阅读说明：本技术 共轭亚油酸异构体在用于制备降低内脏脂肪含量的药物和保健品中的应用 (Application of conjugated linoleic acid isomer in preparation of medicines and health products for reducing visceral fat content ) 是由 缪锦来 曲长凤 刘丽娜 于 2020-12-21 设计创作，主要内容包括：本发明公开了共轭亚油酸异构体在用于制备降低内脏脂肪含量的药物和保健品中的应用。本发明以肥胖小鼠、糖尿病小鼠和高糖高脂饲养小鼠为研究对象,采用生化指标监测、qRT-PCR、组织学切片染色等方法检测其对脂肪代谢作用。经实验证明共轭亚油酸异构体明显促进肝脏内脂肪分解代谢体脂,降低脂肪合成和组织蓄积相关因子表达,从而有效降低动物肝脏内脂肪含量,将该单体应用于制备降脂的药物或保健品,对人体健康具有重要意义,具有良好的市场应用前景。(The invention discloses an application of conjugated linoleic acid isomer in preparing medicaments and health-care products for reducing visceral fat content. The invention takes fat mice, diabetic mice and high-sugar and high-fat fed mice as research objects, and adopts methods of biochemical index monitoring, qRT-PCR, histological section staining and the like to detect the effect of the fat metabolism. Experiments prove that the conjugated linoleic acid isomer obviously promotes fat catabolism body fat in the liver and reduces fat synthesis and tissue accumulation related factor expression, so that the fat content in the animal liver is effectively reduced.)

1. The conjugated linoleic acid isomer is applied to the preparation of medicines and health care products for reducing the visceral fat content.

2. The use of isomers of conjugated linoleic acid in the manufacture of medicaments and nutraceuticals for the reduction of visceral fat content according to claim 1, characterized in that: the conjugated linoleic acid isomers are c9, t11-CLA, t10 and c12-CLA, and the chemical structural formulas are respectively shown as formula 1 and formula 2:

3. use of isomers of conjugated linoleic acid in the manufacture of medicaments and nutraceuticals for the reduction of visceral fat content according to claim 2, characterized in that: the conjugated linoleic acid isomer promotes the expression of LPL gene and UCP1 gene in adipose tissues and inhibits the expression of FAS gene and ACC1 gene.

4. Use of isomers of conjugated linoleic acid in the manufacture of medicaments and nutraceuticals for the reduction of visceral fat content according to claim 2, characterized in that: the conjugated linoleic acid isomer promotes the expression of PKA, Adiponectin and LPL genes in the liver and inhibits the expression of FAS genes.

5. Use of isomers of conjugated linoleic acid in the manufacture of medicaments and nutraceuticals for the reduction of visceral fat content according to claim 2, characterized in that: the conjugated linoleic acid isomer can reduce lipid accumulation of internal organs or adipose tissues, reduce fatty acid synthesis, and promote lipolysis and fatty acid beta oxidation.

6. Use of isomers of conjugated linoleic acid in the manufacture of medicaments and nutraceuticals for the reduction of visceral fat content according to claim 2, characterized in that: the effective dosage of the conjugated linoleic acid isomer is 100-400 mg/Kg/d.

7. Use of isomers of conjugated linoleic acid in the manufacture of medicaments and nutraceuticals for the reduction of visceral fat content according to claim 2, characterized in that: the conjugated linoleic acid isomer has obvious feeding inhibition effect on obese mice and reduces the appetite of the mice.

8. The use of isomers of conjugated linoleic acid in the manufacture of medicaments and nutraceuticals for the reduction of visceral fat content according to claim 6, characterized in that: the conjugated linoleic acid isomer is dose-dependent on the weight reducing effect of obese mice.

9. The use of isomers of conjugated linoleic acid in the manufacture of medicaments and nutraceuticals for the reduction of visceral fat content according to claim 1, characterized in that: the viscera are liver, kidney, pancreas, stomach and spleen.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of conjugated linoleic acid isomers in preparation of medicines and health-care products for reducing visceral fat content.

Background

Conjugated Linoleic Acid (Conjugated Linoleic Acid CLA) is a fatty Acid with high physiological activity, and is a general name of positional isomers and spatial isomers of Linoleic Acid (LA). CLA is abundant in isomer type, and the existence of up to 25 isomers has been found in nature and chemical synthesis preparations, wherein the researches on isomers c9, t11-CLA and t10, c12-CLA are most concentrated, the main function of c9, t11-CLA isomer is cancer resistance, t10, c12-CLA are body fat reduction, blood fat reduction and the like, and the unique physiological function of CLA makes CLA become a star nutrient which is researched in recent years.

In recent years, nutritional diseases related to obesity and the like have become a major public health problem endangering human health in developed and developing countries. At present, the obesity population in China is in a straight-line rising trend, the latest data of the lancets show that the average obesity rate in China reaches 12%, and the obesity population exceeds the first position in the United states. Excessive accumulation of fat in liver can easily induce hepatitis, liver cell swelling, inflammatory cell infiltration, and normal liver lobule structure damaged, and can be developed into liver cirrhosis, even liver cancer. Therefore, the development of a drug for reducing visceral fat is particularly important.

Disclosure of Invention

The invention aims to provide the application of conjugated linoleic acid isomer in preparing medicaments and health-care products for reducing visceral fat content, research the lipid-lowering effect of high-purity conjugated linoleic acid and provide support for the application of the high-purity conjugated linoleic acid isomer in lipid lowering.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the invention provides an application of conjugated linoleic acid isomer in preparing medicaments and health-care products for reducing visceral fat content.

Further: the conjugated linoleic acid isomers are c9, t11-CLA, t10 and c12-CLA, and the chemical structural formulas are respectively shown as formula 1 and formula 2;

further: the conjugated linoleic acid isomer promotes the expression of LPL gene and UCP1 gene in adipose tissues and inhibits the expression of FAS gene and ACC1 gene.

Further: the conjugated linoleic acid isomer promotes the expression of PKA, Adiponectin and LPL genes in the liver and inhibits the expression of FAS genes.

Further: the conjugated linoleic acid isomer can reduce lipid accumulation of viscera or adipose tissues, reduce fatty acid synthesis, and promote lipolysis and fatty acid beta oxidation.

Further: the effective dosage of the conjugated linoleic acid isomer is 100-400 mg/Kg/d. When the conjugated linoleic acid isomer with the dosage of more than 400mg/Kg/d is used, although the difference of high dosage is obvious in the aspect of reducing blood fat, the liver section shows that the high dosage is easy to form fatty liver, and the side effect on the liver is large.

Further: the conjugated linoleic acid isomer has obvious feeding inhibition effect on obese mice and reduces the appetite of the mice.

Further: the conjugated linoleic acid isomer is dose-dependent on the weight reducing effect of obese mice.

Further: the viscera are liver, kidney, pancreas, stomach and spleen.

Compared with the prior art, the invention has the advantages and beneficial effects that: the conjugated linoleic acid isomer (t10, c12-CLA) has no obvious influence on the ingestion of normal mice, has obvious ingestion inhibiting effect on obesity and hyperglycemia model mice with metabolic disorder, and obviously promotes the decomposition of TG and the heat production of white fat; FAS of related lipid metabolism genes in the liver is obviously reduced relative to the model group, and PKA, Adiponectin and LPL are obviously increased. Has significant activity for reducing TG (triglyceride) in liver, can reduce accumulation of lipid in liver and adipose tissue, reduce fatty acid synthesis, promote lipolysis, and promote fatty acid beta oxidation; promoting weight and fat reduction. The high-purity conjugated linoleic acid has important significance for human health and has good market application prospect.

Drawings

FIG. 1 shows the results of gas phase analysis of isomers of conjugated linoleic acid.

FIG. 2 is a graph showing the effect of CLA on food intake in ob/ob mice.

FIG. 3 is a graph showing the effect of CLA on body weight in ob/ob mice.

FIG. 4 is a graph of the effect of CLA on the size of ob/ob mouse adipocytes.

FIG. 5 is a graph of the effect of CLA on the liver of ob/ob mice.

FIG. 6 is the effect of CLA on the pancreas of ob/ob mice.

FIG. 7 is a graph showing the effect of Conjugated Linoleic Acid (CLA) on the obesity index and organ index of ob/ob mice.

FIG. 8 is a graph showing the effect of CLA on the expression of mRNA, a factor involved in lipid metabolism in adipocytes.

FIG. 9 shows the effect of CLA on the expression of mRNA, a factor involved in lipid metabolism in the liver.

Detailed Description

The following embodiments better illustrate the present invention. However, the present invention is not limited to the following examples.

The invention mainly takes fat mice, diabetic mice and high-sugar and high-fat fed mice as research objects, and adopts methods of biochemical index monitoring, qRT-PCR, histological section staining and the like to detect the effect of the fat metabolism. The CLA used in the embodiment of the invention is a conjugated linoleic acid isomer with the purity of 98 percent prepared by the known technical means, and the gas phase analysis result is shown in figure 1. Wherein the two isomers c9, t 11-CLA: t10, c12-CLA are equal to 1: 1; the structural formula is shown as formula 1 and formula 2.

Example 1: measurement of food intake and body weight

1. Experimental procedure

(1) Selecting ob/ob mice as obesity model mice, intervening with CLA of different dosages (low, medium and high dosage concentrations are respectively 100mg/Kg/d, 600mg/Kg/d and 1800mg/Kg/d), respectively being a low dosage group (CLA-L), a medium dosage group (CLA-M) and a high dosage group (CLA-H), and taking a control group as a negative control group (CLA-C) and a normal group (WT) as a positive control group;

(2) weighing the mice in 3 days to reflect the weight change of the mice in different treatment groups and compare the intervention effect of different test substances;

(2) the influence on food intake is monitored once a week, and the change of the food intake of mice in different treatment groups is reflected and the difference between the different groups is compared;

(4) influence on visceral weight and ratio, visceral (liver), fat weighing, calculation:

(ii) Lee's index 3 √ m × 103/L m is the mouse body mass/g; l is the length/cm of the mouse body;

(ii) FC/% ═ m1/m × 100m 1 is fat mass/g; m is the mouse mass/g;

③ the organ coefficient/% ═ m1/m × 100m 1 is the organ mass/g; m is the mouse mass/g.

2. Results of the experiment

(1) Effect of CLA on mouse food intake

CLA-C, CLA-L, CLA-M, CLA-H four groups of food intake before wild type mouse treatment are respectively 2.59 + -0.40, 2.28 + -0.49, 2.20 + -0.75 and 2.13 + -0.71, and CLA is treated with low, medium and high dose in5 weeks.

The food intake of the CLA-C, CLA-L, CLA-M, CLA-H group before the ob/ob mice are not treated is 3.19 +/-0.54, 3.48 +/-1.03, 4.13 +/-0.77 and 4.12 +/-1.04 g/d respectively, and the food intake record shows that the food intake of the CLA-H group mice is 0.87 +/-0.22 g, which is obviously lower than that of the CLA-C group (P is less than 0.05), and the food intake of both the CLA-L group and the CLA-M group is slightly reduced (figure 2). The CLA has obvious feeding inhibition effect on obese model mice and can reduce the appetite of the mice.

(2) Effects of CLA on mouse body weight

The ob/ob mice are obesity model mice, obesity is the main characteristic, the weights of untreated CLA-C, CLA-L, CLA-M, CLA-H groups are 47.18 +/-1.05, 46.78 +/-1.92, 46.54 +/-1.86 and 46.650 +/-0.79 g respectively, the dose dependency of a weighing scale is reduced after the low, medium and high dose CLA preparation is taken for 5 weeks, and the weights of the groups are respectively as follows: 54.00 +/-1.33 g, 51.94 +/-1.75 g, 48.34 +/-2.41 g and 39.22 +/-1.58 g, the body weight of ob mice in the medium and high dose groups is obviously reduced (P is less than 0.05, P is less than 0.05 vs CLAC group, and figure 3). The results of body weight records show that a certain dosage of CLA preparation has the effect of reducing body weight of obese mice and is dose-dependent.

Example 2: research experiment of fat, liver and pancreas

1. Test method

I. Tissue section staining

(1) After a period of intervention, the mice are sacrificed, the liver and adipose tissue of the mice are sliced and analyzed, and the conditions of intrahepatic lipid drop size and white lipid vacuole size are observed;

(2) fresh tissue blocks of fat, liver, pancreas, etc. are fixed with 4% paraformaldehyde, dehydrated, transparent, embedded in paraffin, and sliced to a thickness of 5-10 μm. After the section is flattened, xylene is dewaxed, hematoxylin is dyed, flushing and dehydrating are carried out, then eosin is dyed, dehydrating and transparent are carried out, and gum is sealed;

(3) after HE staining of adipose tissues, 15 high-power fields were randomly selected under an optical microscope to take pictures (the cell membrane of the adipose cells in the fields is smooth and clear). The adipocyte cell membranes were carefully delineated with a brush tool and the Image J Image software calculated the area of all delineated adipocytes.

II. Taking 400mg of fresh liver tissue, adding 4ml of lipid extract (n-heptane: isopropanol ═ 2: 3.5), homogenizing at 4 ℃ in a mortar, shaking to extract lipid, centrifuging and precipitating, taking supernatant, and determining the content of triglyceride in liver by using a kit.

III, fixing mouse pancreatic tissues by paraformaldehyde, and then carrying out conventional HE slicing. The images of the HE sections of the tissue were analyzed using Image J Image software. The percentage of islet area was calculated as follows: under the low power mirror, a painting brush tool is used for delimiting a pancreas area in a visual field, the total area is calculated, the area of each islet is carefully defined by a light pen, and the software obtains the delimited islet area. All islets and total pancreatic area were measured step by step and the percentage of islets to the total pancreatic area was calculated.

2. Results of the experiment

(1) CLA effects on adipose tissue, liver and pancreatic tissue

Fig. 4 is a section of adipose tissues of mice in each group, which shows that the area of single adipose cells of ob/ob mice is remarkably increased (fig. 4A, B), the adipose cells are reduced after CLA treatment, and the medium-high dose group is more remarkable (P < 0.05, fig. 4F), which indicates that CLA promotes the catabolism of fat in the adipose cells. The liver cells of ob/ob obese mice exhibited fat vacuole-like changes (fig. 5B), and the degree of vacuole-like changes in the liver of mice of CLA-M and CLA-H groups was reduced (fig. 5D, F), indicating that Conjugated Linoleic Acid (CLA) was effective in reducing the size of fat vacuoles in ob/ob mice; it was shown that CLA also promotes fat catabolism in the liver. The number of islets in pancreatic tissue of ob/ob mice was significantly increased compared to WT mice (fig. 5A, B), the number of islets was decreased after CLA treatment (fig. 6C-F), and the area occupied by islets was also significantly decreased (table 1), indicating that CLA affected pancreatic function. As shown in fig. 7, Conjugated Linoleic Acid (CLA) can reduce organ index of ob/ob mice, and changes of liver are significant; the CLA with medium and high dose can obviously reduce the visceral fat content of the model mouse, and the change of the liver is obvious.

TABLE 1 CLA vs ob/ob mouse pancreatic histology changes

Note：##：P＜0.01 VS WT；*：p＜0.05 VS CLA-C，**：p＜0.01 VS CLA-C。

Example 3: mRNA expression assay

1. Experimental methods

Fat, liver, muscle and other tissues are extracted by Trizol method to obtain total RNA, and the total RNA is reverse transcribed to obtain cDNA with regulated concentration of 100 ng/ul. The qRT-PCR reaction system is 20 uL: 100ng cDNA, 10uL 2XFaststart Universal SYBR GreenMaster, 0.6uL Primer F/R, 8.4uL RNase Free Water. The reaction conditions are as follows: 10min at 95 ℃, 15s at 95 ℃, 1min at 57 ℃ and 40 cycles. The experiment was repeated 3 times for each sample, using 2-^ΔΔCtThe method calculates the relative expression quantity of the target gene, and the beta-actin is an internal reference. The primer sequences are shown in Table 2.

TABLE 2 summary of primer sequences

2. Results of the experiment

(1) CLA effects on fat tissue and liver fat metabolism factor mRNA expression

The gene expression of fat synthase (FAS) and acetyl coenzyme A carboxylase 1(ACC1) in adipose tissues of ob/ob mice is remarkably increased (figure 8), and the expression of UCP1 is reduced, which indicates that the fat anabolism in model mice is enhanced, and the catabolism is reduced. After CLA is perfused, the expression of LPL and UCP1 mRNA in the fat of the model mouse is increased, which indicates that the fat catabolism is enhanced; FAS and ACC1 inhibited mRNA expression in the CLA group at medium and low doses, and the high dose group was elevated but still significantly lower than the model control group (FIG. 8), indicating that the fat synthesis and tissue accumulation related factor expression of model mice after CLA treatment was significantly reduced compared with the untreated group, which is beneficial to the reduction of fat content in animals.

The expression change of the gene related to the fat metabolism of the liver tissue of the ob/ob mouse is similar to that of the fat tissue. Compared with a model group, the related fat synthesis gene FAS in the liver is obviously reduced, and the PKA, Adiponectin and LPL are obviously increased (figure 9), which shows that CLA can also inhibit fat synthesis in liver cells, enhance fat catabolism and promote weight loss of obese mice.

As shown in fig. 8 and 9, further combining gene analysis of liver and fat, expression of relevant lipid metabolism genes LPL, Plin5, UCP1 and AP2 in white fat is significantly increased in CLA group compared to model group, which indicates that CLA significantly promotes TG decomposition and white fat thermogenesis; compared with a model group, the related lipid metabolism gene FAS in the liver is obviously reduced, and the PKA, the Adiponectin and the LPL are obviously increased, which shows that CLA can obviously promote the oxidative decomposition of liver lipid and reduce the accumulation of the lipid. CLA with different dosages is adopted to intervene an ob/ob mouse, so that the weight of the mouse can be obviously reduced, the lipid accumulation of liver and adipose tissue can be reduced, the synthesis of fatty acid can be reduced, and lipolysis and beta oxidation of fatty acid can be promoted. Different doses of CLA exhibited a dose effect.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.