Synergistic intervention application of nitraria sibirica pall leaf extract based on flavonoid components in resisting obesity and metabolic syndrome

Abstract

The invention provides application of a flavonoid component of Nitraria sibirica Pall. Leaves as a characteristic ethnic medicinal material in Xinjiang of China in the aspects of lowering lipid and resisting obesity, and belongs to the field of biological medicines. It is found for the first time that the nitraria sibirica pall leaf flavonoid substance remarkably relieves obesity induced by high-fat diet by regulating an AMPK / NF-kappa B signal channel, has the effects of resisting hyperlipidemia and non-alcoholic fatty liver diseases, improving the chronic inflammation state of an organism, repairing the intestinal barrier function and regulating intestinal microflora, and animal experiments prove that the nitraria sibirica pall leaf flavonoid substance has a significant effect on relieving obesity induced by high-fat diet and has a significant effect on relieving obesity induced by high-fat diet by regulating the AMPK / NF-kappa B signal channel. The blood fat level can be recovered to a normal range under the intervention of high dose (400mg / kg / d), and no obvious toxic or side effect exists. The sibirica nitraria tangutorum leaf flavonoid component can be prepared into the medicine for treating obesity and related metabolic syndromes, a new candidate scheme is provided for the field of natural medicine anti-obesity, and the sibirica nitraria tangutorum leaf flavonoid component has safety and industrial potential.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an application of a flavonoid component derived from the leaves of Nitrariasibirica Pall. in the preparation of an anti-obesity, lipid-lowering and metabolic syndrome improving drug, wherein the application comprises achieving multi-target intervention in metabolic disorders by regulating the AMPK / NF-κB signaling pathway, repairing the intestinal barrier function and regulating the diversity of intestinal flora. Background Art

[0002] Obesity is a chronic metabolic disease caused by the interaction of multiple factors such as genetics, environment and endocrine system, with abnormal accumulation of body fat and overweight as its main characteristics. According to the 1997 disease classification standard of the World Health Organization (WHO), obesity is clearly defined as an independent disease. Globally, the incidence of obesity is showing a significant upward trend. Epidemiological studies have shown that the risk of obese patients suffering from type 2 diabetes, cardiovascular disease, non-alcoholic fatty liver disease (NAFLD) and malignant tumors is 2-5 times higher than that of people with normal weight, which has become a major challenge in the field of global public health. The current treatments for obesity mainly include lifestyle intervention, metabolic surgery, and drug therapy. However, lifestyle intervention has limited effect due to low patient compliance; although metabolic surgery (such as gastric bypass) can significantly reduce weight, there is a risk of postoperative complications (such as malnutrition and intestinal adhesions), and the indications are strict. Therefore, drug therapy has become the main force that needs to be developed in the field of obesity treatment. At present, the most commonly used anti-obesity drugs in my country are orlistat and semaglutide. Orlistat is a gastric lipase inhibitor and the only OTC weight loss drug approved by the State Food and Drug Administration, but it is often accompanied by adverse reactions such as gastrointestinal discomfort, vitamin deficiency, and even liver damage; semaglutide is a new long-acting insulin-like peptide-1 receptor agonist, mainly used for blood sugar control in adult patients with type 2 diabetes, but it is inconvenient to use as an injection and also has the possibility of causing gastrointestinal discomfort. The development of natural drugs has become a research hotspot in the field of anti-obesity due to its advantages such as multiple targets and few side effects.

[0003] Nitraria sibirica Pall., a plant of the Nitrariaceae family, is an excellent wild tree species in the arid desert area of ​​Xinjiang. Both its leaves and fruits can be used as medicine. It is also a Uyghur ethnic medicinal material with a long history of use in Northwest China and a typical plant for both medicine and food. As a characteristic Uyghur ethnic medicinal material in Xinjiang, my country, studies have confirmed that its leaves are rich in active ingredients such as flavonoids (such as rutin, cloven, diosmin, diosmin, kaempferol), alkaloids and polysaccharides, and have anti-hypertensive (inhibit angiotensin converting enzyme activity), antioxidant (scavenging DPPH free radicals IC50 ≤ 10μg / mL) and inhibiting adipocyte growth (3T3-L1 cells) effects; however, existing literature has not reported systematic studies on the effects of Siberian Nitraria extracts in anti-obesity, regulating lipid metabolism and improving intestinal flora disorders, and its potential for multi-target intervention in metabolic syndrome remains to be explored. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a multi-target anti-obesity solution based on the flavonoid components of the leaves of Nitraria sibiricum, in view of the significant side effects (such as the risk of liver damage), limited administration methods (injection required), and single-target effects of existing anti-obesity drugs. The components can achieve safe and efficient intervention in obesity and metabolic syndrome by synergistically regulating lipid metabolism, inflammatory response and intestinal flora balance, filling the application gap of natural drugs in this field.

[0005] Technical effect and innovation verification Through systematic in vivo experiments, it is confirmed that the flavonoid components of the present invention have the following core advantages:

[0006] Significant lipid reduction and weight control

[0007] Weight intervention: After 4 weeks of administration of the high-dose group (400 mg / kg / d), the weight growth rate of obese rats decreased by 48.0% ( Figure 1 ), body fat mass returned to normal levels (abdominal fat: 2.75±0.67g vs. normal group 2.41±0.5g) ( Figure 2 );

[0008] Blood lipid regulation: serum total cholesterol (TC), triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) decreased by 20.7%, 37.8% and 15% respectively, and high-density lipoprotein cholesterol (HDL-C) increased by 72.3% ( Figure 3 ).

[0009] Improve non-alcoholic fatty liver disease (NAFLD)

[0010] Liver pathological repair: Oil red O staining showed that the area of ​​liver lipid droplets in the high-dose group was reduced to the level of the blank group, and H&E staining confirmed the disappearance of hepatocyte fat vacuoles ( Figure 5 );

[0011] Molecular mechanism: Activation of AMPK signaling pathway (p-AMPK expression increased by 1.56 times), inhibition of NF-κB inflammatory pathway (p-NF-κB p65 decreased by 41.8%) ( Fig.10 ).

[0012] Intestinal flora and barrier function restoration

[0013] Improved flora diversity: Shannon index of intestinal flora increased to 3.8 (vs. 0.6 in the model group), Chao1 index recovered to 480 (vs. 190 in the model group) ( Fig.12 );

[0014] Intestinal barrier repair: The expression of colon tight junction protein Occludin, Zo-1 and mucin Muc-2 increased by 5.7 times, 1.5 times and 5.8 times respectively ( Fig. 9 ), the total content of short-chain fatty acids (acetic acid, propionic acid, butyric acid) recovered to 5471.14 μg / g feces ( Fig.16 ).

[0015] High security verification

[0016] Acute toxicity: No animal died or had abnormal behavior after a single oral administration of 400 mg / kg;

[0017] Subchronic toxicity: Continuous administration for 4 weeks (400 mg / kg / d) did not cause abnormalities in liver function indicators (ALT, AST).

[0018] Application prospects

[0019] The flavonoid components of the present invention can be developed into oral anti-obesity drugs through conventional preparation techniques (such as tablets and capsules), and have the following industrial advantages:

[0020] Multi-target synergy: Simultaneous intervention of lipid metabolism, inflammation and intestinal microecology;

[0021] Natural source: relying on the regional resource advantage of Siberian Nitraria, the ingredients are clear (total flavonoids ≥ 60%) and the quality control is stable;

[0022] Outstanding safety: no hepatotoxicity and gastrointestinal side effects, suitable for long-term use. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Figure 2 shows the weight changes of rats in each group during the experimental period. The trend of body weight changes of rats in different experimental groups during the 14-week experimental period. The horizontal axis is the experimental period (0-14 weeks), and the vertical axis is body weight (g), which are the blank group (NC), high-fat diet obesity model group (DIO), positive drug orlistat group (Orlistat) and low, medium and high doses of Siberian Nitraria leaf flavonoids intervention groups (NSLF-L / M / H).

[0024] Figure 2 Figure 2 shows the body fat mass of rats in each group. The weights of liver, abdominal fat and epididymal fat of rats in different experimental groups before dissection. The horizontal axis represents the group and the vertical axis represents the weight (g).

[0025] Figure 3 The blood lipid profile of rats in each group The levels of TC, TG, LDL-C and HDL-C in the serum of rats in different experimental groups. The horizontal axis is the group and the vertical axis is the concentration (mmol·L-1).

[0026] Figure 4 The liver appearance of rats in each group Appearance morphology of fresh livers after dissection from rats in different experimental groups.

[0027] Figure 5 Oil red O and H&E staining images of the liver of rats in each group Liver tissue staining of rats in different experimental groups.

[0028] Figure 6 H&E staining of fat and colon of rats in each group H&E staining images of fat and colon tissues of rats in different experimental groups indicate the pathological conditions of rats in each group.

[0029] Figure 7 Figure 2 is the mRNA expression level of inflammatory factors and lipid metabolism related factors in the liver of rats in each group The mRNA expression levels of TNF-α, IL-6, IL-1β, MCP-1, AMPK, PPARα, Leptin R, TLR4, and GLUT4 in the liver tissues of rats in different experimental groups. The horizontal axis is the group and the vertical axis is the expression level.

[0030] Figure 8 This is the mRNA expression level of inflammatory factors and lipid metabolism-related factors in the adipose tissue of rats in each group. The mRNA expression levels of PPARα, PPARγ, GLUT4, Leptin, Leptin R, Adiponectin, AMPK, JNK-1, and Lpl in the adipose tissue of rats in different experimental groups. The horizontal axis is the group and the vertical axis is the expression level.

[0031] Fig. 9 This is the mRNA expression level of intestinal barrier function markers in the colon tissue of each group of rats. The mRNA expression levels of Zo-1, Occludin, Claudin-1, Muc-2, Muc-3, and NF-κB p65 in the colon tissue of rats in different experimental groups. The horizontal axis is the group and the vertical axis is the expression level.

[0032] Fig.10 The protein expression levels of inflammatory factors in the liver tissue of each group of rats and the expression levels of lipid metabolism-related proteins in adipose tissue show the protein expression levels of p-NF-κB p65, NF-κB p65, p-IκB, IκB, p-AMPK, AMPK in the liver tissue of rats in different experimental groups and PPARγ, C / EBPβ, and UCP-1 in the adipose tissue.

[0033] Fig.11 The Venn diagram of intestinal microbiota of rats in each group Venn diagram of OTUs with species differences among rats in different experimental groups.

[0034] Fig.12 Figure 2 shows the α diversity index of intestinal microbiota in each group of rats. Simpson, Shannon, Chao1, and Goods indices in alpha diversity of rats in different experimental groups.

[0035] Fig.13 NMDS and PCoA analysis of intestinal microbiota in each group of rats NMDS and PCoA results in β diversity of rats in different experimental groups.

[0036] Fig.14 The activity of LPS in the serum of recipient mice These are the LPS levels in the serum of the untreated group, the pseudo-sterile group and the recipient mice that received fecal transplantation. The horizontal axis represents the group and the vertical axis represents the enzyme activity (U / L).

[0037] Fig.15 The graph shows the changes in the expression levels of intestinal barrier function genes in the colon of recipient mice. The graph shows the mRNA expression levels of Muc-2, Muc-3, and Zo-1 in the colon tissues of the untreated group, pseudo-sterile group, and recipient mice receiving fecal transplantation.

[0038] Fig.16 Figure 2 shows the content of short-chain fatty acids in the feces of recipient mice. These are the contents of acetic acid, butyric acid, isovaleric acid, valeric acid, isobutyric acid and propionic acid in the feces of the untreated group, pseudo-sterile group and recipient mice receiving fecal transplantation. The horizontal axis is the group and the vertical axis is the concentration (μg / g). DETAILED DESCRIPTION

[0039] Anti-obesity Effect of Flavonoid Components from Nitraria sibirica Pall. Leaves on Obese Rats

[0040] 1 Experimental Materials

[0041] 1.1 Experimental Animals

[0042] Five-week-old male specific pathogen free Sprague Dawley rats were purchased from Henan Sk贝斯 Biotechnology Co., Ltd., with the license number: SCXK(Yu)2020-0005.

[0043] 1.2 Experimental Drugs

[0044] Total flavonoids of the ethanol extract of Nitraria sibirica Pall. leaves of the present invention.

[0045] Orlistat (Solarbio Science & Technology Co., Ltd.).

[0046] 1.3 Experimental Reagents

[0047] Tribromoethanol (Sigma); absolute ethanol; isopropanol; chloroform; TG, TC, HDL-C, LDL-C kits (Nanjing Jiancheng Bioengineering Institute); reverse transcription kit (Takara); Q-PCR kit (Takara); Orlistat (Solarbio Science & Technology Co., Ltd.); antibodies p-NFκB p65, NFκBp65, IκBα, p-AMPK, AMPK (Proteintech), p-IκBα (Bioss).

[0048] 1.4 Experimental Instruments

[0049] Electronic balance; rotary evaporator; low-temperature grinder; high-speed refrigerated centrifuge; Jena qTower fluorescence quantitative PCR instrument; electrothermal constant temperature water bath; microplate reader;

[0050] 2 Experimental Methods

[0051] 2.1 Modeling method: Sixty five-week-old male specific pathogen free Sprague Dawley rats were divided into 6 groups, namely blank control group (NC), model control group (DIO), positive drug control group (Orlistat), low-dose NSLF group (NSLF-L), medium-dose NSLF group (NSLF-M), and high-dose NSLF group (NSLF-H); the blank group was fed with conventional feed, and the rest of the groups were all fed with high-fat feed (3% cholesterol, 0.3% sodium cholate, 15% lard, 81.3% basal feed) for 10 consecutive weeks, and the body weight was monitored weekly during this period.

[0052] 2.2 Administration method: After the modeling period, the low, medium and high dose groups were given oral administration of 100 mg / kg / d, 200 mg / kg / d and 400 mg / kg / d of the extract, respectively; the positive drug group was given oral administration of orlistat 20 mg / kg / d; the blank group and the model group were given oral administration of an equal amount of distilled water. The drugs were administered continuously for 4 weeks, during which body weight was monitored every week.

[0053] After administration, all rats were fasted and deprived of water for 12 h, weighed before anesthesia, and injected with sodium pentobarbital intraperitoneally according to their body weight. After anesthesia, the abdominal cavity was opened, blood was collected from the abdominal aorta and stored in sodium heparin anticoagulant tubes, and samples of heart, liver, spleen, lung, kidney, intestine, adipose tissue and feces were collected, weighed and temporarily stored at -80°C.

[0054] The collected rat blood was centrifuged at 4°C and 3000 rpm for 15 min, and the upper serum was collected.

[0055] The blood lipids of rats in each group were detected, mainly the contents of TC, TG, LDL-C, and HDL-C in serum. TC, TG, LDL-C, and HDL-C kits (Nanjing Jiancheng Bioengineering Institute) were used to detect blood lipid contents and analyze blood lipid deposition.

[0056] The external morphological changes of the liver of rats in each group were observed.

[0057] The rat livers were taken for Oil Red O staining and Hematoxylin and eosin (H&E) staining to detect the lipid deposition in the rat liver.

[0058] The fat and colon tissues of rats were taken for hematoxylin and eosin (H&E) staining to detect the pathological changes of fat and colon tissues.

[0059] Obesity is closely related to the body's inflammatory state. Obesity is generally considered to be a chronic, low-grade inflammatory state of adipose tissue. Inflammation in adipose tissue is an important factor inducing various obesity-related risks. Revealing its occurrence mechanism will help prevent the occurrence of obesity-related diseases.

[0060] The inflammatory status of the liver, fat and colon tissues of rats in each experimental group was tested.

[0061] RNA was extracted from liver, fat and colon tissue homogenates, and Q-PCR (Quantitative Real-time polymerase chain reaction) was used to detect changes in the expression levels of various inflammatory factors, chemokines and other genes related to lipid metabolism in liver, fat and colon tissues.

[0062] Liver proteins were extracted, and Western Blot method was used to detect proteins related to AMPK / NF-κB signaling pathway in liver tissue.

[0063] Adipose protein was extracted and the lipid metabolism-related proteins in adipose tissue were detected by Western Blot method.

[0064] 16S rRNA sequencing was performed on 6 groups of samples (blank control group, model control group, positive control group, low-dose group, medium-dose group, and high-dose group) to analyze the improvement effect of NSLF on intestinal microbiota.

[0065] 2.3 Fecal transplantation trial

[0066] 30 c57 mice were divided into 3 groups, namely, no treatment group (CON), pseudo-sterile group (PGF), and fecal transplantation group (FMT). The mice in the pseudo-sterile model group (PGF) and fecal transplantation group (FMT) were orally gavaged with 100 μL of an antibiotic mixture for 7 days, the antibiotic mixture contained neomycin (0.5 g / L), metronidazole (1 g / L), vancomycin (0.5 g / L), and ampicillin (1 g / L); rat feces (50 mg) temporarily stored at -80°C were suspended in 1.5 mL of PBS to prepare a fecal bacterial suspension, which was transferred to the fecal transplantation group (FMT) recipient mice by gavage for 4 weeks, and the mouse serum, colon, and feces were temporarily stored.

[0067] The serum LPS content of mice was detected to analyze the changes in intestinal inflammation.

[0068] Colon RNA was extracted from recipient mice, and the expression level changes of intestinal barrier function genes in the colon tissue of mice were detected.

[0069] Short-chain fatty acids (SCFA) are produced by anaerobic bacteria or yeast glycolysis in the large intestine (especially the colon) through the digestion of fiber. Beneficial intestinal microorganisms break down fiber and produce short-chain fatty acids to provide energy for cells, regulate the balance of intestinal flora, improve intestinal function, regulate immunity, and maintain the health of the colon. Detecting the content of short-chain fatty acids in the feces of each group of mice is crucial to the health of the intestinal flora and colon.

[0070] The feces of the recipient mice were mixed with PBS, sulfuric acid was added to extract SCFA, and the SCFA concentration was subsequently determined using a gas chromatography method.

[0071] 3 Results and discussion Figure 1The experimental results showed that the body weight of rats in the DIO group increased significantly linearly due to continuous high-fat diet feeding; while the body weight growth rate of the NSLF-H group slowed down significantly since the 4th week of administration. By the 14th week, the body weight growth rate of rats in the NSLF-H group was 48.0% lower than that of the model group (P<0.01), and its body weight curve was close to that of the NC group, indicating that the extract can effectively inhibit the abnormal weight gain induced by a high-fat diet. Although Orlistat also showed a weight control effect, the slowdown in growth rate (20.5%) was lower than that of NSLF-H, suggesting that the extract of the present invention has better potential in weight management.

[0072] from Figure 2 It can be seen that compared with the blank group, the liver, abdominal fat, and epididymal fat weights of obese rats also showed an increase, indicating that the high-fat diet caused obesity in rats. After high-dose NSLF treatment, the liver weight of obese rats decreased by 26.7%, and the weight of abdominal fat and epididymal fat in the high-dose treatment group decreased by 57.5% and 43.6%, respectively, even reaching the level of blank control group rats, indicating that NSLF has the effect of reducing body fat and improving obesity in obese rats.

[0073] like Figure 3 As shown in the results, compared with the blank control group, the blood lipid level of obese rats induced by high-fat diet was significantly increased. Compared with the obesity model group, NSLF treatment was able to reduce the levels of triglycerides, cholesterol and LDL-C in the serum of obese rats, which were reduced by 20.7%, 37.8% and 15%, respectively, and HDL-C was increased by 72.3%, making it close to the normal level. This shows that the Siberian Nitraria extract can significantly reduce hypercholesterolemia and hypertriglyceridemia in rats caused by long-term high-fat diet.

[0074] observe Figure 4 The external morphology of rat livers was available, and the external morphology of rat organs also showed obvious changes. The livers of obese rats were pink and white and filled with fat. After administration, as the dosage increased, it gradually returned to bright red, approaching the level of normal blank group rats. The surface of the livers of blank group rats was smooth, while the high-fat diet caused the edges of the rat livers to be rounded and blunt, with diffuse small nodules visible on the surface.

[0075] Oil red O staining results Figure 5The results showed that a large number of hepatocytes were visible in the obesity model group. The high-fat diet caused severe fat deposition in the rat liver, and there were many fat vacuoles. The accumulation of fat enlarged the hepatic sinusoids and squeezed the cell nuclei to one side. After drug intervention, the fat deposition condition improved, and the high-dose group even returned to the blank level. The results of liver H&E staining also showed the same results as those of Oil Red O staining. The liver sinusoidal structure of the blank group rats was normal, and the hepatocytes were neatly arranged. Many transparent fat vacuoles were seen in the liver field of the model group rats, and the hepatocytes were also squeezed to one side of the cell. After treatment, the hepatic sinusoidal structure was restored, and the fat vacuoles were significantly reduced, indicating that drug administration reduced liver lipid deposition in obese rats. The above results show that the Siberian white thorn leaf extract has a significant effect on improving non-alcoholic fatty liver in rats caused by a high-fat diet.

[0076] The results are as follows Figure 6 The results showed that the blank group had normal adipocyte morphology. After high-fat diet induction, the adipocytes of obese rats expanded significantly and became hypertrophic. After treatment, the adipocytes gradually returned to normal in a dose-dependent manner. The results of H&E staining of colon tissue are shown in the figure. The number and length of intestinal villi in the colon tissue of the blank group rats were normal, and there were many goblet cells. However, the colon of obese rats showed goblet cell loss and epithelial cell damage, and mucosal edema occurred. After NSLF treatment, the infiltration of mucosal inflammatory cells was alleviated, the swelling of the crypts disappeared, and the damage was alleviated.

[0077] The results are as follows Figure 7 The results showed that compared with the blank group, the expression levels of inflammatory factors such as TNF-α, IL-6, and IL-1β in the liver of obese rats were significantly increased, indicating that long-term high-fat diet led to liver inflammation in rats. After intervention with the extract, the expression levels of inflammatory factors in the liver of obese rats were significantly and generally decreased, and the expression of Glut-4, Leptin, and Adiponectin also showed upregulation, indicating that the extract alleviated the inflammatory state of obese rats and improved lipid metabolism.

[0078] Figure 8 It can be seen that the inflammatory state of adipose tissue was also improved. Compared with obese rats, the adipogenic transcription factors PPARα, PPARγ and JNK-1 in the adipose tissue of rats in the NSLF treatment group showed downregulation after the intervention of the extract, while Glut-4, Leptin and Adiponectin showed upregulation.

[0079] Intestinal epithelial barrier function markers in colon tissue indicate the permeability of colon tissue and the richness of intestinal flora. Fig. 9It can be observed that long-term high-fat diet leads to impaired intestinal barrier function and intestinal permeability in obese rats. After NSLF administration, markers such as Occludin, Claudin1, Zo-1, Muc-2, and Muc-3 showed significant upregulation, and inflammatory-related factors showed significant downregulation.

[0080] The above results indicate that NSLF treatment significantly improved the inflammatory state of obese rats, improved lipid metabolism, improved colon epithelial barrier function, and maintained the integrity of the intestinal barrier.

[0081] like Fig.10 The results were consistent with those of Q-PCR. The expression levels of phosphorylated NF-κB p65 and IκB in obese rats were significantly increased, indicating that a high-fat diet caused liver inflammation in rats. After administration, phosphorylated NF-κB p65 and IκB showed downregulation, among which the expression of phosphorylated NF-κB p65 was downregulated by 41.8%. The expression of phosphorylated AMPK was also significantly increased, upregulated by 1.56 times, indicating that NSLF inhibited the NF-κB pathway and activated the AMPK pathway through AMPK / NF-κB, thereby improving the body inflammation caused by a high-fat diet and exerting an anti-obesity effect.

[0082] In addition, compared with the rats in the blank control group, the expression of adipogenic transcription factors PPARγ and C / EBPβ in adipose tissue of obese rats was significantly reduced, but increased after treatment, by 31.3% and 42.0%, respectively. The expression of mitochondrial uncoupling protein UCP-1 in adipose tissue was reduced in the adipose tissue of obese rats, but significantly upregulated after NSLF treatment, indicating that mitochondrial heat production increased in rats after NSLF treatment, thereby exerting an anti-obesity effect.

[0083] The above results indicate that NSLF intervention improved the inflammatory state of obese rats and played a positive role in lipid metabolism, thereby exerting an anti-obesity effect.

[0084] Analysis of species differences and marker species showed that Fig.11 The Venn diagram in the middle shows the number of common and unique bacterial OTUs between samples in each group. The number of unique bacterial OTUs in the blank group, positive drug group, low, medium and high dose administration groups were 465, 719, 807, 302 and 713, respectively, and the number of common OTUs was 20. The number of unique bacterial OTUs in the obesity model group was the least, at 196. The Venn diagram intuitively shows the similarity and specificity of the OTU composition in the samples.

[0085] Depend on Fig.12The alpha diversity of the rat intestinal flora can be observed, among which the Shannon and Simpson indexes reflect the diversity of the microbial community, and the Chao1 index reflects the species richness. Compared with the blank control group, the diversity and species richness of the intestinal microbial community in obese rats were significantly decreased. After NSLF treatment, both the richness and diversity increased in a dose-dependent manner, indicating that NSLF can improve the intestinal microbial community environment of obese rats.

[0086] Fig.13 The beta diversity of rat intestinal flora was displayed. Beta diversity reflects the differences in species in the community, and together with alpha diversity, it constitutes the overall diversity. The smaller the value, the more similar the species between communities. The results showed that long-term high-fat diet caused the difference in intestinal flora species composition between obese rats and blank control group rats to widen. After treatment, it became closer to the blank control group.

[0087] from Fig.14 It can be seen that the LPS content in the serum of the recipient mice in the pseudo-sterile model group (PGF) was significantly higher than that of the untreated mice, indicating that the intestinal environment may be disordered, the endotoxin content is high, causing inflammation. After fecal microbiota transplantation, the LPS activity decreased, reducing metabolic endotoxemia. Fig.15 The results showed that compared with the untreated mice (CON), the expression of intestinal barrier function markers Zo-1, Muc-2, and Muc-3 in the colon of the recipient mice in the pseudo-sterile model group was downregulated, and the expression levels were significantly restored after fecal microbiota transplantation. The above results indicate that feces transplanted from rats treated with NSLF into recipient mice significantly improved the intestinal environment of the recipient mice (FMT) and protected the mouse intestine from the effects of endotoxemia.

[0088] Fig.16 is the content of short-chain fatty acids in the feces of each group of recipient mice. The short-chain fatty acids with the highest content in mouse feces are acetic acid, propionic acid and butyric acid, accounting for about 90% of the total short-chain fatty acids. As shown in the figure, the content of acetic acid, propionic acid and butyric acid in the feces of PGF mice decreased, and showed an increasing trend after rat fecal transplantation, indicating that rat feces containing NSLF improved the colon environment and restored intestinal function. NSLF regulates the intestinal flora by regulating the metabolism of short-chain fatty acids, thereby reducing inflammation in the body and playing an anti-obesity role.

[0089] In summary, the Nitraria sibirica leaf extract NSLF provided by the present invention has significant effects on resisting hypercholesterolemia, hypertriglyceridemia and non-alcoholic fatty liver disease, as well as improving inflammation, colon damage and intestinal endotoxemia.

[0090] Although the above embodiments describe the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. Any other embodiments that are changed or modified without departing from the spirit and principle of the present invention belong to the protection scope of the present invention.

Claims

1. A use of flavonoid components from the leaves of Nitraria sibirica in the preparation of a drug for treating obesity and related metabolic syndromes, characterized in that: The ethanol extract is prepared by the following method: (a) extracting Nitraria sibirica Pall. with ethanol to obtain a powder extract having a total flavonoid content of ≥ 60%; (b) The application includes that the flavonoid components of Siberian Nitraria sibiricum leaves regulate the AMPK / NF-κB signaling pathway, reduce serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) levels, increase high-density lipoprotein cholesterol (HDL-C) levels, improve hyperlipidemia, non-alcoholic fatty liver disease (NAFLD) pathological characteristics, repair the expression of intestinal barrier function markers (Occludin, Zo-1, Muc-2), and regulate the α diversity index of the intestinal microbiota (Shannon index ≥4.0, Chao1 index ≥500).

2. The use according to claim 1, characterized in that: The flavonoid component of Nitraria sibirica is in powder form, with a daily dosage of 100-400 mg / kg and a continuous administration period of 4-8 weeks.

3. The use according to claim 1, characterized in that: The treatment of obesity and related metabolic syndrome includes at least one of the following: (i) reduce the weight gain induced by a high-fat diet by ≥30%; (ii) reduction of abdominal fat mass to 80-120% of normal group levels; (iii) inhibiting the mRNA expression of TNF-α and IL-6 inflammatory factors in liver tissue by ≥50%; (iv) up-regulated the expression of lipid metabolism-related proteins (UCP-1, Adiponectin) in adipose tissue to 1.5-2.0 times the level of the normal group; (v) Restore the total content of short-chain fatty acids (acetic acid, propionic acid, butyric acid) in the intestinal flora to ≥80 μg / g feces.

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