Application of Zhongwinning in preparation of medicine for treating or improving obesity

Abstract

The invention belongs to the technical field of new application of medicines, and provides application of Zhongwinning in preparation of medicines for treating or improving obesity. Experimental research proves that after Zhongwu Ning is adopted to treat zebrafish fed with high-fat feed, the survival rate of the zebrafish can be increased, the body weight and the body length of the zebrafish can be reduced, the TC content, the TG content and the LDL-c content can be reduced, and meanwhile the HDL-c content can be increased; the Zhongwinning can significantly reduce the blood sugar level in serum of obese mice, and significantly reduce the levels of serum inflammatory factors such as TNF-alpha, IL-1alpha, IL-1beta, IFN-beta and the like. The Zhongwinning has the application prospect of preparing the medicine for treating obesity or improving obesity.

Description

Technical Field

[0001] The invention belongs to the technical field of new drug applications, and in particular relates to the application of Zhongwuning in preparing drugs for treating or improving obesity. Background Art

[0002] Obesity is a chronic lipid metabolism disease caused by the accumulation of body fat, resulting in overweight and obesity due to multiple factors such as genetics and environment. When the body's nutrient storage system is exposed to excess energy for a long time, the body's energy storage is greater than the effective utilization of energy, leading to obesity. Obesity is closely related to many chronic diseases.

[0003] Obesity is now a global public health problem, and it is becoming younger. The proportion of overweight / obese adults in China has exceeded 50%, and by 2030, the incidence of overweight / obesity among Chinese adults will reach 65.3%.

[0004] Obesity has become the number one chronic disease threatening human health. It has many causes, including abnormal energy balance and weight regulation, genetic factors, environmental factors, endocrine regulation disorders, intestinal flora disorders, and inflammation. In addition, obesity can be accompanied or complicated by hypertension, diabetes, obstructive sleep apnea syndrome, gallbladder disease, hyperuricemia, gout, osteoarthritis, non-alcoholic fatty liver, cardiovascular and cerebrovascular diseases, neurodegenerative diseases, and tumors. Chronic low-grade inflammatory state is considered to be an important mechanism for the occurrence of obesity and related complications. Reduction or inhibition of inflammation helps to improve insulin resistance and metabolic function.

[0005] At present, the commonly used Western medicines for the treatment of obesity include: orlistat, chitosan, thyroid tablets, etc. However, the above drugs have significant side effects. For example, orlistat is prone to gastrointestinal diarrhea; thyroid tablets can cause adverse reactions such as palpitations, insomnia, and tachycardia. In addition, Western medicine treatment is very easy to produce drug resistance, which poses a potential risk to the health of patients. Traditional Chinese medicine used to treat obesity often contains cathartic drugs such as rhubarb and senna leaves, which can easily lead to intestinal dysfunction if taken for a long time.

[0006] Mesaconine (CAS NO.: 6792-09-2; molecular formula: C 24 H 39 NO9) is a new type of alkaloid with medicinal value. It has a clear chemical structure and can be obtained by raw material extraction or chemical synthesis. Zhongwu Ning has a significant effect on improving cardiac function and can effectively prevent and treat or improve acute heart failure caused by myocardial ischemia-reperfusion injury. However, there is no report on the treatment or improvement of obesity by Zhongwu Ning. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide the application of Zhongwu Ning in the preparation of drugs for treating or improving obesity. Studies have shown that the use of Zhongwu Ning to treat zebrafish fed with high-fat diets can improve the survival rate of zebrafish, reduce the weight and body length of zebrafish, and reduce the content of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-c) and high-density lipoprotein cholesterol (HDL-c); Zhongwu Ning can significantly reduce the blood sugar level in the serum of obese mice, and significantly reduce the levels of serum inflammatory factors such as TNF-α, IL-1α, IL-1β, IFN-β, etc. Zhongwu Ning has an application prospect for preparing drugs for treating obesity or improving obesity.

[0008] In order to achieve the above object, the solution adopted by the present invention is:

[0009] Application of Zhongwuning or its pharmaceutically acceptable salt in preparing medicine for treating or improving obesity.

[0010] Furthermore, in a preferred embodiment of the present invention, the obesity drug is a preparation prepared with Zhongwuning or a pharmaceutically acceptable salt thereof as an active ingredient and adjuvants or auxiliary ingredients commonly used in pharmacy.

[0011] Furthermore, in a preferred embodiment of the present invention, the pharmaceutically acceptable salt is an inorganic salt or an organic salt.

[0012] Furthermore, in a preferred embodiment of the present invention, the dosage form of the preparation includes a liquid preparation, a solid preparation or a semisolid preparation.

[0013] The beneficial effects of the application of Zhongwuning provided by the present invention in preparing drugs for treating or improving obesity are:

[0014] (1) This application first discovered the use of Zhongwu Ning in the preparation of drugs for treating or improving obesity. Studies have shown that after treating zebrafish fed with high-fat diets, Zhongwu Ning can increase the survival rate of zebrafish, reduce the weight and length of zebrafish, and reduce the content of TC, TG, and LDL-c while increasing the content of HDL-c, thereby significantly improving the lipid metabolism of hyperlipidemia zebrafish.

[0015] (2) This application confirms through research that Zhongwu Ning can improve obesity-related glucose and lipid metabolism disorders, significantly reduce the blood sugar level in the serum of obese mice, and significantly reduce the levels of serum inflammatory factors such as TNF-α, IL-1α, IL-1β, and IFN-β. The above test results show that Zhongwu Ning has the application prospect of preparing drugs for treating obesity or improving obesity. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0017] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0018] Example 1

[0019] 1. Experimental animals: Wild-type AB strain zebrafish (provided by the Zebrafish Laboratory of the School of Pharmacy, Chengdu University of Traditional Chinese Medicine). Cultivated in water temperature of 28℃, pH value of 7.4, and conductivity of 400-600μs / cm. Feed twice a day, irradiate with fluorescent light for 14h, and keep in dark environment for 10h. The zebrafish fry required for the experiment were selected from healthy and mature male and female zebrafish in the afternoon of the day before the experiment, and moved into the spawning box in the ratio of male to female = 1:1-2. The male and female zebrafish were separated by a transparent partition. The partition was removed at 9 o'clock the next day, and the embryos were collected after the male fish chased the female fish for 1h. The collected embryos were cleaned with culture medium and moved into a sterile culture dish, placed in a constant temperature incubator at 28℃, and clean water was changed every day. After the fertilized eggs were incubated to 5dpf (days postfertilization), healthy individuals were selected for subsequent experiments.

[0020] 2. Experimental methods

[0021] 2.1 Animal grouping and drug administration

[0022] When the embryos developed to 20 hpf and throughout the experimental period thereafter, 0.03 g / L of 1-phenyl2-thiourea (PTU) was added to the feeding water. Fresh PTU culture water was replaced regularly every day and dead eggs and egg shells were removed in time. When the Tg fertilized eggs were incubated to 5 dpf, healthy individuals were selected and randomly divided into a common feed group, a model group, and a Zhongwuning group. They were raised in a 6-well plate, with 50 fry in each well and 3 replicate wells in each group.

[0023] Preparation of high cholesterol feed: immerse ordinary zebrafish fry feed in anhydrous ether dissolved with a certain amount of cholesterol, stir thoroughly until the ether is completely volatilized, so that the cholesterol is attached to the ordinary feed, and prepare a high cholesterol feed containing 4% (w / w).

[0024] Normal control group: fed with ordinary feed.

[0025] Model group: fed with high cholesterol feed.

[0026] Zhongwu Ning group: The mice were fed with high cholesterol feed and 2 μg / mL of Zhongwu Ning was added every morning.

[0027] Each group of zebrafish was fed with a fixed amount of feed twice a day, and the water was changed once at regular intervals for 10 consecutive days.

[0028] 2.2 Measurement indicators and methods

[0029] 2.2.1 Survival rate measurement: 10 days after modeling and drug administration, the number of surviving zebrafish in each group was observed and recorded, and the percentage was calculated.

[0030] 2.2.2 Measurement of body weight and length: The juveniles were anesthetized with tricaine solution, and the surface water of the juveniles was dried with absorbent paper. The weight of each group of zebrafish was weighed and recorded on an analytical balance. The distance from the front end of the juvenile head to the end of the tail was measured with a vernier caliper, and this length represented the body length of the juveniles.

[0031] 2.2.3 Determination of biochemical indicators of lipid metabolism in zebrafish larvae

[0032] At the termination stage of the experiment, 30 zebrafish in each group were exposed to a high concentration of tricaine solution for a long time until euthanasia, and the undigested food in the animal's stomach was removed. The zebrafish larvae were mechanically homogenized in an ice-water bath at a ratio of body weight (g): volume (mL) = 1:9, and centrifuged at 2500rpm for 10 minutes. The supernatant was aspirated and the concentrations of TC, TG, LDL-c and HDL-c in the larvae were measured according to the instructions of the TC, TG, LDL-c and HDL-c kits.

[0033] 2.3 Statistical methods: The experimental data were analyzed using SPSS 21.0 statistical software. The data results were expressed as mean ± standard deviation (x ± s). One-way ANOVA was used for analysis, and the differences between the groups were compared using t-test. P < 0.05 was considered statistically significant.

[0034] 3. Experimental results

[0035] (1) Table 1 shows the survival rate of zebrafish in each group:

[0036] Table 1 Survival rate of zebrafish in each group

[0037]

[0038]

[0039] Note: Compared with the normal control group, ## P<0.01; compared with the model group, ** P<0.01.

[0040] The data in Table 1 show that the growth and death of zebrafish fry in different groups were analyzed. It can be seen that the normal control group had normal growth, normal body pigmentation, and basically no death; the number of dead fish in the model group increased significantly; a small number of zebrafish fry in the Zhongwu Ning group died, and the mortality rate was significantly lower than that in the model group. Compared with the normal control group, the survival rate of fry in the model group was extremely significantly reduced (P < 0.01); compared with the model group, the survival rate of fry in the Zhongwu Ning group was significantly increased (P < 0.01). This shows that the use of Zhongwu Ning to treat zebrafish fed with high-fat diet can improve the survival rate of zebrafish.

[0041] (2) Table 2 shows the changes in body weight and length of zebrafish fry in each group after 10 days of feeding:

[0042] Table 2 Changes in body weight and length of zebrafish fry in each group after 10 days of feeding

[0043] Group No. Number of items Weight(mg) Body length (cm) Normal control group 30 articles 19.68±0.45 10.62±0.25 Model Group 30 articles <![CDATA[25.12±0.28 ## ]]> <![CDATA[12.88±0.38 ## ]]> Zhongwuning Group 30 articles <![CDATA[21.52±0.34 ** ]]> <![CDATA[11.21±0.14 ** ]]>

[0044] Note: Compared with the normal control group, ## P<0.01; compared with the model group, ** P<0.01.

[0045] The data in Table 2 show that the weight and length of zebrafish fry in different groups after feeding for 10 days were analyzed. It can be seen that the weight and length of the normal control group grew normally; compared with the normal control group, the weight and length of the fry in the model group increased significantly (P<0.01); compared with the model group, the weight and length of the fry in the Zhongwu Ning group were significantly reduced (P<0.01). This shows that the use of Zhongwu Ning to treat zebrafish fed with high-fat diet can significantly reduce the weight and length of high-fat zebrafish.

[0046] (3) Table 3 shows the TC, TG, LDL-c and HDL-c of each group of zebrafish:

[0047] Table 3 Concentrations of TC, TG, LDL-c and HDL-c in each group of zebrafish

[0048] Group No. Number of items TC (mmol / gprot) TG (mmol / gprot) LDL-c (mmol / gprot) HDL-c (mmol / gprot) Normal control group 30 articles 0.2552±0.0224 1.6842±0.0345 0.0589±0.0145 0.0148±0.0025 Model Group 30 articles <![CDATA[0.5642±0.0186 ## ]]> <![CDATA[5.4236±0.8225 ## ]]> <![CDATA[0.1344±0.0026 ## ]]> <![CDATA[0.0024±0.0005 ## ]]> Zhongwuning Group 30 articles <![CDATA[0.3282±0.0105 ** ]]> <![CDATA[2.1224±0.9242 ** ]]> <![CDATA[0.0733±0.0134 ** ]]> <![CDATA[0.0115±0.0114 ** ]]>

[0049] Note: Compared with the normal control group, ## P<0.01; compared with the model group, ** P<0.01.

[0050] As shown in Table 3, compared with the normal control group, the levels of TC, TG and LDL-c in the model group were significantly increased (P < 0.01), while the level of HDL-c was significantly decreased (P < 0.01). Compared with the model group, the levels of TC, TG and LDL-c in the Zhongwuning group were significantly decreased, while the level of HDL-c was significantly increased (P < 0.01). The results in Table 3 show that Zhongwuning has a certain effect of lowering blood lipids and cholesterol.

[0051] The above experiment in Example 1 shows that after treating zebrafish fed with high-fat diet with Zhonguning, the survival rate of zebrafish can be increased, the weight and length of zebrafish can be reduced, and the TC, TG, and LDL-c contents can be reduced while the HDL-c content can be increased, thereby significantly improving the lipid metabolism of zebrafish with hyperlipidemia.

[0052] Example 2

[0053] 1. Experimental animals: 10 8-week-old C57BL / 6 male mice, SPF grade; 30 8-week-old ob / ob obese mice, male, SPF grade, provided by Chengdu Dashuo Biotechnology Co., Ltd., production license number: SCXK (Chuan) 2015-030. Rats were allowed to eat and drink freely in an artificial 12-h day / night cycle lighting environment at room temperature of 20-24°C, relative humidity of 50-60%.

[0054] 2. Experimental methods

[0055] 2.1 Animal grouping and drug administration

[0056] After 1 week of adaptive feeding of mice, C57BL / 6 mice were set as blank control group, and ob / ob obesity model mice were randomly divided into three groups: (1) model group; (2) positive group: 500 mg / kg body weight metformin was given by gavage once a day; (3) Zhongwuning group: 6.7 mg / kg body weight Zhongwuning was given by gavage once a day. All mice were fed with normal feed.

[0057] The mice were administered the drug for 8 consecutive weeks. Body weight and food intake were measured weekly. At 4 weeks after administration, about 50 μL of blood was collected from the medial canthal vein to detect changes in blood lipids.

[0058] 2.2 Measurement indicators and methods

[0059] 2.2.1 Glucose level and glucose tolerance test:

[0060] After 8 weeks of administration, the mice were fasted for 12 hours, 0.5 ml of blood was collected from the medial canthal vein, the blood was allowed to stand for 30 minutes, and centrifuged at 2000 g for 10 minutes. As much supernatant as possible was aspirated, and the glucose level was tested according to the instructions of the Biosino Lipid Test Kit.

[0061] 2.2.2 Detection of serum inflammatory factors: After 8 weeks of administration, blood was collected from the inner canthus of the eye after fasting for 12 hours. The blood sample was kept at room temperature for more than 30 minutes, and then centrifuged at 2000xg for 10 minutes at 4°C to collect serum. The serum was packaged and stored at -80°C for later use. According to the instructions of the kit, the level of serum inflammatory factors was detected by flow cytometry, and the level of serum inflammatory factors reflected the level of inflammation in mice.

[0062] 2.3 Statistical methods: The experimental data were compared with the two-tailed Student's t-test. The data results were expressed as mean ± standard deviation (x ± s). One-way ANOVA and Tukey's post-hoc test were used to compare and calculate the data of more than two groups. Kruskal-Wallis sum was used to compare the differences between groups of non-normally distributed data. - Rank test and Wilcoxon rank-sum test were used. P<0.05 was considered statistically significant.

[0063] 3. Experimental results

[0064] (1) Table 4 shows the serum glucose levels in each group of mice after 8 weeks of administration

[0065] Table 4 Serum glucose levels in mice in each group after 8 weeks of administration

[0066] Group No. Number Serum glucose level (mmol / L) Blank control group 10 24.12±0.24 Model Group 10 <![CDATA[48.28±0.36 ## ]]> Positive group 10 29.32±0.44 Zhongwuning Group 10 32.36±0.32

[0067] Note: Compared with the normal control group, ## P<0.01; compared with the model group, ** P<0.01.

[0068] The results in Table 4 show that after 8 weeks of administration, the glucose content of mice in the model group was significantly increased compared with the blank control group; compared with the model group, the glucose content was significantly decreased after treatment with the positive drug metformin (P < 0.01), and the glucose content was also significantly decreased after treatment with Zhongwuning (P < 0.01).

[0069] Table 5 shows the serum inflammatory factor levels of mice in each group.

[0070] Table 5 Serum inflammatory factor levels of mice in each group

[0071] Group No. IL-1α(pg / m1) TNF-a (pg / m1) IL-1β (pg / ml) IFN-β (pg / ml) Blank control group 6.25±0.38 15.32±0.84 11.36±0.67 35.88±1.45 Model Group <![CDATA[25.02±0.44 ## ]]> <![CDATA[78.62±0.66 ## ]]> <![CDATA[38.55±0.82 ## ]]> <![CDATA[442.28±3.23 ## ]]> Positive group 8.16±0.33 20.31±0.46 16.74±0.31 40.21±1.22 Zhongwuning Group 9.28±0.21 22.63±0.74 17.66±0.28 68.23±2.34

[0072] Note: Compared with the normal control group, ## P<0.01; compared with the model group, ** P<0.01.

[0073] The results in Table 5 show that after 8 weeks of drug administration, the serum concentrations of IL-1α, TNF-α, IL-1β and IFN-β serum inflammatory factors in the model group mice were significantly increased compared with the blank control group (P < 0.01); compared with the model group, after treatment with the positive drug metformin, the serum concentrations of IL-1α, TNF-α, IL-1β and IFN-β serum inflammatory factors in the mice were significantly reduced (P < 0.01), and after treatment with Zhongwu Ning, the serum concentrations of IL-1α, TNF-α, IL-1β and IFN-β serum inflammatory factors in the mice were also significantly reduced (P < 0.01). This shows that Zhongwu Ning can reduce the levels of serum inflammatory factors such as TNF-α, IL-1α, IL-1β, IFN-β in the serum of obese mice

[0074] In summary, the study confirmed that after treating zebrafish fed with high-fat diet, Zhongwu Ning can improve the survival rate of zebrafish, reduce the weight and length of zebrafish, and reduce the content of TC, TG, LDL-c while increasing the content of HDL-c; Zhongwu Ning can significantly reduce the blood glucose level in the serum of obese mice, and significantly reduce the levels of serum inflammatory factors such as TNF-α, IL-1α, IL-1β, IFN-β, etc. Zhongwu Ning has the application prospect of preparing drugs for treating obesity or improving obesity.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of Zhongwuning or its pharmaceutically acceptable salt in preparing a drug for treating or improving obesity.

2. The use according to claim 1, characterized in that: The obesity medicine is a preparation prepared by taking Zhongwuning or its pharmaceutically acceptable salt as active ingredient and adding auxiliary materials or auxiliary ingredients commonly used in pharmacy.

3. The use according to claim 2, characterized in that: The pharmaceutically acceptable salt is an inorganic salt or an organic salt.

4. The use according to claim 2, characterized in that: The dosage form of the preparation includes a liquid preparation, a solid preparation or a semisolid preparation.

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