Application of vitexin in preparation of medicine for preventing and treating obesity

Abstract

The invention discloses application of vitexin in preparation of medicines for preventing and treating obesity. According to the application of the vitexin in preparation of the medicine for preventing and treating the obesity, the prevention and treatment comprise one or more of prevention, relieving or treatment of the obesity induced by high fat diet, and the medicine can be generally a solution and contains pharmaceutically acceptable auxiliary materials. The vitexin disclosed by the invention can promote the heat production of brown adipocytes and increase the oxygen consumption; the expression of heat production marker protein UCP1 of brown fat and subcutaneous white fat tissues is increased, the metabolic rate of an organism is increased, obesity induced by high fat diet is resisted, and then insulin resistance is improved.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to an application of vitexin in the preparation of a medicine for preventing and treating obesity. Background Art

[0002] Obesity is a major risk factor for metabolic syndrome. It is a major risk factor for type 2 diabetes, fatty liver disease, cardiovascular and cerebrovascular diseases, and even certain cancers. Therefore, the prevention and treatment of obesity has become an increasingly serious public health issue. Although a variety of medications are currently available for the treatment of obesity, they all have numerous side effects and often lead to weight rebound after discontinuation.

[0003] The existing technology currently lacks effective, low-toxicity natural small molecule drugs for solving the problem of preventing and treating obesity. Summary of the Invention

[0004] In order to solve the problems existing in the background technology, the purpose of the present invention is to provide the use of vitexin in the preparation of drugs for preventing and treating obesity.

[0005] The vitexin of the present invention can promote thermogenesis of brown fat cells and increase oxygen consumption; vitexin increases the expression of the thermogenic marker protein UCP1 in brown fat and subcutaneous white adipose tissue, increases the body's metabolic rate, resists obesity induced by a high-fat diet, and thus improves insulin resistance.

[0006] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0007] 1. Application of vitexin:

[0008] The agnoside is used in the preparation of medicines for preventing and treating obesity.

[0009] The prevention and treatment include one or more of the prevention, alleviation or treatment of obesity induced by a high-fat diet.

[0010] The obesity is obesity induced by a high-fat diet.

[0011] The drug is a solution containing pharmaceutically acceptable excipients, such as physiological saline, PEG300, and Tween-80.

[0012] The drug is administered by intraperitoneal injection, and the dosage is preferably 5 mg / kg.

[0013] 2. Application of a drug containing vitexin:

[0014] The medicine contains the vitexin component.

[0015] In the medicine, the mass content of the vitexin is >99% (purity).

[0016] The medicine is one of a solution, a powder, a sustained-release agent, an emulsion, a suspension, a syrup and drops.

[0017] Preferably, the drug is a solution containing pharmaceutically acceptable excipients such as physiological saline, PEG300, and Tween-80.

[0018] 3. An application method of vitexin:

[0019] After vitexatin was made into a solution, it was injected into the abdominal cavity of mice and fed with high-fat diet to create an obesity model. After the 10th week, the obesity of the animals was alleviated.

[0020] The drug was injected intraperitoneally into obese animals.

[0021] Vitex agnuscastus is a natural diterpenoid glycoside compound extracted from Vitex agnuscastus, which is non-toxic. Vitex agnuscastus is a perennial shrub and also a medicinal plant, mainly used to treat irregular menstrual cycles, premenstrual syndrome and menopausal syndrome. Vitex agnuscastus is native to the Mediterranean region, and its fruits, leaves and roots are rich in a variety of bioactive ingredients, including vitex agnuscastus. In addition, vitex agnuscastus can also be extracted from other plants, such as apples, buckwheat, tea and hawthorn. Existing studies have shown that vitex agnuscastus has multiple biological activities such as anti-arthritis, promoting wound healing, anti-inflammatory and analgesic. However, so far, there are no reports in the existing technology on the use of vitex agnuscastus in the preparation of treatment for obesity, nor are there similar studies or attempts.

[0022] In response to the current lack of effective, low-toxic natural small molecule drugs for the prevention and treatment of obesity, the present invention proposes an innovative solution, which is to significantly alleviate obesity induced by a high-fat diet through vitexin, showing its application potential in the field of obesity prevention and treatment, and has broad application prospects in the preparation of drugs for the prevention and treatment of obesity.

[0023] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0024] The present invention demonstrates that, in vitro, vitexin can increase the expression of thermogenic genes in brown adipocytes and increase oxygen consumption in brown adipocytes. In vivo, vitexin can increase thermogenesis in adipose tissue, reduce the weight of abdominal white fat and subcutaneous white fat, inhibit high-fat diet-induced obesity, and improve glucose and insulin sensitivity in high-fat diet-induced obese mice.

[0025] Vitexin is currently marketed primarily for the treatment of menstrual disorders and menopausal syndrome in women. Its safety has been demonstrated through multiple lines of evidence, but its use in the treatment of obesity and non-alcoholic fatty liver disease has not yet been reported. As a natural small molecule, it is readily available and readily available, and its extraction process is sophisticated, offering broad potential for the treatment of obesity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The molecular chemical structure of vitexin and the effect of different concentrations on UCP1 protein in brown fat cells are shown in the figure. Figure 1 A is the molecular chemical structure diagram of vitexin; Figure 1 B is the result of treating brown fat cells with different concentrations of vitexin to detect its effect on UCP1 protein.

[0027] Figure 2 Differentiated mature brown adipocytes were treated with different concentrations of vitexin and the β3-adrenergic receptor agonist CL316,243 (known to activate UCP1 expression) to compare their effects on cellular oxygen consumption. A shows the changes in OCR over time. BD represent basal oxygen consumption (2B), maximal oxygen consumption (2C), and ATP conversion (2D), respectively.

[0028] Figure 3 Schematic diagram of the in vivo experiment of vitexin; Figure 3 A is a schematic diagram of in vivo experiments using vitexin; Figure 3 B is the effect of vitexin on the food intake of mice; Figure 3 C represents the effect of vitexin on the body weight of mice fed with a high-fat diet; Figure 3 D is the weight of brown adipose tissue (BAT), subcutaneous white adipose tissue (iWAT), abdominal white adipose tissue (eWAT), and liver tissue (Liver) of mice treated with high-fat combined with vitexin for 12 weeks; Figure 3 E is the effect of vitexin on lipid accumulation in three adipose tissues and liver tissue of mice.

[0029] Figure 4 Effect of high-fat combined with vitexin treatment on heat production in mice for 12 weeks Figure 4 A) Oxygen consumption ( Figure 4 B) and carbon dioxide emissions ( Figure 4 C) impact, where Figure 4 A represents heat production, Figure 4 B represents oxygen consumption, Figure 4 The C represents carbon dioxide emissions.

[0030] Figure 5 Effects of high-fat diet combined with vitexin on glucose sensitivity (5A), insulin sensitivity (5B), and serum insulin levels (5C) in mice treated with 12 weeks of high-fat diet. Figure 5 A represents glucose sensitivity, Figure 5 B stands for insulin sensitivity. Figure 5 C represents the serum insulin level.

[0031] Figure 6 To investigate the effects of high-fat combined with vitexin treatment on UCP1 in brown adipose tissue (BAT) and subcutaneous white adipose tissue (iWAT) in mice for 12 weeks. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the accompanying drawings in the embodiments of the present invention.

[0033] The embodiments of the present invention are as follows:

[0034] The experimental cells used were immortalized brown fat cells.

[0035] C57BL / 6J mice were purchased from Shanghai Slake Laboratory Animal Co., Ltd.

[0036] The experimental drug vitexin was purchased from Chengdu Purifa Technology Development Co., Ltd. The molecular structure is attached. Figure 1 As shown in A. The drug was dissolved in DMSO solution to treat cells. A solution was prepared according to the ratio of DMSO / vitexin:PEG300:Tween-80:normal saline = 10:40:5:45 and injected intraperitoneally into mice (5 mg / kg body weight).

[0037] Example 1: In vitro technical solution

[0038] In vitro technical plan: By adding vitexin to mature brown adipocytes, its effect on brown adipocyte thermogenesis was studied.

[0039] The in vitro experiments are as follows:

[0040] 1. Effect of vitexin on thermogenesis of brown adipocytes

[0041] The pre-brown adipocytes were plated into 12-well plates and waited for the cells to grow to 70% density before changing to maintenance medium (insulin 20nM, T3 1μM). The next day, the differentiation medium (insulin 20nM, T3 1nM, 125μMindomethacin, 0.5mM IBMX, 0.5μM Dexamethason) was changed to culture for another 2 days. Then the culture medium was changed to maintenance medium for another 4 days. The entire differentiation process lasted for 8 days. When the cells were differentiated to the 8th day, DMSO, 10, 20, 50, 100, 200μM euphorbia cerifera and CL316,243 (10μM) were added and treated for 12 hours, and cell protein samples were prepared. The expression of UCP1 protein was detected by immunoblotting to determine the effect of different concentrations of euphorbia cerifera on thermogenesis.

[0042] 2. Detection of the effect of vitexin on cellular oxygen consumption

[0043] After the pre-brown adipocytes were differentiated and matured, they were treated with DMSO, 20μM, 50μM, and CL316,243 for 12 hours. After the treatment, the cells were digested and counted, and 10,000 cells were inoculated into each well of the microplate and allowed to adhere. The next day, the original culture medium was aspirated, 180μl of freshly prepared XF culture medium (containing 10mM glucose, 1mM sodium pyruvate, and 2mM glutamine) was added to each well, and the microplate was placed in a 37°C, CO2-free incubator for 1 hour to ensure that the temperature and pH of the culture medium were stable. Subsequently, the Seahorse XF analyzer was turned on, the XF calibration solution was added to the calibration plate and calibrated, which usually takes 20-30 minutes. After the calibration is completed, the calibration plate is replaced with a cell culture microplate, and the experiment is started in the software. The instrument automatically performs the mixing, waiting, and measurement steps.

[0044] Example 2: In vivo experimental protocol

[0045] 1. Construction of mouse obesity model and drug administration

[0046] Six-week-old male C57BL / 6J mice were randomly divided into a normal control group and a high-fat intervention group. The high-fat group was continuously fed with a customized feed with a fat content of 60%. From the start of the experiment, the experimental group received a daily intraperitoneal injection of vitexin (5 mg / kg), while the control group received an equal volume of DMSO for 12 consecutive weeks. During the experiment, the temperature (22±1°C), humidity (50±5%) and 12-hour circadian rhythm of the animal room were strictly monitored, and the weight changes and basal metabolic indicators of the mice were recorded regularly every week.

[0047] 2. Determination of metabolic rate in mice

[0048] After the 12-week intervention, energy metabolism was monitored continuously for 72 hours using a metabolic cage system (Columbus Instruments). Mice were fasted for 12 hours with free access to water before the experiment. Mice were individually placed in metabolic cages equipped with an automated monitoring system, which collected real-time oxygen consumption (VO2), carbon dioxide production (VCO2), locomotor activity, food and water intake, and fecal parameters. Data were collected at 15-minute intervals, and respiratory exchange ratio (RER) and energy expenditure were calculated using Oxymax analysis software. All measurements were performed under a constant light cycle and without external interference.

[0049] 3. Glucose and insulin sensitivity testing

[0050] Glucose tolerance and insulin sensitivity tests were performed one week before the end of the intervention. After a 16-hour fast, basal blood glucose levels were measured by tail vein blood sampling. Following intraperitoneal injection of 2 g / kg glucose solution, blood samples were collected at 15, 30, 60, 90, and 120 minutes for glucose tolerance curve analysis. After a 4-hour fast, an insulin tolerance test was performed three days later. Following the injection of 0.75 U / kg recombinant human insulin, blood glucose levels were measured at 0, 15, 30, 60, and 90 minutes.

[0051] 4. Protein detection in mouse adipose tissue

[0052] Mice were anesthetized and sacrificed, and interscapular brown adipose tissue (BAT) and subcutaneous white adipose tissue (iWAT) were rapidly isolated, snap-frozen in liquid nitrogen, and stored at −80°C. 100 mg of adipose tissue was mechanically homogenized in RIPA lysis buffer (containing protease inhibitors) and centrifuged at 4°C (12,000 g for 15 minutes) to obtain the supernatant. Protein concentration was determined using the BCA assay, and equal amounts of protein were transferred to SDS-PAGE electrophoresis membranes. The membranes were incubated with primary antibodies against UCP1 and HSP90 at 4°C overnight, followed by TBST rinses and incubation with HRP-conjugated secondary antibodies for 1 hour at room temperature. Images were obtained using an ECL imaging system, and grayscale values were quantified using ImageJ software.

[0053] Statistical methods

[0054] Statistical analysis was performed using the t-test in GraphPad Prism version 8.0. Data are presented as mean ± standard deviation (SEM). Differences were considered statistically significant if p-value < 0.05, and extremely significant if p-value < 0.01. All experiments were repeated at least three times.

[0055] Experimental results show

[0056] 1. Vitexin increases heat production and oxygen consumption in brown fat cells

[0057] Figure 1 B shows that with the increase of the concentration of vitexin treatment, the UCP1 protein level was significantly upregulated. When the final concentration reached 50 μM, the induction of UCP1 and oxygen consumption was similar to that of CL316,243 ( Figure 1 B, Figure 2 ).

[0058] Specifically, differentiated mature brown adipocytes were treated with different concentrations of vitexin and simultaneously treated with a known β3 adrenergic receptor agonist - CL316,243 (known to activate the expression of UCP1), and the effects of the two on cellular oxygen consumption were compared.

[0059] 2. Vitexin alleviates obesity induced by high-fat diet

[0060] Six-week-old mice were fed a high-fat diet and intraperitoneally injected with vitexin ( Figure 3 A), the food intake of mice did not change significantly ( Figure 3 B), as time went on, the weight of mice injected with vitexin decreased significantly ( Figure 3 C), after 12 weeks of feeding, the weight of subcutaneous white adipose tissue (iWAT), abdominal white adipose tissue (eWAT), and liver tissue (Liver) of the experimental group mice was significantly reduced ( Figure 3 D), including the significant alleviation of cellular lipid droplet accumulation in brown adipose tissue ( Figure 3 E).

[0061] 3. Vitexin increases overall metabolism in mice

[0062] In the embodiment, the effects of high-fat combined with vitexin on heat production, oxygen consumption and carbon dioxide emissions in mice for 12 weeks were tested, and the results were as follows: Figure 4 As shown in A, B, and C.

[0063] From the metabolic cage data ( Figure 4 ), the heat production, oxygen consumption and carbon dioxide emissions of the experimental group mice increased significantly.

[0064] 4. Vitexin alleviates insulin sensitivity in mice

[0065] Example The effects of high-fat combined with vitexin on glucose sensitivity, insulin sensitivity, and serum insulin levels in mice for 12 weeks were tested. The results are as follows: Figure 5 As shown in A, B, and C.

[0066] The glucose and insulin sensitivity of mice were further tested ( Figure 5 ), the figure shows that the mice in the experimental group showed significant improvement, accompanied by a decrease in serum insulin levels.

[0067] 5. Vitexin increases thermogenesis in adipose tissue

[0068] It is speculated that the improvement of obesity and overall metabolism of mice by vitexin is mainly achieved by mediating thermogenesis of brown fat. Therefore, the expression of UCP1 in brown adipose tissue (BAT) and subcutaneous white adipose tissue (iWAT) was detected, and it was found that both were significantly increased ( Figure 6 ).

[0069] In this specific implementation, the present invention is verified and tested in both in vivo and in vitro:

[0070] In vivo experiments: Vitexin ( Figure 1 ) Increase UCP1 protein levels and cellular oxygen consumption in brown fat cells ( Figure 1-Figure 2 ).

[0071] In vitro experiments: Vitexin combined with high-fat feeding mice ( Figure 3 A), it was observed that the food intake of the two groups of mice did not change ( Figure 3 B). Starting from the 10th week, the mice lost weight significantly. By the 16th week, the weight difference between the mice and the control mice was close to 5g ( Figure 3 C), further dissection revealed that the weight of subcutaneous white fat (iWAT), abdominal white fat (eWAT) and liver of mice was significantly reduced ( Figure 3 D), lipid accumulation in cells was significantly alleviated ( Figure 3 E). And the heat production, oxygen consumption, and carbon dioxide emissions of the experimental group mice ( Figure 4 ) was significantly improved, and insulin sensitivity was alleviated ( Figure 5 ).

[0072] From the results of in vivo and in vitro experiments, it can be seen that vitexin has application prospects in the preparation of drugs for preventing and treating obesity.

[0073] In summary, the in vivo and in vitro experiments in the examples of the present invention show that vitexin increases the heat production of brown fat cells, increases the overall metabolism of mice, and alleviates obesity induced by a high-fat diet.

[0074] The above specific embodiments are used to illustrate the present invention rather than to limit the present invention. Any modifications and changes made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

[0075] The above description is only a preferred embodiment of the present invention. Therefore, any equivalent changes or modifications made according to the structure, characteristics and principles described in the scope of the patent application of the present invention are included in the scope of the patent application of the present invention.

Claims

1. An application of vitexin, characterized in that: The vitexin is used in the preparation of medicines for preventing and treating obesity.

2. The use of vitexin according to claim 1, characterized in that: The prevention and treatment include one or more of the prevention, alleviation or treatment of obesity induced by a high-fat diet.

3. The use of vitexin according to claim 1, characterized in that: The medicine is a solution containing pharmaceutically acceptable excipients.

4. The use of vitexin according to claim 1, characterized in that: The drug is administered by intraperitoneal injection, and the dosage is preferably 5 mg / kg.

5. An application of a medicine containing vitexin, characterized in that: The medicine contains the vitexin component.

6. The use of a medicament containing vitexin according to claim 5, characterized in that: In the medicine, the mass content of vitexin is greater than 99%.

7. The use of a medicament containing vitexin according to claim 5, characterized in that: The medicine is one of a solution, a powder, a sustained-release agent, an emulsion, a suspension, a syrup and drops.

8. The method for using vitexin according to claim 1, characterized in that: After vitexatin was made into a solution, it was injected into the abdominal cavity of mice and fed with high-fat diet to create an obesity model. After the 10th week, the obesity of the animals was alleviated.

9. The method for using vitexin according to claim 1, characterized in that: The drug was injected intraperitoneally into obese animals.

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