Application of 3-hydroxyanthranilic acid in preparation of fat-reducing and weight-reducing products

Abstract

The invention discloses an application of 3-hydroxy o-aminobenzoic acid in preparation of fat-reducing and weight-reducing products. According to the present invention, the first research results show that the 3-hydroxyanthranilic acid can significantly improve the secretion amount of leptin in the mouse body and influence the food intake of the mouse so as to achieve the purposes of reducing the mouse body weight increase, reducing the mouse body weight and improving the mouse obesity; therefore, the 3-hydroxy o-aminobenzoic acid can be used for preparing a fat-reducing and weight-reducing product which is high in biological safety and does not cause any adverse reaction.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of 3-hydroxy-o-aminobenzoic acid in the preparation of weight loss products. Background Technology

[0002] Obesity is a chronic metabolic disease caused by excessive fat accumulation in the body, leading to overweight and obesity. Obesity increases the risk of type 2 diabetes and heart disease, affects bone health and the reproductive system, and increases the risk of certain cancers. In recent years, the number of overweight and obese people in my country has been growing rapidly, with children gaining weight at a rate even faster than adults.

[0003] Currently, obesity treatment primarily focuses on diet control and increased exercise, supplemented by medication or surgery when necessary. Drug treatment mainly includes orlistat capsules, an intestinal lipase inhibitor, and hypoglycemic drugs that also have weight-loss effects (such as metformin and liraglutide). However, these drugs are all synthetically produced, and adverse reactions are inevitable during clinical use. Nausea and diarrhea are very common, while vomiting, constipation, abdominal pain, and indigestion are also frequent, making it difficult for many patients to adhere to their treatment regimens.

[0004] 3-Hydroxyanthranilic acid (3-HAA) is a tryptophan metabolite in the kynurenine pathway. Compared to the drugs mentioned above, 3-Hydroxyanthranilic acid has a higher biocompatibility and does not cause adverse reactions. Currently, there are no reports of 3-Hydroxyanthranilic acid being used for weight loss. Summary of the Invention

[0005] The purpose of this invention is to provide a new and safe way to lose fat and weight.

[0006] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: This invention first provides the application of 3-hydroxy-o-aminobenzoic acid in the preparation of weight loss products.

[0007] This invention is the first to discover that 3-hydroxy-an-aminobenzoic acid can improve the overall health of mice, affect their food intake, and thus reduce weight gain, decrease body weight, and improve obesity in mice. This indicates that 3-hydroxy-an-aminobenzoic acid can be used to prepare a weight-loss product with high biosafety and no adverse reactions.

[0008] This invention also provides the application of 3-hydroxy-o-aminobenzoic acid in the preparation of hypoglycemic drugs.

[0009] 3-Hydroxy-anaminobenzoic acid (3-HAN) significantly improves insulin sensitivity and enhances insulin's ability to regulate blood glucose, thereby reducing the risk of obesity and diabetes, especially when combined with reduced food intake. Treatment of type 2 diabetic mice with 3-HAN resulted in a significant reduction in the incidence of diabetes, indicating that 3-HAN has a significant protective effect on the pancreas. These experimental results suggest that 3-HAN can be used to prepare a hypoglycemic drug with high biosafety and no adverse reactions.

[0010] Compared with the prior art, the technical effects of the present invention are reflected in: (1) This invention is the first to discover that 3-hydroxy-an-aminobenzoic acid can reduce the amount of food consumed in mice, thereby reducing the weight gain of mice, reducing the weight of mice, and improving obesity in mice. This indicates that 3-hydroxy-an-aminobenzoic acid can be used to prepare a weight loss product with high biosafety and no adverse reactions.

[0011] (2) 3-hydroxy-anaminobenzoic acid can significantly improve insulin sensitivity and enhance insulin's ability to regulate blood sugar, thereby reducing the risk of obesity and diabetes, based on the reduction of food intake. After treating type 2 diabetic mice with 3-hydroxy-anaminobenzoic acid, the incidence of diabetes in the mice was significantly reduced, indicating that 3-hydroxy-anaminobenzoic acid has a significant protective effect on the pancreas. All the above experimental results indicate that 3-hydroxy-anaminobenzoic acid can be used to prepare a hypoglycemic drug with high biosafety and no adverse reactions. Attached Figure Description

[0012] Figure 1 The effect of 3-hydroxy-o-aminobenzoic acid on the incidence of diabetes in a mouse model of type 2 diabetes; Figure 2 The effect of 3-hydroxy-o-aminobenzoic acid on glucose tolerance in a type 2 diabetic mouse model; Figure 3 The experimental procedure for Example 2; Figure 4 The effect of 3-hydroxy-o-aminobenzoic acid on the magnitude of changes in mouse body weight; Figure 5 The effect of 3-hydroxy-o-aminobenzoic acid on daily food intake in mice on a high-fat diet; Figure 6 The effect of 3-hydroxy-o-aminobenzoic acid on the daily food intake of mice on a normal diet; Figure 7 The effect of 3-hydroxy-anaminobenzoic acid on lipid metabolism-related genes in mice; Figure 8The effect of 3-hydroxy-o-aminobenzoic acid on the leptin gene versus the adiponectin gene in mice. Detailed Implementation

[0013] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0014] Example 1: 3-Hydroxy-o-aminobenzoic acid reduces the incidence of diabetes. Healthy C57 / 6J mice were selected and randomly divided into two groups (n=8 per group): a model group and a treatment group. The treatment group received two intraperitoneal injections of 3-HAA (10 mg / kg each) using a 1 mL syringe, with a 24-hour interval between the two injections. Two hours later, the mice were injected with the same amount of solvent, followed by an intraperitoneal injection of STZ (125 mg / kg) using a 1 mL syringe to establish a type 2 diabetes model. Initial blood glucose levels in each type 2 diabetes mouse model were measured as follows: 8.9, 7.4, 9.2, 7.9, 8.1, 7.1, 6.8, 7.0, 7.2, 6.4, 7.5, 7.7, 6.8, 7.1, 6.6, 9.0 (mmol / L), all less than 10 mmol / L. Both groups of mice were fed the same high-fat diet and were normally fed for five days. The treatment group received a 10 mg / kg injection of STZ on days 1 and 3 after the initial injection. 3-HAA was injected into the mice, while the model group received the same dose of the solvent. Blood glucose levels were continuously monitored for five days after feeding, and the incidence of diabetes (defined as blood glucose > 11.1 mmol / L) was statistically analyzed for each group. The results are as follows: Figure 1 As shown.

[0015] from Figure 1 It can be seen that after 5 days of feeding, all mice in the model group (STZ + high-fat diet) developed diabetes, with a diabetes incidence rate of 100%, and this rate remained at 100% throughout the 5-6 week monitoring period. In contrast, only 2 mice in the drug-treated group (STZ + high-fat diet + 3-HAA) developed diabetes after 5 days of feeding, with a diabetes incidence rate of only 25%. Although the number of cases fluctuated during the 5-6 week monitoring period, it ultimately remained at 25%. These experimental results indicate that 3-HAA has a significant protective effect on the pancreas and can effectively reduce the incidence of diabetes in a type 2 diabetic mouse model.

[0016] Example 2: ipGTT Experiment Healthy C57 / 6J mice were selected and, after 8 weeks of HFD feeding, were divided into two groups (n=2 per group): a model group and a drug-treated group. Mice in the drug-treated group received two intraperitoneal injections of 3-HAA (10 mg / kg each) with a 24-hour interval between injections. Mice in the model group received the same dose of the drug. An ipGTT test was then performed: mice were fasted for 16 hours before the experiment. At the start of the experiment, the initial blood glucose level after fasting was measured at 0 min. Subsequently, 0.4 mL of a 20% glucose solution (prepared with PBS and 99.8% anhydrous glucose) was injected intraperitoneally into each mouse using a 1 mL syringe. Blood glucose levels were then measured at 15 min, 30 min, 60 min, and 120 min. The results are shown below. Figure 2 As shown.

[0017] from Figure 2 It can be seen that the blood glucose level of the model group mice (high-fat diet) rose sharply 15 minutes after glucose injection, and remained at a high level at 30 and 60 minutes after administration; while the blood glucose level of the drug-treated group mice only increased slightly after glucose injection and remained at a relatively low level throughout the monitoring process, showing a significant difference from the model group mice (p < 0.0001). These experimental results indicate that HFD feeding reduces insulin function in mice, while 3-HAA can significantly improve insulin sensitivity.

[0018] Example 3: 3-Hydroxy-o-aminobenzoic acid improves obesity in mice and its mechanism analysis 1. 3-Hydroxy-o-aminobenzoic acid improves obesity in mice like Figure 3 As shown, healthy C57 / 6J mice were randomly divided into three groups (n=4 per group): normal diet group, high-fat diet group, and high-fat diet + 3-HAA group. The normal diet group was given a normal diet, the high-fat diet group was given a high-fat diet, and the high-fat diet + 3-HAA group was given a high-fat diet plus intraperitoneal injection of 3-HAA (the normal diet group and the high-fat diet group were given the same amount of solvent) in addition to the high-fat diet. The injection dose was 10 mg / kg, and the injection time was once a day between 3 pm and 4 pm.

[0019] Mice body weight was continuously monitored for 60 days during the rearing period, and the results were as follows: Figure 4 As shown.

[0020] Depend on Figure 4As can be seen, with the extension of feeding time, the body weight of mice in both the normal diet group and the high-fat diet group showed an increasing trend, and there was a significant difference in body weight between the two groups (p=0.0239), indicating that the high-fat diet led to obesity in mice: at 60 days after feeding, the body weight of mice in the high-fat diet group was approximately 1.35 times that before the experiment; while the body weight of mice in the high-fat diet + 3-HAA group also increased with the extension of feeding time, at 60 days after feeding, the body weight of mice in the high-fat diet + 3-HAA group was only approximately 1.15 times that before the experiment, lower than that of mice in both the normal diet group and the high-fat diet group, and there was a significant difference compared with mice in the high-fat diet group (p=0.0004). The above experimental results indicate that 3-hydroxy-an-aminobenzoic acid can significantly improve obesity in mice.

[0021] 2. Mechanism analysis of 3-hydroxy-o-aminobenzoic acid in improving obesity in mice (1) 3-Hydroxy-o-aminobenzoic acid affects food intake in mice First, healthy C57 / 6J mice were randomly divided into four groups (n=5 per group): a high-fat diet group, a high-fat diet + 3-HAA group, a normal diet group, and a normal diet + 3-HAA group. The high-fat diet group received a high-fat diet, and the normal diet group received a normal diet. The high-fat diet + 3-HAA group and the normal diet + 3-HAA group received an intraperitoneal injection of 3-HAA (1 mL syringe) in addition to the high-fat diet or normal diet (the high-fat diet group and the normal diet group received the same amount of the solvent). The injection dose was 10 mg / kg, administered once daily between 3 PM and 4 PM. The mice were then observed for 10 consecutive days, and the average daily food intake per mouse was analyzed. The results are as follows: Figure 5 and Figure 6 As shown.

[0022] Depend on Figure 5 As can be seen, compared with the high-fat diet group, the daily food intake of mice in the high-fat diet + 3-HAA group was significantly reduced (p=0.0002); Figure 6 As can be seen, compared with the normal diet group, the daily food intake of mice in the normal diet + 3-HAA group was also significantly reduced (p=0.0040); the above experimental results all indicate that 3-hydroxy-an-aminobenzoic acid can reduce the food intake of mice, thereby improving mouse obesity.

[0023] (2) 3-Hydroxy-o-aminobenzoic acid significantly improved the health status of mice. First, eukaryotic mRNA sequencing analysis was performed on the white epididymal adipose tissue of mice in two groups: high-fat diet and high-fat diet + 3-HAA. The analysis method was as follows: After feeding the mice in both groups for two weeks, they were euthanized, and the white epididymal adipose tissue was removed to extract total RNA. The concentration and purity of the extracted RNA were detected using Nanodrop 2000, RNA integrity was detected by agarose gel electrophoresis, and the RQN value was determined by Agilent 5300. After passing the quality control, since eukaryotic mRNA has a polyA tail at the 3' end, mRNA can be isolated from the total RNA by using magnetic beads with Oligo(dT) to pair with polyA bases. This mRNA was used to analyze transcriptome information. Under the action of reverse transcriptase, one-stranded cDNA was synthesized using random primers with mRNA as a template, followed by two-stranded synthesis to form a stable double-stranded structure. The double-stranded cDNA structure has sticky ends. End Repair Mix was added to create blunt ends, followed by the addition of an A base at the 3' end to facilitate the subsequent addition of an adapter sequence. The product after adapter ligation was purified and fragment sorted. The sorted products were then subjected to PCR amplification, and the purified final library was obtained. Analysis of the library identified genes involved in lipid metabolism, and a heatmap was created based on their expression levels to obtain a comprehensive characterization of lipid metabolism. Figure 7 As shown.

[0024] from Figure 7 It can be seen that, compared with the high-fat diet group, the expression levels of genes related to lipid synthesis were generally downregulated and the expression levels of genes related to lipid breakdown were generally upregulated in the high-fat diet + 3-HAA group mice, indicating that 3-HAA improved the lipid metabolism disorder caused by the high-fat diet.

[0025] As is well known, adiponectin typically promotes fatty acid oxidation, reduces systemic chronic inflammation, and improves insulin resistance. Adiponectin levels are usually lower in obese individuals. While leptin has an appetite-suppressing effect, obese individuals often exhibit leptin resistance, meaning that leptin circulation is high but it does not suppress appetite. Therefore, in the process of obesity, the lower the leptin-to-adiponectin ratio, the better the overall health status. Thus, this embodiment focuses on analyzing the expression changes of the leptin and adiponectin genes from eukaryotic mRNA sequencing results. The results are as follows... Figure 8 As shown.

[0026] from Figure 8 It can be seen that, compared with the high-fat diet group, the high-fat diet + 3-HAA group had a lower leptin-to-adiponectin ratio, which indicates that 3-HAA significantly improved the lipid metabolism disorder caused by the high-fat diet.

Claims

Application of 1,3-hydroxy-o-aminobenzoic acid in the preparation of weight loss products. Application of 2,3-hydroxy-o-aminobenzoic acid in the preparation of hypoglycemic drugs.

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