Application of DL-norvaline in preparation of anti-obesity preparation

Abstract

The invention provides an application of DL-norvaline in preparation of an anti-obesity preparation, the DL-norvaline is tried to be applied to anti-obesity research, and it is found that the DL-norvaline has an obvious relieving effect on obesity induced by high fat diet and related diseases of the obesity. The application of the DL-norvaline to the preparation of anti-obesity preparations induced by high fat diet and the prevention, alleviation and treatment of related diseases thereof has a prospect, and a new direction is provided for the development of anti-obesity preparations.

Description

Technical Field

[0001] The present invention relates to a new application of DL-norvaline, and in particular to an application of DL-norvaline in the preparation of an anti-obesity preparation. Background Art

[0002] Obesity, defined as the accumulation of fat cells and lipids when nutrition is unbalanced or energy intake exceeds energy expenditure, has become a serious social health problem. Metabolic abnormalities caused by obesity can lead to many complications, such as hypertension, diabetes, fatty liver, cardiovascular disease, and hyperlipidemia. Summary of the invention

[0003] The present invention constructs an obese mouse model by inducing C57BL / 6J male mice with a high-fat diet. After 6 weeks of high-fat diet (HFD), the obese group of mice gained 20.61% more weight than the control group of mice, which is greater than 20%, indicating that the obesity model was successfully established. However, DL-norvaline supplementation can significantly reduce the weight gain and tissue weight of mice on a high-fat diet. The degree of weight gain by eating a specific amount of food is called the Food Efficiency Ratio (FER), and lipid accumulation in abdominal fat organs leads to an increase in the food efficiency ratio. DL-norvaline intervention reduced the FER value of obese mice, indicating that DL-norvaline supplementation allows more fat in mice to be used as an energy source.

[0004] Dyslipidemia and hyperglycemia caused by obesity are independent risk factors for the development of chronic liver and kidney diseases. At the same time, obesity may increase the burden on the liver and kidneys, leading to kidney damage. The intervention of DL-norvaline can significantly improve dyslipidemia and liver and kidney damage in obese mice, and the above results are further confirmed by hematoxylin-eosin (H&E) analysis.

[0005] Lipid metabolism disorder is one of the hallmarks of obesity-induced metabolic disorders, which will lead to inefficient lipid metabolism, thus causing the development of obesity and dyslipidemia. DL-norvaline can significantly improve lipid metabolism disorders in HFD-induced obese mice through sphingolipid metabolism and phospholipid metabolism.

[0006] The present study found that for high-fat fed mice, H&E staining showed disordered arrangement of colon epithelial cells, damaged villi, and increased infiltration of a large number of inflammatory cells, while dietary supplementation with DL-norvaline partially reversed this situation, resulting in orderly cell arrangement, reduced villus damage and inflammatory cell infiltration, and higher microstructural integrity, suggesting that DL-norvaline may be beneficial to intestinal health.

[0007] Intestinal barrier damage is considered to be one of the causes of obesity, metabolic disorders, inflammatory responses and oxidative stress. Intestinal goblet cells are able to produce a large number of complex secretory proteins, such as mucins MUC2 and MUC3 and tight junction proteins Claudin-1, Occludin and ZO-1, which can transmit signals intracellularly and form a protective intestinal mucus layer to protect barrier function and maintain intestinal health. The present study found that the expression of tight junction proteins Claudin-1, Occludin and ZO-1 in the colon group of high-fat-fed mice supplemented with DL-norvaline was significantly increased, indicating that DL-norvaline can effectively alleviate HFD-induced intestinal barrier damage in mice.

[0008] Unhealthy diet may lead to imbalance of intestinal flora homeostasis, also known as "intestinal microbial dysbiosis", which further leads to obesity and its related diseases. The present study found that DL-norvaline intervention can improve the imbalance of intestinal microbiota structure induced by HFD in mice, increase the relative abundance of beneficial bacteria (Mollicutes_RF39, Ruminococcaceae, Bacteroidaceae, Lactobacillaceae, Clostridiaceae_1, uncultured_bacterium_f_Muribaculaceae and Rikenellaceae_RC9_gut_group), and reduce the relative abundance of harmful bacteria (Fusobacteriia, Desulfovibrionales, Enterobacteriaceae, Burkholderiaceae, Helicobacteraceae and Veillonellacea).

[0009] In summary, DL-norvaline intervention can alleviate HFD-induced obesity and its related diseases, improve glucose and lipid metabolism, and regulate the structural changes of intestinal microbiota in obese mice, thereby further alleviating intestinal barrier damage.

[0010] Based on the above research, the present invention provides the use of DL-norvaline in the preparation of anti-obesity preparations, wherein the anti-obesity preparations refer to the prevention, alleviation or treatment of obesity caused by a high-fat diet.

[0011] Based on the same inventive concept, the present invention also applies DL-norvaline to the preparation of a preparation for preventing and / or alleviating glucose and lipid metabolism disorders caused by a high-fat diet.

[0012] Based on the same inventive concept, the present invention also applies DL-norvaline to the preparation of a preparation for preventing and / or alleviating fatty liver caused by a high-fat diet.

[0013] Based on the same inventive concept, the present invention also applies DL-norvaline to the preparation of a preparation for preventing and / or alleviating liver and kidney damage caused by a high-fat diet.

[0014] Based on the same inventive concept, the present invention also applies DL-norvaline to the preparation of a preparation for preventing and / or alleviating intestinal barrier function damage caused by a high-fat diet.

[0015] Based on the same inventive concept, the present invention also applies DL-norvaline to the preparation of a preparation for regulating intestinal flora imbalance caused by a high-fat diet.

[0016] Based on the same inventive concept, the present invention also applies DL-norvaline to the preparation of a preparation for regulating lipid metabolism imbalance caused by a high-fat diet.

[0017] The above-mentioned sugar and lipid metabolism caused by high-fat diet, fatty liver caused by high-fat diet, liver and kidney damage caused by high-fat diet, lipid metabolism disorder caused by high-fat diet, intestinal barrier function caused by high-fat diet, and intestinal flora imbalance caused by high-fat diet are all related diseases caused by high-fat diet.

[0018] Based on the same inventive concept, the present invention also provides a preparation for preventing and / or alleviating obesity and related diseases caused by a high-fat diet, which comprises an effective amount of DL-norvaline.

[0019] Preferably, the above-mentioned preparation is food, medicine or health product.

[0020] Preferably, the DL-norvaline is the only active ingredient.

[0021] The present invention has been verified by scientific mouse experiments. ① DL-norvaline can significantly inhibit the increase of the weight of mice and the weight of their liver, spleen, kidney and other tissues, indicating that DL-norvaline can alleviate the fat deposition of mice induced by a high-fat diet and has a significant anti-obesity effect; ② DL-norvaline can significantly inhibit the increase of adipose tissue weight and alleviate liver and kidney damage, thereby indicating that DL-norvaline has the effect of preventing and alleviating fatty liver induced by a high-fat diet; ③ and according to the relevant experimental results on the glucose metabolism and blood lipid levels of mice, it can be shown that DL-norvaline has a significant alleviating effect on the glucose and lipid metabolism disorders induced by a high-fat diet; ④ and according to the relevant lipid metabolism The results of the experiment and its detection showed that DL-norvaline improved lipid metabolism disorders in obese mice by changing sphingolipid metabolism, especially ceramide; ⑤ and according to the results of the detection and analysis of the mouse intestine and flora, it was obvious that the supplementation of DL-norvaline could improve the intestinal barrier damage of mice, increase the relative abundance of beneficial bacteria such as Lachnospiracea and Lactobacillaceae in the intestinal flora of mice, and reduce the relative abundance of harmful bacteria such as Fusobacteriia and Desulfovibrionale. This shows that DL-norvaline has a significant regulating effect on the intestinal barrier damage and intestinal flora imbalance induced by a high-fat diet. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Effects of DL-norvaline on weight gain and tissue weights of obese mice induced by high-fat diet: (A) weight gain; (B) liver weight; (C) epididymal fat weight; (D) kidney weight; (E) spleen weight; The results are expressed as mean ± standard error (n = 10), different letters indicate significant differences among the groups (P < 0.05).

[0023] Figure 2 Effects of DL-norvaline on food intake in high-fat diet-induced obese mice: (A) food intake; (B) energy intake; (C) food efficiency ratio; the results are expressed as mean ± standard error (n = 10), and different letters indicate significant differences among the groups (P < 0.05).

[0024] Figure 3 Effects of DL-norvaline on glucose metabolism in high-fat diet-induced obese mice: (A) fasting blood glucose; (B) 2 h postprandial blood glucose; (C) fasting insulin; (D) HOMA-IR; (E) glucose tolerance test; (F) glucose tolerance test AUC; (G) insulin tolerance test: (H) insulin tolerance test AUC; The results are expressed as mean ± standard error (n = 10), and different letters indicate significant differences among the groups (P < 0.05).

[0025] Figure 4 Effects of DL-norvaline on tissue morphology of high-fat diet-induced obese mice: (A) liver H&E staining (100X); (B) fat H&E staining (200X).

[0026] Figure 5 These are potential lipid biomarkers that differ between the HFD group and the LFD group.

[0027] Figure 6 Potential lipid biomarkers that differed between the L, M, and H groups and the LFD group: (A) sphingolipid metabolism and phospholipid metabolism; (B) sunitinib levels.

[0028] Figure 7 The effect of DL-norvaline on colon tissue morphology in high-fat diet-induced obese mice; the magnification of each figure is 100X.

[0029] Figure 8 The effect of DL-norvaline on the expression of tight junction proteins in colon tissue of obese mice induced by high-fat diet: (A) ZO-1 (tight junction protein ZO-1); (B) Claudin-1 (tight junction protein Claudin-1); (C) Occudin (tight junction protein Occudin); The results are expressed as mean ± standard error, and different letters indicate significant differences among the groups (P<0.05); DAPI: 4',6-diamidino-2-phenylindole; Merge: merge, i.e., the picture after the antibody staining fluorescence and DAPI staining overlap.

[0030] Fig. 9 Effects of DL-norvaline on the α-diversity of intestinal flora in high-fat diet-induced obese mice: (A) Shannon index; (B) Chao1 index; (C) ACE index; (D) Simpson index.

[0031] Fig.10 The effect of DL-norvaline on the β-diversity of intestinal flora in mice with high-fat diet-induced obesity: (A) 3D PCA score graph; (B) 2D PCA score graph (PC1 vs. PC2).

[0032] Fig.11 The effect of DL-norvaline on the species distribution of intestinal flora in mice with high-fat diet-induced obesity: (A) phylum level; (B) class level; (C) order level; (D) family level; (E) genus level; The English on the left side of each figure is: relative abundance (%).

[0033] Fig.12The effects of DL-norvaline on the physiological indices of mice in the acute toxicity test: A and F are the water intake of female and male mice, respectively; B and G are the food intake of female and male mice, respectively; C and H are the weight gain of female and male mice, respectively; D and I are the food efficiency ratios of female and male mice, respectively; E and J are the tissue weights of female and male mice, respectively. The results are expressed as mean ± standard error (n = 6). Different letters indicate significant differences among the groups (P < 0.05).

[0034] Fig.13 Effects of DL-norvaline on histopathology in acute toxicity studies: H&E sections of brain, heart, liver, spleen, lungs, kidneys, stomach, thymus, colon, jejunum, and uterus / testes of (A) female and (B) male mice.

[0035] Fig.14 The effects of DL-norvaline on the physiological indices of mice in a subacute toxicity test: A and F are the water intake of female and male mice, respectively; B and G are the food intake of female and male mice, respectively; C and H are the weight gain of female and male mice, respectively; D and I are the food efficiency ratios of female and male mice, respectively; E and J are the tissue weights of female and male mice, respectively; the results are expressed as mean ± standard error (n = 6), and different letters indicate significant differences among the groups (P < 0.05).

[0036] Fig.15 Effects of DL-norvaline on histopathology in subacute toxicity studies: H&E sections of brain, heart, liver, spleen, lung, kidney, stomach, thymus, colon, jejunum, and uterus / testis of (A) female and (B) male mice. DETAILED DESCRIPTION

[0037] Unless otherwise specified, the scientific and technical terms used herein are understood according to the knowledge of ordinary technicians in the relevant fields.

[0038] Norvaline is an isomer of valine and a non-protein, non-natural branched-chain amino acid. Norvaline is divided into D-norvaline, L-norvaline and DL-norvaline. It is an important raw material and intermediate for some chiral drugs. By splitting racemic DL-norvaline, norvaline with a single optical rotation can be obtained. L-norvaline is a key intermediate for the synthesis of perindopril, a drug for the treatment of various hypertension and congestive heart failure. D-norvaline can be used as a D-type chiral source to synthesize some macrolides with a D-type structure. However, there are few or even no reports on the application of DL-norvaline in anti-obesity research.

[0039] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0040] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. The DL-norvaline used in the following examples was purchased from Aladdin Biochemical Technology Co., Ltd., with a CAS number of 760-78-1.

[0041] Embodiment 1:

[0042] 1. Research plan

[0043] (1) Grouping and treatment of experimental animals

[0044] C57BL / 6J mice (8 weeks old) were purchased from SpEffect (Beijing) Biotechnology Co., Ltd. (Beijing, China).

[0045] The experimental animals were strictly in accordance with the Chinese national standard requirements for experimental animal environment and facilities (GB

[0046] 14925-2010) were kept in a standard animal room: temperature 22 ± 2 ° C, humidity 50% ± 15%, light and dark cycle 12h / 12h cycle, free access to food and water. Before the start of the experiment, the experimental animals were fed adaptively for one week. All animal studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (8th Edition, ISBN-10: 0-309-15401-4) and approved by the Animal Ethics Committee of Northwest Agriculture and Forestry University (SP20220503).

[0047] C57BL / 6J males were randomly divided into 5 groups (n=10 / group):

[0048] ① Low-fat diet group (LFD group): fed with low-fat feed (TP2330055AC, 10% fat, 75% carbohydrate, 15% protein, Nantong Trofi Feed Technology Co., Ltd., China) and water; the low-fat feed was 2.0-2.5 g / kg per day, as shown in the following table. Figure 2 shown.

[0049] ② High-fat diet group (HFD group): fed with high-fat feed (TP2330055A, 60% fat, 25% carbohydrate, 15% protein, Nantong Trophy Feed Technology Co., Ltd., China) and water; the high-fat feed was 1.5-2.5 g / kg per day, see Figure 2 shown.

[0050] ③ Low-dose DL-norvaline intervention group (L group): fed with high-fat diet and 50 mg / kg DL-norvaline per day;

[0051] ④Medium-dose DL-norvaline intervention group (M group): fed with high-fat diet and 300 mg / kg DL-norvaline per day;

[0052] ⑤ High-dose DL-norvaline intervention group (H group): fed with high-fat diet and 700 mg / kg DL-norvaline per day.

[0053] The dosage method was as follows: the DL-norvaline intake in the above experimental groups was set according to the maximum amino acid intake recommended by the Food and Agriculture Organization of the United Nations and the World Health Organization multiplied by the mouse / human mass ratio [1,2]. Oral gavage was performed once a day for 6 consecutive weeks, and the body weight and food intake of the mice were recorded every two days.

[0054] The energy intake and food efficiency ratio of mice during the experiment were calculated according to the corresponding formulas: Energy intake (kcal) = food intake (g) * feed energy content (kcal / g); Food efficiency ratio (FER) = weight gain (g) / energy intake (kcal).

[0055] After 6 weeks of feeding, mice in each group were euthanized and blood was collected from the eyeballs. The blood was centrifuged at 4°C for 15 min (2000 r / min) to collect the supernatant. The liver, spleen, kidney and adipose tissue of the mice were collected and stored at -80°C for subsequent determination.

[0056] (2) Biochemical index testing (blood lipid levels and liver and kidney function)

[0057] The serum levels of aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine (CRE), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were measured in mice using an automatic biochemical analyzer.

[0058] (3) Glucose tolerance and insulin tolerance analysis

[0059] Glucose tolerance test: In the last week of DL-norvaline intervention, mice were gavaged with 2 g / kg glucose solution after fasting for 10 h. Blood glucose was tested from the tail vein at 0, 15, 30, 60, 90 and 120 min, and the curve was drawn. The area under the blood glucose curve (AUC) was calculated.

[0060] Insulin tolerance test: In the last week of DL-norvaline intervention, mice were intraperitoneally injected with 0.5 IU / kg insulin solution after fasting for 8 hours, and blood glucose was tested from the tail vein at 0, 15, 30, 60, 90 and 120 minutes. The curve was drawn and the blood glucose AUC was calculated.

[0061] (4) Fasting blood glucose level, fasting insulin level and insulin stability coefficient testing

[0062] The mice were fasted overnight but not water-deprived, and the fasting blood glucose levels of the mice were tested; 2h postprandial blood glucose (2h PBG) levels of the mice were tested 2h after the diet and water were restored. The insulin levels in the serum samples of the mice were detected using the ELISA kit according to the operating instructions. Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) = fasting glucose (mg / dL) × fasting insulin (mU / L) / 22.5.

[0063] (5) Hematoxylin-eosin staining

[0064] The mouse tissues were fixed in 4% paraformaldehyde for 24 hours, and after washing away the fixative, they were placed in 70%, 80%, 95%, and 100% ethanol for dehydration, and then immersed in xylene twice to make it transparent, and then embedded in molten paraffin. The embedded wax blocks were cut into 4 μm thick slices on a microtome, spread flat in water at 42°C, picked up with a slide, and placed in a constant temperature incubator at 60°C for 4 hours to prepare paraffin sections. Xylene, anhydrous ethanol, 95% ethanol, 85% ethanol, 75% ethanol, 50% ethanol, pure water, phosphate buffered saline (PBS) buffer (pH 7.4) were used for rehydration, and then H&E was used for staining. After sealing with neutral gum, the slices were observed and photographed under a microscope.

[0065] (6) Lipidomics analysis

[0066] 100 mg of adipose tissue from mice in each experimental group was added with 500 μL of 50% methanol solution, ground in a freezing grinder (-30°C, 20 m / s) for 3 min, and centrifuged at 12000 r / min for 10 min at 4°C. After discarding the supernatant, 500 μL of lysis solution (dichloromethane: methanol = 3:1) was added, vortexed and centrifuged at 12000 r / min for 20 min at 4°C. The supernatant was discarded again, diluted 2-fold with the lysis solution, and filtered with a 0.22 μm filter for subsequent determination. An equal amount of supernatant was taken from each processed sample and mixed as a quality control (QC) sample, and a QC mixed sample was run every five samples for quality monitoring.

[0067] The samples were analyzed using a liquid chromatography-mass spectrometry system (LC-MS / MS). The main parameters are as follows.

[0068] Chromatographic conditions: the chromatographic column was C18 CSH column (100mm×2.1mm, 1.7μm); the column temperature was 45℃; the flow rate was 0.4mL / min; the injection volume was 2μL; the mobile phase A was 60% acetonitrile containing 10mM ammonium acetate and 0.1% formic acid; the mobile phase B was acetonitrile-isopropanol solution (1:9, v / v) containing 10mM sodium acetate and 0.1% formic acid; the elution procedure was as follows: 60% A (0min); 40% A (2min); 57% A (2.1min); 50% A (12min); 46% A (12.1min); 30% A (18min); 1% A (18.1min); 60% A (20min).

[0069] Mass spectrometry conditions: In positive ion acquisition mode: ion source temperature is 100°C; capillary voltage is 2.5kv; reduced pressure gas flow rate: 800L / h; cone gas flow rate: 50L / h; cone voltage: 24kv; acquisition rate: 0.2sec / cycle. In negative ion acquisition mode, the mass spectrometry parameters are set as follows: ion source temperature is 100°C; capillary voltage is 2.5kv; reduced pressure gas flow rate: 600L / h; cone gas flow rate: 10L / h; cone voltage: 25kv; acquisition rate: 0.2sec / cycle.

[0070] Data analysis: The raw data files were peak processed using Progenesis QI software and compared with Lipidmaps (http: / / www.lipidmaps.org), HMDB (http: / / www.hmdb.ca) and NIST (https: / / chemdata.nist.gov) self-built lipid databases to further analyze the lipid data results.

[0071] (7) Immunofluorescence staining

[0072] After paraffin sectioning and dewaxing and rehydration according to the method described in (5), mouse colon tissue sections were treated with EDTA antigen retrieval buffer for 25 minutes and incubated with 5% bovine serum albumin for 2 hours to block nonspecific binding. The slides were incubated with the corresponding primary antibody solution in a wet box at 4°C overnight. Subsequently, the slides were washed 3 times with PBS and incubated with the corresponding secondary antibody solution at room temperature for 60 minutes, and then washed 3 times with PBS. Finally, the slides were fixed with DAPI anti-fading reagent, photographed under a microscope after sealing, and quantified using ImageJ (version 1.8.0) software.

[0073] (8) 16S rRNA sequencing analysis of intestinal flora

[0074] use DNA of microorganisms in mouse feces was extracted using a DNA extraction kit, degradation and contamination were monitored on a 1% agarose gel, and purity and concentration were tested using a micro-spectrophotometer. Then, primers (upper primer 338F and lower primer 806R, 5′-ACTCCTACGGGAGGCAGCAG-3′ and 5′-GGACTACHVGGGTWTCTAAT-3′, respectively) were used to amplify the variable region V3-V4 of the 16S rRNA gene.

[0075] PCR was performed under the following conditions: DNA template 5-50ng, upper primer (10μM) 0.3μL, lower primer (10μM) 0.3μL, KOD FX Neo buffer 5μL, dNTP (2mM) 2μL, KOD FX Neo reagent 0.2μL, and finally double deionized water was used to make up to 20μL system. After initial denaturation at 95℃ for 5min, denaturation at 95℃ for 30s, annealing at 50℃ for 30s, extension at 72℃ for 40s, 20 cycles, and finally extension at 72℃ for 7min. The amplified product was purified by DNA purification kit and quantified using Qsep-400 high-throughput biological fragment analyzer, and the amplicon library was sequenced (2×250bp) on Illumina novaseq6000.

[0076] The raw data of 16S rRNA gene sequencing reads were filtered, assembled and screened to obtain high-quality reads. The remaining sequences were divided into operational taxonomic units (OTUs) according to different similarity levels, and bioinformatics analysis was performed at 97% similarity OTUs. The Greengenes database was used for alignment, and the sequencing data were processed on the BMKCloud platform (www.biocloud.net).

[0077] 2. Results

[0078] (1) Body weight and tissue weight: based on Figure 1 The effects of DL-norvaline on body weight gain and tissue weight in high-fat diet-induced obese mice are shown in Figure 2. Figure 1 As shown in A, the weight gain of mice in the HFD group was significantly higher than that in the LFD group, and the supplementation of DL-norvaline could significantly inhibit the weight gain of mice induced by HFD (P<0.05). In addition, compared with the LFD group, the weights of the liver, epididymal white fat, spleen, and kidney in the HFD group were significantly increased (P<0.05), while the weights of each tissue were significantly decreased after DL-norvaline intervention (P<0.05) ( Figure 1 BE).

[0079] (2) Food intake and energy intake: Figure 2 As shown in A and B, compared with the LFD group, the food intake of the HFD group and the DL-norvaline intervention group was significantly reduced (P<0.05), but the energy intake and FER value were significantly increased (P<0.05); compared with the HFD group, the food and energy intake of the mice in the DL-norvaline intervention group did not change significantly, but the FER value was significantly reduced (P<0.05) ( Figure 2 C).

[0080] (3) Glucose and insulin tolerance and blood sugar stability: Figure 3 The effect of DL-norvaline on glucose metabolism in high-fat diet-induced obese mice was shown in Figure 2. Compared with the LFD group, the fasting blood glucose level, 2h postprandial blood glucose level, and fasting insulin level of mice in the HFD group were significantly increased (P<0.05), while DL-norvaline intervention effectively downregulated the blood glucose and insulin levels of mice (P<0.05) ( Figure 3 AC). Consistently, the HOMA-IR value in the DL-norvaline intervention group was significantly decreased compared with the HFD group (P<0.05) ( Figure 3 D).

[0081] like Figure 3 As shown in E and F, after mice were gavaged with glucose solution, the blood glucose levels of each group increased significantly, reaching a peak at 15 minutes, and the blood glucose levels of mice in the HFD group at each time point and the blood glucose AUC from 0 to 120 minutes were significantly higher than those in the LFD group (P<0.05). Compared with the HFD group, the blood glucose levels and AUC of mice in the DL-norvaline intervention group at each time point were significantly reduced (P<0.05), and the high-dose group had the best intervention effect ( Figure 3 E and F). In addition, after intraperitoneal injection of insulin in mice, the HFD group mice showed obvious insulin resistance and significantly increased AUC compared with the LFD group (P<0.05), while DL-norvaline supplementation significantly prolonged the insulin response intensity of obese mice and significantly reduced AUC (P<0.05) ( Figure 3 G and H).

[0082] (4) The effects of DL-norvaline on blood lipid levels and liver and kidney function in high-fat diet-induced obese mice can be seen in Table 1.

[0083] Table 1: Effects of DL-norvaline on blood lipid levels and liver and kidney function in high-fat diet-induced obese mice

[0084]

[0085] It can be clearly seen from the above table that compared with LFD, the TC, TG and LDL-C levels of mice in the HFD group were significantly increased, and the HDL-C level was significantly decreased (P<0.05). However, after 6 weeks of DL-norvaline intervention, the TC, TG and LDL-C levels were significantly decreased, and the HDL-C level was significantly increased (P<0.05). The levels of liver injury markers, including AST, ALT and ALP levels, in mice in the HFD group were significantly increased compared with those in the LFD group (P<0.05), while DL-norvaline intervention significantly reduced the serum AST, ALT and ALP levels of obese mice (P<0.05) (Table 1). Compared with the LFD group, the levels of renal injury markers CRE and BUN in mice in the HFD group were significantly increased (P<0.05), and DL-norvaline intervention significantly reversed this trend (P<0.05).

[0086] (5) The above H&E staining analysis method was used to obtain Figure 4 The results shown are based on Figure 4 The effect of DL-norvaline on the tissue morphology of high-fat diet-induced obese mice shown in the figure shows that the liver structure of the LFD group mice was normal, with curved euchromatic nuclei radiating from the central vein. However, the liver of the HFD group mice had an increase in small fat vacuoles, dilated central veins, disappearance of some hepatocyte nuclei, and slightly disordered arrangement of hepatocytes. After DL-norvaline intervention, the fat vacuoles in the liver of obese mice were significantly reduced, and the hepatocyte structure was improved ( Figure 4 A). In addition, compared with the LFD group, the size and diameter of adipocytes in the HFD group increased, while the adipocytes of obese mice after 6 weeks of DL-norvaline intervention had a more complete morphology, decreased size and diameter, and increased nuclear density ( Figure 4 B).

[0087] (6) Lipidomics analysis

[0088] 1038 lipids were determined in positive ion mode, and 689 lipids were determined in negative ion mode. They can be classified into three major lipid categories: sphingolipids (SPs), glycerophospholipids (GLs), and glycerolipids (GPs), including ceramides (Cers), sphingomyelins (SMs), phosphatidylcholines (PCs), phosphatidylglycerols (PGs), and phosphatidylethanolamine (Phosphatidyle thanolamines, PEs), phosphatidylinositols (PIs), phosphatidylserines (PSs), lysophosphatidylinositols (LPIs), triacylglycerols (TAGs), cardiolipins (CLs), phosphatidylethanol (PEtOH), triacylglycerols (TAGs), oxidized phosphatidylcholines (OxPCs) and oxidized phosphatidylethanolamines (OxPEs), etc.

[0089] like Figure 5 As shown, compared with the LFD group, the adipose tissue of the HFD group mice contained higher levels of Cers (Cer d18:1 / 18:0, Cer d18:1 / 9Z-18:1, Cer-NS d18:2 / 18:0, Cer-NS d18:2 / 24:2), PCs (PC 18:0 / 20:2, PC 22:6e / 17:2), PEs (PE 18:0 / 22:4, PE 18:1e / 22:4, PE 18:4e / 22:1, PE 20:2 / 18:3), SMs (SM d14:0 / 22:0, SM d21:0 / 13:1, SM d21:2 / 15:1, SM d23:2 / 13:0, SM d24:0 / 12:0, SMd24:1 / 12:0, SMd26:2 / 12:0, SMd18:0 / 20:2 11Z,14Z), PEtOH 18:1-20:1, PI 18:0 / 22:5, PSs (PS20:0 / 20:4, PS18:0 / 22:5 (7Z,10Z,13Z,16Z,19Z)) and TAGs (TAG 12:0-14:0-18:1, TAG

[0090] 15:0-16:0-22:6,TAG 15:0-16:1-16:1,TAG 16:0-16:0-18:2,TAG 16:0-18:1-22:4,TAG 16:0-18:2-21:5,TAG 17:0-18:1-18:1,TAG 17:0-18:1-20:4,TAG

[0091] 18:0-18:1-20:2,TAG 18:0-18:1-20:4,TAG 18:0-20:1-20:4,TAG 18:1-18:2-22:4,TAG 18:1-20:1-20:4,TAG 18:1-20:4-22:1,TAG 18:2-18:3-22:4).

[0092] However, lipid compounds in adipose tissue of obese mice were significantly downregulated after DL-norvaline intervention, especially sphingolipid metabolism and phospholipid metabolism. Among sphingolipids, the levels of Cers (Cer-ADS d22:0 / 16:2, Cer-AS d18:1 / 22:0, Cer-AS d18:1 / 24:0, Cer-AS d18:2 / 23:0, Cer-AS d18:2 / 24:0, Cer-AS d18:2 / 24:1, Cer-BDS d14:0 / 20:1, Cer-NS d18:2 / 19:0, Cer-NS d18:2 / 20:0, Cer-NS d18:2 / 23:0) and SM d26:2 / 12:0 in adipose tissue of obese mice were significantly reduced after intervention with medium and high concentrations of DL-norvaline compared with the HFD group. Among phospholipids, compared with the HFD group, the levels of OxPCs (OxPC 16:0-18:2+2O, OxPC 18:1-18:1+1O), OxPEs (OxPE 18:1-18:2+2O, OxPE 18:1-20:5+3O(2Cyc)), PI 18:0 / 22:5 and PC 19:1 / 19:2 in the adipose tissue of obese mice were significantly decreased after low and high concentrations of DL-norvaline intervention ( Figure 6 ). It is noteworthy that compared with the HFD group, the levels of sunitinib in the adipose tissue of obese mice were significantly increased after intervention with low, medium and high levels of DL-norvaline ( Figure 6 ).

[0093] Phospholipids and sphingolipids are the main components of the lipid metabolism pathway of organisms, showing great complexity and being associated with obesity and metabolic syndrome [3,4]. Phospholipids are not only structural molecules of the plasma membrane, but also important bioactive mediators of various cellular processes and have been identified as potential biomarkers of obesity [5]. Sphingolipids can mediate the loss of insulin sensitivity and induce intestinal microbiota dysbiosis and intestinal dysfunction [6]. In particular, ceramide, a bioactive lipid, is involved in intracellular signaling, cell proliferation, migration and apoptosis [7]. Studies have shown that ceramide can promote the production of inflammatory cytokines and subsequently exacerbate inflammatory responses and insulin resistance during the progression of obesity [8]. Choromańska et al. (2019) proposed that ceramide levels in visceral adipose tissue of patients with metabolic syndrome and obesity are positively correlated with lipid accumulation [9]. Lipidomics analysis showed that DL-norvaline improves lipid metabolism disorders through sphingolipid metabolism and glycerophospholipid metabolism, including reducing Cers, SM, OxPCs, OxPEs, PI and PC levels. Compared with the HFD group, low, medium and high doses of DL-norvaline intervention significantly increased the level of sunitinib in the adipose tissue of obese mice. Sunitinib is a small molecule multikinase inhibitor that can inhibit multiple related receptor tyrosine kinases and has anti-angiogenic and anti-tumor activities

[10] . In addition, studies have reported that sunitinib intervention can significantly reduce the adipose tissue weight of obese mice

[11] . Therefore, DL-norvaline can regulate lipid composition, especially phospholipid and sphingolipid metabolism, thereby improving obesity-related symptoms.

[0094] In summary, DL-norvaline can significantly improve lipid metabolism disorders in HFD-induced obese mice through sphingolipid metabolism and phospholipid metabolism.

[0095] (7) The above H&E staining analysis method was used to obtain Figure 7 The results shown and Figure 7 The results of H&E staining of the colon tissue of mice showed that compared with the mice in the LFD group, the mice in the HFD group had obvious inflammatory cell infiltration, intestinal mucosal damage, and crypt loss. However, after supplementing with DL-norvaline, these pathological symptoms were improved to a certain extent, for example, the degree of inflammatory cell infiltration and intestinal mucosal damage was low, and the colon tissue structure was relatively intact.

[0096] The above immunofluorescence staining analysis method Figure 8 The results shown are based on Figure 8The effect of DL-norvaline on intestinal barrier damage in high-fat diet-induced obese mice was shown in the figure. Compared with those in the LFD group, the expression levels of tight junction-related proteins ZO-1, Claudin-1 and Occludin in the colon tissue of mice in the HFD group were significantly decreased (P<0.05), but the expression of ZO-1, Claudin-1 and Occludin in the colon tissue of mice in the DL-norvaline intervention group was significantly increased (P<0.05).

[0097] (8) The intestinal flora 16S rRNA sequencing analysis method was used to obtain Fig. 9 The results shown are based on Fig. 9 The effect of DL-norvaline on the intestinal flora of high-fat diet-induced obese mice shown in the figure shows that, compared with the LFD group mice, there were no significant differences in the intestinal flora of HFD-induced obese mice and the low-dose DL-norvaline intervention group mice except for the ACE index (P>0.05). However, compared with the HFD group, the Shannon index, Chao1 index and ACE index of the intestinal flora of the high-dose DL-norvaline intervention group mice were significantly reduced (P<0.05), indicating that DL-norvaline may enrich some specific microorganisms.

[0098] After further analysis, we found Fig.10 The results shown, Fig.10 The PCA score graph showed that there was a significant separation among the five groups, indicating that there were obvious differences in the β diversity of the intestinal flora of mice in different groups, and the distance between HFD and LFD was relatively large. After DL-norvaline intervention, a new aggregation and separation phenomenon appeared: the distance between the samples of the DL-norvaline intervention group and the LFD group was significantly shortened, and the flora structure gradually recovered to that of the LFD group.

[0099] To reveal the changes in the overall bacterial community structure, relative abundance analyses were further performed at the phylum, class, order, family, genus, and species levels ( Fig.11 ).like Fig.11 A, At the phylum level, the mouse intestinal microbiota was mainly composed of Firmicutes and Bacteroidetes. HFD significantly altered the intestinal microbial structure, characterized by an increase in the relative abundance of Firmicutes and a decrease in the relative abundance of Bacteroidetes, which in turn led to a significant increase in the Firmicutes / Bacteroidetes ratio ( Fig.11 A). However, low-dose DL-norvaline intervention decreased the Firmicutes / Bacteroidetes ratio compared with the HFD group ( Fig.11A). In addition, the relative abundance of Proteobacteria and Fusobacteriota was higher in the HFD group compared with the LFD group, but was reduced after DL-norvaline intervention ( Fig.11 A). At the class level, HFD decreased the relative abundance of Bacteroidia and increased the relative abundance of Alphaproteobacteria and Fusobacteriia compared with the LFD group, while DL-norvaline intervention reversed this trend ( Fig.11 B). At the order level, the relative abundances of Desulfovibrionales, Pseudomonadales, and Mycoplasmatales in the feces of mice in the HFD group increased from 2.64% to 7.33%, from 0.12% to 0.36%, and from 0.02% to 0.06%, respectively, compared with those in the LFD group, and Mollicutes_RF39 decreased from 0.25% to 0.17% ( Fig.11 C). However, after DL-norvaline intervention, the relative abundance of Desulfovibrionales, Pseudomonadales, and Mycoplasmatales decreased, and the relative abundance of Mollicutes_RF39 increased in the feces of mice ( Fig.11 C). In addition, lower relative abundances of Ruminococcaceae, Bacteroidaceae, Rikenellaceae, Lactobacillaceae, and Clostridiaceae_1 and higher relative abundances of Enterobacteriaceae, Helicobacteraceae, Burkholderiaceae, and Veillonellaceae were observed at the family level in the HFD group ( Fig.11 D), but the opposite trend was observed in the DL-norvaline intervention group ( Fig.11 D) At the same time, at the genus level, compared with the LFD group, the HFD group

[0100] The relative abundance of uncultured_bacterium_f_Muribaculaceae, Bacteroides, Mucispirillum, Fournierella, and was suppressed after mice were fed a HFD ( Fig.11 E), but was significantly upregulated after DL-norvaline intervention ( Fig.11 E).

[0101] As probiotics, Ruminococcacea and Lachnospiracea are known for their ability to produce the short-chain fatty acid butyrate. Lactobacillaceae, in particular, plays an important role in human health and has anti-tumor, liver-protective and anti-inflammatory effects

[12] . It has been reported that Lactobacillus and Clostridiaceae_1 can reshape the intestinal microbiota and produce short-chain fatty acids (SCFAs) to improve obesity. In addition, Mollicutes_RF39 is a beneficial microorganism

[13] that is significantly positively correlated with the expression of ZO-1. Compared with the HFD group, the relative abundance of Lachnospiracea, Lactobacillaceae, Clostridiaceae_1 and Mollicutes_RF39 increased after DL-norvaline intervention. In addition, the relative abundance of Bacteroidaceae, Ruminococcaceae and Rikenellaceae was negatively correlated with obesity. The present study found that DL-norvaline intervention upregulated the relative abundance of these bacteria. In particular, as a harmful bacterium, Desulfovibrionales was enriched in obese mice. Obese mice fed with HFD showed a higher relative abundance of Desulfovibrionales, and Desulfovibrionales was positively correlated with fatty liver-related markers. Similarly, Fusobacteriia, as a harmful bacterium, is a characteristic microorganism of diabetic patients. DL-norvaline intervention significantly reduced the relative abundance of Desulfovibrionales and Fusobacteriia in mice fed a high-fat diet. In addition, the abundance of Enterobacteriaceae was positively correlated with weight gain, total cholesterol, insulin concentrations, and inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Burkholderiaceae is also reported to be a pathogenic bacterium that can cause intestinal balance disorders and aggravate obesity. At the same time, Veillonellacea and Helicobacteraceae are important microorganisms that cause gastrointestinal diseases and have a higher relative abundance in overweight individuals. Lower relative abundances of Enterobacteriaceae, Burkholderiaceae, and Veillonellacea were observed in the DL-norvaline intervention group compared with the HFD group.

[0102] Importantly, the relative abundance of Rikenellaceae_RC9_gut_group was low in obese individuals, and was negatively correlated with blood lipids, glucose and insulin levels, and positively correlated with SCFAs. In particular, Muribaculaceae plays an important regulatory role in anti-diabetes and anti-inflammation, and is negatively correlated with lipid indices, HOMA-IR values ​​and inflammatory markers. Compared with the HFD group, DL-norvaline intervention upregulated the relative abundance of uncultured_bacterium_f_Muribaculaceae and Rikenellaceae_RC9_gut_group in the intestine of high-fat mice, further indicating that DL-norvaline intervention regulated the composition of the intestinal microbiota of HFD-induced mice.

[0103] Example 2: Toxicity Experiment

[0104] (1) Acute toxicity test

[0105] Acute oral toxicity studies were performed according to the Organisation for Economic Co-operation and Development (OECD) Guideline-425.

[0106] Twenty-four C57BL / 6J mice were randomly divided into two groups, 12 mice in each group, half of them were male and half were female. The intervention dose of DL-norvaline was designed according to the maximum tolerated dose method to be 5000 mg / kg (equivalent to 200 times the recommended dose for humans).

[0107] After one week of adaptive feeding, one group was gavaged with 5000 mg / kg DL-norvaline dissolved in 0.5% carboxymethylcellulose sodium (CMC-Na), and the other group was gavaged with 0.5% CMC-Na as a control for 14 consecutive days. The mortality and signs of toxicity of the animals were monitored at least twice a day, and the weight, food intake and water intake of the mice were recorded every two days.

[0108] After the mice were euthanized, blood was collected from the eyeballs of the mice, centrifuged at 2000 r / min for 15 min at 4°C, and the supernatant was collected and stored at -80°C for subsequent determination. The blood, brain, heart, liver, spleen, lung, kidney, stomach, thymus, colon, jejunum, and uterus / testis of the mice were collected and weighed.

[0109] (2) Subacute toxicity test

[0110] Subacute toxicity studies were conducted according to OECD Guideline-407. Forty-eight C57BL / 6J mice were randomly divided into four groups, 12 mice in each group, half male and half female. The DL-norvaline intervention group was divided into three dose groups, 500 mg / kg, 1000 mg / kg and 2000 mg / kg. After one week of adaptive feeding, the three groups were gavaged with different concentrations of DL-norvaline dissolved in 0.5% CMC-Na, and the other group was gavaged with 0.5% CMC-Na as a control for 28 consecutive days. The mortality and signs of toxicity of the animals were monitored at least twice a day, and the body weight, food intake and water intake of the mice were recorded every two days. After the mice were euthanized, blood was collected from the eyeballs of the mice, centrifuged at 2000r / min for 15min at 4℃, and the supernatant was collected and stored at -80℃ for subsequent determination. The blood, brain, heart, liver, spleen, lung, kidney, stomach, thymus, colon, jejunum and uterus / testis of the mice were collected and weighed.

[0111] (3) Biochemical index detection

[0112] The levels of serum aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), blood urea nitrogen (BUN), creatinine (CRE), glucose (GLU), total cholesterol (TC), triglyceride (TG), total protein (TP), albumin (ALB), globulin (GLO), ALB / GLO ratio (ALB / GLO Ratio, A / G), lactate dehydrogenase (LDH), creatine kinase (CK) and amylase (AMS) were measured by automatic biochemical analyzer.

[0113] (4) Hematoxylin-eosin staining

[0114] The mouse tissues were fixed in 4% paraformaldehyde for 24 hours, and after washing away the fixative, they were placed in 70%, 80%, 95%, and 100% ethanol for dehydration, and then immersed in xylene twice to make it transparent, and then embedded in molten paraffin. The embedded wax blocks were cut into 4 μm thick slices on a microtome, spread flat in water at 42°C, picked up with a slide, and placed in a constant temperature incubator at 60°C for 4 hours to prepare paraffin sections. Xylene, anhydrous ethanol, 95% ethanol, 85% ethanol, 75% ethanol, 50% ethanol, pure water, phosphate buffered saline (PBS) buffer (pH 7.4) were used for rehydration, and then H&E was used for staining. After sealing with neutral gum, the slices were observed and photographed under a microscope.

[0115] (5) Results

[0116] ① Evaluation of acute toxicity of DL-norvaline

[0117] During the acute toxicity test, no mortality or toxicity-related clinical symptoms were observed in female / male mice, and there were no significant differences in water intake, food intake, body weight gain, food utilization, and organ weights in female / male mice in the DL-norvaline intervention group compared with the control group (P>0.05)( Fig.12 ).

[0118] As shown in Table 2, after female / male mice were treated with 5000 mg / kg DL-norvaline for 14 days, there were no significant differences in their organ ratios (heart to body ratio, liver to body ratio, spleen to body ratio, lung to body ratio, kidney to body ratio, stomach to body ratio, uterus to body ratio / testis to body ratio, thymus to body ratio, brain to body ratio) compared with those of the control group (P>0.05).

[0119] Table 2: Effects of DL-norvaline on the organ-to-body ratio of mice in acute toxicity tests

[0120]

[0121] The results are expressed as mean ± standard error (n = 6). Different letters indicate significant differences among the groups (P < 0.05).

[0122] The results of hematological and biochemical analyses are shown in Table 3. There were no significant differences in the levels of AST, ALP, GGT, TP, ALB, GLO, A / G, LDH, CK, BUN, CRE, GLU, TC, TG, and AMS in female / male mice in the 5000 mg / kg group compared with those in the control group (P>0.05).

[0123] Table 3: Effects of DL-norvaline on hematological and biochemical parameters in mice in acute toxicity studies

[0124]

[0125] The results are expressed as mean ± standard error (n = 6). Different letters indicate significant differences among the groups (P < 0.05).

[0126] like Fig.13 The results of H&E staining of mouse tissues showed that after supplementation with 5000 mg / kg DL-norvaline, there were no obvious pathological changes in the brain, heart, liver, spleen, lung, kidney, stomach, thymus, colon, jejunum, uterus / testis of female / male mice compared with the control group, which was consistent with the results of serum analysis.

[0127] ② Evaluation of subacute toxicity of DL-norvaline

[0128] For the subacute oral toxicity study, there were no abnormal behaviors, deaths, or other adverse events in female / male mice after intervention with 500mg / kg, 1000mg / kg, and 2000mg / kg of DL-norvaline. Compared with the control group, there were no significant differences in water intake, food intake, weight gain, food utilization, and tissue weight in female / male mice after intervention with different doses of DL-norvaline for 28 days (P>0.05)( Fig.14 ).

[0129] Consistently, after intervention with different doses of DL-norvaline (500, 1000 and 2000 mg / kg), the organ ratios of female / male mice showed no significant differences compared with those of the control group (P>0.05) (Table 4).

[0130] Table 4: Effects of DL-norvaline on the organ-to-body ratio of mice in subacute toxicity tests

[0131]

[0132] The results are expressed as mean ± standard error (n = 6). Different letters indicate significant differences among the groups (P < 0.05).

[0133] At the end of the intervention with different doses of DL-norvaline, the histopathological evaluation of major organs was performed by H&E staining. Fig.15 Compared with the control group, no significant changes were found in the histopathological examination of the brain, heart, liver, spleen, lung, kidney, stomach, thymus, colon, jejunum, and uterus / testis in the DL-norvaline intervention groups at various doses.

[0134] The serum parameter levels of female / male mice in the subchronic oral toxicity study are shown in Table 5 (the results in the table are expressed as mean ± standard error (n = 6); different letters indicate significant differences between the groups (P < 0.05)). Compared with the control group, intervention with different doses of DL-norvaline had no significant effect on most hematological and biochemical parameters (P>0.05). However, the A / G level of male mice in the high-dose (2000 mg / kg) DL-norvaline intervention group and the AMS level of mice in the low-dose (500 mg / kg) DL-norvaline intervention group were significantly increased compared with the control group (P<0.05). However, no significant differences were found in the hematological and biochemical parameters in female mice compared with the control group (P>0.05).

[0135]

[0136] During the acute toxicity test, after a single daily oral administration of DL-norvaline (5000 mg / kg, equivalent to 200 times the recommended human dose) for 14 days, the female / male mice were in good condition and showed no obvious symptoms of toxicity or death. There were no significant changes in the growth parameters (water intake, food intake, weight gain, food efficiency ratio and organ-to-body ratio), hematological and biochemical parameter analysis, and histopathological signs of the mice, indicating that 5000 mg / kg DL-norvaline intervention had no toxic effects on mice

[14] .

[0137] During the subacute toxicity test, no death, behavioral abnormalities, or histological signs were observed after 28 days of treatment with 500 mg / kg, 1000 mg / kg, and 2000 mg / kg of DL-norvaline. The A / G levels of male mice treated with high-dose (2000 mg / kg) DL-norvaline were significantly increased compared with the control group. However, since ALB and GLO levels did not differ significantly from those of the control group, and no abnormalities were observed in the female mice treated with high-dose DL-norvaline, the results of increased A / G levels in male mice treated with high-dose DL-norvaline may not be meaningful

[15] . In addition, although AMS levels in male mice treated with low-dose (500 mg / kg) DL-norvaline were significantly increased, histopathological examination of organ samples obtained from mice treated with 500 mg / kg DL-norvaline showed no abnormalities, and no abnormalities were observed in the high-dose treatment group. At the same time, the levels of A / G and AMS in male mice after DL-norvaline treatment were classified within the normal range of mouse indicators

[16] . In toxicological safety evaluation, occasional changes found in the examined organs can be considered to have no biological or toxicological relevance

[17] . The above safety test results provide a scientific basis for the general toxicity characteristics of DL-norvaline and its further clinical application.

[0138] In this example, the safety of DL-norvaline was evaluated through acute toxicity test and subacute toxicity test.

[0139] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

[0140] References

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[0144] Lipid Metabolism and Cognitive Performance in Mice. Molecular Nutrition & Food Research, 2020.

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[0148] 5.Phung, NV, et al., Nervonic acid and its sphingolipids: Biological functions and potential

[0149] food applications.Critical Reviews in Food Science and Nutrition,2023.3:p.1-20.

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[0153] aggravates metabolic derangement during obesity.Molecular Metabolism,2023.73:p.101741.

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Claims

1. Application of DL-norvaline in the preparation of anti-obesity preparations.

2. The use according to claim 1, characterized in that: The obesity is caused by a high-fat diet.

3. Application of DL-norvaline in the preparation of preparations for preventing, regulating and / or alleviating diseases related to high-fat diet.

4. The use according to claim 3, characterized in that: The high-fat diet-induced related diseases include at least one of the following: ① Disorders of glucose and lipid metabolism caused by high-fat diet; ②Fatty liver caused by high-fat diet; ③ Liver and kidney damage caused by high-fat diet; ④ Damage to intestinal barrier function caused by high-fat diet; ⑤ Intestinal flora imbalance caused by high-fat diet; ⑥Lipid metabolism imbalance caused by high-fat diet.

5. The use according to any one of claims 1 to 5, characterized in that: The preparation is food, medicine or health product.

6. A preparation for preventing and / or alleviating obesity and related diseases caused by a high-fat diet, characterized in that: It contains an effective amount of DL-norvaline.

7. A preparation for preventing and / or alleviating obesity and related diseases caused by a high-fat diet according to claim 6, characterized in that: The related diseases include at least one of the following: ① Disorders of glucose and lipid metabolism caused by high-fat diet; ②Fatty liver caused by high-fat diet; ③ Liver and kidney damage caused by high-fat diet; ④ Damage to intestinal barrier function caused by high-fat diet; ⑤ Intestinal flora imbalance caused by high-fat diet; ⑥Lipid metabolism imbalance caused by high-fat diet.

8. A preparation for preventing and / or alleviating obesity and related diseases caused by a high-fat diet according to claim 6 or 7, characterized in that: The preparation is food or oral medicine or oral health care product.

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