Bupleurum leaf extract, preparation method thereof and application of bupleurum leaf extract in preparation of products for preventing and treating metabolic syndrome

Abstract

The invention belongs to the technical field of resource development of non-medicinal parts of traditional Chinese medicines, and provides a bupleurum leaf extract, a preparation method thereof and application of the bupleurum leaf extract in preparation of medicines for preventing and treating metabolic syndromes. The extract comprises the following components in percentage by weight: 233.68 + / -20.27 mg / g of total polysaccharide, 62.41 + / -0.61 mg / g of total flavone, 48.40 + / -2.96 mg / g of total phenol, 0.87 + / -0.055% of radix bupleuri total saponin, 0.17 + / -0.006% of saikoside A, 0.22 + / -0.006% of saikoside B1, 0.05 + / -0.037% of saikoside C and 0.43 + / -0.006% of saikoside D. According to the present invention, the traditional Chinese medicine composition can significantly reduce body weight increase caused by high-fat and high-sugar diet, reduce blood fat, fasting blood glucose and blood uric acid level, improve body metabolic disorder induced by high-fat and high-sugar diet, has a callback effect on various organ lesions, has a regulation effect on complications such as diabetic retinopathy, and can be used as a functional product for preventing and treating metabolic syndrome.

Description

Technical Field

[0001] The invention belongs to the technical field of development of resources of non-medicinal parts of traditional Chinese medicine, and specifically relates to a bupleurum leaf extract and a preparation method thereof and application thereof in preparing products for preventing and treating metabolic syndrome. Background Art

[0002] Metabolic syndrome ( metabolic syndrome , MS) is a group of metabolic disorders including high blood pressure, high blood sugar, abnormal cholesterol levels and abdominal obesity. With the accelerated pace of life and changes in lifestyle in modern society, excessive intake of high-fat and high-sugar diets may lead to metabolic disorders such as obesity, increased blood lipids and blood sugar. Dyslipidemia is mainly manifested by increased serum total cholesterol (T-CHO) or triglyceride (TG) levels. At present, the prevalence of diabetes and dyslipidemia in adults aged ≥18 years in my country is 12.4% and 40.4%, respectively. A study on blood glucose and blood lipids in Chinese type 2 diabetes patients showed that 29.8% of outpatient diabetic patients had both abnormal blood glucose and blood lipids. According to previous clinical studies, obesity, blood lipids, and blood glucose metabolism abnormalities can be accompanied by abnormal uric acid metabolism. A prospective clinical study in my country found that abnormal uric acid metabolism can increase the risk of type 2 diabetes and promote the occurrence and development of its complications, typical of which are diabetic retinopathy ( Diabetic Retinopathy , DR), DR is a type of microvascular disease. Its entire pathological process includes important pathological changes such as loss of retinal capillary pericytes, thickening of the basement membrane, loss of endothelial barrier function, and destruction of the blood-retinal barrier, leading to retinal ischemia. Its blood vessels belong to the terminal vascular system. Any pathological damage and tissue ischemia caused by vascular occlusion can lead to tissue necrosis, causing it to lose the ability to sense and transmit light stimulation. Therefore, vascular changes are an important feature of retinopathy, and retinal ischemia will increase the level of vascular endothelial growth factor (VEGF). Studies have shown that excessive expression of VEGF is related to retinal neovascularization, which can cause retinal hemorrhage, macular edema, retinal detachment, and neovascular glaucoma, causing severe visual impairment and ultimately blindness. At present, surgical treatment is still the first-line treatment for DR, but it is very easy to cause cell lethal damage and affect the patient's visual function. If the prognosis is poor, complications are likely to occur.

[0003] The pathogenesis of MS has not been fully elucidated. Patients often present with symptoms such as obesity, thirst, limb fatigue, and heaviness of the body. Based on individual specific constitutions, due to improper diet and dysfunction of the spleen in transportation and transformation, other visceral diseases occur. However, since Western medicine often focuses on drug treatment for MS, and the action targets of Western drugs are single, there are limitations in the treatment of single metabolic imbalances. When taken for a long time, drug resistance and various side effects are likely to occur. For example, current first-line hypoglycemic drugs such as biguanide drugs, α-glucosidase inhibitors, sulfonylurea drugs, etc. may cause gastrointestinal discomfort, gastrointestinal dysfunction, and kidney damage in patients during the taking period; lipid-lowering drugs such as statins, fibrates, cholesterol inhibitors, etc. Among them, statins may cause liver damage because their main metabolism site is the liver, and other Western medicine lipid-lowering drugs may cause gastrointestinal symptoms such as myopathy, constipation, abdominal distension, and belching; Allopurinol is the most commonly used uric acid-lowering drug in clinical practice for the longest time. Its main effect is to inhibit uric acid production. During the taking period, it may cause gastrointestinal discomfort such as abdominal pain, diarrhea, nausea, vomiting, etc., as well as side effects such as kidney damage such as edema, oliguria, proteinuria, renal failure, and liver function impairment.

[0004] However, when medicated diet is complemented with specific production methods, it is more easily accepted by the public and can achieve the effects of preventing and treating diseases and rehabilitating and strengthening the body. Bupleurum leaf is the leaf of the aerial part of the Umbelliferae plant Bupleurum chinense DC. There are records in ancient Chinese books that Bupleurum leaf can be used as food. For example, "Supplementary Records of Famous Physicians" states: Bupleurum, the leaf is called Yunhao, pungent and fragrant and can be eaten. There are relevant records of Bupleurum leaf in a large number of ancient books such as "M博物志", "Shennong Ben Cao Jing", "Ben Cao Chong Yuan", and "Jiuhuang Ben Cao". "Chinese Medical Dictionary" once recorded: "Bupleurum, belonging to the category of mountain herbs, has two types, southern and northern. Its rhizome, stem, and leaf can all treat diseases." It clearly points out that the root, stem, and leaf of Bupleurum can all be used as medicine. Long-term practice has also proved its food safety: According to research, Bupleurum leaf has a long history of being eaten in Shanxi Province. It is mostly eaten by soaking in water, soaking in wine, cooking dishes, etc. It is more concentrated in areas such as Yuncheng, Linfen, Jincheng, and Changzhi in Shanxi Province. The local people all have the habit of using Bupleurum leaf as a tea drink. Modern analysis and research show that Bupleurum leaf contains rich essential elements for the human body such as iron, manganese, and zinc, as well as a variety of amino acids beneficial to the human body and rich vitamins. Bupleurum chinense DC. The leaf of the aerial part, and there are records in ancient Chinese books that Bupleurum leaf can be used as food. For example, "Supplementary Records of Famous Physicians" states: Bupleurum, the leaf is called Yunhao, pungent and fragrant and can be eaten. There are relevant records of Bupleurum leaf in a large number of ancient books such as "博物志", "Shennong Ben Cao Jing", "Ben Cao Chong Yuan", and "Jiuhuang Ben Cao". "Chinese Medical Dictionary" once recorded: "Bupleurum, belonging to the category of mountain herbs, has two types, southern and northern. Its rhizome, stem, and leaf can all treat diseases." It clearly points out that the root, stem, and leaf of Bupleurum can all be used as medicine. Long-term practice has also proved its food safety: According to research, Bupleurum leaf has a long history of being eaten in Shanxi Province. It is mostly eaten by soaking in water, soaking in wine, cooking dishes, etc. It is more concentrated in areas such as Yuncheng, Linfen, Jincheng, and Changzhi in Shanxi Province. The local people all have the habit of using Bupleurum leaf as a tea drink. Modern analysis and research show that Bupleurum leaf contains rich essential elements for the human body such as iron, manganese, and zinc, as well as a variety of amino acids beneficial to the human body and rich vitamins. Summary of the Invention

[0005] The present invention provides an extract of Bupleurum leaf, a preparation method thereof, and an application thereof in the preparation of a product for preventing and treating metabolic syndrome.

[0006] The present invention is achieved by the following technical scheme: a bupleurum leaf extract, which is a water extract of bupleurum leaf, and in the bupleurum leaf extract: the total polysaccharide content is 233.68±20.27 mg / g, the total flavonoids content is 62.41±0.61 mg / g, the total phenol content is 48.40±2.96 mg / g, the content of total bupleurum saponins is 0.87±0.055%, wherein the content of bupleurum saponin A is 0.17±0.006%, the content of bupleurum saponin B1 is 0.22±0.006%, the content of bupleurum saponin C is 0.05±0.037%, and the content of bupleurum saponin D is 0.43±0.006%. The method for preparing the bupleurum leaf extract comprises the following steps: mixing bupleurum leaf and water in a mass ratio of 1:10, soaking the bupleurum leaf in water for 12 hours, then decocting with 80-90°C hot water for 3 times, each time for 50 minutes, combining the filtrates, concentrating under reduced pressure, and freeze-drying to obtain the bupleurum leaf extract.

[0007] The present invention also provides the use of the bupleurum leaf extract in preparing a product for preventing and treating metabolic syndrome. Further, the bupleurum leaf extract is used in regulating glutathione metabolism and starch and sucrose metabolism pathways to prepare a product for preventing and treating metabolic syndrome. The metabolic syndrome is obesity, hyperlipidemia, hyperglycemia, diabetes or its complications. Furthermore, the complication is diabetic retinopathy.

[0008] The use of the bupleurum leaf extract in the preparation of a product for reducing hyperglycemia induced by a high-fat, high-sugar diet, the dosage of the bupleurum leaf extract is a low dose of 1.35 g / kg. The use of the bupleurum leaf extract in the preparation of a product for reducing hyperuricemia induced by a high-fat, high-sugar diet, the dosage of the bupleurum leaf extract is a low dose of 1.35 g / kg. The use of the bupleurum leaf extract in the preparation of a product for reducing body weight and blood lipids induced by a high-fat, high-sugar diet, the dosage of the bupleurum leaf extract is a medium-high dose of 4.05-12.15 g / kg. The use of the bupleurum leaf extract in the preparation of a product for reducing retinopathy induced by a high-fat, high-sugar diet, the dosage of the bupleurum leaf extract is a medium-high dose of 4.05-12.15 g / kg.

[0009] The invention adopts the traditional clinical decoction method to extract, obtains the bupleurum leaf extract, and uses liquid chromatography-mass spectrometry (LC-MS) to evaluate the chemical components of the bupleurum leaf in vitro; at the same time, the in vivo efficacy evaluation of experimental animals and human experiments are carried out, and the body weight, fat weight, fasting blood sugar changes and serum-related biochemical indicators of ICR mice are used as the main activity indicators to explore the pharmacological effect of bupleurum leaf on type II diabetes ICR mice induced by a high-fat diet. Combining chemical component identification with pharmacological experiments, it is found that the bupleurum leaf extract has fat-reducing, blood sugar-lowering and uric acid-lowering activities, and has an improving effect on metabolic disorders induced by a high-fat diet. The foundation is laid for the development of bupleurum leaf products and the study of their pharmacological effects. The bupleurum leaf extract of the invention is extracted from the leaves of the traditional Chinese medicine bupleurum, and the main components thereof are quantitatively determined by high performance liquid chromatography, and the contents of four components, namely, total polysaccharides, total flavonoids, total phenols and bupleurum total saponins, are determined, wherein the total polysaccharide content is 233.68±20.27 mg / g, the total flavonoid content is 62.41±0.61 mg / g, the total phenol content is 48.40±2.96 mg / g, and the bupleurum total saponins content is 0.87±0.055%. The chemical components are identified by LC-MS, and a total of 40 chemical components are identified, which mainly include 24 saponins, 6 flavonoids, 4 acids and 6 phenylpropanoids. The extract is administered orally to ICR mice, and the results are shown in Table 1. Animal experiments have shown that the extract can significantly reduce the levels of blood lipid-related indicators such as total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in the serum of type Ⅱ diabetic ICR mice induced by a high-fat diet, and inhibit their weight gain. At the same time, their fasting blood glucose levels and blood glucose-related biochemical indicators such as serum insulin (INS), serum glucagon (GC), and serum glycosylated hemoglobin (GHb) levels have been adjusted. Observation of pancreatic pathological sections found that high-dose Bupleurum leaf extract treatment can adjust its pathological damage, improve diabetic complications of retinopathy, and reduce the production of blood uric acid (UA). And through the observation of its liver function and liver pathological sections, it was found that the Bupleurum leaf extract has a certain adjustment effect on liver damage caused by a high-fat and high-sugar diet. At the same time, its safety can be preliminarily confirmed by observing its kidney pathological sections, and it has also been verified in human experiments that it has the same effect. It suggests that the extract can be used in the development of functional products to improve metabolic syndrome.

[0010] In the present invention, the Bupleurum leaf is dissolved by boiling in hot water and then gavage is given to ICR mice. Pharmacological experiments confirm that the Bupleurum leaf extract obtained can significantly reduce the weight gain of ICR mice, and reduce blood lipids, fasting blood sugar and blood uric acid levels, improve the body metabolic disorder induced by a high-fat and high-sugar diet, and has therapeutic significance for diabetic complications DR. It is suggested that the extract can be used in the development of functional products for preventing and treating metabolic syndrome and its complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the HPLC chromatogram of the chemical components of saponins from Bupleurum leaves; Figure 2 This is the percentage of total polysaccharides, total flavonoids, total phenols and total saponins in Bupleurum chinense leaves; Figure 3 This is the extracted ion chromatogram of the chemical components of the Bupleurum leaf extract; Figure 4 The results of dynamic monitoring of the body weight of mice in each group by Bupleurum leaf extract for 12 weeks (compared with the HFD group: * P<0.05, **** P<0.001); Figure 5 The results of epididymal fat weight and fat cell data measurement of each group of mice (AC). In the figure: A is the measurement results of epididymal fat weight of each group of mice on a high-fat diet; B is the measurement results of fat cell data of each group of mice on a high-fat diet; C is the measurement results of the average area of ​​fat cells of each group of mice on a high-fat diet; compared with the HFD group: * P<0.05, **** P < 0.001; Figure 6 The inguinal fat weight measurements of mice in each group (compared with the HFD group: * P<0.05, **** P<0.001); Figure 7 The results of shoulder fat weight measurement of each group of mice (compared with HFD group: * P<0.05, **** P<0.001); Figure 8HE-stained pathological sections of epididymal fat of mice in each group (AG), in the figure: A is the pathological section of epididymal fat of mice in the blank group, B is the pathological section of epididymal fat of mice in the model group, C is the pathological section of epididymal fat of mice in the high-fat diet combined with low-dose administration of Bupleurum leaf extract group, D is the pathological section of epididymal fat of mice in the high-fat diet combined with medium-dose administration of Bupleurum leaf extract group, E is the pathological section of epididymal fat of mice in the high-fat diet combined with high-dose administration of Bupleurum leaf extract group, F is the pathological section of epididymal fat of mice in the high-fat diet combined with Cassia seed extract group, G is the pathological section of epididymal fat of mice in the high-fat diet combined with Bupleurum extract group (×200); Fig. 9 The dynamic monitoring results of the effect of Bupleurum leaf extract on fasting blood glucose in each group of mice (compared with the HFD group: * P<0.05, **** P<0.001); Fig.10 Results of oral glucose tolerance test (OGTT) (compared with HFD group: * P<0.05, **** P<0.001); Fig.11 The figure shows the effect of bupleurum leaf extract on the content of blood glucose-related biochemical indexes in each group of mice. In the figure: A shows the effect of bupleurum leaf extract on the content of serum insulin index in each group of mice; B shows the effect of bupleurum leaf extract on the content of serum glucagon index in each group of mice; C shows the effect of bupleurum leaf extract on serum glycosylated hemoglobin in each group of mice (compared with the HFD group: * P<0.05, **** P<0.001); Fig.12 The results of 12-week dynamic monitoring of blood lipid-related biochemical indicators in each group of mice (AD). In the figure: A is the results of 12-week dynamic monitoring of serum total cholesterol in each group of mice; B is the results of 12-week dynamic monitoring of serum triglycerides in each group of mice; C is the results of 12-week dynamic monitoring of serum low-density lipoprotein in each group of mice; D is the results of 12-week dynamic monitoring of serum high-density lipoprotein in each group of mice. (Compared with the HFD group: * P<0.05, **** P<0.001); Fig.13 The effect of Bupleurum leaf extract on the blood uric acid content of each group of mice (compared with the HFD group: * P<0.05); Fig.14The retinal HE staining pathological sections of mice in each group; in the figure: A is the retinal pathological section of the left eye of the blank group mouse, B is the retinal pathological section of the left eye of the model group mouse, C is the retinal pathological section of the left eye of the mouse in the high-fat diet combined with low-dose Bupleurum leaf extract administration group, D is the retinal pathological section of the left eye of the mouse in the high-fat diet combined with medium-dose Bupleurum leaf extract administration group, E is the retinal pathological section of the left eye of the mouse in the high-fat diet combined with high-dose Bupleurum leaf extract administration group, F is the retinal pathological section of the left eye of the mouse in the high-fat diet combined with Cassia seed extract administration group, G is the retinal pathological section of the left eye of the mouse in the high-fat diet combined with Bupleurum extract administration group (×400); Fig.15 Retinal slides (PAS staining) of mice in each group, in the figure: A is the retinal blood vessel slide of the right eye of the blank group mice, B is the retinal blood vessel slide of the right eye of the model group mice, C is the retinal blood vessel slide of the right eye of the mice in the high-fat diet combined with low-dose Bupleurum leaf extract group, D is the retinal blood vessel slide of the right eye of the mice in the high-fat diet combined with medium-dose Bupleurum leaf extract group, E is the retinal blood vessel slide of the right eye of the mice in the high-fat diet combined with high-dose Bupleurum leaf extract group, F is the retinal blood vessel slide of the right eye of the mice in the high-fat diet combined with Cassia seed extract group, G is the retinal blood vessel slide of the right eye of the mice in the high-fat diet combined with Bupleurum extract group (×400); Fig.16 The retinal VEGF immunohistochemical sections of mice in each group, in the figure: A is the retinal VEGF immunohistochemical section of the left eye of the blank group mouse, B is the retinal VEGF immunohistochemical section of the left eye of the model group mouse, C is the retinal VEGF immunohistochemical section of the left eye of the mouse in the high-fat diet combined with low-dose Bupleurum leaf extract administration group, D is the retinal VEGF immunohistochemical section of the left eye of the mouse in the high-fat diet combined with medium-dose Bupleurum leaf extract administration group, E is the retinal VEGF immunohistochemical section of the left eye of the mouse in the high-fat diet combined with high-dose Bupleurum leaf extract administration group, F is the retinal VEGF immunohistochemical section of the left eye of the mouse in the high-fat diet combined with Cassia seed extract administration group, G is the retinal VEGF immunohistochemical section of the left eye of the mouse in the high-fat diet combined with Bupleurum extract administration group (×400); Fig.17 The results of VEGF expression in the retina of each group of mice (compared with the HFD group: * P<0.05, **** P<0.001); Fig.18HE-stained pathological sections of the liver of mice in each group, in the figure: A is the pathological section of the liver of mice in the blank group, B is the pathological section of the liver of mice in the model group, C is the pathological section of the liver of mice in the high-fat diet combined with low-dose administration of Bupleurum leaf extract group, D is the pathological section of the liver of mice in the high-fat diet combined with medium-dose administration of Bupleurum leaf extract group, E is the pathological section of the liver of mice in the high-fat diet combined with high-dose administration of Bupleurum leaf extract group, F is the pathological section of the liver of mice in the high-fat diet combined with Cassia seed extract group, G is the pathological section of the liver of mice in the high-fat diet combined with Bupleurum extract group (×400); Fig.19 The wet weight of liver of mice in each group (compared with HFD group: * P<0.05, **** P < 0.001); Fig. 20 The results of liver function test of each group of mice are shown in Figure 1. A is the serum alanine aminotransferase (ALT) content of each group of mice, and B is the serum aspartate aminotransferase (AST) content of each group of mice. (Compared with the HFD group: * P<0.05, **** P<0.001); Fig.21 The HE-stained renal pathological sections of mice in each group, in the figure: A is the renal pathological section of mice in the blank group, B is the renal pathological section of mice in the model group, C is the renal pathological section of mice in the high-fat diet combined with high-dose administration of Bupleurum leaf extract group, and D is the renal pathological section of mice in the high-fat diet combined with Bupleurum extract administration group (×400); Fig. 22 The following are the pancreatic pathological sections of mice in each group stained with HE. In the figure: A is the pancreatic pathological section of mice in the blank group, B is the pancreatic pathological section of mice in the model group, C is the pancreatic pathological section of mice in the high-fat diet combined with low-dose administration of Bupleurum leaf extract group, D is the pancreatic pathological section of mice in the high-fat diet combined with medium-dose administration of Bupleurum leaf extract group, E is the pancreatic pathological section of mice in the high-fat diet combined with high-dose administration of Bupleurum leaf extract group, F is the pancreatic pathological section of mice in the high-fat diet combined with Cassia seed extract group, G is the pancreatic pathological section of mice in the high-fat diet combined with Bupleurum extract group (×400); Fig.23 Representative total ion current graphs of ICR mouse liver samples; in the figure: A is the total ion current graph of mouse liver samples in positive ion mode, and B is the total ion current graph of mouse liver samples in negative ion mode; Fig.24 The PCA score diagram of the metabolomics data of liver samples of mice in each group; Fig.25 :A is the OPLS-DA graph of the blank group and the model group, B is the S-Plot graph; Fig.26Heat map of liver metabolic markers for Bupleurum chinense leaf extract in the treatment of metabolic syndrome; Fig. 27 KEGG bubble diagram of liver metabolic markers of Bupleurum chinense leaf extract in the treatment of metabolic syndrome; Fig.28 This is the KEGG map after the intersection of the metabolic marker genes of Bupleurum leaf extract and disease genes. DETAILED DESCRIPTION

[0012] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the field to which the invention belongs, and the materials cited herein and those cited by them will be incorporated by reference. Equivalent technologies of the specific embodiments described that can be understood by routine experiments recognized by those skilled in the art will be included in this application. The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0014] Example 1: Preparation and content determination of Bupleurum chinense leaf extract 1. Preparation of Bupleurum leaf extract: Weigh 200 g of Bupleurum leaf, soak it in 10 times the amount of water for 12 hours, boil it in 80-90°C hot water for 3 times, each time for 50 minutes, combine the filtrate and concentrate it by vacuum rotary evaporator, freeze dry it in freeze dryer for later use, and then dilute it with pure water to obtain the Bupleurum leaf extract.

[0015] 2. Determination of the content of Bupleurum leaf extract: Determination of total polysaccharide content: Accurately weigh 5 mg of glucose reference substance, distilled water to a 50 mL volumetric flask to obtain a 0.1 mg / mL reference substance solution, respectively transfer 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mL, add distilled water to 1 mL, add 1 mL of 5% phenol and 5 mL of concentrated sulfuric acid, shake well and cool, place at room temperature for 30 minutes, use a spectrophotometer to measure the absorbance at a wavelength of 490 nm, and use the glucose mass concentration as the horizontal axis and the absorbance as the vertical axis for regression. Accurately weigh 25 mg of Bupleurum polysaccharide dried to constant weight and place it in a 100 mL volumetric flask, add distilled water to dissolve and dilute to the scale, then take 2 mL and place it in 5 25 mL volumetric flasks and dilute to the scale with distilled water. The absorbance was determined according to the standard curve preparation method, and the glucose content and average value of 5 Bupleurum leaf polysaccharides were calculated by the regression equation. Then the conversion factor f=W / (C×D)=3.12 was calculated. Where W is the mass of polysaccharide (mg), C is the glucose concentration in the polysaccharide solution (mg / mL), and D is the dilution multiple of the polysaccharide. Accurately weigh 100 mg of Bupleurum leaf extract powder into a 10 mL centrifuge tube, add distilled water to 10 mL, mix well, heat in a water bath at 85°C for 2 h, centrifuge at 8000 r / min for 10 min, aspirate the supernatant, add 2 times the amount of ethanol, shake well and let stand, centrifuge at 5000 r / min for 10 min, the resulting precipitate is the crude polysaccharide, dissolve it in water and make up to 100 mL, take 1 mL, add 1 mL 5% phenol and 5 mL concentrated sulfuric acid, shake well and cool, place at room temperature for 30 min, then measure the absorbance at a wavelength of 490 nm, and perform three parallel tests according to the method established for the total polysaccharide standard curve to take the average value to calculate the total polysaccharide content in Bupleurum leaf extract.

[0016] Determination of total flavonoids content: Prepare the Bupleurum leaf extract powder and rutin reference substance into a solution with a concentration of 0.200 mg / mL with methanol. Take 1.000mL, 2.000mL, 3.000mL, 4.000mL, 5.000mL and 6.000mL of rutin reference substance solution and place them in a 25mL volumetric flask, first add 1mL of 5% sodium nitrite solution, shake well and place for 6min, then add 1mL of 10% aluminum nitrate solution, also place for 6min, then add 10mL of 10% sodium hydroxide, shake well and make up to 25mL with distilled water, and place for 15min. Then measure its absorbance value in an ultraviolet spectrophotometer, set the absorption wavelength to 510nm, and then draw a standard curve. The horizontal coordinate of the drawn standard curve is the concentration of the rutin reference substance solution, and the vertical coordinate is the absorbance value. Weigh 20 mg of bupleurum leaf freeze-dried powder accurately in a 100 mL volumetric flask, dilute to scale with methanol, take 2 mL each and place in 5 25 mL volumetric flasks and dilute to scale with methanol, determine the absorbance according to the standard curve preparation method, calculate the total flavonoid content and average value by regression equation, and then calculate its conversion factor f = 1.99. Accurately transfer 5 mL of bupleurum leaf extract solution, perform three parallel tests according to the method of establishing the total flavonoid standard curve, take the average value, and calculate the total flavonoid content in bupleurum leaf extract.

[0017] Determination of total phenol content: Prepare the Bupleurum leaf extract powder into a 0.100 mg / mL solution, and prepare the gallic acid reference substance into a 0.500 mg / mL solution. Take 0.200 mL, 0.400 mL, 0.600 mL, 0.800 mL, 1.000 mL and 1.200 mL of gallic acid reference substance solution in a 25 mL volumetric flask and dilute to volume with distilled water. Take 1 mL of gallic acid solution of different concentrations, add 5 mL of distilled water first, then add 2 mL of Folin reagent, and then put it in the dark for 5 minutes, and then continue to add 4 mL of 10% Na2CO3 solution in the dark for 2 hours. Finally, the above solution is measured by ultraviolet spectrophotometer, and its wavelength is set to 765 nm, and then a standard curve is drawn. The horizontal axis of the standard curve is the concentration of gallic acid reference substance, and the vertical axis is the absorbance value. Weigh 10 mg of Bupleurum chinense leaf freeze-dried powder accurately in a 100 mL volumetric flask, dilute to scale with distilled water, take 2 mL each and place in 5 25 mL volumetric flasks and dilute to scale with distilled water, determine the absorbance according to the standard curve preparation method, calculate the total phenol content and average value by regression equation, and then calculate its conversion factor f = 2.32. Accurately transfer 1 mL of Bupleurum chinense leaf extract solution, perform three parallel tests according to the method of establishing the total phenolic acid standard curve, and take the average value, and calculate the content of total phenolic acid in Bupleurum chinense leaf extract.

[0018] Determination of total saponin content: Preparation of test solution: Pass the Bupleurum leaf extract powder through a 100-mesh sieve, take 2.0 g of fine powder and accurately weigh it, put the weighed powder into a 50ML conical flask, accurately measure 10 mL of methanol, add it to the conical flask, seal it with a sealing film, and weigh it. Ultrasonic treatment for 30 min, cool at room temperature, weigh it again, make up for the weight loss, take the supernatant and centrifuge it at 8000r / min at room temperature in a high-speed refrigerated centrifuge for 10 minutes, take the supernatant, filter it through a 0.22 μm microporous filter membrane, transfer the filtered solution to a liquid phase vial to obtain the test solution.

[0019] Preparation of standard solution: Take appropriate amount of saikosaponin A, saikosaponin C, saikosaponin B1, and saikosaponin D reference substances, accurately weigh them, transfer them to volumetric flasks respectively, add methanol to the scale line, and prepare 0.4 mg / mL saikosaponin A, 0.4 mg / mL saikosaponin B1, 0.5 mg / mL saikosaponin C, and 0.5 mg / mL saikosaponin D reference substance solutions.

[0020] The injection volumes were set to 3, 5, 10, 15, and 20 for analysis, and the standard curve was drawn with the injection volume (x) as the horizontal axis and the peak area (y) as the vertical axis. The regression equation and correlation coefficient were obtained, and the contents of saikosaponin A, saikosaponin B1, saikosaponin C, and saikosaponin D in the bupleurum leaf extract were calculated.

[0021] Results of determination of the content of Bupleurum leaf: UV spectrophotometer was used to determine the contents of total polysaccharides, total flavonoids and total phenols in Bupleurum leaf extract. The results showed that the total polysaccharide content was 233.68±20.27 mg / g, the total flavonoid content was 62.41±0.61 mg / g, and the total phenol content was 48.40±2.96 mg / g. High performance liquid chromatography was used to determine the content of total saponins in Bupleurum leaf extract, which was 0.87±0.055%, of which saikosaponin A content was 0.17±0.006%, saikosaponin B1 content was 0.22±0.006%, saikosaponin C content was 0.05±0.037%, and saikosaponin D content was 0.43±0.006%. Figure 1 The figure shows the HPLC chromatogram of saikosaponins in the extract of bupleurum leaf. Figure 2 The content percentage of total polysaccharides, total flavonoids, total phenols and total saponins in the extract of Bupleurum chinense leaves is shown in Table 1. The content of total polysaccharides, total phenols and total flavonoids in Bupleurum chinense leaves is shown in Table 2. The content of saponins A, B1, C and D in Bupleurum chinense leaves is shown in Table 2.

[0022] Table 1 Results of total polysaccharides, total phenols and total flavonoids content in Bupleurum chinense leaves Table 2 Results of saponin A, B1, C, D content in Bupleurum chinense leaves Chemical component identification: Take the test solution for testing, collect LC-MS chromatograms, observe the quasi-molecular ion peaks and multi-level fragments in the mass spectrum corresponding to each chromatographic peak, and identify and identify by searching literature and related databases. The identification results are shown in Table 1 and Figure 3 The results show that a total of 40 chemical components were identified, including 24 saponins, 6 flavonoids, 4 acids, and 6 phenylpropanoids. Figure 3 Among them, the representative component 1 is the flavonoid component isorhamnetin-3-O-glucoside, 2 is the flavonoid component kaempferol-7-O-rhamnoside, 3 is the flavonoid glycoside component rutin, 4 is the saponin component acetyl-saponin C, and 5 is the saponin component malonylhydroxy-saponin A.

[0023] Table 3 Identification results of chemical components of Bupleurum chinense leaves Example 2: Whole animal efficacy evaluation of Bupleurum chinense leaf extract 1. Animal grouping and dosing: 70 SPF male ICR mice were randomly divided into blank group (CON) (n=10), model group (HFD) (n=10), low-dose bupleurum leaf extract group (HFD+BLL) (n=10), medium-dose bupleurum leaf extract group (HFD+BLM) (n=10), high-dose bupleurum leaf extract group (HFD+BLH) (n=10), positive drug cassia seed extract group (HFD+CS) (n=10), and bupleurum extract group (HFD+BR) (n=10). All drugs in the experiment were administered by gavage, among which the dosage of low-dose group of Bupleurum leaf extract was 1.35 g / kg, the dosage of medium-dose group of Bupleurum leaf extract was 4.05 g / kg, the dosage of high-dose group of Bupleurum leaf extract was 12.15 g / kg, the dosage of positive drug Cassia seed extract group was 2.25 g / kg, and the dosage of Bupleurum extract group was 1.35 g / kg. Both the blank group and the model group were given equal volumes of normal saline by gavage.

[0024] 2. Type 2 diabetes model induced by high-fat diet: After 1 week of adaptive feeding, all groups except the blank group were fed with high-fat and high-sugar feed, and the blank group was fed with standard feed throughout the whole process. The experimental period was 12 weeks.

[0025] A. During the whole experimental period, the weight changes of mice in each group were dynamically monitored every 2 weeks; B. During the whole experimental period, the fasting blood glucose level of mice in each group was dynamically monitored every 2 weeks by taking blood from the tail tip; C. During the whole experimental period, blood was collected from the eye sockets of mice in each group every 4 weeks, and the blood was centrifuged at 3500r / min for 15min at 4℃ to separate the serum. The biochemical and ELISA kits were used to detect the biochemical indicators related to blood lipids, blood sugar and blood uric acid, and the serum biochemical indicators were dynamically monitored; D. After 12 weeks, the eyeballs of mice in each group were removed and fixed after fasting for 12h, and the retinal pathological state was observed by HE staining sections, and the retinal vascular network flat mount and PAS staining techniques were used to observe the retinal vascular disease of mice in each group. Observe the changes: fix the eyeball with optic nerve in eyeball fixative for more than 12 hours, wash thoroughly with PBS after 12 hours to remove the fixative, clamp the optic nerve of the eyeball with pointed tweezers to take out the eyeball, gently clamp the eyeball with thumb and index finger, pierce along the serration 4-5 times with a fine needle to make the aqueous humor flow out, cut the iris along the serration with ophthalmic scissors, and then gently peel off the cornea and lens of the front part of the eye with pointed tweezers, immerse in PBS solution and rinse gently, wash off the vitreous body, divide the cup-shaped eyeball wall into 3-4 parts with the optic nerve head as the center, and gently peel off the retinal layer of the inner wall with pointed tweezers, and immerse the peeled retina in PBS solution, digest and blow after immersion, and finally perform vascular network laying and PAS staining; E. After 12 weeks, the eyeballs were removed and blood was collected after fasting for 12 hours. The blood was separated and serum was tested for relevant biochemical indicators. The liver, epididymal fat, inguinal fat, and scapular fat were collected and weighed, and the liver, kidney, pancreas, and epididymal fat were fixed in a fixative for more than 24 hours. After taking them out, the target tissues were trimmed flat with a scalpel in a fume hood, and the trimmed tissues and corresponding labels were placed in an embedding frame for dehydration and embedding operations later. After sectioning, hematoxylin-eosin (HE) staining was performed for microscopic examination and image acquisition and analysis.

[0026] F. After the experiment, ultra-high performance liquid chromatography-tandem quadrupole electrostatic field orbitrap mass spectrometry (UPLC-Q-Exactive Orbitrap-MS / MS) was used to conduct metabolomics research on the liver samples of mice in each drug-treated group, and metabolic pathway enrichment was performed to preliminarily explore its mechanism of action.

[0027] The liver tissue samples were taken out of the -80℃ freezer and thawed at 4℃. 80 mg of liver tissue samples were homogenized in pre-cooled methanol / water (1:1, 300 μL) for 1 min, vortexed for 2 min, and then centrifuged at 13 000 r·min at 4℃. -1Centrifuge for 15 min, take the supernatant and transfer it to a 1.5 mL EP tube. Extract the precipitate again in the same way, combine the supernatants of the two extractions, and blow dry the mixed supernatant under nitrogen. Before injection analysis, the dried extract was re-dissolved in 300 μL methanol-water (4:1), vortexed for 5 min, and centrifuged at 13000 r·min-1 at 4°C for 15 min, and 250 μL of supernatant was taken for injection analysis. Take 10 μL of the supernatant of each of the above test samples and mix them thoroughly for quality control (QC) samples. During injection, insert a QC sample for every 5 samples to check the stability and performance of the instrument.

[0028] Liquid phase conditions: mobile phase: A (containing 0.1% formic acid in water), B (containing 0.1% formic acid in acetonitrile); chromatographic column: ACQUITY UPLC HSS T3 (2.1 mm×100 mm, 1.8μm). Mass spectrometry conditions: heated electrospray ionization (HESI) method; positive and negative ion switching acquisition mode, positive and negative ion switching acquisition mode, scanning mode: Full Scan / dd-MS 2 ;The positive and negative spray voltages were 3.5 kV and 2.5 kV respectively; capillary temperature, 320℃; sheath gas flow rate: 35 arb, auxiliary gas flow rate: 10 arb; m / z acquisition range: 100~1500 Da.

[0029] SIMCA software was used to select and find the differential metabolites between the blank group and the model group. The obtained differential metabolites were restricted by VIP value, P value and FC value to screen out key potential biomarkers. The screening conditions were that compounds that simultaneously met VIP>1, P<0.05, and FC>1.5 were considered to be significantly up-regulated metabolites, and compounds that simultaneously met VIP>1, P<0.05, and FC<0.5 were considered to be significantly down-regulated metabolites. With the help of the KEGG database, MetaboAnalyst was used to perform pathway enrichment analysis on the screened differential metabolites.

[0030] 3. Experimental results A. Dynamic monitoring of body weight: The results of dynamic monitoring of body weight of mice in each group for 12 weeks are shown in Table 4. Figure 4 As shown, the weight gain of mice in the high-dose group of Bupleurum leaf extract was the smallest, and the degree of inhibition of their weight gain was the highest, and the effect was seen after 4 weeks of administration, indicating that high-dose Bupleurum leaf extract can have a slimming effect on the weight gain of mice fed a high-fat diet.

[0031] Table 4 Results of dynamic monitoring of body weight Note: Different letters after the values ​​in the same column indicate significant differences (P<0.05).

[0032] The epididymal adipose tissue of each group of mice was weighed and the density and average area of ​​adipocytes were measured. Figure 5 As shown in the results, it was found that after treatment with Bupleurum leaf extract, the epididymal adipocyte density and average area of ​​each group of mice were lower than those of the model group, indicating that Bupleurum leaf extract has slimming and fat-reducing activity. At the same time, the weight of scapular fat and inguinal fat was measured, and the results are shown in Table 5. Figure 6 , 7 As shown, all of them indicate that the extract of Bupleurum chinense leaves has the activity of reducing body weight and fat. The epididymal fat pathological sections of mice in each group were stained with hematoxylin-eosin (HE) to observe their pathological state. The results are shown in Figure 8 As shown, compared with the model group, the adipocytes in the epididymal fat of the blank group were closely arranged, uniform in size, and honeycomb-shaped; while the adipocytes in the model group were of different sizes and irregularly arranged, with few adipocyte fragmentation, adjacent adipocytes fused with each other, local adipocytes squeezed, and irregular in shape. After treatment with low-dose Bupleurum leaf extract, it can be seen that the adipocytes are closely arranged, uniform in size, and honeycomb-shaped, with a large lipid droplet in the center of the cell, a thin layer of cytoplasm located at the periphery of the cell, surrounding the lipid droplet, and the nucleus is oblate and squeezed to one side of the cell by the lipid droplet, but no obvious abnormality in the tissue was observed. After treatment with medium-dose Bupleurum leaf extract, it can be seen that the adipocytes are closely arranged, uniform in size, and honeycomb-shaped, but adipocyte fragmentation is occasionally observed. After treatment with high-dose Bupleurum leaf extract, the adipocytes are in good condition, closely arranged, uniform in size, regular in shape, and honeycomb-shaped, which is significantly improved compared with the model group. The positive drug Cassia seed extract group and Bupleurum extract group also have this effect, but according to the weight of epididymal fat, inguinal fat, and scapular fat, the effect of the high-dose Bupleurum leaf extract group is better than that of the positive drug group.

[0033] Table 5 Measurement results of fat weight and epididymal adipocyte data Note: Different letters after the values ​​in the same column indicate significant differences (P<0.05).

[0034] B. Fasting blood glucose and glucose tolerance test (OGTT) and dynamic monitoring of blood glucose-related biochemical indicators: After the feeding was completed every 2 weeks, the mice in each group were fasted at 21:00 on the same day. After 12 hours, the fasting blood glucose value was measured by blood sampling from the tail tip. The dynamic monitoring results are shown in Table 6. Fig. 9 As shown, it was found that the effect of Bupleurum leaf extract began to take effect from the 4th week after administration, indicating that Bupleurum leaf extract can have a certain correction effect on the increase in fasting blood glucose in type 2 diabetic mice induced by a high-fat diet, and the correction effect is most obvious under low-dose Bupleurum leaf extract treatment.

[0035] Table 6 Dynamic monitoring of fasting blood glucose Note: Different letters after the values ​​in the same column indicate significant differences (P<0.05).

[0036] After the last treatment with Bupleurum chinense leaf extract, mice in each group were fasted for 12 h, and then given 10% glucose solution by gavage. Blood glucose levels were measured at 0, 30, 60, and 120 min, and blood glucose curves were drawn. The results are shown in Table 7. Fig.10 As shown in the figure, compared with the blank group, the blood glucose levels of the mice in the model group increased at each time point and were significantly higher than those of the mice in the blank group. The blood glucose levels of the mice in the Bupleurum leaf extract treatment group were lower than those of the model group at each time point, but there was no significant difference between the dose groups. The results showed that Bupleurum leaf extract can improve the glucose metabolism disorder caused by a high-fat and high-sugar diet. The blood glucose levels of the two groups of mice treated with Cassia seed extract and Bupleurum extract also decreased, but the blood glucose levels of the mice in the Bupleurum leaf extract group were slightly lower than those in the Bupleurum extract group after 120 minutes.

[0037] Table 7 Oral glucose tolerance test (OGTT) results Note: Different letters after the values ​​in the same column indicate significant differences (P<0.05).

[0038] The results of blood glucose related biochemical indexes of mice in each group are shown in Table 8. Fig.11 As shown in the figure, serum insulin (INS), serum glucagon (GC) and serum glycosylated hemoglobin (GHb) levels were measured to evaluate the fasting blood glucose regulatory effect of Bupleurum leaf extract on type II diabetic mice induced by high-fat diet. It was found that compared with the blank group, serum INS, GC and GHb levels in the model group were significantly increased. After intervention with Bupleurum leaf extract, serum INS, GC and GHb in each group were adjusted back, among which the low-dose Bupleurum leaf extract group had significant difference compared with the model group (P<0.05), indicating that Bupleurum leaf extract has hypoglycemic activity and can regulate the fasting blood glucose level of type II diabetic mice induced by high-fat diet (P<0.05).

[0039] Table 8 Results of blood sugar related biochemical indicators Note: Different letters after the values ​​in the same column indicate significant differences (P<0.05).

[0040] C. Dynamic monitoring of lipid-related biochemical indicators: Biochemical kits were used to measure the total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) levels in the serum of each group of mice. Blood was collected from the orbits and serum was separated for dynamic monitoring every 4 weeks. The dynamic monitoring results are shown in Table 9. Fig.12 As shown in the results, it was found that Bupleurum leaf extract could significantly reduce the levels of total cholesterol (TC) and triglyceride (TG) in the serum of mice fed a high-fat diet, and adjust the levels of low-density lipoprotein (LDL) and high-density lipoprotein (HDL), indicating that Bupleurum leaf extract has lipid-lowering activity and can regulate the blood lipid levels of mice fed a high-fat diet (P<0.05). After 12 weeks of feeding, the high-dose Bupleurum leaf extract group was significantly different from the model group (P<0.01).

[0041] Table 9 Results of blood lipid related biochemical indicators Note: Different letters after the values ​​in the same column indicate significant differences (P<0.05).

[0042] D. Blood uric acid content: The blood uric acid (UA) content of each group of mice was measured. The results are shown in Table 10. Fig.13 As shown, it was found that the low-dose group of Bupleurum leaf extract could significantly reduce the uric acid content in the serum of mice fed a high-fat diet, indicating that Bupleurum leaf extract has uric acid-lowering activity on mice fed a high-fat diet and can regulate their blood uric acid levels (P<0.05).

[0043] Table 10 Blood uric acid content Note: Different letters after the values ​​in the same column indicate significant differences (P<0.05).

[0044] E. Observation of retinal pathological status: Observation of retinal pathological sections of mice in each group (HE staining), such as Fig.14 As shown, it was found that different dose groups of Bupleurum leaf extract can reduce the pathological damage of the retina in type II diabetic mice. HE staining results showed that the retinal tissue surface of the blank group mice in Figure A was smooth, the structure of each layer was clear, the cells were arranged tightly and regularly, there was no edema and loose changes in the inner plexiform layer and the outer plexiform layer, and no obvious pathological changes were found; in Figure B, the number of retinal ganglion cells in the model group mice decreased, the inner plexiform layer and the outer plexiform layer showed obvious loose changes, the distribution of cells in the inner nuclear layer and the outer nuclear layer was disordered, and the number was significantly reduced; compared with the model group, Bupleurum leaf extract and positive drugs Cassia seed extract and Bupleurum extract can reduce the pathological damage of the retina in type II diabetic mice. Among them, the treatment effects of the medium and high dose groups of Bupleurum leaf extract in Figures D and E were more obvious ( Fig.11 , GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer).

[0045] The retinal vascular network was observed by staining with PAS (Periodic Acid-Schiffstain) staining method. Fig.15 As shown in Figure A, the retinal blood vessels of the blank group mice run smoothly, the diameter of the tubes is uniform, the endothelial cell nuclei of the tube wall are oval and lightly stained, and the round pericyte nuclei, darkly stained, and evenly distributed around the blood vessels can be seen; while the retinal blood vessels of the model group mice in Figure B are twisted, irregular, the number of pericytes is significantly reduced, the endothelial cells proliferate, and are oval, fusiform or polygonal, with darkly stained nuclei, and acellular capillaries can be seen, and lumen occlusion occurs; compared with the model group, the treatment of Bupleurum leaf extract and positive drugs Cassia seed extract and Bupleurum extract can reduce the pathological damage of retinal blood vessels in type II diabetic mice. Among them, the improvement effect of the medium and high dose groups of Bupleurum leaf extract in Figures D and E is more obvious.

[0046] The results of retinal VEGF immunohistochemical staining are shown in Table 11. Fig.16 , and the expression of retinal VEGF was statistically analyzed. The results are as follows Fig.17 As shown, it was found that VEGF expression in the retina of blank group mice was very low; in the retinal ganglion cell layer of model group mice, VEGF expression was significantly increased (P<0.001); compared with the model group, both Bupleurum leaf extract and positive drug treatment could significantly reduce the expression of VEGF in the mouse retina, but except for the low-dose Bupleurum leaf extract group, there were no significant differences among the other drug-treated groups.

[0047] Table 11 Retinal VEGF expression Note: Different letters after the values ​​in the same column indicate significant differences (P<0.05).

[0048] F. Observation of liver, kidney and pancreas pathological status: The pathological status of the liver of each group of mice was observed. Fig.18As shown in the figure, there was no obvious watery deformation of hepatocytes around the central vein of the hepatic lobule of mice in the blank group in Figure A, the cytoplasm was obviously stained, and no obvious inflammatory cell infiltration was observed. In the figure B, the cells in the hepatic lobule of mice in the model group were swollen, the cytoplasm was loose and lightly stained, a large number of hepatocytes were watery (black arrows), and more hepatocytes were balloon-like swollen (black arrows), the nuclei were in the middle or biased to one side, and the cytoplasm was sparse or fine reticular structure; after treatment with low-dose bupleurum leaf extract, there was less watery degeneration of hepatocytes, but it was still accompanied by balloon-like swelling of hepatocytes, cell swelling, loose and lightly stained cytoplasm, after treatment with medium-dose bupleurum leaf extract, a small amount of central vein and portal vein congestion was observed, and there was less congestion and dilation of hepatic sinusoids, after treatment with high-dose bupleurum leaf extract, it was found that the central vein of the hepatic lobule was surrounded by hepatocytes and hepatic sinusoids arranged roughly radially, and the hepatocytes were round and plump, without obvious inflammatory cell infiltration, which was significantly improved compared with the model group. After treatment with Cassia seed extract, the hepatocytes were round and plump, but congestion and dilatation of the hepatic sinusoids were rare. After treatment with Bupleurum extract, a small number of hepatocytes showed mild watery degeneration, cell swelling, loose and lightly stained cytoplasm, but no obvious inflammatory cell infiltration was observed.

[0049] The wet weight of the liver of each group of mice was weighed when the samples were collected. The results are shown in Table 12. Fig.19 As shown in the figure, compared with the blank group, the wet weight of liver in the model group was significantly higher than that in the blank group. After treatment with bupleurum leaf extract, the wet weight of liver was adjusted back, and the adjustment effect was most significant in the high-dose bupleurum leaf treatment group. The cassia seed extract and bupleurum extract groups also adjusted the wet weight of liver. The liver function indexes of mice in each group were measured using a biochemical kit, and the results are shown in Table 13, Fig. 20 As shown in the results, compared with the blank group, the levels of AST and ALT in the serum of mice in the model group were significantly increased. After treatment with Bupleurum chinense leaf extract, the levels of AST and ALT decreased, and the callback effect was most significant in the high-dose Bupleurum chinense leaf treatment group. The results showed that Bupleurum chinense leaf extract has a certain liver-protective activity.

[0050] Table 12 Liver wet weight Note: Different letters after the values ​​in the same column indicate significant differences (P<0.05).

[0051] Table 13 Serum aspartate aminotransferase and alanine aminotransferase levels Note: Different letters after the values ​​in the same column indicate significant differences (P<0.05).

[0052] The pathological state of the kidneys of mice in each group was observed to evaluate its preliminary safety, such as Fig.21As shown, in the blank group mice in Figure A, the glomeruli are evenly distributed in the cortex of the kidney tissue, and the renal tubular epithelial cells are round and plump. In the model group mice in Figure B, the renal cyst wall is occasionally hyperplastic and thickened (black arrow), and a large number of renal tubular epithelial cells are hydropic (black arrow), and the cells are swollen, the cytoplasm is loose and lightly stained. After high-dose treatment with Bupleurum leaves, the glomeruli are evenly distributed in the cortex of the kidney tissue, and the number of cells and matrix in the glomeruli are uniform, that is, the physiological state of the kidney does not change significantly after high-dose treatment with Bupleurum leaves. After treatment with Bupleurum extract, the glomeruli are evenly distributed in the cortex of the kidney tissue, and the renal cyst wall is occasionally hyperplastic and thickened, and a large number of renal tubular epithelial cells are hydropic, the cells are swollen, the cytoplasm is loose and lightly stained, a small number of renal tubular epithelial cells are vacuolated, a small number of renal tubular atrophy, and interstitial vascular congestion is rare (black arrow).

[0053] The pathological state of the pancreas of mice in each group was observed. Fig. 22 As shown in the figure, the pancreatic islets of the blank group mice were normal in morphology and regular in arrangement of cells; the number of acinar cells was abundant, closely arranged, with clear boundaries, eosinophilic at the top and basophilic at the base, and the cell morphology was normal. However, the pancreatic islets of the model group mice were wrinkled and irregular in shape, and acinar cells could be seen invading the islets (black arrows). The acinar cytoplasm was lightly stained and loose, with blurred boundaries, and the number of alveolar heart cells was significantly reduced. After low-dose treatment with Bupleurum leaves, the islets were found to be of different sizes, and a small amount of interstitial vascular congestion could be observed. After medium-dose treatment with Bupleurum leaves, the islets were still found to be irregular in morphology, and multiple interstitial vascular congestion could be observed. After high-dose treatment with Bupleurum leaves, the islets were found to be normal in morphology, cylindrical, with abundant acinar cells, closely arranged, and clear boundaries, which was significantly improved compared with the model group. After treatment with Cassia seed extract, the islets were found to be irregular in shape, and acinar cells could be seen invading the islets. After treatment with Bupleurum extract, the islets were found to be normal in morphology, but a small amount of interstitial vascular congestion could be observed. The callback effects of all efficacy indicators are summarized in Table 14.

[0054] Table 14 Summary of the callback effects of Bupleurum chinense leaf extract and positive drugs Note: In the table, “#” means significant difference compared with the HFD group at the initial measurement, “#” means P<0.05, “##” means P<0.01, “###” means P<0.001, “####” means P<0.0001, “-” means P>0.05, and “—” means unknown.

[0055] G. Metabolomics study of liver samples of mice in each drug-treated group was performed using ultra-high performance liquid chromatography tandem quadrupole electrostatic field orbitrap mass spectrometry (UPLC-Q-Exactive Orbitrap-MS / MS). Typical representative total ion current graphs are shown in Figure 2. Fig.23 The collected data of each experimental group were subjected to unsupervised PCA analysis. The results are shown in Fig.24 . The results showed that the QC samples were well clustered, indicating that the results were stable and reliable, and the model group and the blank group could be clearly separated, that is, the animal experiment model was successful; each drug-treated group had a clear separation trend from the model group, and the trend was adjusted back to the blank group, indicating that the 12-week drug treatment of the Bupleurum leaf extract group was effective, and similar to the positive drug group, indicating that its drug administration effect was close to that of the positive drug group. Overall, the Bupleurum leaf extract had a significant pharmacodynamic effect, and the effects of the medium and high dose groups were better than those of the low dose group.

[0056] The OPLS-DA method was further used to verify the model. Fig.25 As shown in the figure, the model validation results show that the model has a larger slope than the negative semi-axis with respect to the vertical axis, indicating that the model has no overfitting phenomenon and meets the experimental requirements. It can be used for the screening of potential biomarkers, and the potential biomarkers can be analyzed by heat map. Fig.26 As shown, the results showed that these potential markers can be used to evaluate the efficacy of drugs among different groups.

[0057] Table 15 Liver metabolic markers in the treatment of metabolic syndrome with Bupleurum chinense leaves MetaboAnalyst metabolic pathway enrichment analysis was performed on the key metabolic markers of Bupleurum leaf extract in the treatment of metabolic syndrome. Fig. 27 As shown in the figure, the enriched pathways include glutathione metabolism, cysteine ​​and methionine metabolism, starch and sucrose metabolism, etc. The gene targets obtained from the intersection metabolic markers of Bupleurum chinense leaf extract on the Swiss target prediction website and the disease gene targets related to metabolic syndrome and diabetic retinopathy found in Gene cards were then analyzed for intersection, further focusing on the core pathways for treating metabolic syndrome. A total of 153 intersection targets were screened, and KEGG pathway analysis was performed on the intersection targets using metscape. The results are as follows: Fig.28As shown in the figure, the results of KEGG pathway analysis showed that the possible pathways for Bupleurum leaf extract to treat metabolic syndrome include lipid and atherosclerosis, insulin resistance, and glutathione metabolism, etc. This suggests that glutathione metabolism and starch and sucrose metabolism may be the main metabolic pathways for Bupleurum leaf extract to play a role in treating metabolic syndrome.

[0058] In summary, UPLC-Q-Exactive Orbitrap-MS / MS technology was used to conduct metabolomics research on liver samples of mice in each drug-treated group, and KEGG pathway analysis was performed to preliminarily explore its mechanism of action. The results showed that the metabolic pathways of Bupleurum leaf extract in the liver of mice fed a high-fat and high-sugar diet mainly included glutathione metabolism, starch and sucrose metabolism, etc. Glutathione can help maintain a normal immune function system and has antioxidant, integrated detoxification effects. The weight loss, fat reduction and liver protection effects of Bupleurum leaf extract may be related to this; the metabolic process of starch and sucrose is essential for maintaining the energy supply of the organism and is closely related to the metabolism of glucose in the body. It is speculated that the function of Bupleurum leaf extract in reducing fasting blood sugar in mice fed a high-fat and high-sugar diet may be related to this process.

[0059] Example 3: Human experiment of Bupleurum leaf extract: 6 subjects aged 25-30 with high fasting blood sugar, blood lipids and blood uric acid were selected, and the Bupleurum leaf extract prepared by the present invention was given with water for drinking every day, 5g each time, twice a day, and both groups were observed for 2 weeks. After 2 weeks, the human experiment results of Bupleurum leaf extract were measured, and the results showed that Bupleurum leaf extract had good palatability, no discomfort, and slightly adjusted body weight and fasting blood sugar.

[0060] In summary, the bupleurum leaf extract is extracted from bupleurum leaves. The chemical composition is identified by LC-MS, and it is found that the extract contains saponins, flavonoids, acids, phenylpropanoids and other components. Pharmacological experiments have proved that the bupleurum leaf extract has a correction effect on the weight gain induced by a high-fat diet, and has a certain inhibitory effect on the increase in blood sugar caused by type 2 diabetes induced by a high-fat diet. At the same time, through the observation of the pancreatic state, it was found that the bupleurum leaf extract has a correction on the pancreatic pathological state, and it is particularly obvious under the treatment of high-dose bupleurum leaf extract. Through dynamic monitoring, it was found that taking a low dose of bupleurum leaf extract can significantly reduce the increase in fasting blood sugar and blood uric acid levels induced by a high-fat and high-sugar diet, and taking a high dose of bupleurum leaf extract can significantly reduce blood lipid levels and has therapeutic significance for diabetic complications. In the observation of its liver pathological sections, it was found that the bupleurum leaf extract can improve liver lesions caused by a high-fat and high-sugar diet, and has a certain degree of liver protection. A preliminary safety evaluation was carried out through the observation of its renal pathological sections, and it was found that there was no obvious change in the morphology and number of glomeruli after high-dose bupleurum leaf treatment. Therefore, the extract of bupleurum leaf may be of great significance in improving metabolic syndrome-like diseases induced by a high-fat diet, and its effect is close to that of the positive control drug Cassia seed. Metabolomics studies were conducted on the liver samples of mice in each drug-treated group in the experiment, and KEGG pathway analysis was performed on the metabolic markers of the bupleurum leaf extract group. The results suggested that the bupleurum leaf extract does have a significant pharmacodynamic effect, and the main metabolic pathways for the treatment of metabolic syndrome may be glutathione metabolism (Glutathione metabolism) and starch and sucrose metabolism (Starch and sucrosemetabolism). At the same time, the target effect can still be observed in a small-scale human experiment lasting 2 weeks. This shows that the extract can be used in the development of functional products to improve metabolic syndrome.

Claims

1. A Bupleurum leaf extract, characterized in that: The bupleurum leaf extract is a water extract of bupleurum leaves. In the bupleurum leaf extract, the total polysaccharide content is 233.68±20.27 mg / g, the total flavonoids content is 62.41±0.61 mg / g, the total phenol content is 48.40±2.96 mg / g, the total saponin content of bupleurum is 0.87±0.055%, of which the content of saikosaponin A is 0.17±0.006%, the content of saikosaponin B1 is 0.22±0.006%, the content of saikosaponin C is 0.05±0.037%, and the content of saikosaponin D is 0.43±0.006%.

2. A method for preparing the Bupleurum chinense leaf extract according to claim 1, characterized in that: The method comprises the following steps: mixing bupleurum leaf and water in a mass ratio of 1:10, soaking the bupleurum leaf in water for 12 hours, then decocting with 80-90℃ hot water for 3 times, each time for 50 minutes, combining the filtrate, concentrating under reduced pressure, and freeze-drying to obtain the bupleurum leaf extract.

3. Use of the Bupleurum chinense leaf extract according to claim 1 in the preparation of products for preventing and treating metabolic syndrome.

4. The use according to claim 3, characterized in that: The bupleurum leaf extract is used in regulating glutathione metabolism and starch and sucrose metabolism pathways to prepare products for preventing and treating metabolic syndrome.

5. The use according to claim 3, characterized in that: The metabolic syndrome is obesity, hyperlipidemia, hyperglycemia, diabetes or its complications.

6. The use according to claim 5, characterized in that: The complication is diabetic retinopathy.

7. The use according to claim 5, characterized in that: The bupleurum leaf extract is used in the preparation of a product for reducing hyperglycemia induced by a high-fat and high-sugar diet, and the dosage of the bupleurum leaf extract is a low dose of 1.35 g / kg.

8. The use according to claim 5, characterized in that: The bupleurum leaf extract is used in the preparation of a product for reducing hyperuricemia induced by a high-fat and high-sugar diet, and the dosage of the bupleurum leaf extract is a low dose of 1.35 g / kg.

9. The use according to claim 5, characterized in that: The bupleurum leaf extract is used in the preparation of a product for reducing body weight and blood lipids induced by a high-fat and high-sugar diet, and the dosage of the bupleurum leaf extract is a medium-high dose of 4.05-12.15 g / kg.

10. The use according to claim 5, characterized in that: The bupleurum leaf extract is used in the preparation of a product for reducing retinopathy induced by a high-fat and high-sugar diet, and the dosage of the bupleurum leaf extract is a medium-high dose of 4.05-12.15 g / kg.

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