Application of Atractylodin

Abstract

The present invention discloses an application of atractylodin, an additive and a pharmaceutical preparation. The application of atractylodin includes its application in promoting the browning of white adipocytes. Through white adipocytes differentiated from white adipose mesenchymal cells isolated in vitro, it is found that atractylodin can effectively promote the browning of white adipocytes, that is, enhance the expression of uncoupling protein 1, so that atractylodin can be used to treat metabolic diseases such as obesity, type 2 diabetes and non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention relates to the field of biological technologies, and particularly to an application of atractylodin. Background Art

[0002] With the development of the national economy, the number of obese people in the country has been on the rise year by year. Obesity has always been one of the important factors threatening human health, and the essence of its occurrence is energy imbalance. Reducing energy intake or enhancing energy consumption can effectively treat obesity.

[0003] Nonalcoholic fatty liver disease (NAFLD) refers to a clinicopathological syndrome characterized by abnormal deposition of liver fat, excluding other clear factors such as alcohol, drugs, and genetic diseases. According to the degree of its lesions and whether it is accompanied by inflammation and fibrosis, it is divided into nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), liver fibrosis or cirrhosis, and may further develop into hepatocellular carcinoma (HCC). In order to more accurately understand the pathogenesis of nonalcoholic fatty liver disease related to overweight, obesity, type 2 diabetes, or multiple metabolic disorders, in 2020, the International Fatty Liver Nomenclature Expert Group reached a consensus to replace NAFLD with metabolic associated fatty liver disease (MAFLD). The incidence of NAFLD has been increasing year by year and has become the world's number one chronic liver disease. At present, the etiology of NAFLD has not been fully clarified, and there is a lack of effective treatment measures in clinical practice. Medical workers are urgently needed to find effective intervention means according to its pathogenesis. Summary of the Invention

[0004] The main object of the present invention is to propose an application, an additive, and a pharmaceutical preparation of atractylodin, aiming to promote the browning of white adipocytes, thereby solving the problem of nonalcoholic fatty liver disease.

[0005] To achieve the above object, the present invention proposes an application of atractylodin in promoting the browning of white adipocytes.

[0006] To achieve the above object, the present invention provides a pharmaceutical preparation, and the pharmaceutical preparation includes atractylodin that promotes the browning of white adipocytes.

[0007] Optionally, the pharmaceutical preparation is used for treating obesity, type 2 diabetes, and nonalcoholic fatty liver disease.

[0008] In the technical solution of the present invention, an application of atractylodin in promoting the browning of white adipocytes is proposed. Atractylodin is a broad-spectrum antiparasitic drug and a broad-spectrum antiviral drug, which is mainly used for anti-inflammatory and diuretic effects in medicine. However, the present invention proposes a new use of atractylodin. Through white adipocytes differentiated from white adipose mesenchymal cells isolated in vitro, it is found that atractylodin can effectively promote the browning of white adipocytes, that is, enhance the expression of uncoupling protein 1, so that atractylodin can be used to treat metabolic diseases such as obesity, type 2 diabetes, and non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the results shown in these drawings.

[0010] Figure 1 For the promotion of the expression of uncoupling protein 1 in white adipocytes in vitro by atractylodin at different concentrations in Example 1 of the present invention;

[0011] Figure 2 Schematic diagram of the resistance to high-fat diet-induced obesity in mice in Example 1 and the control group;

[0012] Figure 3 Comparison chart of fat weights in mice in Example 1 and the control group;

[0013] Figure 4 Schematic diagram of enhancing the browning of white adipocytes in mice in Example 1 and the control group;

[0014] Figure 5 Schematic diagram of improving insulin sensitivity in mice in Example 1 and the control group;

[0015] Figure 6 Schematic diagram of reducing lipid accumulation in the liver in mice in Example 1 and the control group.

[0016] The realization, functional characteristics, and advantages of the object of the present invention will be further described with reference to the embodiments and the drawings. DETAILED DESCRIPTION OF THE INVENTION

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not specified by the manufacturer are all conventional products that can be obtained through commercial purchase. In addition, the meaning of "and / or" appearing throughout the text includes three parallel scenarios. Taking "A and / or B" as an example, it includes Scenario A, or Scenario B, or the scenario where both A and B are satisfied simultaneously. In addition, the technical solutions between various embodiments can be combined with each other, but it must be based on the ability of those of ordinary skill in the art to implement. When the combination of technical solutions results in contradictions or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0018] With the development of the national economy, the incidence of obesity, type 2 diabetes, and non-alcoholic fatty liver disease is increasing day by day. Among them, obesity has always been one of the important factors threatening human health.

[0019] In view of this, the present invention provides an application of atractylodin in promoting the browning of white adipocytes.

[0020] It should be noted that atractylodes rhizome is the dried rhizome of Atractylodes lancea (Thunb.) DC. or Atractylodes chinensis (DC.) Koidz. of the genus Atractylodes in the Compositae family, and has the effects of drying dampness and strengthening the spleen, expelling wind and cold, and improving eyesight. Atractylodes rhizome mainly contains volatile oil, with a content of about 5% - 9%. The main components of the volatile oil are atractylol, β-eudesmol, hinesol, atractylone, atractylodin, etc. As one of the active ingredients of atractylodes rhizome, atractylodin has great clinical potential and pharmacological activities such as promoting gastric emptying, anti-inflammatory, anti-tumor, and diuretic effects.

[0021] It should be noted in advance that classical adipocytes are divided into two types: white adipocytes and brown adipocytes. Inside white adipocytes, there is a huge round lipid droplet, and the nucleus is flat and located at the edge of the cell. The lipid droplet occupies almost 99% of the cell space. White adipocytes mainly store energy in the lipid droplet in the form of triglycerides and cholesterol. The nucleus of brown adipocytes is round, and the lipid droplets in the cells are distributed in multiple compartments throughout the cell. Brown adipocytes contain a large amount of cytoplasmic matrix and have a high content of mitochondria in the cells. The main characteristic that brown adipocytes are different from white adipocytes is that the expression level of uncoupling protein 1 (UCP1) on the inner mitochondrial membrane is very high. UCP1 can convert the proton motive force generated by the oxidative respiratory chain into heat without generating ATP. The non-shivering thermogenesis function of brown adipocytes can maintain a constant body temperature, enhance the body's energy consumption, and reduce the occurrence of obesity. Mice transplanted with brown adipose tissue can effectively resist obesity and insulin resistance induced by a high-fat diet.

[0022] In recent years, studies have found that under freezing or other stimulating conditions, some cells similar to brown adipocytes with high expression of UCP1 and more mitochondria appear in white adipose tissue. Such cells are called beige adipocytes, and this process is called the browning of white adipocytes. The most important thing in the process of white adipocyte browning is the significant increase in the expression level of UCP1. Beige adipocytes also exist in some parts such as the deep neck of adults, and the number of human beige adipocytes and brown adipocytes is negatively correlated with body weight. A large number of experiments have proved that enhancing the expression of UCP1 in the body can effectively resist obesity, non-alcoholic fatty liver disease, and type 2 diabetes.

[0023] Atractylodin can promote the browning of white adipocytes through white adipose mesenchymal cells differentiated from in vitro isolated white adipocytes. Through detection, it is found that atractylodin can be used to treat obesity, non-alcoholic fatty liver, or type 2 diabetes.

[0024] In some embodiments, atractylodin can prevent the occurrence of some diseases and has an improvement effect on certain diseases. Atractylodin has the effect of treating obesity, non-alcoholic fatty liver, or type 2 diabetes.

[0025] The present invention also provides a pharmaceutical preparation, and the pharmaceutical preparation includes atractylodin that promotes the browning of white adipocytes.

[0026] Furthermore, the pharmaceutical preparation can be used to treat metabolic diseases such as obesity, type 2 diabetes, and non-alcoholic fatty liver.

[0027] The technical solutions of the present invention will be further described in detail below in combination with specific embodiments and the accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0028] Example 1: Atractylodin promotes the browning of white adipocytes in a dose-dependent manner in vitro

[0029] I. Isolation of primary adipose mesenchymal cells from inguinal white fat

[0030] 1) A male mouse at 8 weeks old with a C57BL6 background was sacrificed by cervical dislocation, soaked in 75% alcohol for 5 minutes, then transferred to the operating table, and the inguinal white adipose tissue was taken out;

[0031] 2) It was washed 3 times with PBS, the adipose tissue was minced with scissors, 1 g of adipose tissue was added to 10 ml of collagenase I solution (prepared with D-Hanks solution, 0.1 g of collagenase I was added to 100 ml), and digested at 37 °C for 40 minutes;

[0032] 3) It was filtered through a 250 µm filter membrane, cell culture medium was added, then transferred to a centrifuge tube, and centrifuged at 1000 rpm for 3 minutes;

[0033] 4) The cells at the bottom after the first centrifugation were adipose mesenchymal cells. After discarding the supernatant, the adipose mesenchymal cells were suspended with fresh culture medium, plated onto a culture plate, and the medium was changed the next day.

[0034] 6) The obtained adipose mesenchymal cells grew to confluence in a culture medium supplemented with 10% fetal bovine serum (Hyclone) and high-glucose DMEM (Hyclone);

[0035] 7) The culture medium was changed to an induction medium supplemented with MDIR (0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, 87 nM insulin, and 0.5 μM rosiglitazone: Isobutylmethylxanthine + Dexamethasone + Insulin + Rosiglitazone) (a culture medium supplemented with 10% fetal bovine serum and high-glucose DMEM), and cultured for 2 days.

[0036] 8) After 2 days, the culture medium was changed to an induction medium supplemented only with IR (87 nM insulin and 0.5 μM rosiglitazone: Insulin + Rosiglitazone) (a culture medium supplemented with 10% fetal bovine serum and high-glucose DMEM), and changed every two days until the cells were completely differentiated into adipocytes.

[0037] 9) The culture medium was changed to a normal medium (a culture medium supplemented with 10% fetal bovine serum and high-glucose DMEM), and at the same time, 4 different concentrations of atractylodin (1, 3, 10, and 30 μM) were added to stimulate the differentiated and mature adipocytes for 24 hours. After 24 hours, the cells were lysed for subsequent qPCR analysis of gene expression in the cells.

[0038] II. Gene Expression Identification of Inguinal Fat Cells

[0039] RNA extraction: RNA was extracted using the Trizol method. The specific operations are as follows:

[0040] 1) Take out the atractylenolide-stimulated fat cells from step (I), remove the culture medium, wash once with PBS, then add 1 ml of Trizol, lyse for 30 minutes, and then transfer to an EP tube;

[0041] 2) Add 200 μl of chloroform / 1 ml of Trizol to the EP tube, shake vigorously for 15 seconds, let stand at room temperature for 3 minutes, and centrifuge at 4 °C, 12,000 g for 15 minutes;

[0042] 3) The solution is divided into three layers: a colorless upper aqueous phase, a middle white layer, and a pink lower organic phase. Carefully aspirate the upper aqueous phase into a new centrifuge tube;

[0043] 4) Add 500 μl of isopropanol, mix gently, let stand at room temperature for 10 minutes, and centrifuge at 4 °C, 12,000 g for 10 minutes;

[0044] 5) Carefully remove the supernatant, add 1 ml of 75% ethanol (prepared with DEPC water), mix gently to suspend the precipitate, and centrifuge at 4 °C, 7500 g for 5 minutes;

[0045] 6) Remove the supernatant, air-dry naturally for about 5 minutes, and add 40 μl of DEPC water to dissolve the RNA precipitate;

[0046] 7) Take 5 μl for electrophoresis to detect the quality of the extracted RNA. RNA with a 28S band brightness twice that of the 18S band is of better quality. The gel and electrophoresis buffer need to be freshly prepared;

[0047] 8) Take 2 μl to measure the RNA concentration. An OD 260 nm / OD 280 nm between 1.8 and 2.0 indicates a very high purity of the extracted RNA.

[0048] RNA reverse transcription: cDNA was synthesized using the SuperScriptTM II RT reverse transcription kit;

[0049] Fluorescence real-time quantitative PCR: The SYBR® RT-PCR Kit (Perfect Real Time) quantitative PCR kit from Takara was used to perform the quantitative PCR reaction according to the instructions.

[0050] Reaction conditions:

[0051] Pre-denaturation: 95 °C for 20 s; Denaturation: 95 °C for 10 s; Annealing: 60 °C for 20 s; Incubation: 70 °C for 1 s;

[0052] Repeat 39 cycles according to the gene expression level. Make a melting curve at 65 - 95 °C, read the plate every 0.5 °C, with a time of 1 s, and measure the qPCR results in real-time quantification.

[0053] The experimental results show that: Figure 1 It can be seen that as the concentration of atractylodin increases, the expression level of Ucp1 in adipocytes also increases, indicating that the expression of Ucp1 is dose-dependent on atractylodin.

[0054] Experimental Example

[0055] This example will verify the direct effect of atractylodin on enhancing the expression of uncoupling protein 1 in organisms. The operation method is as follows:

[0056] After feeding 12-week-old mice with high-fat diet for 7 weeks while gavage with atractylodin, detect the body weight, adipose tissue weight, browning of inguinal white adipose tissue in vivo, and glucose tolerance in vivo of the mice.

[0057] Experimental Example 1 Mouse Body Weight

[0058] Male C57BL6-background mice at 8 weeks old (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were fed with high-fat diet (High fat diets, HFD; 60% fat, D12492, Research Diets) for 4 weeks. After the body weight reached about 30 g, they were gavaged with 200 mg / kg of atractylodin, with the solvent being normal saline, and the control group was gavaged with normal saline. At the same time, the mice continued to be fed with high-fat diet. The body weight of the mice was measured once a week.

[0059] The experimental results are as Figure 2 shown: The body weight of the control group mice without gavage of atractylodin continued to increase in the following weeks, while on the contrary, the body weight of the mice gavaged with atractylodin only increased slightly, indicating that atractylodin can resist the occurrence of obesity in vivo.

[0060] Experimental Example 2 Mouse Tissue Weight

[0061] Same as in Experimental Example 1, the control group was gavaged with normal saline for mice, and the experimental group was gavaged with 200 mg / kg of atractylodin for mice. After the gavage, the mice were sacrificed by cervical dislocation, and the white adipose tissue (IngWAT, inguinal adipose tissue) and epididymal white adipose tissue (EpiWAT, epidydymal adipose tissue) at the inguinal region were separated and weighed respectively.

[0062] The detection results are as follows Figure 3 shown: The weight of white adipose tissue at the groin of mice in the control group was about 0.5 g, and the weight of white adipose tissue at the epididymis was about 0.9 g. While in the experimental group, after intragastric administration of atractylodin, the weight of white adipose tissue at the groin of mice was about 0.3 g, and the weight of white adipose tissue at the epididymis was about 0.6 g. This indicates that atractylodin can reduce the weight of adipose tissue.

[0063] Experimental Example 4 Identification of Gene Expression in Adipose Tissue

[0064] RNA extraction: It was extracted using the Trizol method, and the specific operation is as follows:

[0065] Same as in Experimental Example 1, in the control group, mice were intragastrically administered normal saline, and in the experimental group, mice were intragastrically administered 200 mg / kg of atractylodin. After the intragastric administration was completed, the mice were sacrificed by cervical dislocation. The white adipose tissue at the groin of the mice was separated respectively, and 100 mg was weighed respectively, then 1 ml of Trizol was added respectively, ground for 30 minutes, and then transferred to an EP tube; the remaining steps were the same as those for the identification of gene expression in the inguinal fat cells above.

[0066] The detection results are as follows Figure 4 shown: The relative mRNA expression levels of Ucp1, Tbx1, Prdm16, Cidea, and Dio2 in the mice of the control group were all 1, while the relative mRNA expression levels of Ucp1, Tbx1, Prdm16, Cidea, and Dio2 in the mice of the experimental group were approximately 4.2, 2.4, 1.9, 2.4, and 3 respectively. This indicates that atractylodin can promote the browning of white adipocytes in vivo.

[0067] Experimental Example 5 Glucose Tolerance Test

[0068] Same as in Experimental Example 1, the difference is that this experiment started from the 6th week after the intragastric administration of mice. A 20% glucose solution was prepared with normal saline, and then mice were injected at a dose of 2 g of glucose / kg body weight. The mice that had been fasted for 12 hours (from 9 pm to 9 am the next day) were intraperitoneally injected. The blood glucose concentrations of the mice were monitored with a blood glucose meter before injection (0 minute) and at different time points after injection (15 minutes, 30 minutes, 60 minutes, 120 minutes), and the time error was controlled within 5 seconds.

[0069] The detection results are as follows Figure 5As shown: The trend lines of the control group and the group added with atractylodin are similar, but the overall blood glucose concentration of the control group is higher than that of the atractylodin group. At the same time, around 30 minutes, the blood glucose concentration of the mice in the control group reached about 25 mM, while that of atractylodin was about 18 mM. This indicates that atractylodin can improve insulin sensitivity in vivo and reduce blood glucose concentration, and thus can be applied to the treatment of type 2 diabetes.

[0070] Experimental Example 6 Oil Red staining of the liver

[0071] Same as in Experimental Example 1, except that: After the gavage of the mice was completed, the liver tissue was separated and stained with Oil Red O. The change in lipid accumulation in the liver was reflected by observing the amount of Oil Red O under an optical microscope.

[0072] The test results are as Figure 6 shown: The lipid accumulation in the liver of the control group is more than that of the experimental group. This indicates that atractylodin can reduce lipid accumulation in the liver, and thus can be applied to the treatment of metabolic diseases such as non-alcoholic fatty liver.

[0073] Conclusion

[0074] In summary, the present invention proposes a new use of atractylodin. Through white adipocytes differentiated from white adipose mesenchymal cells isolated in vitro, it is found that atractylodin can effectively promote the browning of white adipocytes, that is, enhance the expression of uncoupling protein 1, so that atractylodin can be used to treat metabolic diseases such as obesity, type 2 diabetes and non-alcoholic fatty liver.

[0075] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. Application of atractylodin as a monomer in the preparation of a preparation for treating related diseases that promote browning of white adipocytes, wherein, the related disease is non-alcoholic fatty liver disease.

2. The application according to claim 1, characterized in that, the atractylodin treats and / or prevents non-alcoholic fatty liver disease by enhancing the expression of UCP1.

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