Compound lipid-lowering tea and preparation method thereof

Abstract

The invention discloses compound lipid-lowering tea and a preparation method, and belongs to the technical field of lipid-lowering tea. The compound lipid-lowering tea comprises 10 g of semen cassiae, 5-15 g of gynostemma pentaphylla, 5-15 g of salvia miltiorrhiza, 5-15 g of hawthorn and 5-15 g of sticktight. According to the application disclosed by the invention, lipid-lowering active ingredients such as tanshinone, flavone, ursolic acid and anthraquinone in medicinal materials are researched, so that the lipid-lowering effect of the ingredients through a plurality of signal pathways such as a targeted PI3K-AKT signal pathway, a cancer morbidity pathway and an estrogen signal pathway is definite. Wherein flavone is combined with a core target PPAGR, anthraquinone and ursolic acid are respectively combined with HSP90AA and ESR1, lipid synthesis and metabolism are regulated and controlled, then the fat index and the serum low-density lipoprotein and triglyceride level are reduced, and synthesis of high-density lipoprotein is promoted. Animal experiments prove that the lipid-lowering tea has a remarkable lipid-lowering effect, has no adverse effect on visceral organs of mice, and provides a new safe and effective lipid-lowering choice for hyperlipidemia patients.

Description

Technical Field

[0001] The invention relates to the technical field of lipid-lowering tea, and in particular to a compound lipid-lowering tea and a preparation method thereof. Background Art

[0002] Hyperlipidemia, often referred to as high blood lipids, is also known as dyslipidemia in medicine. It usually refers to the increase of triglyceride (TG) and / or total cholesterol (TC) in plasma, and also includes the increase of low-density lipoprotein cholesterol (LDL-C) and the decrease of high-density lipoprotein cholesterol (HDL-C). According to a survey by the World Health Organization, more than 2 billion people in the world have high blood lipids, and this number is still growing. High blood lipids can lead to the occurrence of diseases such as atherosclerosis, coronary heart disease, and stroke, and also increase the risk of diseases such as diabetes and hypertension, posing a serious threat to human health. Therefore, the prevention and treatment of hyperlipidemia are of great significance for improving human health and promoting economic development.

[0003] At present, the commonly used drugs for treating hyperlipidemia include statins, fibrates, niacin, bile acid chelators, cholesterol absorption inhibitors, PCSK9 inhibitors, etc. However, Western medicine has the following shortcomings: 1. Drug addiction and dependence: Once patients start taking Western medicine to treat hyperlipidemia, they must take it for a long time, otherwise blood lipids will rebound. Therefore, patients will become addicted to and dependent on drugs. 2. Drug complications: Western medicine treatment of hyperlipidemia has certain side effects, such as causing muscle damage, leading to rhabdomyolysis, or causing liver and kidney damage, intestinal discomfort, etc. 3. High treatment costs: The price of lipid-lowering Western medicine is not affordable, and it needs to be taken for a long time, which puts a certain burden on the patient's economy. 4. Limited treatment effect: Some patients use Western medicine to treat hyperlipidemia ineffectively, especially the elderly, and the adverse reactions of the drugs are more prominent and difficult to tolerate. 5. Negative effects on health: Long-term use of Western medicine to treat hyperlipidemia may have an adverse effect on the patient's immune system and increase the risk of other diseases. Therefore, it is urgent to find a lipid-lowering drug with good efficacy, low cost, green and safe. Summary of the invention

[0004] The present invention aims to provide a preparation method and application of a compound lipid-lowering tea, so as to solve the problems of easy addiction, complications and low safety of existing drugs for treating hyperlipidemia.

[0005] To solve the above technical problems, the present invention provides the following technical solutions: a compound lipid-lowering tea, comprising 10g of Cassia seed, 5-15g of Gynostemma pentaphyllum, 5-15g of Salvia miltiorrhiza, 5-15g of Crataegus pinnatifida, and 5-15g of Bidens pilosa.

[0006] Furthermore, the Cassia seed is 10g, the Gynostemma pentaphyllum is 5g, the Salvia miltiorrhiza is 5g, the Crataegus pinnatifida is 5g, and the Bidens pilosa L. is 5g.

[0007] Furthermore, the Cassia seed is 10g, Gynostemma pentaphyllum is 5g, Salvia miltiorrhiza is 10g, Crataegus pinnatifida is 10g, and Bidens pilosa is 10g.

[0008] Furthermore, the Cassia seed is 10g, the Gynostemma pentaphyllum is 5g, the Salvia miltiorrhiza is 15g, the Crataegus pinnatifida is 15g, and the Bidens pilosa L. is 15g.

[0009] Furthermore, the Cassia seed is 10g, the Gynostemma pentaphyllum is 10g, the Salvia miltiorrhiza is 5g, the Crataegus pinnatifida is 10g, and the Bidens pilosa L. is 15g.

[0010] Furthermore, the Cassia seed is 10g, the Gynostemma pentaphyllum is 15g, the Salvia miltiorrhiza is 15g, the Crataegus pinnatifida is 10g, and the Bidens pilosa L. is 5g.

[0011] Furthermore, the Cassia seed 10g, Gynostemma pentaphyllum 10g, Salvia miltiorrhiza 15g, Crataegus 5g, Bidens pilosa 10g.

[0012] Furthermore, the ingredients include 10g of Cassia seed, 15g of Gynostemma pentaphyllum, 10g of Salvia miltiorrhiza, 5g of Crataegus pinnatifida, and 15g of Bidens pilosa.

[0013] Further, a method for preparing the compound lipid-lowering tea according to any one of the above items comprises the following steps:

[0014] S1, pretreatment: clean and dry each Chinese medicine, and crush it into powder using a Chinese medicine grinder;

[0015] S2. Weighing: Weigh the required weights of Cassia seed, Gynostemma pentaphyllum, Salvia miltiorrhiza, Crataegus pinnatifida, and Bidens pilosa respectively by weighing equipment, and add appropriate amount of water to the mixed powder and soak for a period of time;

[0016] S3, using a high fire to boil the soaking liquid in S2 for a period of time, and then using a low fire to boil the soaking liquid for a period of time;

[0017] S4, filter the decoction in S3 using gauze and collect the filtrate;

[0018] S5. Add appropriate amount of water to the filtered medicinal residue and boil it over high heat for a period of time, then boil it over low heat for a period of time;

[0019] S6, filtering the decoction in S5 using gauze, and combining the filtrate;

[0020] S7. Place the filtrate in a water bath to concentrate and obtain the compound lipid-lowering tea.

[0021] Furthermore, in step S2, the mixed powder is added with 1L of pure water and soaked for half an hour; in step S2, the soaking liquid is first decocted on high heat for 30 minutes, and then decocted on low heat for 1 hour; in step S5, the filtered medicinal residue is added with 0.5L of pure water and then decocted on high heat for 30 minutes, and then decocted on low heat for 1 hour; in step S7, the filtrate is placed in a 100°C water bath and concentrated to 100mL to obtain the compound lipid-lowering tea.

[0022] Beneficial effects of the present invention: 1. Through network pharmacology analysis and molecular docking technology, the main lipid-lowering active ingredients (tanshinone, flavonoids, ursolic acid and anthraquinone) in the compound lipid-lowering tea and their mechanisms of action are clearly defined. These active ingredients can exert lipid-lowering effects in multiple ways and targets, such as inhibiting fatty acid synthase activity, activating PPARs to enhance fatty acid oxidation metabolism, regulating intestinal microbiota to affect lipid absorption metabolism, antioxidant to reduce ox-LDL levels, affecting bile acid metabolism and excretion, etc. The binding relationship with the core targets (PPAGR, HSP90AA, ESR1, etc.) is also accurately determined. This not only provides a solid theoretical basis for the lipid-lowering effect of lipid-lowering tea, but also lays the foundation for subsequent in-depth research and optimization of formulas, which is helpful to promote the development of the field of lipid-lowering in traditional Chinese medicine. 2. Through animal experiments, the lipid-lowering effect of the compound lipid-lowering tea was verified from multiple aspects such as body weight, organ index, four indicators of blood lipids, and liver tissue pathology. The experimental results show that lipid-lowering tea can not only significantly reduce the weight of mice, but also regulate four indicators of blood lipids, reduce fat formation in liver cells, and improve liver tissue pathological characteristics. At the same time, it has a positive effect on the kidney index, improves the phenomenon of kidney atrophy caused by a high-fat diet, and has no adverse effects on other organs. This fully proves the effectiveness and safety of lipid-lowering tea in vivo, and increases the credibility and application value of the product. 3. The compound lipid-lowering tea of ​​the present invention uses Cassia seed, Gynostemma pentaphyllum, Salvia miltiorrhiza, Crataegus pinnatifida to form a formula, and clarifies the dosage range of each component. Compared with other comparative documents, the formula combination and ratio are unique. This unique formula has been verified by experiments and can accurately target hyperlipidemia. In animal experiments, the fat index of mice in the lipid-lowering tea group was significantly reduced, and the effect was better than simvastatin. It effectively accelerated the decomposition of fat in high-fat mice, reduced hoarding, and showed good lipid-lowering effects, providing a new safe and effective choice for patients with hyperlipidemia. 4. Most of the medicinal materials in the formula are medicinal and edible, and the safety of long-term drinking is guaranteed. Animal experiments showed that compared with the normal control group, the lipid-lowering tea group had no significant adverse effects on the liver, spleen, heart, testicles and epididymis of mice (p>0.05), indicating that it would not cause damage to the growth and development of these important organs, reducing the risks of liver and kidney damage that may be caused by traditional lipid-lowering Western medicines, and improving the safety of users. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1This is the analysis diagram of the common target genes of compound lipid-lowering tea and hyperlipidemia;

[0024] Figure 2 A is the intersection gene protein interaction network relationship diagram;

[0025] Figure 2 B is the drug-ingredient-target-disease interaction PPI network relationship diagram;

[0026] Figure 3 The top 8 PPI network core targets in degree ranking;

[0027] Figure 4 A is the GO functional enrichment analysis result of common target genes;

[0028] Figure 4 B is the result of KEGG signaling pathway enrichment analysis of common target genes;

[0029] Figure 5 3D structures of the core active ingredients, A: 3D structure of anthraquinone, B: 3D structure of ursolic acid, C: 3D structure of tanshinone, D: 3D structure of flavonoids;

[0030] Figure 6 The ability of the core active ingredient to bind to the core protein;

[0031] Figure 7 The docking modes of target molecules and small molecule ligands are shown in Figure 1. A: docking mode of ERS1 target and ursolic acid from hawthorn, B: docking mode of HSP90AA1 target and anthraquinone from cassia seed, and C: docking mode of PPARG target and flavonoids.

[0032] Figure 8 is the weight change trend of mice;

[0033] Fig. 9 The effect of lipid-lowering tea on the organ index of mice;

[0034] Fig.10 These are the results of four blood lipid tests in mouse serum;

[0035] Fig.11 This is the Oil Red O staining step;

[0036] Fig.12 Oil red O staining results of liver tissues of mice in each group, A: normal control group, B: high fat model group, C: simvastatin group, D: lipid-lowering drug group;

[0037] Fig.13 HE staining step;

[0038] Fig.14HE staining results of liver tissues of mice in each group: A: normal control group, B: hyperlipidemia model group, C: simvastatin group, D: lipid-lowering tea group. DETAILED DESCRIPTION

[0039] The following is further described in detail through specific implementation methods:

[0040] Example:

[0041] The present invention uses hawthorn, gynostemma pentaphyllum, cassia seed, salvia miltiorrhiza and bidentae to make compound lipid-lowering tea in different proportions, and analyzes the above five traditional Chinese medicine active ingredients and their lipid-lowering molecular networks by network pharmacology methods to preliminarily understand the lipid-lowering mechanism of the compound lipid-lowering tea. Then, volunteers are recruited to score the taste, color, etc. of lipid-lowering teas with different proportions, and the formula with the highest score is selected for animal experiments. The lipid-lowering effect and mechanism of the formula are explored from four indicators of body weight, organ index, blood lipids, and pathology, respectively, to provide a compound lipid-lowering tea with good taste, high safety and good effect for people with hyperlipidemia, which can be taken in daily life to achieve the purpose of lowering blood lipids, and also provide a scientific basis for the development and promotion of traditional Chinese medicine theory.

[0042] 1. Experimental Materials

[0043] 1.1 Experimental drugs

[0044] Cassia seed, Salvia miltiorrhiza, Gynostemma pentaphyllum, Crataegus pinnatifida, Bidens pilosa, Simvastatin.

[0045] 1.2 Experimental animals

[0046] This experiment used Kunming mice, SPF grade, male, weighing 18-20g, and the breeding conditions of experimental animals were: air-conditioned environment, natural light, temperature at 23-25℃, humidity controlled at 40%-60%, and all mice could eat and drink freely.

[0047] 1.3 Main reagents and instruments

[0048] Kits for measuring serum biochemical indexes: TC and TG, HDL-C and LDL-C. TGL-16 centrifuge; ACS electronic balance; spectrophotometer.

[0049] 2. Experimental Methods

[0050] 2.1 Network pharmacology analysis of active ingredients of compound lipid-lowering tea and common target genes with hyperlipidemia

[0051] Drug component collection: Literature review was conducted to collect and organize the names of active chemical components with lipid-lowering effects in each traditional Chinese medicine component. All component names were retrieved in the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP, https: / / tcmspw.com / ). All detected chemical components were screened using oral bioavailability (OB) ≥ 30% and drug-like property (DL) ≥ 0.18 as thresholds to obtain potential active ingredients in the compound lipid-lowering tea.

[0052] Target collection: In order to obtain the intersection of drug ingredient targets and disease gene targets, the collected tanshinone (TanshinoneⅠ), flavonoids (Flavone), ursolic acid (Ursolic acid), and anthraquinone (Anthraquinone) are used as active ingredients in the compound Chinese medicine lipid-lowering tea for the effective treatment of hyperlipidemia. The above active ingredients can play a lipid-lowering role through multiple pathways and multiple targets, such as reducing the synthesis of fatty acids by inhibiting the activity of fatty acid synthase and other key enzymes; activating peroxisome proliferator-activated receptors (PPARs), etc., enhancing the oxidation and metabolism of fatty acids and promoting lipid transport; regulating intestinal microbiota, thereby affecting the absorption and metabolism of lipids; the antioxidant effect can reduce oxidative stress and reduce the level of oxidized low-density lipoprotein cholesterol (ox-LDL) in the blood; by affecting the metabolism and excretion of bile acids, it changes the digestion and absorption of lipids. These effects may be interrelated and jointly regulate blood lipid levels.

[0053] The SMILES numbers of the four ingredients were collected in the Pubchem database, and the collected results were used to search for targets with probability>0.1 in the Swisstargetprediction database. The GeneCards database was used to search for hyperlipidemia-related targets and targets related to the effective active ingredients of the compound Chinese medicine lipid-lowering tea, and the intersection was taken, and a Venn diagram was drawn (such as Figure 1 ). Figure 1 It can be seen that a total of 1,300 disease targets for hyperlipidemia were screened, of which 115 targets were found for the four active ingredients of traditional Chinese medicine, and 49 targets were common to both, accounting for 3.3% of all targets. These 49 common target genes provide a molecular basis for lipid-lowering drugs to exert their lipid-lowering effects.

[0054] Upload 49 targets that intersect hyperlipidemia and drugs to the STRING database to obtain an interaction network diagram ( Figure 2 A), and then use Cytoscape 3.7.2 to draw the PPI network ( Figure 2 B). The cytoNCA plug-in was used to analyze the core genes, and the top 8 degree rankings were PPAGR, ESR1, HSP90AA1, PTGS2, EGFR, CASP3, BCL2, MMP9 ( Figure 3 ), the above target genes may be the core targets of compound lipid-lowering tea for the treatment of hyperlipidemia. Among them, peroxisome proliferator-activated receptor (PPAGR) can promote the uptake and storage of fatty acids, increase adipocyte differentiation, thereby reducing plasma lipid levels, and regulate the expression of transport proteins and lipases related to fat metabolism, thereby affecting lipid metabolism. Therefore, PPAGR is an important target for lipid-lowering tea to exert its lipid-lowering effect.

[0055] GO function and KEGG signaling pathway enrichment analysis: DAVID platform was used to conduct GO function enrichment analysis on potential targets of traditional Chinese medicine compound lipid-lowering tea for the treatment of hyperlipidemia. Figure 4 A shows that the biological process (BP) entries mainly include the cell's response to hormones, the response to organic cyclic compounds, and the response to steroid hormones; the cellular component (CC) entries mainly include membrane rafts, membrane microdomains, plasma membrane rafts, and myelin sheaths; the molecular function (MF) entries mainly involve nuclear receptor activity, ligand-activated transcription factor activity, steroid binding, estrogen response element binding, and transcription factor binding. In order to gain a deeper understanding of the specific mechanism of lipid-lowering tea in lowering blood lipids, the KEGG signaling pathway was further enriched for 49 targets. The results showed that the PI3K-AKT signaling pathway, cancer pathogenesis, estrogen signaling pathway, endocrine resistance, tyrosine kinase inhibitor resistance and other pathways were significantly enriched ( Figure 4 B). This indicates that the active ingredients in lipid-lowering tea can play a lipid-lowering role mainly by intervening in these signal pathways. Among them, the PI3K-Akt signal pathway has important connections with multiple lipid metabolism-related genes, multiple lipid metabolism organs, and multiple lipid metabolism disorder-related diseases. This signal pathway directly or indirectly participates in the molecular mechanism of lipid synthesis, transport, uptake and decomposition, as well as signal transduction regulation under pathological conditions. The active ingredients in lipid-lowering drugs can regulate blood lipid levels and play a lipid-lowering role by intervening in the PI3K-Akt signal pathway.

[0056] 2.2 Molecular docking

[0057] The 3D structures of the compounds Anthraquinone, Ursolicacid, TanshinoneⅠ, Flavone, etc. in energy-minimized format were obtained by processing with Chem3D software. Figure 5 The AutoDockVina software was used to dock the three small molecule ligands with the protein receptor genes for the first time, and the pattern diagram and binding energy table were obtained ( Figure 6), the results showed that there was no hydrogen bond in the binding mode of ESR1 and Flavone, ESR1 and Anthraquinone, so the result was discarded. The second docking of the three groups of results of PPARG and Flavone, ESR1 and Ursolic acid, and HSP90AA1 and Anthraquinone obtained the names of the residues in the binding sites and the hydrogen bond energies, see Figure 7 The analysis showed that the binding energy required for PPARG and Flavone group was the lowest, the binding affinity between small molecule ligand and protein receptor was the best, and the binding was more stable.

[0058] 2.3 Preparation of compound lipid-lowering tea

[0059] An orthogonal table with 4 factors (cratweed, Gynostemma pentaphyllum, Salvia miltiorrhiza and Bidens pilosa) and 3 levels (5, 10, 15 g) was designed with the daily dosage of 10 g of Cassia seed as the standard, and 9 lipid-lowering teas with different ratios were determined, as shown in Table 1. Lipid-lowering tea was brewed according to the ratio. First, each Chinese medicine was cleaned and dried, crushed into powder with a Chinese medicine grinder, and then the dosage of each Chinese medicine was weighed according to the ratio, and then soaked in pure water for half an hour, first decocted on high heat for 30 minutes, and then decocted on low heat for 1 hour. After the filtrate was poured out and filtered, it was concentrated to 100 mL in a water bath. 100 volunteers were recruited to taste the lipid-lowering tea and score the color (5 points), aroma (5 points) and taste (5 points) of the lipid-lowering tea, of which taste and aroma accounted for 40% and color accounted for 20%. The 4 lipid-lowering teas with higher scores were selected. 117 volunteers were recruited again to score the 4 lipid-lowering teas with higher scores.

[0060] Group Gynostemma pentaphyllum / g Salvia miltiorrhiza / g Hawthorn / g Bidens pilosa / g Cassia seed / g 1 5 5 5 5 10 2 5 10 10 10 10 3 5 15 15 15 10 4 10 5 10 15 10 5 10 10 15 5 10 6 10 15 5 10 10 7 15 5 15 10 10 8 15 10 5 15 10 9 15 15 10 5 10 total 90 90 90 90 90

[0061] Table 1

[0062] The results are shown in Table 2. Lipid-lowering Tea 8 scored the highest, so Lipid-lowering Tea 8 was selected as the formula for subsequent animal experiments. The formula was Cassia seed (10 g), Gynostemma pentaphyllum (15 g), Salvia miltiorrhiza (10 g), Crataegus pinnatifida (5 g), Bidens pilosa (15 g).

[0063]

[0064] Table 2

[0065] The database is queried to determine the maximum intake for humans and the dosage for mice is obtained through the conversion relationship between animal and human dosages, and the dosage for each mouse is determined according to its weight. The total dosage required for oral administration is calculated based on the final formula, and the Chinese medicine decoction is boiled and then filtered, freeze-dried and ground into fine powder for later use.

[0066] 2.4 Animal Experiment Grouping and Dosing

[0067] 40 Kunming mice were randomly divided into 4 groups, 10 mice in each group, namely normal control group, hyperlipidemia model group, compound lipid-lowering tea group and simvastatin group. The normal group was fed with Vitt feed, and the other 3 groups were fed with high-fat feed, with each mouse fed with 7g feed per day. All mice were free to eat and drink water. After the mice were adaptively fed, the normal control group was still fed with ordinary feed, while the hyperlipidemia model group, simvastatin group and lipid-lowering tea group were fed with high-fat feed for 9 weeks. Blood was collected from the tail vein at the end of the 9th week, and serum was obtained to detect the concentration of TC in the serum of fasting mice, and the fasting TC value greater than 7mmol / L was considered a successful hyperlipidemia model.

[0068] Depend on Figure 8 It can be seen that as the feeding time increases, the weight of mice gradually increases. After 2 weeks of high-fat feeding, the weight of mice in the hyperlipidemia model group, simvastatin group and lipid-lowering tea group gradually increased compared with the normal control group. By the 10th week, the weight of mice fed with a high-fat diet was significantly higher than that of the normal control group. After the 10th week, the normal control group and the hyperlipidemia model group were gavaged with distilled water, and the simvastatin group and lipid-lowering tea group were gavaged with simvastatin and lipid-lowering tea, respectively. Figure 8 It can be seen that after the 10th week, the weight of mice in the normal control and hyperlipidemia model groups remained basically stable, while the weight of mice in the simvastatin and lipid-lowering tea groups showed a significant downward trend. At the 13th week, the weight of these two groups of mice was significantly lower than that of mice in the normal control group. The above results show that the intervention of simvastatin and lipid-lowering tea can significantly reduce the weight of mice and has a significant weight loss effect.

[0069] 2.5 Effects of lipid-lowering tea on the indexes of various organs in mice

[0070] Under normal growth conditions, the ratio of each organ to body weight is relatively constant. After an animal becomes ill, the weight of the damaged organ may change, so the organ index also changes accordingly. An increase in the organ index indicates organ congestion, edema, or hyperplasia; a decrease in the organ index indicates organ atrophy and other degenerative changes. Compared with the normal control group, the fat index of the hyperlipidemia model group was significantly increased (p < 0.05), indicating that the body fat content of the hyperlipidemia model mice increased significantly, and the high-fat diet led to the accumulation of adipose tissue in the body, thereby causing the mice to gain weight. Compared with the hyperlipidemia model group, the fat index of the simvastatin group mice was significantly decreased (p < 0.05), indicating that under the intervention of the drug, the fat accumulation in the mice was significantly reduced, and the drug could effectively decompose the adipose tissue in the mice and inhibit the fat accumulation caused by the hyperlipidemia diet. Compared with the hyperlipidemia model group, the fat index of the lipid-lowering tea group was extremely significantly reduced (p < 0.01), indicating that the lipid-lowering tea has an extremely significant inhibitory effect on the accumulation of fat in mice (see Fig. 9 A), its effect is better than simvastatin, indicating that the compound lipid-lowering tea can effectively accelerate the decomposition of fat in high-fat mice, reduce fat accumulation, and show a good lipid-lowering effect.

[0071] Compared with the normal control group, the renal index of the hyperlipidemia model group was significantly reduced (p < 0.05), indicating that a high-fat diet has an inhibitory effect on the growth and development of the kidneys, which may cause atrophy of renal tissue and cause adverse effects. Compared with the hyperlipidemia model group, the renal index of the simvastatin group and the lipid-lowering tea group was significantly increased (p < 0.05), which was consistent with the renal index of the normal control group ( Fig. 9 B), indicating that simvastatin and lipid-lowering tea can effectively improve kidney atrophy caused by a high-fat diet.

[0072] Compared with the normal control group, the liver index of the hyperlipidemia model group decreased, but there was no significant difference (p>0.05), indicating that a high-fat diet is not conducive to the growth and development of the liver, but it did not cause a significant difference. Compared with the hyperlipidemia model group, the liver index of the simvastatin group and the lipid-lowering tea group increased, but there was no significant difference (p>0.05), indicating that drug intervention did not cause damage to the liver. Fig. 9 C. Compared with the normal control group, the spleen, heart, testis and epididymis indexes of the mice in the hyperlipidemia model group did not change significantly (p>0.05), indicating that the high-fat diet had no adverse effects on the growth and development of the above tissues and organs, and did not cause swelling, congestion or atrophy of the tissues. Compared with the hyperlipidemia model group, the organ indexes of the mice in the simvastatin group and the lipid-lowering tea group did not change significantly (p>0.05), indicating that simvastatin and lipid-lowering tea had no adverse effects on the growth and development of these tissues and organs. Fig. 9 DF.

[0073] 2.6 Effects of lipid-lowering tea on four blood lipids in mouse serum

[0074] Each group of mice fasted for 12 hours before testing to reduce the interference of food on blood lipid test results. Blood was collected by orbital venous plexus blood collection method. The mice were grasped and fixed during blood collection, and a capillary glass tube was inserted into the orbital venous plexus to collect blood. Generally, 0.2-0.5 mL of blood was collected. The collected blood sample was placed in a centrifuge tube and centrifuged at 3000-5000 rpm for 10-15 minutes. The serum was separated and carefully aspirated and placed in a clean and dry centrifuge tube. Then, the blood lipid index in the serum was detected by ultraviolet spectrophotometer. According to the detection instructions of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C), the serum sample was accurately added to the corresponding reagent reaction, and the values ​​of blood lipid index were read under the spectrophotometer and recorded for analysis.

[0075] After the model was successfully established, the drug was administered by oral gavage for 3 weeks to detect the four blood lipid levels in the serum of each group of mice. It was found that compared with the normal control group, TC and LDL in the hyperlipidemia model group were slightly increased, but there was no significant difference (p>0.05), while HDL was significantly decreased, and TG content was significantly increased, both of which were statistically significant (p<0.05), indicating that the high-fat diet caused dyslipidemia in mice, mainly by reducing HDL content and increasing TG content. Compared with the hyperlipidemia model group, the TC, LDL and TG levels of mice in the simvastatin group were significantly decreased, while the HDL content was significantly increased, both of which were statistically significant (p<0.05). This shows that simvastatin has a significant lipid-lowering effect, which mainly exerts its lipid-lowering effect by reducing TC, LDL and TG contents and increasing HDL content. Fig.10 It can be seen that compared with the hyperlipidemia model group, the TC and LDL levels of mice in the lipid-lowering tea group were significantly decreased (p < 0.05), the HDL content was significantly increased (p < 0.05), and the TG content did not change significantly (p > 0.05), indicating that lipid-lowering tea mainly regulates blood lipid components by reducing the synthesis of TC and LDL and increasing the HDL content. The lack of significant change in TG content may be due to the significant increase in HDL content.

[0076] 2.7 Oil Red O staining to detect lipid droplet formation in mouse liver tissue

[0077] Oil Red O dye has good fat dissolving and adsorption effects on tissues and organs with fatty degeneration and lipid deposition. It is easy to operate and has bright colors, which is superior to Sudan III dye. After the 13th week, the mice in each group were killed by cervical dislocation, and the liver tissue was taken. It was first fixed with 40g / L paraformaldehyde solution for 4 hours, and then transferred to 200g / L sucrose solution-PB solution for dehydration (the sucrose solution was changed many times). After 24 hours, it was replaced with 300g / L sucrose-PB solution, placed in embedding agent after 24 hours, and finally frozen at -80℃ for use. Slice in a -20℃ freezing slicer, spread the slides and dry at room temperature for 20 minutes for Oil Red O staining. Stain with Oil Red O dye, differentiate with 60% isopropanol, counterstain with hematoxylin, rinse with running water, and seal with glycerol gelatin or aqueous sealing medium. The changes in lipid droplets in the liver are observed and recorded by oil red staining. The specific staining steps are as follows: Fig.11 The lipolysis and adsorption of Oil Red O dye dyes lipid droplets bright red and cell nuclei blue.

[0078] When liver tissue develops fatty changes, the fat in the cytoplasm aggregates into fat bodies, which then fuse into fat droplets, existing in the form of vacuoles of varying sizes with some cell nuclei biased toward one side of the cell. Fig.12The results showed that in the normal control group (A), uniform red particles appeared in the liver cells, the fat volume was small, and the oil red O staining was light, indicating that the liver fat accumulation was small and uniform. In the hyperlipidemia model group (B), the liver tissue showed obvious cavitation. Compared with the normal control group, the volume of intracellular lipid droplets increased significantly, and the intracellular distribution was uneven. The oil red O staining was deeper, indicating that the number and volume of lipid droplets in the liver cells of the hyperlipidemia model group mice increased, and fat accumulation was high. In the simvastatin group (C), red lipid droplets were also seen in the liver cells, but the number and volume were smaller than those in the hyperlipidemia model group. There were also a small number of small lipid droplets in the liver cells of the Chinese medicine lipid-lowering drug group (D), which was significantly reduced compared with the hyperlipidemia model group. The oil red O staining was uniform and the color was lighter. The above results show that simvastatin and lipid-lowering drugs can significantly inhibit the formation of fat in liver cells, inhibit the accumulation of fat in liver cells by reducing the number and volume of lipid droplets, and thus play a lipid-lowering effect.

[0079] 2.8 Observation of liver histopathological characteristics of mice in each group by HE staining

[0080] The liver tissues of mice in each group were collected according to Fig.13 Paraffin sections were prepared according to the steps and HE staining was performed. Then the sections were observed and compared under a microscope: the structure, arrangement and shape of the liver lobules were observed at low magnification; the nuclei, cytoplasm and their number, size, shape and other surrounding structural tissues of the liver cells were observed at high magnification.

[0081] After HE staining, the results were as follows Fig.14 As shown in the figure, the cytoplasm of hepatocytes in the liver tissue of the normal control group was purple-red, the nucleus was blue, there were one or several nucleoli, the volume was large, round and obvious, the nuclear membrane was clear, and the nuclear structure was relatively loose. The hepatocytes were arranged neatly and regularly, and were arranged radially with the central vein as the center. In the hyperlipidemia model group, obvious vacuoles appeared in the liver tissue, which appeared as round, colorless areas with clear boundaries, and were distributed more near the central vein area. The cytoplasm of hepatocytes became darker, the nucleus became smaller, and the nuclear staining became darker. However, no vacuoles appeared in the liver tissue of mice in the simvastatin group and the lipid-lowering tea group. The cells were arranged tightly, and the cytoplasm was stained darker, the nucleus was stained smaller, and the nuclear staining was also darker. It shows that a hyperlipidemia diet will cause an increase in fat in hepatocytes and accumulate fat content. Excessive fat accumulation affects the normal function of liver tissue and leads to vacuoles. Simvastatin and lipid-lowering tea can reduce the formation of lipid droplets in hepatocytes and improve the phenomenon of vacuoles in liver tissue, showing a good lipid-lowering effect.

[0082] In summary, the present invention explores the lipid-lowering effect and mechanism of lipid-lowering tea from many aspects through network pharmacology analysis and combined with animal experiments. Through research, it is confirmed that the main lipid-lowering active ingredients in lipid-lowering tea are tanshinone, flavonoids, ursolic acid and anthraquinone. These active ingredients play a lipid-lowering role by targeting PI3K-AKT signaling pathway, cancer pathogenesis pathway, estrogen signaling pathway, endocrine resistance, tyrosine kinase inhibitors and other signaling pathways. Among them, flavonoids can bind to the core target PPAGR, anthraquinone and ursolic acid can bind to the core targets HSP90AA and ESR1 respectively, regulate the activity of these target proteins and affect lipid synthesis or metabolism, thereby inhibiting the accumulation of adipose tissue and reducing fat index. At the same time, it can also reduce the synthesis of low-density lipoprotein and triglycerides in serum, promote the synthesis of high-density lipoprotein, inhibit the formation of lipid droplets in liver cells, reduce the growth of lipids, and play a lipid-lowering role in many aspects and pathways.

[0083] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A compound lipid-lowering tea, characterized in that: Including Cassia Seed 10g, Gynostemma Pentaphyllum 5-15g, Salvia miltiorrhiza 5-15g, Crataegus pinnatifida 5-15g, Bidens pilosa 5-15g.

2. The compound lipid-lowering tea according to claim 1, characterized in that: The ingredients include 10g Cassia seed, 5g Gynostemma pentaphyllum, 5g Salvia miltiorrhiza, 5g Crataegus pinnatifida, and 5g Bidens pilosa.

3. The compound lipid-lowering tea according to claim 2, characterized in that: The ingredients include 10g Cassia seed, 5g Gynostemma pentaphyllum, 10g Salvia miltiorrhiza, 10g Crataegus pinnatifida, and 10g Bidens pilosa.

4. The compound lipid-lowering tea according to claim 3, characterized in that: The ingredients include 10g Cassia seed, 5g Gynostemma pentaphyllum, 15g Salvia miltiorrhiza, 15g Crataegus pinnatifida, and 15g Bidens pilosa.

5. The compound lipid-lowering tea according to claim 4, characterized in that: The ingredients include 10g Cassia seed, 10g Gynostemma pentaphyllum, 5g Salvia miltiorrhiza, 10g Crataegus pinnatifida, and 15g Bidens pilosa.

6. The compound lipid-lowering tea according to claim 5, characterized in that: The ingredients include 10g Cassia seed, 15g Gynostemma pentaphyllum, 15g Salvia miltiorrhiza, 10g Crataegus pinnatifida, and 5g Bidens pilosa.

7. The compound lipid-lowering tea according to claim 6, characterized in that: The ingredients include 10g Cassia seed, 10g Gynostemma pentaphyllum, 15g Salvia miltiorrhiza, 5g Crataegus pinnatifida, and 10g Bidens pilosa.

8. The compound lipid-lowering tea according to claim 8, characterized in that: The ingredients include 10g Cassia seed, 15g Gynostemma pentaphyllum, 10g Salvia miltiorrhiza, 5g Crataegus pinnatifida, and 15g Bidens pilosa.

9. The method for preparing the compound lipid-lowering tea according to any one of claims 1 to 9, characterized in that: The following steps are involved: S1, pretreatment: clean and dry each Chinese medicine, and crush it into powder using a Chinese medicine grinder; S2. Weighing: Weigh the required weights of Cassia seed, Gynostemma pentaphyllum, Salvia miltiorrhiza, Crataegus pinnatifida, and Bidens pilosa respectively by weighing equipment, and add appropriate amount of water to the mixed powder and soak for a period of time; S3, using a high fire to boil the soaking liquid in S2 for a period of time, and then using a low fire to boil the soaking liquid for a period of time; S4, filter the decoction in S3 using gauze and collect the filtrate; S5. Add appropriate amount of water to the filtered medicinal residue and boil it over high heat for a period of time, then boil it over low heat for a period of time; S6, filtering the decoction in S5 using gauze, and combining the filtrate; S7. Place the filtrate in a water bath to concentrate and obtain the compound lipid-lowering tea.

10. The method for preparing the compound lipid-lowering tea according to claim 10, characterized in that: In step S2, the mixed powder is added with 1L of pure water and soaked for half an hour; in step S2, the soaking liquid is first decocted on high heat for 30 minutes, and then decocted on low heat for 1 hour; in step S5, the filtered medicinal residue is added with 0.5L of pure water and then decocted on high heat for 30 minutes, and then decocted on low heat for 1 hour; in step S7, the filtrate is placed in a 100°C water bath and concentrated to 100mL to obtain the compound lipid-lowering tea.

Images