阅读说明：本技术 一种用于高脂饮食诱导肥胖小鼠减肥的肋柱花提取物及制备方法 (Extract of lomatogonium carinthiacum for inducing obese mice to lose weight by high-fat diet and preparation method thereof ) 是由 付明海 阿丽沙 巴根那 李慧芳 小平 于 2021-09-27 设计创作，主要内容包括：本发明提供了一种用于高脂饮食诱导肥胖小鼠减肥的肋柱花提取物及制备方法；所述肋柱花提取物为将肋柱花全草经二氯甲烷溶液浸泡,用乙醇提取,干燥,得到的肋柱花提取物。本发明还涉及前述肋柱花提取物的制备方法。本发明所涉及的肋柱花提取物可显著降低小鼠相对体重增长率、摄食量、Lee`s指数、肝脏重量,进而达到有效减肥的目的。(The invention provides a stylosanthes guianensis extract for inducing obese mice to lose weight by high-fat diet and a preparation method thereof; the extract of the lomatogonium carinthiacum is obtained by soaking whole plant of the lomatogonium carinthiacum in dichloromethane solution, extracting with ethanol, and drying. The invention also relates to a preparation method of the extract of the lomatogonium carinthiacum. The extract of the lomatogonium carinthiacum can obviously reduce the relative weight growth rate, the food intake, the Lee's index and the liver weight of a mouse, thereby achieving the purpose of effectively losing weight.)

1. A lomatogonis extract for inducing obesity of high-fat diet is prepared by soaking whole plant of lomatogonis in dichloromethane solution, extracting with ethanol, and drying to obtain lomatogonis extract.

2. The method for preparing the extract of lomatogonium carinthiacum for inducing obesity in a high-fat diet as claimed in claim 1, comprising the steps of:

step 1, airing the whole plant of the Mongolian medicinal material ribbing flower in the shade, and crushing into powder with the particle size of 0.5 cm;

step 2, soaking the mixture in ten times of dichloromethane solution for one night, and extracting the mixture once every 2 hours for 2 times;

and step 3, soaking the medicine residues in 95% ethanol for one night, extracting for 2 times every 3 hours, recovering ethanol, and preparing the medicine residues into powder by using a freeze dryer.

Technical Field

The invention belongs to the field of national medicine; in particular to a lomatogonium carinthiacum extract for inducing obese mice to lose weight by high-fat diet and a preparation method thereof.

Background

Obesity is one of ten chronic metabolic diseases determined by WHO to be caused by the combined action of gene, environment and motion factors, particularly fat accumulation in vivo caused by excessive intake of food nutrients. Obesity not only can bring about changes of body morphology, but also more importantly metabolic syndromes caused by overweight and obesity, namely hypertension, hyperlipidemia, type 2 diabetes mellitus, coronary heart disease, malignant tumor, polycystic ovary syndrome, sleep apnea and other diseases, become healthy invisible killers in our lives. Excess storage of triglycerides in the body of obesity can lead to significant accumulation of macrophages in visceral adipose tissue, resulting in modified release of adipokines and pro-inflammatory cytokines, such as TNF-and IL-6, leading to metabolic abnormalities.

Thus, obesity is characterized by a sustained systemic low level inflammatory response. The current treatment methods mainly comprise Roux-en-Y gastric bypass surgery, adjustable gastric band and sleeve gastrectomy, behavior weight-losing treatment after cognitive remediation, nanoparticle anti-angiogenesis therapy treatment, chemotherapy using Hyperalamic broad-derived neurotrophic factor (BDNF) or Fibroblast growth factor 21(FGF21) gene therapy, intestinal flora and the like.

With the intensive research on the intestinal flora in recent years, it is gradually found that the intestinal flora is closely related to the development and development of obesity. The intestinal flora inhabits the human intestinal tract and is a microbial flora composed of bacteria which depend on the human body to live. The intestinal tract of a human body contains 100-1500 bacteria of nearly hundreds of trillion, the number of the bacteria is close to the total number of cells of the human body, and the ratio is about 1: 1. The intestinal flora has great influence and effect on human health and maintaining human physiological functions, for example, the intestinal flora can regulate the immune system of the body and abnormal metabolic diseases, and can also control the absorption of energy of the human body through the influence on intestinal epithelial cells. Eating high-fat and high-sugar diet for a long time can cause the disturbance of intestinal flora, reduce the proportion of beneficial bacteria and increase the proportion of pathogenic bacteria. Imbalance of the intestinal flora also leads to increased permeability of the intestinal mucosa and the development of chronic inflammation. The low-degree inflammation can cause the body to have insulin resistance and blood fat disorder, further influence the metabolism of sugar and fat, and finally cause the occurrence and development of diabetes and obesity.

Mongolian medicine costal flower (Lomatogeniumrotatum (L.) Fries ex Nym.), Mongolian name Habizugen-Digeda, which was loaded into the drug Standard Mongolian medicine booklet of Ministry of health of the people's republic of China in 1998, has the functions of clearing heat, invigorating stomach, healing wound and the like. Recent research shows that the components of the xanthone compound, swertiamarin, luteolin, oleanolic acid and the like in the Mongolian costcolumn flower have different degrees of liver protection and cholagogic activity and are one of the main active components of the costcolumn flower. Researchers find that Mongolian medicine lomatogonium carinthiacum has the effects of protecting liver and benefiting gallbladder, and flavonoids compounds 1-hydroxy-3, 5, 8-trimethoxyflavone, l-hydroxy-3, 7, 8-trimethoxyflavone, swertiamarin and 6, 8-dihydroxy-1, 2-dimethoxyflavone can treat obesity-related diseases, relieve hypercholesterolemia and hypertriglyceridemia caused by high-sugar food intake, and improve lipid and leptin metabolism of insulin resistant rats. However, whether costal columna acts to regulate gut microbiota to prevent obesity has not been reported.

Disclosure of Invention

The invention aims to provide a lomatogonium extract for inducing obese mice to lose weight by high-fat diet and a preparation method thereof.

The invention is realized by the following technical scheme:

the invention relates to a lomatogonis extract for inducing obese mice to lose weight by high-fat diet.

The invention also relates to a preparation method of the extract of the lomatogonium carinthiacum for inducing the fat mice to lose weight by high-fat diet, which comprises the following steps:

step 1, airing the whole plant of the Mongolian medicinal material ribbing flower in the shade, and crushing into powder with the particle size of 0.5 cm;

step 2, soaking the mixture in a dichloromethane solution for one night, and extracting the mixture for 2 times every 2 hours;

and step 3, soaking the medicine residues in 95% ethanol for one night, extracting for 2 times every 3 hours, recovering ethanol, and preparing the medicine residues into powder by using a freeze dryer.

The main chemical components of the lomatogonium carinthiacum related by the invention are flavonoids, terpenoids and other compounds.

Wherein the terpenes are triterpenes and iridoid glycosides, including: 6 kinds of compounds such as oleanolic acid, swertiamarin, 2 alpha-hydroxy oleanolic acid, ursolic acid, lomacarinoside A and lomacarinoside B.

The flavones are dipyridone and its glycoside, flavone and its glycoside, flavonol, and carbon biflavone glycoside; the method comprises the following steps: 22 compounds such as kaempferol, luteolin, apigenin, luteolin-7-O-D-beta-glucoside, apigenin-7-O-D-beta-glucoside, mangiferin, gentiin, swertisin, isoorientin, isovitexin , swertisin and the like.

Luteolin in the flavonoids compounds can reduce lipid aggregation in 3T3-L1 cells and HepG2 cells, has lipid lowering effect, and can inhibit macrophage polarization related to obesity and reduce obesity.

The swertiamarin in terpenoid compounds can improve lipid deposition and hyperlipidemia induced by high-fat diet, and has effects of reducing blood lipid and blood sugar.

Oleanolic acid in terpenes has remarkable effect of inhibiting lipid formation of 3T3-L1 fat cells, and is beneficial to protecting blood vessels and preventing cardiovascular diseases to a certain extent by reducing lipid accumulation and regulating blood lipid metabolism. Oleanolic acid has effects of resisting obesity, resisting diabetes, and reducing blood lipid.

Ursolic acid in terpenes can promote proliferation and differentiation of 3T3-L1 preadipocyte, increase glucose uptake of adipocyte, inhibit generation of free fatty acid, promote secretion of adiponectin, and improve insulin resistance of adipocyte. Ursolic acid has effects of obviously reducing body weight and adipose tissue quality of obese male mice, and improving sugar tolerance and insulin sensitivity.

Mangiferin in flavone can promote glucose uptake of IR-HepG2 cell, reduce TG and TC content, and improve insulin resistance and abnormal glycolipid metabolism. The mangiferin can also obviously increase the glucose oxidation of C57BL6/J mice without changing the fatty acid oxidation, obviously reduce the weight gain and fat accumulation in the bodies of the mice caused by high-fat diet, promote the energy consumption and the utilization of carbohydrate, and improve the insulin sensitivity.

Kaempferol in the flavonoids can also effectively inhibit oxLDL-induced lipid accumulation in macrophages and has the effect of reducing blood fat.

The invention specifically applies the preparation of the extract of the lomatogonium carinthiacum as follows:

after 8W, the mice of the obesity model (with the ND group as an experimental control group and the body weight more than 20% of the mice of the ND group) were randomly divided into a high fat diet group (HFD), a high dose group (LR 1.8g/kg), a medium dose group (LR 0.9g/kg), a low dose group (LR0.18g/kg) and a positive control group (Orlistat, 0.048g/kg) except for the general diet group (ND). Mouse body weight and food intake were observed and recorded weekly. After 9 weeks of continuous administration, relevant weight loss indexes were examined.

The invention has the following advantages:

(1) the extract of the lomatogonium carinthiacum can obviously reduce the relative weight growth rate, the food intake, the Lee's index and the liver weight of a mouse, thereby achieving the purpose of effectively losing weight;

(2) the extract of the lomatogonium carinthiacum related by the invention also obviously reduces the level of serum Triacylglycerol (TG) and Total Cholesterol (TC) of an LR1.8g/kg group, increases High Density Lipoprotein (HDL), and can also obviously reduce the level of serum alanine Aminotransferase (ALT) at LR0.18g/kg.

Drawings

FIG. 1 is a graph of the effect of extract of ribwort on body weight and food intake;

FIG. 2 is a graph of the effect of extract of ribwort on adipose tissue and liver weight;

FIG. 3 is a graph showing the effect of extract of intercostal flower on serum blood lipid levels and liver levels.

FIG. 4 is a graph showing changes in lipid levels and histopathology of liver tissues observed by HE staining under an optical microscope;

FIG. 5 is a graph showing changes in lipid levels and histopathology of periintestinal adipose tissue observed by HE staining under an optical microscope;

FIG. 6 is a graph showing changes in perirenal adipose tissue lipid levels and histopathology observed by HE staining under an optical microscope;

FIG. 7 is a graph showing changes in lipid level and histopathology in peritesticular adipose tissue observed by HE staining under an optical microscope.

Detailed Description

The present invention will be described in detail with reference to specific examples. It should be noted that the following examples are only illustrative of the present invention, but the scope of the present invention is not limited to the following examples.

Examples

The embodiment relates to a preparation method and application of a lomatogonium carinthiacum extract;

materials used

1.1 Experimental instruments

Varioskan^TMLUX multifunctional microplate reader (Thermo Fisher Scientific-CN), Sigma 3-18KS high speed refrigerated centrifuge, FD-1A-50 freeze dryer (Beijing Bo Yi kang laboratory instruments Co., Ltd.), full automatic biochemical instrument electronic balance (Saedolis Scientific instruments (Beijing) Co., Ltd.).

1.2 Experimental drugs and reagents

The flower of Ribes nigrum is collected from American flag of Nemongolian Siller, West, and identified as dry whole grass of Lomatogoniumcarinthiacaum (Wulf) Reichb. High fat feed (Shandong Hengrong Biotechnology Co., Ltd.) and common feed (Liaoning Changsheng Biotechnology Co., Ltd.).

1.3 Experimental animals

Male and female SPS-grade KM mice are selected for the experiment, the mice are 5 weeks old, the body mass (35 +/-2) g and 80 mice are purchased from Liaoning Changsheng biotechnology GmbH, the animal license number is as follows: SCXK (Liao) 2015-0001.

Second, operation method

2.1 preparation of extract of Crotalaria costata

Drying Mongolian medicinal material, air drying and pulverizing in the shade, soaking in dichloromethane solution overnight, extracting for 4h for 2 times, soaking the residue in 95% ethanol overnight, extracting for 6h for 2 times, recovering ethanol, and making into powder with freeze dryer.

2.2 animal grouping, modeling, dosing and sample Collection

After 80 SPS-grade KM mice are adaptively fed for 1 week, the mice are randomly divided into 2 groups, 10 mice are given common feeds to be common diet groups (ND), the rest mice are given high-fat feeds to establish an obesity model, water is freely drunk and food is taken, the animals are in 12h day-night circulation in the room, the room temperature is (22 +/-2) DEG C, and the relative humidity is 40-50%.

After 8 weeks, the common diet group was used as an experimental control group, anti-obesity mice were removed, and an obesity model was determined using an average body weight of more than 20% as an obesity standard. After successful modeling, the obesity model is randomly divided into a high fat diet group (HFD), a high, medium and low dose group (1.8g/kg, 0.9g/kg and 0.18g/kg) and a positive control group (orlistat, 0.048g/kg) on the basis of high fat feed feeding, wherein each group contains 10 n. ND and HFD were given equal volumes of saline 1 time a day for 9 weeks with continuous gavage. Mice body mass, food intake were observed and recorded weekly during this period. After the last administration of the experiment, fasting was performed for 12 hours without water deprivation, the body length, obesity index (Lee's index) were measured after anesthesia by intraperitoneal injection of 10% chloral hydrate 3ml/kg after measurement of body weight, serum was collected, perirenal, peritesticular, periintestinal fat and liver tissue were weighed, stool samples were collected in the colon part immediately after freezing in liquid nitrogen, and stored at-80 ℃.

2.3 Biochemical analysis of serum and liver indices

2h after the last administration of the experiment, the weight was taken, the amount of anesthesia was calculated, and an anesthetic (10% chloral hydrate: 0.003ml/g) was applied. Rapidly drawing blood with 1ml syringe, standing in ice box for 30min, centrifuging at 3000 r/min for 10min, subpackaging the supernatant, immediately freezing in liquid nitrogen, and storing in-80 deg.C refrigerator. Levels of Triacylglycerol (TG), Total Cholesterol (TC), high-density lipoprotein (LDL), low-density lipoprotein (HDL), alanine Aminotransferase (ALT), and aspartate Aminotransferase (AST) (purchased from shenzhen kueberr biotechnology, ltd) in serum were measured using a fully automatic biochemical analyzer.

2.4 HE staining for pathological changes in liver and adipose tissues

Fixing the tissues of liver, periintestinal fat, perirenal fat and peritesticular fat in 10% paraformaldehyde solution, dehydrating by gradient with 80%, 90%, 95% and 100% ethanol, embedding paraffin after xylene is transparent, cutting into 3 μm slices, placing on a glass slide, and baking at 62 ℃ for one hour. Washing paraffin sections with xylene, anhydrous ethanol, 95%, 90%, 80% ethanol, and tap water, staining the sections with hematoxylin for 1-3min, staining with eosin for 30s, and sealing with neutral gum. The structural state and pathological damage condition of the liver tissue and adipose tissue cells are observed under a microscope and photographed. The nuclei appear blue, and the cytoplasm and other tissue components appear in a red color with varying shades.

Third, experimental results

3.1 Effect of extract of Ixeris carinata on weight gain, Lee's index and food intake in obese mice

The results are shown in FIG. 1, wherein A is a graph of the change in body weight gain in 8 weeks of high fat diet-induced mice; b is a graph comparing body weight to time analysis (n for each group 10) during administration of ribbonfil; c is a result chart of the effect of the costal Styloides on the food intake of the obese mice after 9 weeks of administration,^*p is less than 0.05, D is a result chart of the influence of the costaria on the Lee's index of an obese mouse after 9 weeks of administration,^#p is less than 0.05. As expected, by analyzing the body mass of mice in each group after oral gavage of intercostal ribbon and orlistat, it was found that the body weight, food intake and Lee's index were increased in the HFD group as compared with the ND group, and this was reduced by the intercostal ribbon administration. Results prove that LR1.8g/kg, LR0.9g/kg, LR0.18g/kg and orlistat can obviously reduce the increase of the body weight and food intake of high-fat diet-induced obese mice and reduce the Lee's index.

The Lee's index, the increase in body weight and fat content are important indicators for the evaluation of obesity. The Lee's index, body weight and fat content increased significantly after high fat diet induced obesity, as confirmed again by the model group in this experiment. The experimental results show that the use of the extract of ribbonfil can significantly reduce the HFD-induced Lee's index and body weight gain in obese mice, and reduce the fat mass of periintestinal fat, perirenal fat and epididymal adipose tissue.

3.2 Effect of extract of Crotalaria on adipose tissue and liver weight in obese mice

The results are shown in FIG. 2, in which A is a graph of the effect of extract of lomatogonium on periintestinal fat; b is a graph of the effect of extract of intercostal flower on perirenal fat; c is a graph of the influence of the extract of the lomatogonium on the fat around the testis; d is a graph of the effect of extract of ribbonfil on liver weight. It was found that the HFD group had a significant increase in adipose tissue compared to the ND group^*P is less than 0.05, and the weight of adipose tissue is obviously reduced after the costal columna is administrated compared with that of HFD group^#P is less than 0.05. During the 9-week dosing period after successful modeling, significant increases in periintestinal fat, perirenal fat, peritesticular fat, and liver weight were observed following HFD feeding. Significant reduction in periintestinal, perirenal and peritesticular fat following gavage of the extract of ribbing in comparison to the HFD group). In addition, the LR1.8g/kg and LR0.18g/kg groups significantly reduced liver weight (P < 0.05). In particular, the liver weights of the LR1.8g/kg group were not significantly different from those of the ND group (p > 0.05).

3.3 influence of extract of Rizhua on lipid serum blood lipid index and liver index of obese mice

FIG. 3 is a graph showing the effect of extract of Crotalaria on serum blood lipid and liver indices; wherein A is a TC comparison graph; b is TG contrast diagram; c is HDL contrast map; d is AST contrast diagram; e is ALT comparison. The concentrations of TC, TG and HDL-C, LDL-C in the serum of the mouse are detected by a full-automatic biochemical analyzer. The TC, TG and HDL-C of the HFD group were significantly increased and LDL-C was significantly decreased compared to the ND group. The LR1.8g/kg group significantly reduced TC, TG levels, increases and HDL-C concentrations in HFD-fed mice, as shown in A-C in FIG. 3. ALT and AST are 2 markers for evaluating liver function, and compared with the ND group, the ALT and AST levels in the serum of the model group mice are higher (P is less than 0.001), which indicates that the liver of the model group mice is seriously damaged. Serum ALT levels were significantly down-regulated (P < 0.05) in mice in the LR0.18g/kg group after administration of the extract of intercostal flowers, whereas AST was less significantly affected by the extract of intercostal flowers, see D, E in FIG. 3. These results indicate that the extract of ribcage flower may improve dyslipidemia in obese mice and may repair a certain degree of liver damage in obese mice, but AST concentration is not significantly affected by the extract of ribcage flower.

Studies have shown that obese, particularly abdominal obese, are often accompanied by a disorder of lipid metabolism, i.e., dyslipidemia, manifested by elevated TG, TC, and LDL-C and reduced HDL-C. Dyslipidemia is a major risk factor for coronary heart disease, intermittent claudication and cerebral palsy. Because people's attention to dyslipidemia and atherosclerosis and cardiovascular and cerebrovascular diseases caused by dyslipidemia and atherosclerosis is increased, researches on treating dyslipidemia are more and more paid attention. Therefore, the invention detects the influence of the costal Style on the blood lipid index, and finds that the invention has the effects of reducing TC and TG and obviously increasing the serum HDL level. TC is an important material for synthesizing physiologically active substances such as adrenocortical hormone, cholic acid, sex hormone, vitamin D, etc., and is also a main component of cell membranes, and the degree of fat metabolism is indicated by its serum concentration. TG is an important factor in cardiovascular diseases, and the increase of TG can cause diseases such as diabetes, hypothyroidism, nephrotic syndrome, pancreatitis and atherosclerosis. HDL, as a lipoprotein, is responsible for preventing the development of atherosclerosis, and is a major protective factor in coronary heart disease, and can clear away the accumulation of cholesterol in peripheral tissues to prevent the development of atherosclerosis. In contrast, LDL is likely to cause the formation of arteriosclerosis, so this protein is called "bad cholesterol". These experimental results again confirmed that the extract of costal Styloides has hypolipidemic effect.

3.4 liver and adipose tissue lipid levels and histopathological changes

FIG. 4 is a graph showing changes in lipid levels and histopathology of liver tissues observed by HE staining under an optical microscope. The results showed that ND nuclei were rounded and stained purple, and cytoplasm was stained red-purple. The shape and boundary of HFD cells are not clear, lipid vacuoles with different sizes can be seen in the HFD cells, partial cell lipid vacuoles are fused, and the total lipid vacuoles are large in area. There was significantly more fat deposition in liver tissue compared to ND group. Although the LR group hepatocytes were well-defined in structure and had a small number of vacuoles, the area of cytoplasmic vacuoles was significantly reduced compared to the HFD group. Of these, the LR0.18g/kg group was most pronounced.

FIGS. 5 to 7 are graphs showing the changes in periintestinal fat, perirenal fat, peritesticular fat tissue lipid level and histopathology observed by HE staining under an optical microscope. Compared with ND group, the fat cells of HFD group mouse are bigger, the inside is full of fat droplets, and the number of the fat cells is obviously reduced in the same visual field range of microscope. Compared with the HFD group, the LR group mice have obviously reduced intestinal surrounding fat cell diameter, and the number of fat cells in the same visual field of a microscope is obviously increased. Compared with the HFD group, the diameter of perirenal adipocytes in the LR0.9g/kg and LR0.18g/kg groups is obviously reduced, and the number of adipocytes in the same visual field of a microscope is obviously increased. Compared with the HFD group, the diameter of the fat cells around the testis in the LR0.18g/kg group is obviously reduced, and the number of the fat cells in the same visual field of a microscope is obviously increased. This result is probably due to the fact that LR can reduce the accumulation of lipid droplets in adipocytes, inhibiting the enlargement of adipocytes.

The results of the HE staining also indicate that the extract of the lomatogonium carinthiacum can improve the size of the visceral fat cells, and the number of the fat cells in the same visual field under a microscope is obviously increased. These results indicate that costal Styloides has a good effect against obesity induced by high fat diet.

In conclusion, the invention researches the influence of the costal column flower of the Mongolian medicine on the weight gain, food intake, adipose tissue weight, serum blood lipid index, liver weight, microbiota and the like of the HFD-induced obese mice respectively, and finally proves that the intervention of the costal column flower of the Mongolian medicine can obviously reduce the weight growth rate, the food intake, the Lee's index, the liver, the adipose tissue weight, the TC, TG and ALT index and obviously increase the serum HDL level.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.