Application of pyroglutamic acid in the preparation of products for inhibiting fat deposition

Abstract

This invention belongs to the field of medicine and discloses the use of pyroglutamic acid in the preparation of a product for inhibiting fat deposition. The advantages of this invention are: 1. It prevents obesity induced by a high-fat diet; 2. Pyroglutamic acid is a naturally occurring amino acid derivative, widely found in animals and plants, and has good biosafety. The invention also discloses a product for inhibiting fat deposition.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to application of pyroglutamic acid in preparing a product for inhibiting fat deposition. Background Art

[0002] Obesity is a complex, multifactorial chronic disease, closely linked to genetics, environment, lifestyle, and psychological factors. In recent years, changes in lifestyles driven by globalization have exacerbated the obesity epidemic. According to the World Health Organization (WHO), the number of obese people worldwide has nearly tripled since 1975. In 2016, over 1.9 billion adults were overweight, of whom 650 million were obese. The number of obese people in my country is also rapidly increasing. The obesity epidemic has posed a major challenge to global public health systems and placed a heavy health burden on society.

[0003] The search for safe, effective, and side-effect-free anti-obesity compounds from natural products has become a hot topic and a key focus in obesity research. Natural products possess rich chemical diversity and excellent biocompatibility. Many active substances derived from plants, animals, and microorganisms have been shown to have anti-obesity effects, such as regulating energy metabolism, inhibiting fat synthesis, and promoting lipolysis. These natural compounds can not only serve as the basis for the development of weight-loss drugs but can also be developed into functional foods or health supplements, offering a safer, more convenient, and sustainable means of obesity prevention and control. These compounds hold broad market potential and significant social significance.

[0004] Pyroglutamic acid (Pyroglutamic acid), also known as 5-oxoproline, is a naturally occurring amino acid derivative found widely in plants and animals. Pyroglutamic acid has multiple physiological functions, such as regulating metabolism and enhancing immunity. Recent studies have found that Pyroglutamic acid plays a role in improving insulin resistance, but its role in preventing obesity has been less extensively studied.

[0005] Pyroglutamate participates in a series of enzyme reactions for the synthesis and utilization of glutathione in animals, which is called the γ-glutamyl cycle. The γ-glutamyl cycle consists of six enzyme-catalyzed reactions, such as Figure 8 As shown in Figure 1 . Glutathione, catalyzed by γ-glutamyl transpeptidase on the outer plasma membrane of renal cells, transfers γ-glutamyl to an acceptor amino acid, generating cysteinylglycine. The resulting γ-glutamyl amino acid is then reabsorbed by cells in other organs via γ-glutamyl cyclotransferase to form the intermediate pyroglutamate, simultaneously releasing the transported free amino acid. Pyroglutamate and ATP are further converted to glutamate by the enzyme 5-hydroxyprolinase, which then serves as a raw material for the two-step synthesis of glutathione, thus completing the cycle. The γ-glutamyl cycle is also one of the systems that mediate amino transport in mammals.

[0006] An interesting phenomenon is that there is no report on the use of L-pyroglutamic acid in weight loss in existing literature. However, some literature reports that complexes formed by L-pyroglutamic acid and other drugs through coupling, cocrystal, etc. have weight loss effects, such as:

[0007] Publication number NO2018019I2, the subject is ergliflozin, optionally in crystalline form, in particular a co-crystal with L-pyroglutamic acid, and in particular a patent application for ergliflozin L-pyroglutamic acid, which can be used to prevent and treat obesity and its related complications, in particular type II (type 2) diabetes.

[0008] The patent application with publication number CN104231070A, which is titled as N-terminally modified glucose-dependent insulinotropic polypeptide (GIP) analogs, discloses modification of pyroglutamic acid at the end of a specific peptide.

[0009] Most of the above-mentioned plans are based on the combined effects of diabetes drugs to achieve the effect of weight loss.

[0010] Therefore, the technical problem solved in this case is: how to develop new and safe products that inhibit fat deposition, especially weight loss agents. Summary of the Invention

[0011] The purpose of the present invention is to provide the use of pyroglutamic acid in the preparation of products for inhibiting fat deposition and products containing pyroglutamic acid. The advantages of the present invention are: 1. prevention of obesity induced by a high-fat diet; 2. pyroglutamic acid is a naturally occurring amino acid derivative that is widely present in animals and plants and has good biosafety.

[0012] To achieve the above objectives, this application discloses:

[0013] The application of pyroglutamic acid in preparing a product for inhibiting fat deposition. More generally, the present invention proposes the application of pyroglutamic acid in preparing a product for weight loss.

[0014] More specifically, the product is used to alleviate high-fat diet-induced weight gain, and / or to reduce adipose tissue mass and inguinal white adipose tissue cell size, and / or to inhibit fat accumulation in liver cells.

[0015] In addition, the present invention also discloses a product for inhibiting fat deposition, which contains pyroglutamic acid.

[0016] The above-mentioned product also includes pharmaceutically acceptable excipients or carriers; the dosage form of the weight loss product is oral liquid, powder, tablet, capsule or injection.

[0017] The above-mentioned product contains 0.01~1wt% of pyroglutamic acid.

[0018] This application has at least the following beneficial effects:

[0019] This invention has discovered a new use for pyroglutamic acid. Animal experimental results show that supplementing pyroglutamic acid with drinking water at a dosage of 0.2% for 10 weeks significantly reduced body weight and weight gain in mice fed a high-fat diet, inhibited liver lipid accumulation, reduced adipose tissue mass, and decreased inguinal white adipocyte area, demonstrating that pyroglutamic acid can prevent high-fat diet-induced obesity. Furthermore, genetic analysis has shown that pyroglutamic acid can increase the expression of genes and proteins related to thermogenesis, achieving weight loss by improving digestion and metabolism.

[0020] The present invention provides a new use of pyroglutamic acid in preparing related health-care foods and medicines capable of preventing obesity induced by a high-fat diet. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The effect of pyroglutamate on the body weight of mice fed a high-fat diet;

[0022] Figure 2 The effect of pyroglutamate on adipose tissue mass in mice fed a high-fat diet;

[0023] Figure 3 The effect of pyroglutamate on adipose tissue size in mice fed a high-fat diet;

[0024] Figure 4 The effect of pyroglutamate on inguinal white adipose tissue slices of mice fed a high-fat diet;

[0025] Figure 5 Effects of pyroglutamate on the cell area of ​​inguinal white adipose tissue in mice fed a high-fat diet;

[0026] Figure 6 The effect of pyroglutamate on the mass of non-adipose tissue in mice fed a high-fat diet;

[0027] Figure 7A The effect of pyroglutamate on liver fat accumulation in mice fed a high-fat diet;

[0028] Figure 7B Schematic diagram of the γ-glutamyl cycle;

[0029] Figure 8 The effect of pyroglutamate on the body temperature of mice fed a high-fat diet at room temperature;

[0030] Figure 9 The effect of pyroglutamate on brown fat thermogenesis in mice fed a high-fat diet at room temperature;

[0031] Figure 10 Effects of pyroglutamate on the body temperature of mice fed a high-fat diet under cold stimulation;

[0032] Figure 11 The effect of pyroglutamate on energy expenditure in mice fed a high-fat diet;

[0033] Figure 12 The effect of pyroglutamate on the mRNA level of UCP1 in mice fed a high-fat diet;

[0034] Figure 13 Effects of pyroglutamate on the protein level of UCP1 in mice fed a high-fat diet. DETAILED DESCRIPTION

[0035] Below in conjunction with embodiments of the present invention, the present invention is clearly and completely described, in description of the present invention, it should be noted that, in the embodiment, the unrecited specific conditions person, carry out according to the condition of normal condition or manufacturer's suggestion. Reagents therefor or instrument are not recited manufacturer, are the conventional products that can be obtained by commercial purchase. In the case of not making special instructions, used part in the embodiments of the present invention is all weight part, and % is all weight percentage.

[0036] Example 1 Effects of pyroglutamate on body weight and weight gain in mice fed a high-fat diet

[0037] Sixteen 5-week-old male C57BL / 6J mice were housed under specific conditions (ambient temperature 22±2°C, relative humidity 40-70%, 12h-12h light-dark cycle).

[0038] At the start of the experiment, mice were randomly divided into a control group and a pyroglutamate-treated group, with eight mice in each group. They were fed a high-fat diet (HF60, Catalog No. 112252), with 60% of calories from fat. The pyroglutamate-treated group received 0.2 wt% pyroglutamate supplemented in their drinking water. The experiment lasted 10 weeks, with all mice receiving food and water ad libitum. Body weights were recorded weekly.

[0039] The mice fed the above diet for 10 weeks were fasted for 12 hours and then sacrificed by cervical dislocation. The liver, scapular brown fat, inguinal white fat, and epididymal white fat were removed and weighed (the weight change curve of mice fed a high-fat diet is shown in Figure 2). Figure 1 shown).

[0040] according to Figure 2 and Figure 3 As shown, pyroglutamate can significantly reduce the mass and tissue size of scapular brown fat, inguinal white fat, and epididymal white fat in mice fed a high-fat diet.

[0041] The inguinal white fat of the obtained mice was fixed with formalin fixative, then removed and prepared into pathological sections after dehydration, embedding, sectioning, drying, dewaxing, and hematoxylin-eosin (H&E) staining. The sections were observed under a 10x microscope and photographed. Figure 4 As shown in the figure, the cell volume of inguinal white adipocytes in the pyroglutamate-treated mice was significantly reduced in the same field of view compared with the control group. The cell area of ​​H&E-stained inguinal white adipose tissue was analyzed using ImageJ (National Institute of Health, Bethesda, MD, USA). Figure 5 As shown, pyroglutamate treatment can significantly reduce the average area of ​​inguinal white adipocytes in mice induced by a high-fat diet.

[0042] First, the mouse liver was fixed with formalin fixative, then removed and dehydrated, embedded, sliced, dried, dewaxed, and stained with hematoxylin-eosin (H&E) to prepare pathological sections. The sections were observed under a 10x microscope and photographed. Figure 6 As shown in Figure 2, pyroglutamate had no significant effect on liver mass in high-fat diet-induced obese mice. Figure 7A As shown in the figure, the liver lobules and hepatocyte cords of the control group mice were arranged in disorder, with blurred boundaries, significantly enlarged and deformed cells, and severe fat infiltration in the cytoplasm, manifested as a large number of round fat vacuoles, indicating that long-term high-fat feeding led to a large amount of fat accumulation in the livers of mice. The liver lobules of the control group mice were arranged neatly, with clear structures, and distributed radially around the central vein; compared with the control group, the fat vacuoles in the hepatocytes of the pyroglutamate-treated group mice were significantly smaller and less than those in the control group, and the arrangement of the liver lobules and hepatocyte cords was significantly improved.

[0043] according to Figure 8 As shown, the room temperature of the pyroglutamate-treated group was significantly higher than that of the control group, indicating that pyroglutamate treatment enhanced the energy expenditure of mice.

[0044] according to Figure 9 As shown, the backs of the two groups of mice were photographed using an infrared imager, and it was found that the brown fat temperature of the pyroglutamate-treated group was significantly higher than that of the control group, indicating that pyroglutamate treatment enhanced the heat production of brown fat in mice.

[0045] Cold stimulation is the main way to activate brown fat. Figure 10As shown, the two groups of mice were placed in a 4°C environment and their body temperature was measured every hour. It was found that the body temperature of the pyroglutamate-treated group was significantly higher than that of the control group, indicating that pyroglutamate treatment enhanced the heat production of mice.

[0046] according to Figure 11 As shown, the two groups of mice were placed in metabolic cages to detect energy metabolism, and it was found that the energy metabolism level of the pyroglutamate-treated group was significantly higher than that of the control group, but there was no difference in spontaneous movement distance, indicating that pyroglutamate treatment enhanced the energy consumption of mice and improved the energy metabolism level of mice.

[0047] BAT tissue (brown adipose tissue) of mice was obtained to measure the expression of thermogenic genes and proteins in BAT.

[0048] The total RNA of BAT was extracted using the Trizol method, and chloroform was added for extraction. The supernatant was precipitated with an equal volume of isopropanol, and then the precipitate was washed with 75% ethanol. The RNA precipitate was dissolved in DEPC water for subsequent experiments. Reverse transcription was performed using a cDNA kit. The reaction system was prepared using a qPCR premix kit, and the mRNA level was quantified using ACTB as an internal reference. The results are shown in Figure 2. Figure 12 The results showed that the UCP1 mRNA level in the pyroglutamate group was higher than that in the control group.

[0049] The total protein of BAT was extracted using RIPA lysis buffer containing PMSF, and the protein concentration was adjusted to 1 μg / μL. The protein was denatured by heating at 95°C for 5 minutes. SDS-PAGE 10% separation gel and 5% stacking gel were prepared, protein samples and markers were added, and electrophoresis was performed at 90V for 90 minutes. Methanol was used to activate the PVDP film, and the "wet transfer method" was used for 75 minutes at 180V. The PVDF membrane was removed and blocked with a fast blocking solution for 10 minutes. The primary antibody (1:1000) was incubated at 4°C overnight, washed with TBST, and the secondary antibody (1:25000) was incubated at room temperature for 1 hour, and washed with TBST. ECL chemiluminescent solution was added and imaged under a developer. The results are as follows. Figure 13 As shown, the results showed that the UCP1 protein level in the pyroglutamate group was higher than that in the control group.

[0050] Safety of pyroglutamate

[0051] L-pyroglutamate is an amino acid derivative naturally present in plants and animals. The human body can process this substance through normal metabolism. L-pyroglutamate participates in many physiological processes, including the synthesis of neurotransmitters, protein metabolism, and energy metabolism (its participation in metabolic processes can be found in Figure 7B Schematic diagram of the γ-glutamyl cycle shown in FIG); It is widely used in the food, medicine, cosmetics and other industries. Existing research and the animal experiments of the present invention have shown that this amino acid has good safety.

[0052] The above results show that:

[0053] 1. Pyroglutamate can effectively alleviate lipid accumulation in the liver of mice fed a high-fat diet.

[0054] 2. Pyroglutamate treatment can significantly reduce the average area and volume of inguinal white adipocytes in mice induced by a high-fat diet.

[0055] 3. Pyroglutamate treatment enhanced the energy consumption and heat production of mice, and improved the energy metabolism level of mice.

[0056] 4. Pyroglutamate increased the expression of thermogenesis-related genes and proteins.

[0057] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and it is intended that equivalents falling within the claims be included. All variations within the meaning and scope of the elements are encompassed by the present invention. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

Claims

1. Application of pyroglutamic acid as the sole active ingredient in the preparation of weight loss products.

2. The use according to claim 1, characterized in that The product is used to alleviate weight gain induced by a high-fat diet, and / or reduce the mass of adipose tissue and the size of inguinal white adipose tissue cells, and / or inhibit fat accumulation in liver cells, and / or promote the expression of thermogenic-related genes and proteins in brown adipose tissue.

3. The use according to claim 1, characterized in that The product also includes pharmaceutically acceptable excipients; the dosage form of the weight loss product is oral liquid, powder, tablet, or capsule.

4. The use according to claim 1, characterized in that Contains 0.01~1wt% of pyroglutamic acid.

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