim 1 for the preparation of a product for reducing the vitamin K content in blood, characterized in that, The product is a health food, a medicine or a laboratory preparation.
3. A product for reducing the vitamin K content of blood, characterized in that, The raw material of the product comprises dihydromyricetin.
4. The product for reducing the content of vitamin K in blood according to claim 3, characterized in that, The dosage form of the product comprises any one of tablets, capsules, intravenous injections, intraperitoneal injections, inhalants, atomizers, lyophilized preparations, patches, gels, sprays or suppositories.
5. A method of reducing the vitamin K content in blood not for the purpose of disease diagnosis and treatment, characterized by, The subject is administered with dihydromyricetin or the product of claim 3.
6. The method of reducing the amount of vitamin K in the blood according to claim 5, wherein The amount of dihydromyricetin is 0.1-10 mg / kg.