Sanguisorba officinalis extract and application of functional components of sanguisorba officinalis extract in treatment of microangiopathy

Abstract

The invention belongs to the technical field of biological pharmacy, and provides application of a sanguisorba officinalis extract and functional components thereof in treatment of microangiopathy, and the application is that the sanguisorba officinalis extract and a preparation thereof are applied to preparation of a medicine for treating microangiopathy. According to the invention, a diabetic coronary artery microvessel (diabetic CMVD) injury model is established through combination of STZ and high-fat feed induction, and the treatment effect of the prepared sanguisorba officinalis extract and the functional component ziyuglycoside II thereof on microangiopathy is evaluated; results show that the sanguisorba officinalis extract and the functional components thereof can achieve the purpose of treating microvessels by improving lipid metabolism and insulin secretion of diabetic CMVD mice, myocardial injury of the diabetic CMVD mice, chronic inflammation of the diabetic CMVD mice, kidney microvessel injury of the diabetic CMVD mice and heart microvessel injury of the diabetic CMVD mice.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and relates to an extract of Sanguisorba officinalis, and particularly to the application of an extract of Sanguisorba officinalis in the preparation of a medicament for treating microvascular lesions. Background Art

[0002] Microvascular lesions refer to pathological changes occurring in microvessels other than large, medium and small vessels, mainly involving microcirculation systems such as capillaries, arterioles and venules, and can affect multiple organs and tissues such as the heart, kidneys, nerves and retina. The inducing factors of microvascular lesions include hypertension, hyperlipidemia, hyperuricemia, hyperglycemia, autoimmunity and thrombosis. Single or multiple factors cause damage to vascular endothelial cells, increase the permeability of the vascular wall, lipid deposition in the blood to form plaques, and endothelial hyperplasia of the vascular wall, thereby triggering inflammation and fibrosis, resulting in vascular stenosis or occlusion. Currently, the microvascular diseases with clear diagnostic criteria mainly include thrombotic microangiopathy (TAM) and coronary microvascular disease (CMVD). Among them, TAM is manifested as microangiopathic hemolytic anemia, thrombocytopenia and organ involvement caused by thrombocytopenia in the microcirculation, and the kidney is the most commonly involved organ. TAM includes primary and secondary; drugs, tumors, infections, autoimmunity and malignant hypertension are all risk factors for secondary TAM. CMVD is mainly caused by structural and functional abnormalities of precoronary arterioles, arterioles and capillaries under the action of atherosclerotic and non-atherosclerotic pathogenic factors, resulting in acute and chronic myocardial ischemia. CMVD is an independent risk predictor for cardiovascular events, and the heart is its main involved organ. In addition to being related to atherosclerosis, traditional risk factors such as smoking, hypertension, hyperlipidemia and diabetes can also promote the occurrence and development of CMVD. CMVD not only affects the heart, but may also be a systemic disease, and patients may also have small vessel diseases in the brain, retina or kidneys, such as retinopathy, kidney and heart dysfunction caused by microvascular lesions in diabetic patients. Currently, the treatment of CMVD mainly includes controlling risk factors, improving lifestyle and selecting β-blockers, calcium channel blockers, nicorandil and trimetazidine, etc. according to the symptoms. However, although Western medicine treatment can improve the symptoms of CMVD patients, the treatment effect on microcirculation dysfunction is limited. Traditional Chinese medicine has unique advantages in improving microcirculation dysfunction. Therefore, there is great potential in searching for drugs for treating microvascular lesions from traditional Chinese medicine.

[0003] Sanguisorba officinalis L. or the dried root of Sanguisorba officinalis L. var. longifolia (Bert.) Yü et Li of the Rosaceae family enters the liver and large intestine meridians, and its effects are to cool the blood and stop bleeding, and to detoxify and astringe sores. Modern research has found that Sanguisorba officinalis contains various chemical components such as triterpenoid saponins, flavonoids, and tannins, and has various pharmacological activities such as hemostasis and blood coagulation, antioxidant, anti-inflammatory, antibacterial, and anti-tumor effects.

[0004] Previous studies of the present invention have found that the extract of Sanguisorba officinalis and its main active ingredient, sanguisorboside 2, can improve LPS-induced vascular endothelial cell injury in vitro, inhibit the migration of endothelial cells, and reduce the pro-inflammatory factors TNF-α and IL-6 in the supernatant of endothelial cell culture. And there is no report in the prior art on whether the extract of Sanguisorba officinalis and its active ingredients have a therapeutic effect on CMVD. Hyperglycemia is one of the important pathological factors promoting CMVD. Therefore, the present invention uses STZ combined with a high-fat diet to induce and establish a diabetic coronary microvascular injury model to evaluate the therapeutic effect of the extract of Sanguisorba officinalis and its active ingredient, sanguisorboside 2, on CMVD, providing a new way and means for the treatment of CMVD. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of the extract of Sanguisorba officinalis and its active ingredients in the treatment of microvascular lesions, and to apply the extract of Sanguisorba officinalis to the preparation of drugs for the treatment of CMVD diseases, providing a new way and means for the treatment of microvascular lesions, so as to solve the problem that Western medicine treatment can only improve the symptoms of CMVD patients and has limited therapeutic effects on microcirculation dysfunction.

[0006] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0007] The present invention provides an application of an extract of Sanguisorba officinalis in the preparation of drugs for the treatment of microvascular lesions.

[0008] It should be noted here that microvascular lesions include thrombotic microvascular lesions and coronary microvascular lesions.

[0009] Preferably, the active ingredients contained in the extract of Sanguisorba officinalis include at least sanguisorboside II.

[0010] Preferably, the extract of Sanguisorba officinalis is one of the water extract of Sanguisorba officinalis, the methanol extract of Sanguisorba officinalis, the ethanol extract of Sanguisorba officinalis, or the acid extract of Sanguisorba officinalis.

[0011] Preferably, the extract of Sanguisorba officinalis further includes a preparation of the extract of Sanguisorba officinalis.

[0012] Preferably, the preparation of the extract of Sanguisorba officinalis is made into an oral preparation or an injection preparation by adding excipients permitted in medicine to the extract of Sanguisorba officinalis.

[0013] Preferably, the oral preparation is a tablet, a capsule, a pill, a granule or an oral liquid preparation.

[0014] Preferably, the treatment of microvascular lesions is achieved by improving renal microvascular injury.

[0015] Preferably, the treatment of microvascular lesions is achieved by reducing chronic inflammation.

[0016] Preferably, the treatment of microvascular lesions can improve myocardial injury.

[0017] Preferably, the treatment of microvascular lesions is achieved by improving lipid metabolism and insulin secretion function, and the microvascular lesions are caused by hyperglycemia.

[0018] Preferably, the preparation method of the sanguisorba extract is as follows: Weigh an appropriate amount of sanguisorba medicinal materials and crush them, add an extraction solvent and reflux for 0.5 - 2 h, extract 1 - 3 times, filter and combine the filtrates, concentrate, and spray-dry to obtain the sanguisorba extract; the volume ratio of the extraction solvent to the mass of the sanguisorba medicinal materials is 6 - 15.

[0019] Preferably, the volume fraction of ethanol in the extraction solvent is 0 - 99%, and the mesh number of the crushed sanguisorba medicinal materials is 5 - 20 mesh.

[0020] Advantages of the present invention:

[0021] By using STZ combined with a high-fat diet to induce the establishment of a diabetic coronary microvascular injury model, the present invention evaluates the treatment effect of the sanguisorba extract prepared by the present invention and its active ingredient - sanguisorboside II on microvascular diseases; the results show that the sanguisorba extract and its active ingredient can improve lipid metabolism and insulin secretion in diabetic CMVD mice, improve myocardial injury in diabetic CMVD mice, reduce chronic inflammation in diabetic CMVD mice, reduce renal microvascular injury in diabetic CMVD mice, and improve cardiac microvascular injury in diabetic CMVD mice so as to achieve the purpose of treating microvascular diseases. Description of the Drawings

[0022] Figure 1 is the HPLC chromatogram of the sanguisorba extract in the present invention (A is the mixed standard, B is the sanguisorba extract; 1 is sanguisorboside I, 2 is sanguisorboside II);

[0023] Figure 2These are the result graphs showing the improvement of lipid metabolism and insulin secretion in diabetic CMVD mice by the Sanguisorba officinalis extract and its active ingredients in the present invention (Graph A shows the results of the fasting blood glucose (FBG) content in mice, Graph B shows the results of the triglyceride (TG) content in the liver of mice, Graph C shows the results of the total cholesterol ester (TC) content in the liver of mice, and Graph D shows the results of the secretion of insulin and glucagon in the islets of mice; among them, Normal is the normal group, DM-CMVD is the model group, DYE is the Sanguisorba officinalis extract group, Ziyu II is the ziyu II group, and Metformin is the positive control metformin group; ## P < 0.01, compared with the Normal group; * P < 0.05, ** P < 0.01, compared with the DM-CMVD group);

[0024] Figure 3 These are the result graphs showing the improvement of myocardial injury in diabetic CMVD mice by the Sanguisorba officinalis extract and its active ingredients in the present invention (Graph A shows the activity of creatine kinase (CK) in serum, Graph B shows the activity of creatine kinase isoenzyme (CK-MB) in serum, Graph C shows the activity of lactate dehydrogenase (LDH) in serum; Graph E shows the left ventricular ejection fraction (LVEF); Graph F shows the left ventricular fractional shortening (LVFS); ## P < 0.01, compared with the Normal group; * P < 0.05, ** P < 0.01, compared with the DM-CMVD group);

[0025] Figure 4 These are the result graphs showing the alleviation of chronic inflammation in diabetic CMVD mice by the Sanguisorba officinalis extract and its active ingredients in the present invention (Graph A shows the content of interleukin-1β (IL-1β) in serum, Graph B shows the content of tumor necrosis factor-α (TNF-α) in serum, and Graph C shows the content of interleukin-6 (IL-6) in serum; ## P < 0.01, compared with the Normal group; * P < 0.05, ** P < 0.01, compared with the DM-CMVD group);

[0026] Figure 5 These are the result graphs showing the alleviation of renal microvascular injury in diabetic CMVD mice by the Sanguisorba officinalis extract and its active ingredients in the present invention (Graph A shows the concentration of creatinine (Cr) in serum, Graph B shows the concentration of blood urea nitrogen (BUN) in serum, Graph C shows the mRNA expression level of Collagen IV in the kidney, Graph D shows the microvessels in the kidney labeled with CD31 and Collagen IV, and Graph E shows the fluorescence intensity of Collagen IV; among them, CD31 is a marker of vascular endothelial cells, also known as platelet endothelial cell adhesion molecule (PECAM-1), and Collagen IV is type IV collagen; ## P < 0.01, compared with the Normal group; *P < 0.05, ** P < 0.01, compared with the DM-CMVD group);

[0027] Figure 6 It is the result diagram of the improvement of cardiac microvascular injury in diabetic CMVD mice by the sanguisorba extract and its active ingredients in the present invention (A is the blood vessels in the heart labeled with CD31 and Collagen IV, B is the cross-sectional thickness of the blood vessels labeled with Collagen IV, C is the mRNA expression level of Collagen IV in the heart, D is the mRNA expression level of VEGF in the heart, E is the mRNA expression level of ICAM-1 in the heart, where VEGF is vascular endothelial growth factor and ICAM-1 is intercellular adhesion molecule-1; ## P < 0.01, compared with the Normal group; * P < 0.05, ** P < 0.01, compared with the DM-CMVD group). Detailed implementation manners

[0028] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0029] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0030] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0031] Embodiment

[0032] Taking the contents of sanguisorbin I and sanguisorbin II as the inspection indexes, taking the solid-liquid ratio, crushing particle size, ethanol concentration, extraction time and extraction times as the inspection factors, an L16(4 5 ) orthogonal test table was selected and quasi-level correction was carried out. The factor level table is shown in Table 1.

[0033] Table 1 Orthogonal test factors and levels

[0034]

[0035] The extraction liquids in the orthogonal test were respectively concentrated under reduced pressure, ethanol was recovered until there was no alcohol smell in the concentrated liquid, and then spray-dried to obtain the sanguisorba extract. The following method was used to detect the contents of sanguisorbin I and sanguisorbin II in the sanguisorba extract:

[0036] C18 chromatographic column (250×4.6 mm, 5 μm); the column temperature was 35 °C, the detection wavelength was 203 nm, the injection volume was 10 μL, and the flow rate was 1.0 mL / min. Acetonitrile (A)-0.1% phosphoric acid water (B) was used as the mobile phase, and the gradient elution program was 65% B from 0 to 13 min; 65%→50% B from 13 to 22 min; 50% B from 22 to 40 min.

[0037] Preparation of the mixed standard solution: Weigh appropriate amounts of the reference substances of saponin I and saponin II from Sanguisorba officinalis, and dissolve them in methanol to prepare a mixed standard solution with the concentrations of saponin I and saponin II from Sanguisorba officinalis being 0.5 mg / mL and 1.0 mg / mL;

[0038] Preparation of the sample solution: Weigh an appropriate amount of the Sanguisorba officinalis extract, add methanol and dissolve it by ultrasonic treatment, then cool and make up the volume.

[0039] Using the Analytic Hierarchy Process (AHP), the weight coefficients (ω1) of saponin I and saponin II from Sanguisorba officinalis in the Sanguisorba officinalis extract were assigned as 50% and 50%, and the comprehensive scoring method was used for analysis. The results are shown in Table 2.

[0040] Table 2 Results of the orthogonal experiment

[0041]

[0042]

[0043] It can be seen from the results of the variance analysis that the influence degrees of various factors on the extraction process of Sanguisorba officinalis were C>B>A>E>D. According to the analysis results, the optimal extraction process of Sanguisorba officinalis was determined as A2B3C3D1E2, that is: the Sanguisorba officinalis medicinal materials were crushed to 20 meshes, added with 10 times the amount of 60% ethanol, refluxed for 1 h, extracted twice, and the two filtrates were combined and spray-dried to obtain the product.

[0044] It should be noted that the Sanguisorba officinalis medicinal materials (sliced medicinal materials) used in the examples were purchased from Shaanxi Duoyao Chinese Herbal Pieces Co., Ltd. and were identified as the dried roots of the plant Sanguisorba officinalis of the Rosaceae family. Each 1 g of the Sanguisorba officinalis extract obtained by using the optimal extraction process of Sanguisorba officinalis in the examples was equivalent to 3.3 g of the raw Sanguisorba officinalis medicinal materials. The contents of saponin I and saponin II from Sanguisorba officinalis in this Sanguisorba officinalis extract were 9.80% and 1.95% respectively detected by HPLC (the HPLC chromatogram of the Sanguisorba officinalis extract is shown in Figure 1 )

[0045] Application examples

[0046] A diabetic coronary microvascular injury model was established by combining streptozotocin (STZ) with a high-fat diet. The therapeutic effect of the sanguisorba extract prepared by the optimal extraction process of sanguisorba and its active ingredient, sanguisorboside II, on CMVD was verified in the examples.

[0047] 1. Experimental instruments and materials

[0048] 1.1 Instruments

[0049] DK-S28 constant temperature water bath (Shanghai Jinghong Experimental Equipment Co., Ltd.), ME204 analytical balance (Mettler Toledo, Switzerland), Vevo 770 small animal ultrasound system (Visual Sonics, Canada), Multiskan FC microplate reader (Thermo Fisher, USA), UV ultrapure water system (Merck Millipore, Germany), Nikon Eclipse C1 fluorescence microscope (Nikon, Japan), 480 fluorescence quantitative PCR instrument (Roche, Switzerland), Nikon Eclipse E100 optical microscope (Nikon, Japan).

[0050] 1.2 Materials

[0051] Sanguisorba officinalis extract (obtained by the preparation method of the examples, with the content of saponin II being 1.95%), saponin II (Chengdu Purigen Biotech Co., Ltd., purity > 98.5%), Streptozoci (sigma, product number: S0130), 45% kcal high-fat diet for mice (Dietz Biotech Co., Ltd., product number: 102629), sodium citrate buffer (PH4.5), Roche blood glucose meter (Johnson & Johnson, USA), blood glucose test strips (Johnson & Johnson, USA), Amplex Red triglyceride (TG) detection kit (Beyotime Biotechnology Co., Ltd., product number: S0219S), Amplex Red cholesterol and cholesteryl ester (TC) detection kit (Beyotime Biotechnology Co., Ltd., product number: S0211S), mouse interleukin 1β (IL-1β) enzyme-linked immunosorbent assay kit (Elabscience, product number: E-EL-M0037), mouse tumor necrosis factor α (TNF-α) enzyme-linked immunosorbent assay kit (Elabscience, product number: E-EL-M3063), mouse interleukin 6 (IL-6) enzyme-linked immunosorbent assay kit (E-EL-M0044), Amplex Red creatine kinase (CK) detection kit (Beyotime Biotechnology Co., Ltd., product number: S0287S), lactate dehydrogenase (LDH) detection kit (Beyotime Biotechnology Co., Ltd., product number: P0395S), mouse creatine kinase isoenzyme (CK-MB) detection kit (Beijing Solarbio Science & Technology Co., Ltd., product number: SEKM-0152), Amplex Red creatinine (Cr) detection kit (Beyotime Biotechnology Co., Ltd., product number: S0291S), urea nitrogen (BUN) content detection kit (Beijing Solarbio Science & Technology Co., Ltd., product number: BC1535), anti-Collagen IV (Merck, USA, product number: SAB4500369), anti-CD31 antibody (Sigma, product number: SAB5700639).

[0052] 1.3 Experimental animals

[0053] 75 male C57BL / 6J mice, 5 weeks old, were purchased from the Experimental Animal Center of the Air Force Military Medical University and used in the experiment after one week of adaptive feeding. The lighting was controlled artificially for 12 hours. The bedding was changed once a week before diabetes modeling and once every 2 days after successful modeling. The breeding cages were cleaned and disinfected with alcohol. The feed was added freshly every day, and the drinking water was changed and the water bottles were cleaned every day.

[0054] 2 Experimental methods

[0055] 2.1 Modeling and grouping

[0056] After 30 days of feeding mice with a 45% high-fat diet, they were fasted for 12 h. The next morning, STZ (60 mg / kg) was intraperitoneally injected, and they were continued to be fed with a 45% high-fat diet to induce the establishment of a diabetic CMVD model. Fasting blood glucose was measured one week after STZ injection. Mice with blood glucose greater than 11.1 mmol / L were randomly divided into a model group (DM-CMVD), a sanguisorba officinalis extract group (DYE, 400 mg / kg), a ziyu II group (ZiyuⅡ, 8 mg / kg), a positive control metformin group (Metformin, 200 mg / kg), and a normal group (Normal). Mice in the normal group were intraperitoneally injected with an equal volume of sodium citrate buffer and fed with a normal diet. The remaining operations were the same as those in the model group. After the blood glucose of the mice increased and they were grouped, they were continued to be fed for 30 days to form DM-CMVD. After 30 days, mice in the DYE and ZiyuⅡ groups were respectively intragastrically administered 400 mg / kg of sanguisorba officinalis extract or 8 mg / kg of pure ziyu II, the positive drug group was intragastrically administered 200 mg / kg of metformin, and mice in the normal group and the model group were intragastrically administered an equal volume of deionized water. Random blood glucose of the mice was measured and their body weights were weighed during the drug administration period, and the drug administration continued for 30 days.

[0057] 2.2 Echocardiogram detection

[0058] The mice were fixed in a supine position on a heating pad after being anesthetized by inhaling isoflurane. The hair on the chest and abdomen was removed and ultrasonic coupling agent was applied. The ultrasonic probe was closely attached to the position of the mouse heart for echocardiogram detection to obtain left ventricular cardiac activity images. After continuously measuring 3 cardiac cycles, analysis was performed using Vevo LAB 3.1.0 software, and the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were calculated.

[0059] 2.3 Detection of TC and TG in the liver

[0060] Weighed 20 mg of liver tissue and added 200 μL of isopropanol. Homogenized on ice using a glass homogenizer and centrifuged at 12000 r / min at 4℃ for 5 min, then the supernatant was aspirated. The detection working solution was prepared according to the instructions. 40 μL of the standard product or tissue supernatant was added to a 96-well plate, and then the total cholesterol TC or triglyceride TG detection working solution was added. Reacted at 37℃ in the dark for 60 min, and the absorbance was measured at 570 nm, and the concentrations of TC and TG in the liver were calculated.

[0061] 2.4 Detection of myocardial enzyme spectrum

[0062] Restore the myocardial enzyme spectrum (CK, CK-MB, LDH) detection kit to room temperature and perform the detection according to the instructions. Prepare the Amplex Red reaction working solution, dilute the standard product to 5 concentration gradients, add 20 μL of the diluted standard product or sample to be tested into the 96-well plate respectively, then add 80 μL of the reaction working solution, react at 37 °C in the dark for 3 min, measure the absorbance at 570 nm as A1, continue to react at 37 °C in the dark for 30 min, measure the absorbance at 570 nm as A2, and calculate creatine kinase CK, creatine kinase isoenzyme CK-MB and lactate dehydrogenase LDH respectively.

[0063] 2.5 Detection of Cr and BUN in serum

[0064] Prepare the working solution and standard product according to the instructions of the Cr and BUN detection kit, add the sample to be tested or the standard product into the 96-well plate respectively, then add an appropriate amount of the working solution, measure the absorbance at 570 nm, and calculate the concentration of creatinine Cr or urea nitrogen BUN in the sample.

[0065] 2.6 Detection of inflammatory factors in serum by enzyme-linked immunosorbent assay

[0066] Refer to the enzyme-linked immunosorbent assay kits for IL-1β, TNF-α and IL-6 to detect the pro-inflammatory factors in serum. Thaw and mix the serum on ice and dilute it with the diluent. Take out the kit and restore it to room temperature. Add 100 μL of serum or standard product solution into the antibody pre-coated 96-well plate, incubate at 37 °C in the dark for 1.5 h, wash the plate 3 times with the washing solution, add the biotinylated secondary antibody and horseradish peroxidase-labeled avidin, after the reaction ends, add the chromogenic substrate and react for 15 min, add the stop solution and then measure the absorbance at 450 nm, draw the standard curve and calculate the concentrations of interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) in serum.

[0067] 2.7 Real-time fluorescence quantitative PCR detection

[0068] TRIzol TM Extract the RNA of heart or kidney tissues by TRIzol method and measure the RNA concentration. Reverse transcribe the RNA into cDNA using the ABScript Neo-RT Master Mix kit. Take the 5-fold diluted cDNA as the template, add the primers for Collagen IV, VEGF and ICAM-1 respectively, prepare a 20 μL qPCR reaction system, and perform amplification on the machine to detect the relative expression levels of the 3 genes. The primer information is shown in Table 3.

[0069] Table 3 Primer information

[0070]

[0071] 2.8 Immunofluorescence staining

[0072] Cut the heart or kidney tissue into 3-5 μm sections, dewax and perform antigen repair, wash 3 times with PBS, add 3% BSA and block at room temperature for 1 h, dilute the mixture of anti-Collagen IV antibody and anti-CD31 antibody at 1:200, and incubate overnight at 4°C. After incubation with the primary antibody, wash 3 times with PBS, and continue to incubate with fluorescein-labeled goat anti-rabbit IgG in the dark at room temperature for 1 h. After washing with PBS buffer, label the cell nuclei with DAPI (4',6-diamidino-2-phenylindole) fluorescent dye, add an anti-quenching mounting medium, and then collect images using a Nikon Eclipse C1 fluorescence microscope, and perform fluorescence intensity analysis using Image J.

[0073] 2.9 Data analysis

[0074] The data were analyzed using SPSS 23.0 software, expressed as mean±SD, and one-way analysis of variance (One-way ANOVA) was used for comparison among multiple groups. A significant difference was considered when P < 0.05.

[0075] 3 Experimental results

[0076] 3.1 Sanguisorba officinalis extract and its active components can improve lipid metabolism and insulin secretion in diabetic CMVD mice

[0077] By detecting the blood glucose of mice, it was found that compared with the mice in the Normal group, the blood glucose of DM-CMVD mice increased, and the Sanguisorba officinalis extract (DYE) and its active component (ZiyuII) had no significant effect on the blood glucose of DM-CMVD mice (P > 0.05, Figure 2 A). However, compared with the DM-CMVD group, DYE and ZiyuII could reduce the contents of TC and TG in the liver of mice, indicating that DYE and ZiyuII had a regulatory effect on lipid metabolism in DM-CMVD mice (P < 0.01, Figure 2 B-C). Further detection of insulin and glucagon secreted by the islets showed that the islets of mice in the Normal group secreted more insulin, less glucagon, and there was more insulin distributed around the blood vessels; compared with the Normal group, the islets of DM-CMVD mice mainly secreted glucagon, had defective insulin secretion, and a large amount of glucagon was distributed around the blood vessels; compared with DM-CMVD mice, the DYE, ZiyuII, and metformin treatment groups could improve the islet function of DM-CMVD mice, increase insulin secretion, and there was more insulin distributed around their blood vessels ( Figure 2 D). This result indicated that DYE and ZiyuII could improve lipid metabolism and insulin secretion function in DM-CMVD mice.

[0078] 3.2 Sanguisorba officinalis L. extract and its active components improve myocardial injury in diabetic CMVD mice

[0079] Sustained hyperglycemic state directly damages vascular endothelial cells, thereby causing myocardial function injury. It can be seen from the detection of myocardial injury markers and echocardiogram that, compared with the Normal group, the levels of CK, CK-MB, and LDH in the myocardium of mice in the DM-CMVD group increased, and LVEF and LVFS decreased, indicating that the myocardium of DM-CMVD mice was damaged (P < 0.01, Figure 3 ). Compared with the DM-CMVD group, DYE and ZiyuII could reduce CK, CK-MB, and LDH in the myocardium of DM-CMVD mice, and LVEF and LVFS were higher than those in the DM-CMVD group (P < 0.05 or P < 0.01 Figure 3 ), indicating that DYE and ZiyuII could improve myocardial injury in DM-CMVD mice.

[0080] 3.3 Sanguisorba officinalis L. extract and its active components can alleviate chronic inflammation in diabetic CMVD mice

[0081] Chronic inflammation caused by long-term hyperglycemia in the body is an important factor contributing to DM-CMVD. It can be seen from the detection of pro-inflammatory factors in the serum of mice that, compared with the Normal group, the levels of IL-1β, TNF-α, and IL-6 in the serum of mice in the DM-CMVD group increased, indicating the presence of chronic inflammatory response in the body (P < 0.01, Figure 4 ). DYE and ZiyuII could reduce the pro-inflammatory factors in the serum of DM-CMVD mice, thereby alleviating the chronic inflammatory response in the body.

[0082] 3.4 Sanguisorba officinalis L. extract and its active components alleviate renal microvascular injury in diabetic CMVD mice

[0083] The renal function test of DM-CMVD mice found that the levels of Cr and BUN in the serum of DM-CMVD mice were lower than those in the Normal group, indicating that hyperglycemia damaged the renal function of diabetic mice (P < 0.01, Figure 5 A-B). Further detection of the mRNA of CollagenIV in the kidneys of mice found that the mRNA expression level of Collagen IV in DM-CMVD mice increased, and the fluorescence intensity of CollagenIV in the glomeruli of mice enhanced, indicating that the collagen fibers in the glomeruli of DM-CMVD mice increased and the basement membrane thickened (P < 0.01, Figure 5 C-D). The levels of Cr and BUN in the serum of mice in the DYE and ZiyuII treatment groups were lower than those in the DM-CMVD group, and the mRNA expression and fluorescence intensity of Collagen IV in the kidneys decreased (P < 0.05 or P < 0.01, Figure 5), indicating that DYE and ZiyuII can alleviate renal microvascular injury in DM-CMVD mice.

[0084] 3.5 Sanguisorba officinalis L. extract and its active ingredients improve cardiac microvascular injury in diabetic CMVD mice

[0085] Collagen IV is the main component of the basement membrane and also the main marker of vascular wall thickening. Further detection of Collagen IV in the microvessels of the myocardium shows that, compared with the Normal group, the thickness of the vessels positive for Collagen IV in the myocardium of mice in the DM-CMVD group increased (P < 0.01, Figure 6 A-B), the mRNA expression of Collagen IV increased, and the mRNA expressions of VEGF and ICAM-1 in the DM-CMVD group of mice also increased (P < 0.01, Figure 6 C-E), indicating that the vascular wall of DM-CMVD mice thickened and a large amount of collagen fibers formed. Compared with the DM-CMVD group, the thickness of the vessels positive for Collagen IV in the myocardium of mice in the DYE and ZiyuII groups decreased, and the mRNA expressions of CollagenIV, VEGF, and ICAM-1 in the myocardium decreased (P < 0.05 or P < 0.01, Figure 6 ), indicating that DYE and ZiyuII can improve cardiac microvascular injury in DM-CMVD mice.

[0086] In summary, the Sanguisorba officinalis L. extract and its active ingredient (sanguisorbic acid II) can improve lipid metabolism and insulin secretion in diabetic CMVD mice, improve myocardial injury in diabetic CMVD mice, alleviate chronic inflammation in diabetic CMVD mice, alleviate renal microvascular injury in diabetic CMVD mice, and improve cardiac microvascular injury in diabetic CMVD mice. That is to say, the Sanguisorba officinalis L. extract and its active ingredient - sanguisorbic acid II prepared in the present invention have a certain therapeutic effect on microangiopathy.

[0087] The above-described embodiments only represent the preferred embodiments of the present invention, and the description is relatively specific and detailed, but it should not be construed as a limitation of the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. Use of a Sanguisorba officinalis extract for preparing a drug for treating microangiopathy.

2. The use according to claim 1, characterized in that: The effective ingredients contained in the Sanguisorba officinalis extract at least include Sanguisorba officinalis saponin II.

3. The use according to claim 1, characterized in that: The Sanguisorba officinalis extract is one of a water extract of Sanguisorba officinalis, a methanol extract of Sanguisorba officinalis, an ethanol extract of Sanguisorba officinalis or an acid extract of Sanguisorba officinalis.

4. The use according to claim 1, characterized in that: The Sanguisorba officinalis extract also includes a Sanguisorba officinalis extract preparation.

5. The use according to claim 1, characterized in that: The Sanguisorba officinalis extract preparation is prepared by adding medically permitted auxiliary materials to the Sanguisorba officinalis extract to prepare an oral preparation or an injection preparation.

6. The use according to claim 5, characterized in that: The oral preparation is a tablet, capsule, pill, granule or oral liquid preparation.

7. The use according to claim 1, characterized in that: The treatment of microvascular disease is achieved by improving cardiac microvascular damage.

8. The use according to claim 1, characterized in that: The treatment of microangiopathy is achieved by improving renal microvascular damage.

9. The use according to claim 1, characterized in that The treatment of microangiopathy is achieved by alleviating chronic inflammation.

10. The use according to claim 1, characterized in that: The treatment of microangiopathy can improve myocardial damage.

11. The use according to claim 1, characterized in that: The treatment of microangiopathy is achieved by improving lipid metabolism and insulin secretion functions, and the microangiopathy is caused by hyperglycemia and chronic inflammation.

12. The use according to claim 1, characterized in that: The preparation method of the Sanguisorba officinalis extract is as follows: weigh an appropriate amount of Sanguisorba officinalis medicinal material, crush it, add an extraction solvent, reflux and extract for 0.5 to 2 hours, extract 1 to 3 times, filter and combine the filtrate, concentrate, and spray dry to obtain the Sanguisorba officinalis extract; the volume ratio of the extraction solvent to the mass ratio of the Sanguisorba officinalis medicinal material is 6 to 15.

13. The use according to claim 12, characterized in that: The volume fraction of ethanol in the extraction solvent is 0-99%, and the mesh size of the Sanguisorba officinalis medicinal material after being crushed is 5-20 meshes.

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