An active component with antioxidant, anti-glycation and anti-inflammatory effects, its preparation method and application

Abstract

The present invention discloses an active component with antioxidant, anti-glycation and anti-inflammatory effects, as well as its preparation method and application. This component includes oleuropein, diosmin, neodiosmin, rosmarinic acid, oleuropein, salvianolic acid B, luteolin-7-O-glucoside, pectolinarigenin, ligustroside G13, 6'-O-trans-cinnamoyl-8-epigingkolic acid. Its preparation method uses Zhenzhu Tiaozhi Formula, Tongmai Formula, Baoshen Formula, etc. as raw materials, ultrasonic extraction with methanol, and the extract is separated and purified by preparative liquid chromatography or dynamic axial compression column chromatography, combined with real-time on-line ultraviolet spectrophotometric detection to obtain. Research shows that the active component of the present invention has good in vitro antioxidant, anti-glycation and anti-inflammatory activities, can significantly reduce the levels of inflammatory factors TNF-α and IL-1β in LPS-induced Raw 264.7 cells, and has a protective effect on LPS-induced liver and kidney damage in C57 / BL6J mice, and is suitable for application in the preparation of drugs for preventing or treating metabolic inflammatory diseases.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine research and development, and particularly relates to the development of an active component with antioxidant, anti-glycation and anti-inflammatory effects from hospital preparations, and its preparation method and application. Background Art

[0002] Glycolipid metabolic diseases are a group of diseases characterized by glycolipid metabolism disorders and involving multiple factors such as genetics, environment, and mental state. Oxidative stress and chronic inflammation are typical pathological features of such diseases. Zhenshu Tiaozhi Prescription, Tongmai Prescription, Baoshen Prescription, etc. are mainly composed of medicinal herbs such as bergamot, atractylodes rhizome, glossy privet fruit, thistle, eucommia bark, salvia miltiorrhiza, notoginseng, coptis chinensis, astragalus membranaceus, American ginseng, safflower, and hypericum japonicum, and have the effect of regulating the liver, activating the pivot, and resolving turbidity, and are mainly used for preventing and treating glycolipid metabolic diseases such as hyperlipidemia, non-alcoholic fatty liver, cardiovascular diseases, and diabetic nephropathy.

[0003] Free radicals are the products of normal metabolism in the body. Many intermediate metabolites in biochemical reactions in human tissues are accompanied by the generation and elimination of free radicals, and they have strong oxidation ability. However, when the production of free radicals in the human body is excessive or the elimination is restricted, that is, in a state of oxidative stress, the balance between antioxidant substances and pro-oxidant substances is disrupted, which will lead to cumulative oxidative damage of macromolecular substances (proteins, lipids, carbohydrates) in cells, accelerate the aging rate of the body, induce the occurrence of chronic diseases in the body, and endanger human life and health. In addition, in an environment of persistent hyperglycemia, macromolecular substances such as proteins, lipids, or nucleic acids spontaneously undergo non-enzymatic glycosylation reactions with glucose or other reducing monosaccharides to form stable covalent compounds, and the irreversible polymers formed are called advanced glycation end products (AGEs). The sources of AGEs in the human body can be divided into two categories. One is endogenous AGEs, which are produced by individuals through metabolism or the aging process, and the other is exogenous AGEs, which enter the human body through food, that is, from the outside. Their formation is a part of normal metabolism, but if their content is too high in tissues and the systemic circulation, it may cause diseases. The mechanism is to induce oxidative stress reactions by directly modifying proteins, lipids, nucleic acids, etc. or interacting with their receptors, causing various cells in the body to undergo hyperplasia, inflammation, fibrosis reactions, thrombosis, etc., and thus accelerating human aging or leading to the occurrence of various chronic degenerative diseases. Due to changes in modern living habits and the environment, metabolic disorders and metabolites, including free fatty acids (FFA) and endotoxins, polarize macrophages and induce chronic low-grade inflammation, also known as metabolic inflammation. The latter damages tissues and organs, leading to the occurrence of metabolic diseases such as atherosclerosis, type 2 diabetes (T2DM), non-alcoholic fatty liver, and obesity, and resulting in metabolic diseases.

[0004] Using modern separation analysis and formulation technologies to conduct secondary development and transformation research on hospital preparations with proven clinical efficacy, and then developing component traditional Chinese medicines with good efficacy and clear material basis is one of the important research contents in the modernization research of traditional Chinese medicines. Based on the core pathological mechanisms of oxidative stress, non-enzymatic glycosylation modification, and metabolic inflammation in glycolipid metabolic diseases, combined with in vitro and in vivo activity evaluations of antioxidant, anti-glycation, and anti-inflammatory activities, active ingredients are isolated, purified, and screened from the hospital preparations Zhenshu Tiaozhi Prescription, Tongmai Prescription, or Baoshen Prescription, which is a component traditional Chinese medicine new drug with great potential. Summary of the Invention

[0005] The primary object of the present invention is to provide an active component with antioxidant, anti-glycation, and anti-inflammatory effects, which has good in vitro antioxidant, anti-glycation, and anti-inflammatory activities.

[0006] Another object of the present invention is to provide a preparation method for the above-mentioned active component with antioxidant, anti-glycation, and anti-inflammatory effects. This method uses preparative liquid chromatography or dynamic axial compression column chromatography separation technology, combined with real-time on-line ultraviolet spectrophotometric detection, which has strong separation targeting for the target active ingredient, a compact separation and purification process, reduces the risk of pollution, has good repeatability, and high efficiency.

[0007] The present invention is achieved through the following technical solutions:

[0008] An active component with antioxidant, anti-glycation, and anti-inflammatory effects, by mass percentage, includes the following components:

[0009] Specnuezhenide 1 - 4%, Diosmin 0.5 - 3.5%, Neodiosmin 0.5 - 3.5%, Rosmarinic acid 1 - 4%, Oleuropein 2 - 8%, Salvianolic acid B 7.5 - 17.5%, Buddleoside 17.5% - 37.5%, Pectolinarigenin 1 - 9%, Ligustroside G1 39.5 - 29.5%, 6'-O-trans-cinnamoyl-8-epikingisidic acid 1 - 9%.

[0010] The present invention also provides a preparation method for the above-mentioned active component with antioxidant, anti-glycation, and anti-inflammatory effects, including the following steps:

[0011] (1) Using Zhenshu Tiaozhi Prescription, Tongmai Prescription, or Baoshen Prescription as raw materials, ultrasonic extraction is carried out with methanol. The extract is filtered and concentrated to obtain a methanol extract;

[0012] (2) The methanol extract is separated and purified by preparative liquid chromatography or dynamic axial compression column chromatography with a C18 reversed-phase silica gel bonded phase as the filler, gradient elution is carried out, combined with real-time on-line ultraviolet spectrophotometric detection, the elution fractions at specific time periods are collected, concentrated, and dried to obtain the product.

[0013] Preferably, in step (1), the number of times of ultrasonic extraction with methanol is 1 - 2 times, the extraction time is 20 - 40 min, and the solid - liquid ratio is 1:5 - 10.

[0014] Preferably, in step (2), the conditions for separation and purification are as follows: methanol is used as mobile phase A, and water containing 0.1% acetic acid is used as mobile phase B, with gradient elution. The elution program is as follows: 0 - 20 min, B is 5% - 40%; 20 - 50 min, B is 40% - 80%; 50 - 60 min, B is 80% - 95%; 60 - 70 min, B is 95%; 70 - 75 min, B is 95% - 5%; 75 - 80 min, B is 5%; the flow rate is 6 mL / min; the detection wavelengths are 210 nm and 254 nm.

[0015] Preferably, in step (2), the drying is freeze - drying or vacuum heating drying.

[0016] The present invention also provides the application of the above - mentioned active components with antioxidant, anti - glycation and anti - inflammatory effects in the preparation of drugs for preventing or treating glycolipid metabolic diseases. The metabolic inflammatory diseases include diabetes, non - alcoholic fatty liver, atherosclerosis, diabetic cardiomyopathy, neurodegenerative diseases, etc.

[0017] The present invention screens target separated components through antioxidant, anti - glycation and anti - inflammatory activity evaluations, including: inhibiting the decay of AAPH fluorescence by each separated component at the same concentration within the same time to evaluate the oxygen radical absorption capacity of each component; inhibiting the glycation binding of BSA and MGO by each separated component at the same concentration within the same time, and calculating the inhibition rate of glycation formation; mixing each separated component with cyclooxygenase 2 at the same concentration to inhibit the generation of fluorescence by the COX - 2 probe, and calculating the inhibition rate of COX - 2 enzyme. The results show that the above - prepared active ingredient has an antioxidant ORAC value of 4312 μmol TE / g at a concentration of 0.05 mg / mL, an inhibition rate of 77.78% on the fluorescence - based AGEs in the BSA - MGO system, and an inhibition rate of 84.19% on cyclooxygenase 2 (COX - 2), showing good in vitro antioxidant, anti - glycation and anti - inflammatory activities.

[0018] The present invention further determines the anti - inflammatory effect of the target component through a macrophage anti - inflammatory model. The target component and the specified inflammatory inducer LPS are added to the culture medium of macrophages, and the concentration of inflammatory factors in the culture supernatant is determined after the culture ends. The results show that the above - prepared active ingredient can significantly reduce the levels of inflammatory factors TNF - α and IL - 1β in LPS - induced Raw 264.7 cells.

[0019] The present invention further determines the in vivo anti-inflammatory activity of the target component through an animal model. Specifically, mice are randomly divided into a normal group, a model group, a dexamethasone group, and low- and high-dose F5 groups. The normal group and the model group are given normal saline, and the F5 group is given an F5 solution prepared with normal saline. The administration method is intraperitoneal injection. After continuous administration for 2 days, except for the normal group, the mice in the other groups are intraperitoneally injected with LPS 2 hours after administration to induce inflammation. After modeling, treatment administration is continued for one day. The results show that the active ingredient prepared above has a protective activity against LPS-induced liver and kidney damage in C57 / BL6 mice.

[0020] The dosage form of the drug of the present invention can be tablets, capsules, granules, oral liquids or injections, and pharmaceutically acceptable excipients or carriers can be added and prepared by conventional methods in the art.

[0021] Advantages of the present invention:

[0022] (1) The present invention adopts preparative chromatography or dynamic axial compression column chromatography technology, and through real-time UV-VIS detection and fraction collection, has a strong separation targeting for the target active ingredient, the separation and purification process is compact, reduces the risk of pollution links, has good repeatability, and high preparation efficiency;

[0023] (2) Experimental studies show that the active components screened by the present invention have good in vitro antioxidant, anti-glycation and anti-inflammatory activities, can significantly reduce the levels of inflammatory factors TNF-α and IL-1β in LPS-induced Raw 264.7 cells, and have a protective activity against LPS-induced liver and kidney damage in C57 / BL6 mice, and are suitable for use in the preparation of drugs for preventing or treating glycolipid metabolic diseases. Description of the Drawings

[0024] Figure 1 is the flow chart of the preparation method of the present invention;

[0025] Figure 2 is the semi-preparative liquid chromatogram of the FTZ extract;

[0026] Figure 3 is the screening result of the ORAC activity of the separated components F1-F6 in FTZ ((A) Fluorescence decay curves of Trolox with concentrations from 12.5 μm to 200 μm; (B) Regression equation of Trolox; (C) Fluorescence decay curves of FTZ and F1-F6 components at a concentration of 50 μg / mL; (D) ORAC values of FTZ and F1-F6 components at a concentration of 50 μg / mL);

[0027] Figure 4 is the result of the anti-glycation activity (A) and anti-inflammatory activity (B) of the separated components F1-F6 in FTZ;

[0028] Figure 5 FTZ-F5 improves the expression of inflammatory factors in LPS-induced RAW264.7 cells;

[0029] Figure 6 FTZ-F5 improves the method for establishing an animal model of acute liver and kidney injury induced by LPS;

[0030] Figure 7 The effects of FTZ-F5 intervention on liver function ((A) Detect the ALT level in the mouse liver, (B) Detect the AST level in the mouse liver, *P<0.05, **P<0.01, ***P<0.001, compared with the model group);

[0031] Figure 8 The effects of FTZ-F5 intervention on inflammatory factors ((A) Detect the TNF-α level in the mouse serum, (B) Detect the IL-1β level in the mouse serum, *P<0.05, **P<0.01, ***P<0.001, compared with the model group);

[0032] Figure 9 The effects of FTZ-F5 intervention on oxidative stress ((A) Detect the CAT level in the mouse liver, (B) Detect the GPx level in the mouse liver (C) Detect the MDA level in the mouse liver, *P<0.05, **P<0.01, ***P<0.001, compared with the model group);

[0033] Figure 10 The effects of FTZ-F5 intervention on the histopathology of liver and kidney tissues;

[0034] Figure 11 TIC map of FTZ-F5 in negative ion mode;

[0035] Figure 12 Qualitative mass spectrometry map of FTZ-F5 by LC-MS (A Ligustroside, B Diosmin, C Rosmarinic acid, D Oleuropein, E Neo-diosmin, F Salvianolic acid B, G Ligustroside G13, H Buddleoside, I Pectolinarigenin, J 6'-O-trans-cinnamoyl 8-epikingisidic acid);

[0036] Figure 13 The determination results of the contents of each component of FTZ-F5. Specific embodiments

[0037] The present invention will be further described below through specific embodiments. The following examples are specific embodiments of the present invention, but the embodiments of the present invention are not limited by the following examples.

[0038] Example 1: Preparation method of an active component with antioxidant, anti-glycation and anti-inflammatory effects

[0039] Experimental reagents and instruments: The Zhenzhu Tiaozhi Capsules (FTZ) were provided by the Pharmacy Department of the First Affiliated Hospital of Guangdong Pharmaceutical University (batch number: 200501); methanol (Tianjin Zhiyuan Chemical Reagent Co., Ltd.); formic acid (Tianjin Kemiou Chemical Reagent Co., Ltd.); Newstyle preparative liquid chromatography (Jiangsu Hanbang Technology Co., Ltd.); Alpha 1-2LD plus freeze dryer (CHRIST company); RV10 rotary evaporator (IKA company).

[0040] 1. Raw material treatment and crude extraction

[0041] Take out the contents in the shell of the Zhenzhu Tiaozhi Capsules (batch number: 200501), add methanol for dissolution according to the material-liquid ratio of 1:7, ultrasonically extract for 30 minutes, filter out the supernatant, add 50% methanol to the recovered residue for the second ultrasonic extraction for 30 minutes, then combine the supernatants, and concentrate by rotary evaporation under reduced pressure at 60 °C to obtain the methanol extract of FTZ.

[0042] 2. Component separation and purification

[0043] Take an appropriate amount of the methanol extract of FTZ, filter it through a 0.45 μm nylon filter membrane, and then separate and purify it by preparative liquid chromatography equipped with a Luna C 18 chromatographic column (250×10 mm, 10 μm, Phenomenex company).

[0044] Method 1: Mobile phase A is water (containing 0.1% acetic acid), B is methanol, gradient elution: 0 - 30 min, B is 5% - 30%; 30 - 60 min, B is 30% - 70%; 60 - 71 min, B is 70% - 100%; 71 - 80 min, B is 5%; the injection volume is 200 μL, the wavelengths are 254 nm and 320 nm, and the flow rate is 4 mL / min. The chromatographic peaks of this method are mainly concentrated around 50 min, and the separation effect is not good.

[0045] Method 2: Mobile phase A is water (containing 0.1% acetic acid), B is methanol, gradient elution: 0 - 30 min, B is 5% - 40%; 30 - 70 min, B is 40% - 80%; 70 - 80 min, B is 80% - 95%; 80 - 85 min, B is 95%; 85 - 95 min, B is 95% - 5%; the injection volume is 200 μL, the wavelengths are 254 nm and 210 nm, and the flow rate is 5 mL / min. The chromatographic peaks of this method have a good separation effect, but the separation time is longer.

[0046] Method 3: Mobile phase A is water (containing 0.1% acetic acid); mobile phase B is methanol. The gradient elution program is as follows: from 0 to 20 min, B is 5% - 40%; from 20 to 50 min, B is 40% - 80%; from 50 to 60 min, B is 80% - 95%; from 60 to 70 min, B is 95%; from 70 to 75 min, B is 95% - 5%; from 75 to 80 min, B is 5%. The injection volume is 1.0 mL, the flow rate is 6 mL / min, and the UV detection wavelengths are 210 nm and 254 nm. This method has good separation effect, shorter time, and better repeatability. Therefore, Method 3 is selected as the separation and purification condition of the present invention.

[0047] Collect the effluents of the first 60 minutes every 10 minutes in chronological order. F1 is the effluent from 0 - 10 min, F2 is the effluent from 10 - 20 min, F3 is the effluent from 20 - 30 min, F4 is the effluent from 30 - 40 min, F5 is the effluent from 40 - 50 min, and F6 is the effluent from 51 - 60 min. Inject samples continuously for several times, combine the effluents with the same retention time, obtain six fractions of sample solutions F1 - F6, then remove the organic solvents by rotary evaporation under reduced pressure respectively, and place them in a vacuum freeze dryer for freeze-drying to obtain the freeze-dried powders of the six fractions F1 - F6. The results are as Figure 2 。

[0048] Example 2: Screening target separated components through in vitro antioxidant, anti-glycation and anti-inflammatory activities

[0049] Experimental reagents and instruments: Methanol (Tianjin Zhiyuan Chemical Reagent Co., Ltd.); phosphate (PBS) buffer solution (Xiamen Haibiao Technology Co., Ltd.); fluorescein sodium (FL), 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH), water-soluble vitamin E (Trolox) are all purchased from Shanghai Macklin Biochemical Co., Ltd.; COX-2 inhibitor screening kit (Beyotime Biotechnology Co., Ltd.); Spectra Max i3x type multifunctional microplate reader (Molecular Devices Co., Ltd.);

[0050] 1. Determination of antioxidant activity by ORAC method

[0051] All the reagents required for this experiment were prepared with 10 mM PBS. 25 μL of different sample solutions or Trolox standard solutions (positive control) with gradient concentrations of 50 μg / mL were respectively pipetted into 96-well plates. Then, 150 μL of fluorescein solution (10 nM) was quickly added. After gently shaking to mix, the mixture was incubated at 37 °C for 30 min. Then, 25 μL of 240 mM AAPH was quickly added. The excitation wavelength was set at 480 nm and the emission wavelength was set at 520 nm. The fluorescence intensity was measured every 90 s, and the temperature of the system was kept constant at 37 °C during the measurement. The measurement was stopped after the fluorescence decay was stable. Fluorescence measurements were performed using PBS buffer solution (10 mM) as the blank control, and 3 replicates were set for each sample. The relative fluorescence intensity decay curves were plotted with the relative fluorescence intensity of the sample and the positive control Trolox on the vertical axis and time on the horizontal axis. The ORAC value was expressed in Trolox equivalents, with the unit of μmol TE / g.

[0052] 2. Determination of anti-glycation activity by BSA-MGO method

[0053] All the reagents used in this experiment were freshly prepared with PBS (pH 7.4, containing 0.02% sodium azide) at a concentration of 0.2 M. The positive control AG and the samples were prepared as 0.05 mg / mL solutions with PBS as the solvent. Equal proportions of 15 mg / mL BSA solution, 0.5 mM MGO solution, and AG solution or sample solution were added to test tubes. The blank control was prepared by replacing the sample solution with PBS. The tubes were sealed and incubated in the dark at 37 °C for 7 days. After the reaction ended, the total fluorescence AGEs in the reaction system were measured, and the set program was an excitation wavelength of 340 nm and an emission wavelength of 420 nm.

[0054] 3. Screening of COX-2 anti-inflammatory activity

[0055] The operation was carried out according to the instructions of the COX-2 inhibitor screening kit manufacturer. In a 96-well plate, 75 μL of COX-2 detection buffer, 5 μL of COX-2 cofactor working solution, 5 μL of COX-2 working solution, and 5 μL of the sample solution to be tested were incubated at 37 °C for 10 min. After the incubation ended, 5 μL of COX-2 Probe was added to each well, and then 5 μL of COX-2 Substrate was quickly added to all wells. After incubating again in the dark at 37 °C for 5 min, the fluorescence value of each well was measured using an excitation wavelength of 560 nm and an emission wavelength of 590 nm.

[0056] The results are shown in Figure 3 and Figure 4, when the concentration of component F5 is 0.05 mg / mL, its antioxidant ORAC value is 4312 μmol TE / g, the inhibition rate of fluorescent AGEs in the BSA-MGO system is 77.78%, and the inhibition rate of cyclooxygenase 2 (COX-2) reaches 84.19%. These results indicate that component F5 has the best in vitro activity among the six separated components of FTZ.

[0057] Example 3: Determine the anti-inflammatory effect of the target component F5 through a macrophage anti-inflammatory model

[0058] Cells: Mouse macrophages RAW264.7, purchased from the Chinese Academy of Sciences.

[0059] Experimental reagents and instruments: High-glucose DMEM medium, inactivated fetal bovine serum; double antibodies, DMSO were all purchased from Gibco, lipopolysaccharide (Escherichia coli O55:B5, Sigma-Aldrich), mouse TNF-α, IL-6, IL-1β enzyme-linked immunosorbent assay (ELISA) kits (Wuhan Huamei Bio-Engineering Co., Ltd.), CO2 incubator, inverted microscope, laminar flow hood, Spectra Max i3x multi-functional microplate reader;

[0060] Inoculate RAW264.7 cells in a 96-well plate at an inoculation density of 2x10 5 / mL, 100 μL per well. After the cells are inoculated, culture them in a CO2 incubator for 24 hours. After the culture is completed, divide the cells into an experimental group, a model group, and a control group. Add high-glucose DMEM medium containing 0.1 μg / mL of component F5 to the experimental group. After incubating for 1 hour, add high-glucose DMEM medium containing 1 μg / mL LPS to the model group and the experimental group. After culturing for 24 hours, take the cell supernatants of each group and measure the concentrations of inflammatory factors TNF-α and IL-1β in the supernatants according to the kit instructions.

[0061] The results are shown in Figure 5 , component F5 can significantly reduce the concentrations of inflammatory factors in the cell supernatants induced by LPS.

[0062] Example 4: Determine the in vivo anti-inflammatory activity of the target component F5 through an animal model

[0063] Experimental reagents: FTZ-F5 was prepared in the laboratory; lipopolysaccharide (Escherichia coli O55:B5, Sigma-Aldrich); dexamethasone (Shanghai Macklin Biochemical Co., Ltd.); mouse TNF-α and IL-1β enzyme-linked immunosorbent assay (ELISA) kits (Wuhan Huamei Bio-Engineering Co., Ltd.); AST and ALT (Nanjing Jiancheng Bioengineering Institute); lipid oxidation detection kit, glutathione peroxidase detection kit, catalase detection kit and BCA protein concentration assay kit (Beyotime Biotechnology); hematoxylin and eosin staining solution (LEAGENE); neutral gum (Sinopharm Chemical Reagent Co., Ltd.); 4% paraformaldehyde (Biosharp); sodium chloride injection (Henan Kelun Pharmaceutical Co., Ltd.).

[0064] Experimental instruments: RM2235 paraffin slicer (Leica); ZT-12 automatic tissue dehydrator and YB-LF paraffin embedding machine (Yaguang Medical Electronics Co., Ltd.); Olympus BX53 microscope (Olympus); TissueLyser II tissue grinder (QIAGEN); microplate reader (BioTek); low-temperature high-speed centrifuge (Eppendorf).

[0065] As Figure 6 shown, 50 mice were randomly divided into a normal group, a model group, a dexamethasone group (10 mg / kg), low- and high-dose F5 groups (20, 40 mg / kg). The normal group and the model group were given normal saline, and the F5 groups were given F5 solution prepared with normal saline. The administration method was intraperitoneal injection, and the administration volume was 10 mL / kg. The administration time was every morning. After continuous administration for 2 days, except for the normal group, the mice in the other groups were intraperitoneally injected with 5 mg / kg of LPS 2 hours after administration to induce acute liver and kidney injury. After modeling, treatment and administration were continued for one day. 24 hours after the last administration, blood samples were collected by orbital blood sampling, and then the mice were immediately sacrificed by cervical dislocation. The liver and kidney tissues were isolated. After rinsing the mouse liver and kidney with normal saline, an appropriate amount of liver and kidney was fixed in 4% paraformaldehyde. The remaining liver and kidney tissues were quickly frozen in liquid nitrogen and stored at -80 °C for later use. The blood samples were placed at room temperature for about 2 hours, centrifuged at 3500 rpm for 15 minutes at 4 °C, and the supernatant serum samples were stored at -80 °C for later use. According to the kit instructions, the levels of serum inflammatory factors (TNF-α, IL-1β), oxidative stress indexes MDA, GSH-Px, CAT, and liver function indexes AST and ALT were measured. The liver and kidney tissues of the mice were fixed in 4% paraformaldehyde, dehydrated through gradient ethanol, cleared with xylene, infiltrated with wax and embedded to make sections. After HE staining, the pathological conditions of the liver and kidney tissues were observed under a microscope and photographed.

[0066] The results are shown in Figure 7 , 8 , 9. Compared with the model group, F5 could significantly reduce the levels of inflammatory factors (TNF-a, IL-1β) and oxidative stress (MDA, GSH-Px, CAT) in the serum of mice, and at the same time reduce the levels of AST and ALT in the liver. The pathological results are as Figure 10 shown. The renal tissue morphology of the normal control group was normal, and no pathological changes were observed; while in the LPS group, there were inflammatory cell infiltrations in the kidneys, the glomeruli became larger, the renal tubules dilated, the renal tubular epithelial cells exfoliated, the brush border exfoliated, and vacuoles appeared; compared with the LPS group, the degree of inflammatory cell infiltration in the kidneys of the F5 intervention group and the dexamethasone group was reduced, and the renal tissue damage was alleviated. The hepatic tissue structure of the control group was clear, the hepatocyte morphology was normal, and the arrangement was regular; compared with the control group, the outline of the hepatic lobules in the model group was blurred, the hepatic cords were arranged disorderly, diffuse vacuoles appeared, and there were more inflammatory cell infiltrations; compared with the model group, the degree of inflammatory cell infiltration in the F5 intervention group was slight, the vacuoles decreased, the structure of the hepatic lobules was clearer, and the pathological changes of the liver were improved to a certain extent.

[0067] Example 5: Analysis of the main chemical components and contents of FTZ-F5

[0068] Experimental reagents and instruments: The reference substances such as ligustroside, diosmin, neodiosmin, lithospermic acid, rosmarinic acid, salvianolic acid, oleuropein, buddleoside, pectolinarigenin, and 6'-O-trans-cinnamoyl-8-epigingiberoside acid were all purchased from Chengdu Efar Biotechnology Co., Ltd.; acetonitrile (Sigma-Aldrich); experimental instruments: one over one hundred thousand balance (OHAUS); high performance liquid chromatography (DIONEX); UHPLC Q-TOF-MS liquid chromatography-quadrupole time-of-flight liquid chromatography-mass spectrometry system (model 2695-6840, Agilent Technologies Co., Ltd.).

[0069] LC-MS chromatographic conditions: An Agilent Technologies ZORBAX RRHD C18 chromatographic column (2.1×100mm, 1.8μm) was used; mobile phase A was acetonitrile, and mobile phase B was 0.05% formic acid aqueous solution (0 - 4 min, A was 5% - 10%; 4 - 10 min, A was 10% - 12%; 10 - 11 min, A was 12%; 11 - 16 min, A was 12% - 25%; 16 - 30 min, A was 25% - 95%; 30 - 33 min, A was 95% - 5%; 33 - 35 min, A was 5%); the flow rate was 0.3 mL·min -1 , and the elution temperature was room temperature, and the sample injection volume was 2 μL.

[0070] LC-MS Mass Spectrometry Conditions: Dual AJS ion source (ESI) was used, with positive and negative ion scanning modes, and the nozzle voltage was 1000 V; the sheath gas temperature was 350 °C and the flow rate was 11 L / min; the dryer temperature and flow rate were 320 °C and 8 L·min -1 , the nebulizer gas pressure was 35 psi. The capillary voltage was 4000 V, the MS collision energy was 10, 20, 30 eV; the scanning ion range was m / z 50 - 1000.

[0071] HPLC Quantitative Analysis Chromatography Conditions: Kromasil C18 chromatographic column (Akzonobel, Sweden, 250 mm × 4.6 mm, 5 μm) was used, and the mobile phase was acetonitrile (A) and 0.05% formic acid in water (B). The gradient program was as follows: 0 - 10 min, 80% - 75% of B; 10 - 22 min, 75% - 74% of B; 22 - 35 min, 74% - 72% of B; 35 - 42 min, 28% - 90% of B; the flow rate was 1 mL·min -1 , the column temperature was 30 °C, the sample volume was 10 μL, and the detection wavelength was 254 nm.

[0072] The results are shown in Figure 11 、 12 、13. The main chemical components and contents of F5 are as follows: Ligustroside (1.51%), Diosmin (1.05%), Neodiosmin (1.09%), Rosmarinic acid (1.42%), Oleuropein (3.08%), Salvianolic acid B (12.57%), Buddleoside (27.46%), Pectolinarin (4.02%), Ligustroside G13 (19.53%), 6’-O-trans-cinnamoyl-8-epigingesidic acid (3.84%).

Claims

1. An active component with antioxidant, anti-glycation and anti-inflammatory effects, characterized in that, By mass percentage, it includes the following components: 1-4% of oleuropein aglycone, 0.5-3.5% of diosmin, 0.5-3.5% of neodiosmin, 1-4% of rosmarinic acid, 2-8% of oleuropein, 7.5-17.5% of salvianolic acid B, 17.5%-37.5% of buddleoside, 1-9% of pectolinarigenin, 9.5-29.5% of ligustroside G13, 1-9% of 6’O-trans-cinnamoyl-8-epikingisidic acid; The preparation method of the active component with antioxidant, anti-glycation and anti-inflammatory effects includes the following steps: (1) Using Zhenzhu Tiaozhi Formula as the raw material, adding methanol to dissolve according to the solid-liquid ratio of 1:7, ultrasonically extracting for 30 minutes, filtering out the supernatant, recycling the residue, adding 50% methanol for the second ultrasonic extraction for 30 minutes, then combining the supernatants, and concentrating by rotary evaporation under reduced pressure at 60 °C to obtain a methanol extract; (2) Separating and purifying the methanol extract by preparative liquid chromatography with C18 reversed-phase silica gel bonded phase as the filler, combining with real-time online ultraviolet spectrophotometric detection, collecting the elution fractions at 40-50 min, concentrating and drying to obtain; The conditions for the separation and purification are as follows: methanol as mobile phase B and water containing 0.1% acetic acid as mobile phase A, gradient elution, the elution program is 0-20 min, B is 5%-40%; 20-50 min, B is 40%-80%; 50-60 min, B is 80%-95%; 60-70 min, B is 95%; 70-75 min, B is 95%-5%; 75-80 min; B is 5%; flow rate 6 mL / min; detection wavelengths 210 nm and 254 nm.

2. The application of the active component with antioxidant, anti-glycation and anti-inflammatory effects described in claim 1 in the preparation of drugs for preventing or treating glycolipid metabolic diseases.

3. The application according to claim 2, characterized in that The glycolipid metabolic diseases include diabetes, non-alcoholic fatty liver, atherosclerosis or diabetic cardiomyopathy.

4. The application according to claim 2, characterized in that, The active component can significantly reduce the levels of inflammatory factors TNF-α and IL-1β in LPS-induced Raw264.7 cells.

5. The application according to claim 2, characterized in that, The active component has a protective activity against LPS-induced liver and kidney injuries in C57 / BL6J mice.

6. The application according to claim 2, characterized in that, The dosage form of the drug is tablets, pills, capsules, granules, oral liquids or injections.

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