Cell membrane chromatography screening method of medicine for treating hyperlipidemia

Abstract

The invention discloses a cell membrane chromatography screening method of a medicine for treating hyperlipidemia, and belongs to the technical field of biological medicines. Comprising the following steps: (1) fixing a hyperlipidemia liver cell membrane on the surface of silica gel to prepare a cell membrane stationary phase, and filling a chromatographic column with the prepared stationary phase; and (2) carrying out on-line active component screening from the medicinal and edible material extracting solution to screen out components with blood fat reducing activity. Animal level research shows that the prepared granules can remarkably reduce blood fat and are related to regulation and control of protein expression of AMPK and SREBP-1c, and safety investigation finds that the granules have no obvious damage to livers of mice. The technology can be widely applied to medicine research and development and functional food and natural product development, and an efficient and low-cost active ingredient screening solution is provided for blood fat reducing products.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a cell membrane chromatography screening method for a drug for treating hyperlipidemia. Background Art

[0002] As a core pathological factor of global metabolic diseases, hyperlipidemia has caused a burden on more than 1.87 billion patients (WHO report in 2025), and more than 60% of the patients are accompanied by disorders of liver lipid metabolism and the risk of liver injury. Although current mainstream drugs such as statins and PCSK9 inhibitors can control blood lipid levels in the short term, long-term use is likely to cause abnormal elevation of liver enzymes (incidence rate > 15%), muscle toxicity (such as rhabdomyolysis), and dose-dependent drug resistance (the failure rate of 5-year treatment reaches 30%). At the same time, ingredients homologous to drugs and foods have become a hot direction for the development of lipid-lowering products due to their natural sources and safety advantages. However, the active ingredients in materials homologous to drugs and foods are diverse, and it is difficult for traditional separation and purification technologies to quickly lock in the effective ingredients. Traditional screening methods rely heavily on molecular docking technologies (only targeting single proteins such as HMG-CoA reductase) and animal model verification (the cycle is more than 6 months, and the development cost of a single ingredient exceeds $500,000), resulting in a very low correlation between the in vitro screening results of active ingredients and their true efficacy in vivo (false negative rate > 45%), which severely restricts the industrial application of resources homologous to drugs and foods.

[0003] Conventional cell membrane chromatography (CMC) improves screening accuracy by simulating the interaction between drugs and cell membranes. However, its stationary phase mostly uses healthy liver cell membranes or cell membranes cultured conventionally, and the cholesterol content is only 32 ± 2 μg / mg, which cannot simulate the target microenvironment characteristics (such as changes in receptor expression levels and abnormal lipid raft structures) under pathological conditions such as hyperlipidemia and inflammation, resulting in insufficient activity of the screened components in real disease models. Research shows that due to cholesterol deposition in hyperlipidemic liver cell membranes, the lipid raft structure is reconstructed (the proportion of phosphatidylcholine decreases by 40%), and the membrane fluidity is significantly reduced (the fluorescence polarization value increases by 2.1 times). This pathological difference makes it difficult for the components screened by traditional CMC to penetrate the pathological membrane barrier or effectively bind to the target in vivo. In addition, the membrane protein activity retention rate of the conventional silica gel coupling process is less than 60%, and when combined with high-performance liquid chromatography (HPLC), the column pressure is unstable due to fluctuations in the ionic strength of the buffer solution (blockage rate > 20%), severely limiting the feasibility of high-throughput screening.

[0004] In recent years, materials with homology of medicine and food have become a hot spot for the development of anti - hyperlipidemic active ingredients due to their natural sources, high safety, and potential for multi - target regulation. However, traditional methods for screening active ingredients (such as in vitro enzyme inhibition experiments, etc.) have limitations such as low efficiency, high cost, and high false - positive rates, and it is difficult to dynamically reflect the true interaction between drugs and target cell membrane receptors or transporters. In addition, the components of materials with homology of medicine and food are complex, and conventional separation methods are difficult to quickly lock in the key components with biological activity, resulting in a long R & D cycle and serious waste of resources. Summary of the Invention

[0005] The object of the present invention is to provide a cell membrane chromatography screening method for drugs treating hyperlipidemia to solve the problems existing in the above - mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention, a cell membrane chromatography screening method for drugs treating hyperlipidemia, includes the following steps:

[0008] (1) Fix hyperlipidemic liver cell membranes on the surface of silica gel to prepare a cell membrane stationary phase, and fill the prepared stationary phase into a chromatographic column;

[0009] (2) Conduct on - line screening of active ingredients from the extract of materials with homology of medicine and food, and screen out the ingredients with anti - hyperlipidemic activity.

[0010] Another technical solution of the present invention, a medicine - food homologous composition for treating hyperlipidemia, includes the following raw materials in parts by weight: 10 - 15 parts of hawthorn, 10 - 15 parts of egg yolk lecithin, 10 - 15 parts of honeysuckle, 10 - 15 parts of grapefruit, 10 - 15 parts of cherry, 5 - 10 parts of dried tangerine peel, 5 - 10 parts of poria cocos, 5 - 10 parts of cassia seed, 5 - 10 parts of rattan tea, 5 - 10 parts of licorice, 2 - 5 parts of polygonatum sibiricum, 2 - 5 parts of blueberry, 2 - 5 parts of waxberry, 2 - 5 parts of tremella fuciformis, and 2 - 5 parts of lotus seed.

[0011] Another technical solution of the present invention, a preparation method of the medicine - food homologous composition, includes the following steps:

[0012] (1) Dry honeysuckle, egg yolk lecithin, dried tangerine peel, poria cocos, cassia seed, rattan tea, licorice, and polygonatum sibiricum in the shade or in the sun, and powder them to obtain powder; wash, peel, and pit hawthorn, grapefruit, cherry, blueberry, waxberry, tremella fuciformis, and lotus seed, and add water to make a thick fruit pulp;

[0013] (2) Conduct alcohol extraction on the powder and the thick fruit pulp, centrifuge to collect the supernatant, and rotary evaporate and spray - dry to obtain an extract powder;

[0014] (3) Add excipients for granulation to obtain granules.

[0015] Based on the above technical solutions, the present invention has the following technical effects:

[0016] The present invention for the first time constructs a pathological liver cell membrane using a high-fat diet-induced ApoE gene knockout mouse model, retains the abnormal expression characteristics of lipid metabolism-related targets (such as proteins in the AMPK / SREBP-1c pathway) under pathological conditions, and significantly improves the disease relevance of screening. High-purity membrane components (membrane protein purity > 95%) are extracted by sucrose density gradient centrifugation technology, and membrane proteins are covalently fixed to silica gel carriers using a silanization-directed coupling process. By optimizing the activation conditions of 3-aminopropyltriethoxysilane (APTES) and the ratio of bifunctional thiol crosslinkers, the membrane protein loading reaches 2.1 mg / g silica gel, and the retention rate of ATPase activity exceeds 90%. Verified by lipidomics, the cholesterol / phospholipid molar ratio in the stationary phase is stable at 0.85 ± 0.05, accurately simulating the pathological membrane characteristics of hyperlipidemic hepatocytes.

[0017] Regarding the problems of the stability and release efficiency of active ingredients, the present invention develops a maltodextrin-erythritol co-embedding system and achieves high bioavailability through wet granulation technology, filling the deficiency of traditional tablets or capsules in compliance.

[0018] The present invention has been successfully applied to the development of lipid-lowering granules. Its formula covers the ingredients newly added to the "List of Medicinal and Edible Homologous Substances 2025". Animal experiments show that the granules can significantly reduce the serum LDL-C level of model mice (the reduction rate reaches 54.3%), and there is no obvious damage in the histopathology of liver tissue. The 15 selected ingredients all belong to the list of medicinal and edible homologous substances issued by the National Health Commission. By promoting the analysis of the action mechanism of traditional Chinese medicine compounds through CMC technology, it helps the modernization of traditional Chinese medicine. Brief Description of the Drawings

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0020] Figure 1 It is a schematic diagram of the process for treating hyperlipidemic mice by cell membrane chromatography.

[0021] Figure 2 It is a scanning electron microscope (SEM) image of aminated silica gel and silica gel loaded with cell membranes.

[0022] Figure 3 It is a hyperlipidemic cell membrane chromatography column.

[0023] Figure 4For the detection of physicochemical indexes related to blood lipid. Among them, A is TC, B is TG, C is LDL, and D is HDL. **: Compared with the blank group, p < 0.01; ##: Compared with the high-fat model group, p < 0.01.

[0024] Figure 5 For the results of HE staining of the liver.

[0025] Figure 6 For the results of the detection of protein expression. Among them, A is the result diagram of the WB experiment, B is the expression of p-AMPK protein, and C is the expression of SREBP-1c protein. **: Compared with the blank group, p < 0.01; ##: Compared with the high-fat model group, p < 0.01. Detailed implementation manners

[0026] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing particular implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0029] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of this application are only exemplary.

[0030] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0031] The technical solutions described in the present invention are conventional solutions in the art unless otherwise specified. The reagents or raw materials used are purchased from commercial channels or are publicly available unless otherwise specified.

[0032] The embodiments of the present invention provide a method for screening drugs for treating hyperlipidemia by cell membrane chromatography, comprising the following steps:

[0033] (1) Fix the hyperlipidemic liver cell membrane on the surface of silica gel to prepare a cell membrane stationary phase, and fill the prepared stationary phase into a chromatographic column;

[0034] (2) Perform on-line screening of active ingredients from the extract of medicated and edible homologous materials to screen out the ingredients with lipid-lowering activity.

[0035] In some specific embodiments, the method for preparing the hyperlipidemic liver cell membrane is: extracting liver cells from the liver tissue of a hyperlipidemic mouse model, and then extracting the hyperlipidemic liver cell membrane after ultrasonic disruption.

[0036] In some specific embodiments, the silica gel is amino-modified SiO2;

[0037] The method for preparing the amino-modified SiO2 is: performing reflux acidification treatment on the silica gel, and then placing the silica gel in an ethanol solution of (4-aminobutyl)triethoxysilane for shaking reaction and curing treatment to obtain the amino-modified SiO2.

[0038] In some specific embodiments, the chromatographic conditions for screening are: column temperature 30°C, flow rate 0.2 mL / min, mobile phase is 10 mM ammonium acetate (pH 7.2 - 7.4), injection volume 20 μL, using an ELSD1260 detector: evaporation temperature 60°C, drift tube temperature 60°C, high-purity nitrogen flow rate 1.6 L / min.

[0039] The embodiments of the present invention also provide a medicated and edible homologous composition for treating hyperlipidemia, comprising the following raw materials in parts by weight: 10 - 15 parts of hawthorn, 10 - 15 parts of egg yolk lecithin, 10 - 15 parts of honeysuckle, 10 - 15 parts of grapefruit, 10 - 15 parts of cherry, 5 - 10 parts of dried tangerine peel, 5 - 10 parts of poria cocos, 5 - 10 parts of cassia seed, 5 - 10 parts of rattan tea, 5 - 10 parts of licorice, 2 - 5 parts of polygonatum sibiricum, 2 - 5 parts of blueberry, 2 - 5 parts of waxberry, 2 - 5 parts of tremella fuciformis, and 2 - 5 parts of lotus seed.

[0040] The embodiments of the present invention also provide a method for preparing the medicated and edible homologous composition, comprising the following steps:

[0041] (1) Dry or sun-dry honeysuckle, egg yolk lecithin, tangerine peel, poria cocos, cassia seed, rattan tea, liquorice and polygonatum sibiricum, and powder them to obtain a powder; wash, peel and pit hawthorn, grapefruit, cherry, blueberry, waxberry, tremella and lotus seeds, and add water to make a thick fruit pulp by pulping;

[0042] (2) Subject the powder and the thick fruit pulp to alcohol extraction, centrifuge to collect the supernatant, and perform rotary evaporation and spray drying to obtain an extract powder;

[0043] (3) Add excipients for granulation to obtain a granule.

[0044] In some specific embodiments, the conditions for the alcohol extraction are as follows: mix the powder and the thick fruit pulp with an ethanol solution at a mass ratio of 1:20, extract in a water bath with ultrasonic waves at 60°C - 90°C for 1 h, and repeat 3 - 4 times;

[0045] The volume fraction of the ethanol solution is 30% - 80%.

[0046] In some specific embodiments, the excipients include erythritol and maltodextrin; the weight fraction of erythritol is 45% - 55%; the weight fraction of maltodextrin is 5% - 10%.

[0047] The cell membrane chromatography stationary phase provided by the present invention is a high-fat liver cell membrane silica gel, in which the silica gel is aminated with (4-aminobutyl)triethoxysilane; connect this chromatographic column to HPLC, and online screen 15 components with lipid-lowering activity from 50 medicine and food homologous materials, namely hawthorn, honeysuckle, grapefruit, cherry, egg yolk lecithin, tangerine peel, poria cocos, cassia seed, rattan tea, liquorice, polygonatum sibiricum, blueberry, waxberry, tremella and lotus seeds; after the above components are extracted and concentrated, granule is prepared with erythritol and maltodextrin as adjuvants. Studies at the animal level show that the granule can significantly reduce blood lipid, and is related to the regulation of the protein expression of AMPK and SREBP-1c. Safety investigation finds that the granule has no obvious damage to the liver of mice. This technology can be widely applied to pharmaceutical R & D, functional food and natural product development, and provides an efficient and low-cost solution for screening active ingredients of lipid-lowering products.

[0048] The granule prepared by the present invention can significantly reduce the contents of triglyceride (TG), total cholesterol (TC) and low-density lipoprotein (LDL-C) in hyperlipidemic mice, and increase the content of high-density lipoprotein (HDL-C) in hyperlipidemic mice. The mechanism of reducing hyperlipidemia is related to the regulation of the protein expression of signal pathway-related proteins of AMPK and SREBP-1c.

[0049] Amino-functionalized silica gel was prepared by using (4-aminobutyl)triethoxysilane to modify silica gel, forming amino-surface-modified silica gel, which enhanced the covalent binding force between cell membranes and carriers, solved the problems of easy shedding and loss of activity of the stationary phase in traditional cell membrane chromatography (CMC), and improved the stability and service life of chromatographic columns. Subsequently, the liver cell membranes of hyperlipidemic mice were immobilized on the surface of silica gel through electrostatic adsorption and covalent cross-linking to construct a bioactive cell membrane stationary phase.

[0050] Source of cell membranes: Primary hepatocytes were extracted from the liver tissues of hyperlipidemic model mice, and high-fat cell membrane fragments were obtained through ultrasonic disruption, homogenization, and purification.

[0051] The cell membrane stationary phase chromatographic column was coupled with a high-performance liquid chromatography (HPLC) system to achieve online screening of active ingredients in extracts of medicated and edible homologous materials. The extracts of natural products could be continuously screened online, which was suitable for high-throughput analysis of complex natural components. The following parameters were used to accurately screen active ingredients:

[0052] Chromatographic conditions were as follows: column temperature 30°C, flow rate 0.2 mL / min, mobile phase 10 mM ammonium acetate (pH 7.2 - 7.4), injection volume 20 μL, and ELSD1260 detector was used: evaporation temperature 60°C, drift tube temperature 60°C, and high-purity nitrogen flow rate 1.6 L / min.

[0053] Activity determination criteria: Components showing characteristic chromatographic peaks with a signal-to-noise ratio of the chromatographic peak > 15 were regarded as potential active materials.

[0054] Fifteen components with synergistic lipid-lowering effects were screened out from 50 medicated and edible homologous materials.

[0055] The extracts of the 15 active ingredients accounted for 40% - 50% of the total mass, among which egg yolk lecithin, as a fat-soluble carrier, accounted for 5% - 10%, supplemented with erythritol (45% - 55%) and maltodextrin (5% - 10%).

[0056] Animal experiment data: After continuous administration to hyperlipidemic model mice for 28 days, the changes in serum indicators of the granule treatment group compared with the model group were as follows:

[0057] Triglyceride (TG) decreased by 27.7% - 55.0%, and total cholesterol (TC) decreased by 37.0% - 58.7%.

[0058] Low-density lipoprotein (LDL-C) decreased by 29.3% - 52.8%, and high-density lipoprotein (HDL-C) increased by 43.0% - 56.6%.

[0059] Molecular mechanism: Western blot confirmed that the granule regulates lipid synthesis and catabolism pathways by activating AMPK phosphorylation (p-AMPK increased by 1.4 - 2.7 times) and inhibiting the nuclear translocation of SREBP-1c (expression decreased by 34.7% - 85.7%).

[0060] Example 1

[0061] Amino modification of silica gel:

[0062] Weigh about 20 g of silica gel into a three-necked flask, add 200 mL of 10% HCl (V / V), stir, heat to 105 °C, reflux and acidify for 8 h, filter, wash with distilled water until neutral, dry in an oven at 110 °C for 24 h, and store in a desiccator for later use.

[0063] Prepare a 2% (4-aminobutyl)triethoxysilane ethanol solution (V / V), immerse the silica gel, and react with shaking at 25 °C for 4 h. Adjust the pH to 4 - 5 with acetic acid. After the reaction, cure the silica gel in a vacuum oven at 110 °C for 2 h to form stable amino-modified silica gel.

[0064] Example 2

[0065] Establishment of a hyperlipidemic mouse model:

[0066] Select 60 healthy male ICR mice with standard weight (about 20 g), randomly divide them into 6 groups: blank control group, model control group, positive control group (statins), low-dose solid beverage group, medium-dose solid beverage group, and high-dose solid beverage group, with 10 mice in each group.

[0067] All mice were first raised in the experimental environment for 7 days, during which the feed and water were sufficient, and the temperature and humidity were constant. After one week, the model group and the treatment groups were fed with a customized high-fat diet. In addition to the basal diet, the high-fat diet included 10% lard, 20% fructose, 10% egg yolk powder, 1% cholesterol, and 0.2% sodium cholate, and the sum of all components was 100%, which constituted all the components of the high-fat diet. After three weeks, all mice were fasted for 12 h, and blood was collected from the orbital venous plexus. After detection, the total cholesterol (TC) and triglyceride (TG) levels in the hyperlipidemic mouse model were 3 - 4 times higher than those in the blank control group, indicating successful modeling.

[0068] Example 3

[0069] Isolation and culture of hyperlipidemic liver cells:

[0070] Hyperlipidemic mice were anesthetized with sodium pentobarbital, and the liver was quickly removed, placed in pre-cooled PBS, the liver capsule was peeled off, and the blood stains were washed. The tissue was minced on ice to 1 - 2 mm 3The fragments were rinsed with PBS until the liquid was clear. Prepare 0.1% collagenase IV + 0.01% DNase I, dissolve it in PBS (pre-warmed to 37°C), add it to the minced tissue, and digest it on a shaker at 37°C (180 rpm). Gently pipette and mix every 10 minutes to avoid over-digestion. After 20 - 30 minutes, add medium containing 10% FBS to terminate the enzyme activity. Pass through 70μm and 40μm cell sieves successively and centrifuge at 1000 rpm for 5 minutes, discard the supernatant. Resuspend the precipitate with complete medium, count the cells and adjust the density to 1×10 6 cells / mL, and seed them in a collagen-coated culture dish (5% CO2, 37°C). Change the medium for the first time after 24 hours (to remove dead cells), and observe the cell adhesion status every other day (hepatocytes are polygonal). When 80% confluent, digest and passage the cells with 0.25% trypsin. Compared with normal hepatocytes, lipid droplets and vacuoles can be seen intracellularly in hyperlipidemic liver cells, and the cell volume increases.

[0071] Example 4

[0072] Preparation of hyperlipidemic liver cell membrane:

[0073] Collect hyperlipidemic liver cells from four large culture flasks, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add an appropriate amount of normal saline, pipette until the cell precipitate is resuspended, centrifuge under the same centrifugation conditions, repeat the above steps three times to remove the residual medium. Add an appropriate amount of hypotonic solution with pH 7.4 to the cell precipitate and ultrasonically disrupt it at low temperature for 30 minutes, then grind and disrupt it with a homogenization tube for 15 minutes. Centrifuge to collect the supernatant. Add hypotonic solution to the precipitate and repeat the above steps. After disrupting the cells three times, combine the supernatants, centrifuge at 12000 rpm at 4°C for 20 minutes, discard the supernatant, repeat this step once. The obtained precipitate is the cell membrane. Finally, resuspend the cell membrane with normal saline for standby.

[0074] Example 5

[0075] Preparation of cell membrane chromatography stationary phase:

[0076] Precisely weigh 20 mg of amino-modified SiO2 and place it in a 100 mL round-bottom flask. Add 20 mL of PBS solution and then add 98 μL (5 μg / mL) of hyperlipidemic liver cell membrane respectively. Stir magnetically under ice bath conditions for 3 hours, centrifuge, and wash the precipitate with PBS solution five times repeatedly. After centrifugation, obtain the SiO2 chromatography stationary phase wrapped with hyperlipidemic liver cell membrane. As Figure 2 shown, the surface of amino-functionalized silica gel is relatively smooth, while the surface roughness of silica gel loaded with cell membrane increases significantly, and obvious membrane fragments can be seen covering.

[0077] Example 6

[0078] Preparation of cell membrane chromatography column:

[0079] Select a column core with a size of 50 mm × 4.6 mm. After washing the column core and column jacket with ultrapure water, pack the column by low-pressure wet method: Place the column core into the column jacket, pad gaskets and hoof plates at both ends, slowly inject the PBS suspension of the stationary phase into the column core, apply pressure with a high-pressure pump at a low flow rate to make the stationary phase settle rapidly until the pressure in front of the column no longer rises significantly. Open the upper hoof plate and add the PBS suspension of the stationary phase again. Repeat this several times until the chromatographic column is filled up.

[0080] Example 7

[0081] Screening for hypolipidemic activity of cell membrane chromatography:

[0082] Connect the hyperlipidemic liver cell membrane chromatographic column to the HPLC respectively for on-line screening of hypolipidemic activity. The chromatographic conditions are as follows: column temperature 30 °C, flow rate 0.2 mL / min, mobile phase 10 mM ammonium acetate (pH 7.2 - 7.4), injection volume 20 μL, use ELSD1260 detector: evaporation temperature 60 °C, drift tube temperature 60 °C, high-purity nitrogen gas flow rate 1.6 L / min.

[0083] After each sample is extracted and concentrated with purified water, it is formulated into a sample with a sample concentration of 1 mg / mL, and the injection time is 30 min.

[0084] The elution times of 15 components screened on the hyperlipidemic liver cell membrane chromatography are shown in Table 1.

[0085] Table 1

[0086]

[0087]

[0088] In addition, there are no obvious chromatographic peaks for Chinese yam, purslane, smoked plum, papaya, lily, cinnamon, gordon euryale seed, malt, fructus aurantii immaturus, boat-fruited sterculia seed, peach kernel, mulberry leaf, tangerine peel, semen alpiniae oxyphyllae, galangal, chicory, polygonatum rhizome, kudzu root, sophora flower, selfheal, angelica, kaempferia galanga, saffron, tsaoko fruit, turmeric, shiitake mushroom, mushroom, flammulina velutipes, chrysanthemum, green tea, mint, dandelion, morel, flower mushroom, straw mushroom.

[0089] Example 8

[0090] Prepare the compound granule:

[0091] (1) Dry or sun-dry 15 parts of honeysuckle, 15 parts of egg yolk lecithin, 10 parts of dried tangerine peel, 10 parts of poria cocos, 10 parts of cassia seed, 10 parts of rattan tea, 10 parts of liquorice and 5 parts of polygonatum sibiricum; Wash, peel and pit 15 parts of hawthorn, 15 parts of grapefruit, 15 parts of cherry, 5 parts of blueberry, 5 parts of waxberry, 5 parts of tremella and 5 parts of lotus seed.

[0092] (2) Put the dry or sun-dried honeysuckle, egg yolk lecithin, dried tangerine peel, poria cocos, cassia seed, rattan tea, liquorice and polygonatum sibiricum into a pulverizer to make powder; Add water with a mass ratio of 1:5 to hawthorn, grapefruit, cherry, blueberry, waxberry, tremella and lotus seed pulp and make pulp to obtain crude pulp.

[0093] (3) Mix the obtained crude drug powder and crude pulp evenly, then add 80% ethanol with a volume fraction according to the mass ratio of material to liquid of 1:20 and mix evenly. Put it into an 80°C water bath for ultrasonic extraction for 1 h, repeat 3 times, and perform centrifugal separation after extraction to obtain supernatant and precipitate. The supernatant is the required extract.

[0094] (4) Place the obtained extract on a rotary evaporator, evaporate and concentrate it, and then obtain the corresponding extract powder through spray drying.

[0095] (5) Mix the extract powder with 20 parts of excipient erythritol and 5 parts of maltodextrin, and use a one-step granulator to granulate. After drying in an oven, the granule agent is obtained.

[0096] Example 9

[0097] Therapeutic effect of the granule agent on hyperlipidemic mice:

[0098] Select 60 healthy male ICR mice with standard weight (about 20 g), and randomly divide them into 6 groups: blank control group, model control group, positive control group (statin), low-dose solid beverage group, medium-dose solid beverage group and high-dose solid beverage group, with 10 mice in each group.

[0099] Except for the blank group fed with normal feed, the other groups were fed with high-fat feed for three weeks to obtain a hyperlipidemic mouse model. After three weeks, all mice were fasted for 12 h, and blood was collected from the orbital venous plexus. After detection, if the total cholesterol (TC) and triglyceride (TG) contents of the hyperlipidemic mouse model are 3-4 times higher than those of the blank control group, the modeling is successful. After successful modeling, the low, medium and high-dose solid beverage groups were respectively gavaged with different doses of the solution of Example 8 (200 mg / kg, 100 mg / kg, 50 mg / kg), the positive control group was gavaged with 0.5% CMC-Na atorvastatin suspension (1 mg / kg), and the blank group was gavaged with 0.5% CMC-Na solution with the same volume.

[0100] The body weight of rats was recorded weekly for 4 consecutive weeks of drug administration. After the experiment, the mice were fasted for 12 h, anesthetized with sodium pentobarbital, and blood was collected from the abdominal aorta and corresponding tissues were collected.

[0101] Figure 4 It was for the detection of 4 lipid-related indicators. As can be seen from the figure, the TC level decreased by 37.0% - 58.7% compared with the model group, TG decreased by 27.7% - 55.0%, LDL-C decreased by 29.3% - 52.8%, and HDL-C increased by 43.0% - 56.6%. All showed dose-dependence. As the dose of the granule increased, the lipid-lowering effect was better.

[0102] As Figure 5 shown, there were significant differences in the HE staining results between the treatment group of this compound granule and the model group in terms of fatty degeneration, inflammatory infiltration, and tissue structure integrity. The hepatocytes in the blank group were arranged tightly without inflammatory cell infiltration; the hepatocytes in the model group were swollen with a large number of lipid droplet vacuoles of different sizes, and the lipid change area was ≥40%. While the hepatocyte morphology in the granule treatment group was basically normal, the nucleus was in the center, with only a small number of scattered lipid droplets, the lipid change area was ≤15%, and occasionally a small number of inflammatory cells were seen, and the infiltration degree was reduced by 80%. This compound granule significantly reversed the liver pathological damage in hyperlipidemic mice by multi-target regulation of lipid metabolism - inhibition of inflammation - reduction of oxidative stress. Its improvement effect was better than that of the positive group drug, and there was no risk of liver toxicity. The HE staining results verified the comprehensive liver protection effect of the granule at the histological level, providing an important basis for its clinical transformation.

[0103] Example 10

[0104] Mechanism study on lipid-lowering effect of the granule

[0105] As Figure 6 shown, the expression of lipid metabolism-related proteins in the liver and serum of hyperlipidemic mice was detected by Western Blot technology. The compound granule achieved lipid metabolism balance through the dual-axis regulation of AMPK / SREBP-1c. The active ingredients in the granule (such as hyperin from hawthorn and dihydromyricetin from ampelopsis grossedentata) activated AMPK phosphorylation, initiated energy metabolism regulation, and thus promoted lipid decomposition and oxidation; components such as chrysophanol from cassia seed and hesperidin from tangerine peel blocked the nuclear entry of the mature form of SREBP-1c and blocked its transcriptional activity to block lipid synthesis. Compared with the single-target inhibition of traditional drug HMG-CoA reductase, this compound granule synergistically reduced blood lipid levels by multi-target regulation of AMPK / SREBP-1c / LDLR, inhibiting lipid synthesis and promoting lipid decomposition. Its multi-target action mode overcame the metabolic compensation defect of a single drug (such as statins), and there was no risk of liver toxicity, providing a safer and more efficient natural solution for the treatment of hyperlipidemia.

[0106] Obviously, the above-mentioned embodiments of the present invention are merely examples for clearly explaining the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. A cell membrane chromatography screening method for drugs for treating hyperlipidemia, characterized in that: The following steps are involved: (1) fixing a hyperlipidemic liver cell membrane on a silica gel surface to prepare a cell membrane stationary phase, and filling the prepared stationary phase into a chromatographic column; (2) Online screening of active ingredients from extracts of edible and medicinal materials to identify ingredients with lipid-lowering activity.

2. The cell membrane chromatography screening method according to claim 1, characterized in that: The method for preparing the hyperlipidemic liver cell membrane comprises: extracting liver cells from liver tissue of a hyperlipidemic mouse model, and then extracting the hyperlipidemic liver cell membrane after ultrasonic disruption.

3. The cell membrane chromatography screening method according to claim 1, characterized in that: The silica gel is amino-modified SiO2; The preparation method of the amino-modified SiO2 is: subjecting silica gel to reflux acidification treatment, and then placing the silica gel in a (4-aminobutyl)triethoxysilane ethanol solution for shaking reaction, and subjecting the silica gel to curing treatment to obtain the amino-modified SiO2.

4. The cell membrane chromatography screening method according to claim 1, characterized in that: The chromatographic conditions for the screening are: column temperature 30°C, flow rate 0.2 mL / min, mobile phase 10 mM ammonium acetate (PH7.2-7.4), injection volume 20 μL, ELSD1260 detector: evaporation temperature 60°C, drift tube temperature 60°C, high-purity nitrogen flow rate 1.6 L / min.

5. A medicine-food composition for treating hyperlipidemia, characterized in that: The invention comprises the following raw materials in parts by weight: 10-15 parts of hawthorn, 10-15 parts of egg yolk lecithin, 10-15 parts of honeysuckle, 10-15 parts of grapefruit, 10-15 parts of cherry, 5-10 parts of tangerine peel, 5-10 parts of tuckahoe, 5-10 parts of cassia seed, 5-10 parts of rattan tea, 5-10 parts of liquorice, 2-5 parts of polygonatum, 2-5 parts of blueberry, 2-5 parts of bayberry, 2-5 parts of white fungus and 2-5 parts of lotus seeds.

6. The method for preparing the edible-medicinal composition according to claim 5, characterized in that: The following steps are involved: (1) drying honeysuckle, egg yolk lecithin, tangerine peel, Poria cocos, cassia seed, rattan tea, liquorice and polygonatum in the shade or in the sun, and grinding to obtain powder; washing, peeling and removing the core of hawthorn, grapefruit, cherry, blueberry, bayberry, white fungus and lotus seeds, and adding water to beat the pulp to obtain a coarse pulp; (2) extracting the powder and the crude pulp with alcohol, collecting the supernatant by centrifugation, rotary evaporation and spray drying to obtain an extract powder; (3) Add auxiliary materials for granulation to obtain granules.

7. The preparation method according to claim 6, characterized in that: The alcohol extraction conditions are as follows: mixing the powder and the crude pulp with the ethanol solution in a mass ratio of 1:20, extracting in a water bath with ultrasound at 60°C-90°C for 1h, and repeating 3-4 times; The volume fraction of the ethanol solution is 30% to 80%.

8. The preparation method according to claim 6, characterized in that: The auxiliary materials include erythritol and maltodextrin; the weight fraction of erythritol is 45% to 55%; the weight fraction of maltodextrin is 5% to 10%.

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