CN114392292A

CN114392292A - Extraction method of rape pollen total flavonoids and application of rape pollen total flavonoids in preparation of drugs for preventing or treating fatty liver

Info

Publication number: CN114392292A
Application number: CN202210165358.4A
Authority: CN
Inventors: 贾安; 黄小强; 郭志刚
Original assignee: Huanghe Science and Technology College
Current assignee: Huanghe Science and Technology College
Priority date: 2022-02-23
Filing date: 2022-02-23
Publication date: 2022-04-26

Abstract

The invention relates to a prevention effect of rape pollen total flavonoids on high fat induced non-alcoholic fatty liver disease. A non-alcoholic fatty liver mouse model is established by raising a mouse with high fat for 10 weeks, the mouse is subjected to intragastric administration intervention while molding, the levels of AST and ALT in serum of the mouse, the contents of TG, TC and LDL-C, HDL-C and the contents of TG and TC in liver tissues are measured 1 time a day after 10 weeks, and the morphological change of the liver tissues is observed by an HE staining method. Compared with a model group, the rape pollen total flavone can obviously reduce the weight of a mouse, the AST and ALT levels in serum, the TG, TC and LDL-C contents in serum, the TC and TG contents in liver tissues, the HDL-C content in serum and the pathological morphology of the liver tissues of the mouse. The rape pollen total flavone can prevent the high fat induced non-alcoholic fatty liver through improving liver function, regulating blood lipid level, improving liver tissue pathological changes and the like.

Description

Extraction method of rape pollen total flavonoids and application of rape pollen total flavonoids in preparation of drugs for preventing or treating fatty liver

Technical Field

The invention relates to a method for extracting rape pollen total flavonoids, belonging to the technical field of fatty liver prevention or treatment.

Background

Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases in metabolic syndrome, due to abnormal hepatic fat metabolism and excessive lipid accumulation. The liver is an important organ for whole body lipogenesis, gluconeogenesis and cholesterol metabolism. The development of NAFLD is primarily characterized by abnormal liver function and can progress to more severe liver diseases such as nonalcoholic steatohepatitis, liver fibrosis and cirrhosis. NAFLD has become a serious health problem worldwide due to its increased morbidity and mortality. However, no safe and effective drug for NAFLD is available clinically at present. NAFLD patients often require long-term treatment, which may lead to serious or multiple side effects. Therefore, it is imperative to develop drugs with better biological efficacy and safety.

Disclosure of Invention

Aiming at the problems, the invention provides an extraction method of rape pollen total flavonoids and application thereof in preparing a medicament for preventing or treating fatty liver, and the specific scheme is as follows:

the extraction method of rape pollen total flavone comprises the following steps:

(1) breaking cell wall of rape pollen, drying, and pulverizing;

(2) weighing the rape pollen after wall breaking, adding ethanol, and performing ultrasonic treatment to extract the rape pollen total flavonoids.

Preferably, in (1), an 80 mesh screen is passed.

Preferably, 80% ethanol is added in (2) according to the feed-liquid ratio of 1:10 (g: mL).

Preferably, in the step (2), the ultrasonic temperature is 60 ℃, and the ultrasonic time is 60 min.

Preferably, in (2), the residue is extracted once more, the two filtrates are combined, the solvent is recovered, and the filtrate is lyophilized under vacuum and stored at-20 ℃.

Application of rape pollen total flavone in preparing medicine for preventing or treating fatty liver is disclosed.

A medicine for preventing or treating fatty liver comprises rape pollen total flavone.

The beneficial effects of the invention are as follows:

there is no clinically effective drug for NAFLD, and therefore, research on nonalcoholic fatty liver disease has been a hot spot. In the research, a NAFLD animal model is established by feeding mice with high fat for a long time, and the weight of the mice is obviously increased in the process, and the liver steatosis, the liver function and the lipid metabolism are abnormal. In the course of this study, at the end of the experiment, the weight and liver weight of the model group mice increased significantly due to long-term high fat diet, and the canola pollen total flavonoids were able to inhibit the weight and liver weight increase of the mice. Liver pathology shows that liver cells of mice in a model group are obviously degenerated, a large number of fat vacuoles appear in liver tissues, and the model building success of the non-alcoholic fatty liver animal model is proved.

Long-term high fat diet can cause the FFA in serum to be increased, and the continuous accumulation of the FFA in the body can cause liver injury. The AST and ALT level is usually used to directly reflect the degree of liver injury, and once the liver is injured, intracellular AST and ALT are induced to flow into blood, so that the content of blood is increased, and the AST and ALT indexes are commonly used for evaluating the degree of liver function injury. The research result shows that the AST and ALT levels in the blood serum of the mice in the model group are obviously higher than those in the normal group, and the AST and ALT levels in the blood serum of the mice in the drug intervention group are lower than those in the model group in different degrees. The rape pollen total flavone is suggested to have a certain liver protection effect.

High-fat diet can cause the body to have hyperlipidemia, which is an important factor caused by fatty liver, and about 90 percent of patients with hyperlipidemia are accompanied by fatty liver. Therefore, the content of TC, TG, HDL-C and LDL-C in serum is closely related to NAFLD. The research result shows that the contents of TC, TG and LDL-C in the serum of the mouse in the model group are obviously higher than those in the normal group, the content of HDL-C is obviously lower than that in the normal group, the contents of TC, TG and LDL-C in the serum of the mouse in the drug intervention group are lower than those in the model group in different degrees, and the content of HDL-C is higher than that in the model group. It is suggested that the rape pollen total flavone is related to the improvement of NAFLD disease and the regulation of blood fat.

The main organ of fat synthesis and metabolism is the liver, and a high fat diet causes an increase in calories in the diet, causes an increase in the biosynthesis of TC, TG and FFA in liver tissues, and reduces the degradation of TG and TC and the oxidation of FFA in liver tissues, thereby causing TG, TC and FFA to continuously accumulate in the liver to form fatty liver. The research result shows that the contents of TC and TG in the liver of the mouse in the model group are obviously higher than those in the normal group, and the contents of TC and TG in the liver of the mouse in the drug intervention group are lower than those in the model group in different degrees. It is suggested that the improvement of NAFLD disease by rape pollen total flavone is related to the reduction of lipid content in liver tissue.

In conclusion, the rape pollen total flavonoids can obviously reduce the weight of the mouse with high fat diet and the weight of the liver, regulate lipid metabolism disorder, improve liver steatosis and fat accumulation, play a certain role in preventing the incidence of NAFLD, have important value in preventing or treating fatty liver, and are expected to have great influence on drug development and disease treatment.

Drawings

FIG. 1 shows the effect of total flavonoids of rape pollen on the liver weight and liver weight ratio of mice. Compared with the normal group, # # P is less than 0.01; p <0.05, P < 0.01 compared to model group.

FIG. 2 shows the effect of total flavonoids from canola pollen on AST and ALT levels. In comparison with the normal group,^##p is less than 0.01; in comparison with the set of models,^*P＜0.05,^**P＜0.01。

FIG. 3 shows the effect of total flavonoids of canola pollen on TC, TG, LDL-C and HDL-C contents. Compared with the normal group, # # P is less than 0.01; p <0.05, P < 0.01 compared to model group.

FIG. 4 is a graph showing the effect of canola pollen on TC and TG content in mouse liver tissue. Compared with the normal group, # # P is less than 0.01; p <0.05, P < 0.01 compared to model group.

FIG. 5 shows the effect of rape pollen on the histopathological morphology of mouse liver.

Detailed Description

The following describes the embodiments of the present invention in detail with reference to specific examples.

The experimental materials adopted by the invention are as follows:

medicine preparation: rape pollen, Henan Mei le Yuan bee-keeping professional cooperative; metformin, Shanghai-derived Phyllotechnisch, Inc.

Animals: 60C 57BL/6j mice with the age of 6 weeks are male, and the weight is 20 +/-2 g, and the mice are purchased from the animal experiment center of the medical college of the yellow river science and technology school.

Reagent: AST, ALT, TG, TC and HDL-C, LDL-C kit (purchased from Nanjing institute of bioengineering); hematoxylin-eosin (available from Beijing Solebao Biotech, Inc.); high fat feed and general feed (Guangzhou center for laboratory animals); other reagents are all in-home analytical grade.

Multifunctional microplate reader (Infine, Austria); high speed bench refrigerated centrifuge (Sammer Feisha, USA, science and technology Co., Ltd.); ultraviolet visible spectrophotometer (shanghai instrument electric analyzer limited); DHP-9052 electric heating constant temperature incubator (Shanghai-Hengshi Co., Ltd.); a BS-3000A series electronic balance (shanghai friend sound weighing apparatus ltd); optical microscopes (OLYMPUS corporation); model RM2125 Paraffin microtome (Leica, Germany).

The extraction process of rape pollen total flavone includes breaking rape pollen wall, drying, crushing and 80 mesh sieving. Weighing 100g of wall-broken rape pollen, adding 80% ethanol according to a material-liquid ratio of 1:10 (g: mL), performing ultrasonic treatment at 60 ℃ for 60min, extracting total flavonoids of rape pollen, performing suction filtration to obtain supernatant, repeatedly extracting filter residue once, combining filtrates, recovering solvent, and performing vacuum freeze drying at-20 ℃ for later use.

Using NaNO₂-Al(NO₃)₃-NaOH color development, taking rutin as reference substance, taking absorbance at 510nm as ordinate, taking rutin standard solution concentration as abscissa, drawing working curve, wherein Y is 0.0094X +0.0104, R is²When the total flavone content of the rape pollen extract is determined to be 0.9993, the total flavone content of the rape pollen extract is determined to be 17.81 percent.

A proper amount of metformin standard substance is taken and placed in a 50mL centrifuge tube, and a proper amount of distilled water is added to prepare a solution with the concentration of 0.02g/mL for standby.

Experimental grouping and administration

After 60C 57BL/6j male mice were acclimatized for one week, they were randomized into 6 groups: normal group, model group, metformin group (200mg/kg.d), rape pollen low, medium and high dose group (5,10,20g/kg.d, crude drug), each group comprises 10 mice, normal group comprises common feed, other groups comprise high fat feed, the gavage is carried out according to the body mass of 10mL/kg.d, the gavage is carried out for 1 time/d, normal group and model group comprise physiological saline with the same volume as the gavage, and other groups comprise corresponding drugs. Mice were allowed free access to water for 10 weeks during the experiment, weighed once per week, and recorded.

Obtaining experimental animal materials

After the experiment is finished, all mice are fasted for 12 hours without water prohibition, weighed, picked with eyeballs for blood sampling, dislocated and killed, then the liver of the mice is taken quickly, weighed, stored at ultralow temperature for later use, and the liver index is calculated.

Liver index%

Determination of biochemical indicators in serum

And (3) after blood is taken, slightly standing, centrifuging at the low temperature of 4 ℃ for 10min, taking supernatant, and measuring the AST, ALT, TG, TC, HDL-C and LDL-C contents in the mouse serum according to the operation of a kit instruction.

Determination of Biochemical indicators in liver tissue

Taking a proper amount of liver tissue, weighing, and weighing: adding 9 times volume of physiological saline into the mixture according to the volume (mL) ratio of 1:9, fully grinding the mixture in an ice water bath, centrifuging the mixture for 10min at a speed of 2500r/min, taking supernatant, and measuring the content of TC and TG in the homogenate of the liver of the mouse according to the operation of a kit instruction.

Liver tissue slice observation

Cutting soybean liver tissue, fixing in 4% paraformaldehyde for 16 hr, washing with distilled water until there is no alcohol smell, gradient dehydrating with ethanol, embedding in paraffin, slicing, dewaxing, staining with hematoxylin-eosin, sealing with neutral gum, air drying, observing under optical microscope, and taking photograph.

Statistical analysis of data

Statistical analysis was performed on the experimental data using SPSS20.0 software, with the results expressed as mean (X ± s), significance analysis using univariate analysis, multiple comparisons between groups using t-test, statistical significance of differences with P <0.05, and mapping using graphpad5.0 software.

TABLE 1 Effect of Total Flavonoids of canola pollen on mouse body weight: (

n＝10)

As can be seen from Table 1, the mice in the model group had a significantly larger body weight from the second week end than the normal group, as compared with the normal group (^**P is less than 0.01), and at the end of the experiment, the average weight of the mice in the model group is 9g greater than that of the mice in the normal group; at the end of the experiment, the weight of mice in each administration group was less than that in the model group, and the weight of mice in the metformin group, the rape pollen total flavone medium dose group and the high dose group was significantly less than that in the model group (as compared with the model group) ((^*P＜0.05,^**P is less than 0.01); the experimental result indicates that the rape pollen total flavone has the function of slowing down the weight gain, thereby preventing the occurrence and the development of the non-alcoholic fatty liver.

As can be seen from FIG. 1, the liver weight of the mice in the model group was significantly higher than that in the normal group (^**P is less than 0.01); compared with the model group, the weight of the liver of each mouse in the administration group is less than that in the model group, wherein the metformin group and the rape pollen total flavone high dose group have statistical difference (^*P＜0.05,^**P is less than 0.01); meanwhile, the liver weight ratio of each group of mice is not different. The experimental result indicates that the rape pollen total flavone can inhibit the weight increase of the liver of a mouse and prevent the occurrence of fatty liver.

As can be seen from FIG. 2, the serum AST and ALT levels in the model group mice were significantly increased (P < 0.01) compared with the normal group, indicating abnormal liver function of the mice; the serum levels of AST and ALT were reduced in mice from each group, with significant reductions in AST levels in mice from each group (P <0.05, P < 0.01), and ALT levels in mice from both the dose and high dose groups, metformin and canola pollen total flavonoids (P <0.05, P < 0.01). The experimental result indicates that the rape pollen total flavone has a certain liver protection effect.

As can be seen from FIG. 3, the serum contents of TC, TG and LDL-C in the model group mice were significantly increased as compared with the normal group (^**P is less than 0.01, and the content of HDL-C is obviously reduced (^**P is less than 0.01); compared with the model group, the serum contents of TC, TG and LDL-C of mice in each administration group are reduced to different degrees, wherein the serum contents of TC and LDL-C of the serum in each dosage group of metformin and rape pollen total flavonoids are obviously reduced (the content of TC and LDL-C in the serum of mice in each dosage group is obviously reduced by the formula (I))^*P＜0.05,^**P is less than 0.01), and the content of TG in serum of the medium-dose group and the high-dose group of the metformin group and the rape pollen total flavonoids is obviously reduced (^*P＜0.05,^**P is less than 0.01), HDL-C is increased in different degrees in each administration group, wherein metformin, rape pollen total flavone and high dose group are significantly increased (^**P is less than 0.01). The experimental result indicates that the rape pollen total flavone has a certain function of reducing blood fat, thereby reducing the occurrence and development of non-alcoholic fatty liver.

As can be seen from FIG. 4, the amounts of TG and TC in liver tissues of the model group mice were significantly increased as compared with those of the normal group (^**P is less than 0.01); compared with the model group, the liver tissues of mice in each administration group have different reductions of TG and TC contents, wherein the TG content in the metformin group, the rape pollen total flavone middle and high dose group is significantly reduced, and the TC content in the metformin group and the rape pollen total flavone high dose group is significantly reduced (the)^**P is less than 0.05); the experimental result indicates that the rape pollen total flavone can reduce the content of TC and TG in liver tissues, so that the occurrence of non-alcoholic fatty liver can be slowed down to a certain extent.

From the left to the right in the first row of FIG. 5, the normal group, the model group, and the metformin group are shown in sequence; the second row, from left to right, is low dose, medium dose, high dose.

The liver cells of the normal group of mice have complete and clear structure and regular arrangement, the morphology of cell nuclei has no abnormal change, and liver cell steatosis is not seen. The shape of the liver cells of the mice in the model group is obviously changed, a large number of bubble-like fat vacuoles with different sizes appear in the liver cells, and the cell nucleus is extruded to one side of the cell by larger fat particles. Compared with the model group, the mice of each administration group have the advantages of reduced liver fatty degeneration, reduced fat vacuole number and reduced volume, wherein the medium dose and high dose of rape pollen total flavonoids are relieved obviously. The experimental result indicates that the rape pollen total flavone is further proved to have the improvement effect on the nonalcoholic fatty liver injury.

Claims

1. The extraction method of rape pollen total flavonoids is characterized by comprising the following steps:

(1) breaking cell wall of rape pollen, drying, and pulverizing;

2. The method for extracting total flavonoids from rape pollen as claimed in claim 1, wherein the method comprises the following steps:

in (1), pass through a 80 mesh screen.

3. The method for extracting total flavonoids from rape pollen as claimed in claim 1, wherein the method comprises the following steps:

in (2), 80% ethanol was added in a feed-to-liquid ratio of 1:10 (g: mL).

4. The method for extracting total flavonoids from rape pollen as claimed in claim 1, wherein the method comprises the following steps: and (2) carrying out ultrasonic treatment at the ultrasonic temperature of 60 ℃ for 60 min.

5. The method for extracting total flavonoids from rape pollen as claimed in claim 1, wherein the method comprises the following steps: and (2) repeatedly extracting filter residues once, combining the two filtrates, recovering the solvent, and performing vacuum freeze drying at-20 ℃ for storage.

6. Application of rape pollen total flavone in preparing medicine for preventing or treating fatty liver is disclosed.

7. A medicament for preventing or treating fatty liver, which is characterized in that: comprises rape pollen total flavonoids.