CN111544440A

CN111544440A - Application of diosmin and composition in preparation of anti-obesity product

Info

Publication number: CN111544440A
Application number: CN202010428692.5A
Authority: CN
Inventors: 孙华; 张梦迪; 李明媛; 郝思雨; 吴岩; 郁彭
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2020-05-20
Filing date: 2020-05-20
Publication date: 2020-08-18

Abstract

The invention relates to application of diosmin and a composition in preparation of anti-obesity products. The diosmin has simple extraction and separation method and lower cost, and the food-borne substances have small toxic and side effects, so the application of the diosmin or the composition thereof in the aspect of preparing anti-obesity functional foods, health-care products and medicines has huge market potential.

Description

Application of diosmin and composition in preparation of anti-obesity product

Technical Field

The invention belongs to the technical field of medicines and functional foods, and relates to a new application of diosmin and a composition thereof, in particular to an application of diosmin and a composition thereof in the aspect of preparing anti-obesity products.

Background

Diosmin is a medicine which is already on the market, and the new application of old medicine is an important way for finding new medicine. In addition, diosmin is simple and easy to extract and synthesize as a flavonoid compound from natural citrus plants (such as hesperidin and lemon peel) or chrysanthemum, can be extracted from natural plants in large quantity, and can also be obtained by semi-synthesis of natural product hesperidin. In recent years, diosmin has been found to have various biological activities, such as antishock, anti-apoptosis, anti-tumor, anti-diabetic and the like. For example, diosmin can reduce hepatic gluconeogenesis and reduce hyperglycemia of diabetic rats, diosmin also has a positive effect on improving diabetic complications and oxidative stress of diabetic rats, and a novel zinc and diosmin complex has an activity of maintaining glucose homeostasis of diabetic rats.

With the rapid increase of economy and the prevalence of western diet, the obesity metabolic syndrome is rapidly spreading worldwide, and the rising incidence rate and the growing age of onset make the obesity lesion a serious problem to face. Obesity is a disease caused by a combination of excessive diet, lack of exercise and neurotransmitters, and the like. In order to improve the risk and effect of obesity, a large number of anti-obesity drugs have been marketed, but since these anti-obesity drugs involve various side effects such as diarrhea and vomiting during the treatment, development of anti-obesity drugs from natural products having few side effects is a new direction. The research finds that the flavone compound has potential activity for treating obesity. The polyphenol compounds rutin and quercetin can be used for treating obesity caused by dyslipidemia. The loquat and burdock plants also have weight loss activity on adipocytes and obese mice. Obesity is a disease associated with energy imbalance, and the AMP-dependent protein kinase (AMP) pathway is a key cellular energy sensor pathway. Once the AMPK pathway is activated, the anabolic processes that consume adenosine triphosphate are inhibited, allowing cellular and body energy to be balanced. More and more researches prove that the AMPK pathway can be used as a potential target for treating metabolic disorder, and the activation of the AMPK pathway can inhibit the expression of SREBP-1c, fatty acid and PPAR gamma in cells and adipose tissues and reduce lipogenesis to generate the anti-obesity activity effect.

Through searching, the following three publications related to the patent application of the invention are found:

1. patent application No. CN105613828A, a chrysanthemum health tea bag for reducing weight, the patent discloses a health tea bag, the tea bag has multiple components of chrysanthemum, hawthorn, radix bupleuri, honeysuckle and green tea, the weight reducing activity does not need to be derived from chrysanthemum or chrysanthemum, the patent is that the tea bag is not an aqueous extract or an alcohol extract.

2. Journal 1: synthesis, SpectraLCharacterisation, and Biochemical Evaluation of the biological Properties of an aNew Zinc-Diosymin Complex study in High Fat Diet Fed Type-Low Dose streptozoctonia induced Experimental Type 2 Diabetes in rates biochem Res Int 2015, 11, 50829 this study paper investigated a new Type 2 diabetic rat Fed a Zinc-Diosmin Complex on a High Fat Diet, with improved glucose homeostasis and increased insulin sensitivity in diabetic rats for the treatment of Diabetes, but without direct relation to anti-obesity.

3. And 2, journal: Chia-Chen Hsu et al, Diosmin, a Citrus Nutrient, Activate Imidazoline Receptors to Alleviate Blood Glucose and Lipids in Type 1-like diabetes rates, Nutrients, 2017, 9, 684 this study was primarily directed to the effects of Diosmin on β -endorphin (BER) and Blood Glucose in the plasma of diabetic Rats, and found that Diosmin increased BER levels and improved hyperglycemia in a dose-dependent manner. The study mainly demonstrated the ability of diosmin to lower blood glucose and blood lipids, and did not mention activity in treating obesity.

By contrast, the present invention is substantially different from the above-mentioned publications.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides the application of diosmin and a composition in preparing anti-obesity products.

The technical scheme adopted by the invention for solving the technical problems is as follows:

the use of diosmin in the preparation of anti-obesity products.

Furthermore, the natural plant molecule diosmin has the structural formula:

moreover, the product is a functional food, a health product or a medicament.

A natural plant molecular composition containing diosmin for treating obesity is provided.

Moreover, the components and the parts by weight are as follows: 0.5-5 parts of diosmin, and/or 1-15 parts of chrysanthemum aqueous extract, and/or 1-15 parts of chrysanthemum alcohol extract.

Moreover, the preparation method of the chrysanthemum aqueous extract comprises the following steps:

weighing chrysanthemum, crushing, adding water in a volume ratio of 1: 1-1: 5, soaking for 4-8 hours, leaching for 6-10 hours at 80-120 ℃, filtering, collecting a water layer, and freeze-drying the water layer to obtain a chrysanthemum water extract;

the preparation method of the chrysanthemum alcohol extract comprises the following steps:

weighing chrysanthemum, crushing, adding 95% ethanol water solution, wherein the volume ratio of the chrysanthemum to the ethanol water solution is 1: 10-1: 40, refluxing and extracting for 3-5 hours, filtering, collecting an ethanol layer, and evaporating to dryness to obtain the chrysanthemum alcohol extract.

Moreover, the preparation method of the natural plant molecular composition comprises the following steps:

mixing diosmin and flos Chrysanthemi water extract, or diosmin and flos Chrysanthemi ethanol extract, or diosmin, flos Chrysanthemi water extract and flos Chrysanthemi ethanol extract uniformly according to weight parts.

Use of a diosmin composition as described above for the preparation of an anti-obesity product.

Moreover, the product is a functional food, a health product or a medicament.

The invention has the advantages and positive effects that:

1. the invention provides application research of diosmin and a composition thereof in the aspect of resisting obesity, and no report about application of diosmin or the composition thereof in treating anti-obesity diseases is available. The diosmin is simple in extraction and separation or synthesis method and low in cost, and the food-borne substances have small toxic and side effects, so that the application of the diosmin or the composition thereof in the aspects of preparing anti-obesity functional foods, health-care products and medicines has great market potential.

2. The diosmin or the composition of the diosmin and chrysanthemum has the activity of treating obesity, is prepared into a corresponding preparation which is easy to control the quality, has the characteristic of small food-borne toxic and side effects, can be applied to functional foods, health-care products and medicines, and is used for treating related diseases of obesity.

3. The invention discloses diosmin and a composition thereof, which have good treatment effect on obesity, and the evaluation result of the activity in vivo of a mouse shows that the diosmin and the composition thereof can obviously reduce the body weight of the obese mouse and inhibit the generation of white fat of epididymis, perirenal mucosa and intestinal mucosa and the increase of liver, kidney and spleen organ indexes. In vitro cell level evaluation results also show that diosmin and the composition thereof have the activities of relieving lipid accumulation in HepG2 cells and 3T3-L1 fat cells, inhibiting the content of triglyceride in cells, activating AMPK (adenosine monophosphate kinase) pathways and the like. Therefore, diosmin and the composition thereof can be used as functional food, health-care products or medicines for treating obesity, and have great market potential.

Drawings

FIG. 1 is a graph of the effect of diosmin and compositions thereof on the body weight of obese mice in the present invention (A) the change in body weight over days 0-27; (B) comparison of body weights on day 27. Wherein Diosmin is Diosmin (400mg/kg), DMZH1 is Diosmin (150mg/kg) + chrysanthemum aqueous extract (250mg/kg), DMZH2 is Diosmin (150mg/kg) + chrysanthemum alcohol extract (250mg/kg), DMZH3 is Diosmin (150mg/kg) + chrysanthemum aqueous extract (125mg/kg) + chrysanthemum alcohol extract (150 mg/kg): l-carnitine is L-carnitine (400mg/kg)

FIG. 2 is a graph showing the effect of the present invention on white adipose tissue (A) on the weight of each portion of white adipose tissue; (B) the morphological influence on white fat of epididymis and kidney periphery. Wherein Diosmin is Diosmin (400mg/kg), DMZH1 is Diosmin (150mg/kg) + chrysanthemum aqueous extract (250mg/kg), DMZH2 is Diosmin (150mg/kg) + chrysanthemum alcohol extract (250mg/kg), DMZH3 is Diosmin (150mg/kg) + chrysanthemum aqueous extract (125mg/kg) + chrysanthemum alcohol extract (150 mg/kg); l-carnitine is L-carnitine (400 mg/kg).

FIG. 3 is a graph of the effect of diosmin and compositions thereof on lipid deposition in HepG2 cell models (A) on the intracellular lipid content of HepG 2; (B) influence on triglyceride content in HepG2 cells; (C) fluorescence staining pattern for intracellular lipid content. Wherein Diosmin is Diosmin (20 mu M), DMZH1 is Diosmin (10 mu M) + chrysanthemum aqueous extract (10 mu M), DMZH2 is Diosmin (10 mu M) + chrysanthemum alcohol extract (10 mu M), DMZH3 is Diosmin (10 mu M) + chrysanthemum aqueous extract (5 mu M) + chrysanthemum alcohol extract (5 mu M), and lovastatin is lovastatin (20 mu M); p < 0.1, P < 0.01, P < 0.001 vs model group;

FIG. 4 is a graph of the effect of diosmin and compositions thereof on lipid deposition in 3T3-L1 cell models in accordance with the present invention (A) on the lipid content in 3T3-L1 preadipocytes; (B) effect on lipid content in 3T3-L1 mature adipocytes; (C) influence on triglyceride content in 3T3-L1 mature fat cells. Wherein Diosmin is Diosmin (20 mu M), DMZH1 is Diosmin (10 mu M) + chrysanthemum aqueous extract (10 mu M), DMZH2 is Diosmin (10 mu M) + chrysanthemum alcohol extract (10 mu M), DMZH3 is Diosmin (10 mu M) + chrysanthemum aqueous extract (5 mu M) + chrysanthemum alcohol extract (5 mu M), and lovastatin is lovastatin (20 mu M); p < 0.1, P < 0.01, P < 0.001 vs model group;

FIG. 5 is a graph of the effect of diosmin and compositions thereof on the AMPK pathway in HepG2 cells in accordance with the present invention; (A) effects on the AMPK pathway in HepG2 cells; (B) and (5) gray level analysis. Wherein Diosmin is Diosmin (20 μ M), DMZH1 is Diosmin (10 μ M) + chrysanthemum aqueous extract (10 μ M), DMZH2 is Diosmin (10 μ M) + chrysanthemum alcohol extract (10 μ M), DMZH3 is Diosmin (10 μ M) + chrysanthemum aqueous extract (5 μ M) + chrysanthemum alcohol extract (5 μ M), and AICAR (250 μ M); p < 0.1, P < 0.01, P < 0.001 vs model group;

FIG. 6 is a graph of the effect of diosmin and compositions thereof on the 3T3-L1 cell AMPK pathway in accordance with the present invention; (A) effects on the 3T3-L1 preadipocyte AMPK pathway; (B) gray scale analysis of 3T3-L1 preadipocytes; (C) effects on the 3T3-L1 mature adipocyte AMPK pathway; (D) gray scale analysis of 3T3-L1 mature adipocytes; wherein Diosmin is Diosmin (20 μ M), DMZH1 is Diosmin (10 μ M) + chrysanthemum aqueous extract (10 μ M), DMZH2 is Diosmin (10 μ M) + chrysanthemum alcohol extract (10 μ M), DMZH3 is Diosmin (10 μ M) + chrysanthemum aqueous extract (5 μ M) + chrysanthemum alcohol extract (5 μ M), and AICAR (250 μ M); p < 0.1, P < 0.01, P < 0.001 vs model group;

Detailed Description

The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.

The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.

Application of diosmin in preparing anti-obesity product is provided.

Preferably, the product is a functional food, a health product or a medicament.

Preferably, the composition and the parts by weight are as follows: 0.5-5 parts of diosmin, and/or 1-15 parts of chrysanthemum aqueous extract, and/or 1-15 parts of chrysanthemum alcohol extract.

Preferably, the preparation method of the chrysanthemum aqueous extract comprises the following steps:

weighing chrysanthemum, crushing, adding water and chrysanthemum in a volume ratio of 1: 1-1: 5, soaking for 4-8 hours, leaching for 6-10 hours at 80-120 ℃, filtering, collecting a water layer, and freeze-drying the water layer to obtain a chrysanthemum water extract;

Preferably, the preparation method of the natural plant molecular composition comprises the following steps:

The application of the natural plant molecular composition in preparing anti-obesity products.

Preferably, the product is a functional food, a health product or a medicament.

More specifically, the preparation and activity evaluation were as follows:

preparation of Diosmin composition

(1) The preparation method of the chrysanthemum aqueous extract comprises the following steps: weighing chrysanthemum, crushing, adding water in a volume ratio of 1: 1-1: 5, soaking for 4-8 hours, leaching for 6-10 hours at 80-120 ℃, filtering, collecting a water layer, and freeze-drying the water layer to obtain the chrysanthemum aqueous extract.

(2) The preparation method of the chrysanthemum alcohol extract comprises the following steps: weighing chrysanthemum, crushing, adding 95% ethanol water solution in a volume ratio of 1: 10-1: 40, performing reflux extraction for 3-5 hours, filtering, collecting an ethanol layer, and evaporating to obtain the chrysanthemum alcohol extract.

(3) The preparation method of the diosmin and chrysanthemum aqueous extract composition comprises the following steps: 5 g of diosmin and 25 g of chrysanthemum aqueous extract are mixed uniformly.

(4) The preparation method of the diosmin and chrysanthemum alcohol extract composition comprises the following steps: 5 g of diosmin and 25 g of chrysanthemum alcohol extract are mixed uniformly.

(5) The preparation method of the diosmin and chrysanthemum aqueous extract and chrysanthemum alcoholic extract composition comprises the following steps: 5 g of diosmin, 12.5 g of chrysanthemum aqueous extract and 12.5 g of chrysanthemum alcohol extract, and uniformly mixing.

Diosmin and diosmin composition for in vivo activity evaluation of mice for treating obesity

(I) test materials

Male C57BL/6J mice (6 weeks old), purchased from Experimental animals technology, Inc., Weitonghua, Beijing, were housed in the animal House, Tianjin technology university, with ambient temperature 18-23 deg.C, alternating light and shade at 12 hours/12 hours, and all mice had free access to water. L-carnitine is purchased from Jiangtian chemical technology limited company in Tianjin; acadesine purchased from melphalan organisms; TP23302 low-fat feed, TP23300 (60% high-fat obesity model feed) product, the composition and content of which are shown in Table 1, were purchased from Nantong Telofia feed science and technology Co.

TABLE 1 feed composition and content

(II) Experimental method

1. Evaluation of Activity for treating obesity in animal body

(1) Establishing an obese mouse model:

after one week of adaptive feeding of male C57BL/6J mice with normal diet, 7 mice were randomly selected as normal group and fed with TP23302 low-fat diet, and the remaining 42 mice were fed as model group and fed with TP23300 (60% high-fat obesity model diet). After the mice are fed with the high-fat feed for 12 days, the weight of the mice in the model group is more than 20 percent of that of the mice in the normal group, namely the mice in the obese model are established.

(2) The preparation and administration modes of the medicine are as follows:

diosmin and its composition are dissolved in physiological saline and administered by intragastric administration.

(3) Grouping animals

49 male C57BL/6J mice were acclimatized with the normal diet for one week and then randomized into 7 groups of 7 mice each.

1) Normal mouse group (Low fat feed)

2) Model mouse group (high fat feed)

3) Diosmin group (Diosmin 400mg/kg)

4) Diosmin composition 1 group (diosmin 150mg/kg + chrysanthemum aqueous extract 250mg/kg)

5) Diosmin composition 2 group (diosmin 150mg/kg + chrysanthemum alcohol extract 250mg/kg)

6) Diosmin composition 3 groups (diosmin 150mg/kg + chrysanthemum water extract 125mg/kg + chrysanthemum alcohol extract 125mg/kg)

7) Positive control group (L-carnitine 400mg/kg)

(4) Mouse in vivo experiment evaluation process

After adaptive feeding for one week, feeding the model group mice with high-fat feed for 12 days, and then building an obesity model, wherein the weight of the model group mice is 20% higher than that of the normal group mice. Gavage drug intervention was performed for the next 15 days. The diosmin group is given 400 mg/kg; the diosmin composition 1 is administered with 150mg/kg of diosmin and 250mg/kg of chrysanthemum aqueous extract; the diosmin composition 2 is prepared by taking diosmin 150mg/kg and chrysanthemum alcohol extract 250 mg/kg; the diosmin composition 3 groups are administered with diosmin 150mg/kg, chrysanthemum aqueous extract 125mg/kg and chrysanthemum alcohol extract 125 mg/kg; the positive control group is administrated with 400mg/kg L-carnitine for intragastric administration; the model group was given a saline intragastric lavage. Weighing every 3 days, after 15 days, taking blood from the mouse by taking eyeballs, collecting serum, quickly dissecting out tissues such as liver, kidney, fat and the like, cleaning and weighing. And (3) putting part of liver and adipose tissue into 4% paraformaldehyde fixing solution, and staining fat and liver oil red O. And (4) placing the residual adipose tissues into liquid nitrogen for precooling, and then placing the tissues in a refrigerator at the temperature of minus 80 ℃ for standby.

(III) evaluation of Activity

1) Effects on body weight in obese mice

The model group mice were fed with 12 days of high fat diet, and the normal group mice were fed with 12 days of low fat diet. When 12 days are finished, the body weight of the mice in the model group is more than 20% higher than that of the mice in the normal group, namely the obesity model is successfully modeled. And (4) grouping randomly. The administration is continued for 15 days, and the body weight of each group of mice is recorded every three days, as shown in fig. 1, the body weight of the model group is significantly increased by 26.88% compared with that of the normal group of mice during the previous period of non-administration of high fat diet; in the later period of administration, the body weight of the mice in the model group is continuously improved, but the body weight of the administration group is obviously reduced, the Diosmin and Diosmin composition 1 group is most obvious and is lower than 20.98% and 22.71% of the body weight of the obesity model group, the experimental result shows that the Diosmin and the composition thereof have the activity of reducing the body weight, the application of the composition is favorable for reducing the Diosmin, and the similar effect can be achieved by adding the food-borne chrysanthemum component.

2) Effect on white adipose tissue

White fat is used as the origin of obesity, has great harm to the body, and is a new direction for treating obesity by controlling the generation of white fat. White fat of epididymis, white fat around kidney and white fat of intestinal mucosa of mice are respectively dissected, weighed and morphologically photographed, and it is found that diosmin and the composition thereof both significantly reduce white lipogenesis (fig. 2A), and in white fat of epididymis, white fat around kidney and white fat of intestinal mucosa, dosage administration group of diosmin is reduced by 62.67%, 38.38% and 37.55% compared with model group, dosage of diosmin is reduced by 63.40%, 36.19% and 39.49% compared with positive control L-carnitine group, and both significantly effect is achieved. As shown in FIG. 2B, white fat in epididymis and perirenal area was photographed morphologically, and the results of the experiment showed that diosmin and its composition both inhibited white adipose tissue in each part significantly. The experimental result shows that the diosmin and the composition thereof can improve the generation of white fat, the application of the composition is beneficial to reducing the diosmin, and similar effects can be achieved by adding the food-borne chrysanthemum component.

3) Influence on organs of obese mice

After the mice are dissected, the indexes of organs such as liver, heart, spleen, kidney and the like are analyzed, and the weight of the liver, the kidney and the spleen of the obese mice after administration of the diosmin and the composition thereof is found to be remarkably different from that of a model group, and as shown in table 2, the increase of the indexes of the liver, the kidney and the spleen is inhibited. The experimental result shows that the diosmin and the composition thereof have the effect of relieving the organ enlargement caused by the obesity. The experimental data show that the diosmin and the composition thereof have the effect of relieving organ enlargement caused by obesity after oral administration.

Table 2 effect of diosmin and its compositions on mouse organ index (n-7). P < 0.01 compared to model group. Diosmin is Diosmin, L-carnitine is L-carnitine, DMZH is Diosmin and its composition abbreviation.

Tetrastigmastine and diosmin composition for in vitro activity evaluation of anti-obesity

(I) test materials

The human liver cancer cell line (HepG2) and the mouse embryo fibroblast cell line (3T3-L1) are both from the basic medical research institute of Beijing medical academy of sciences. Oleic acid, palmitic acid, linoleic acid, arachidonic acid, dexamethasone, and 3-isobutyl-1-methylxanthine are all available from sigma. Insulin was purchased from Novonide (China) pharmaceutical Co., Ltd., diosmin was purchased from Annagi chemical reagents Ltd., and lovastatin was purchased from Tianjin Soromen Biotech Ltd. The antibodies AMPK, p-AMPK, ACC, p-ACC and β -action were purchased from Cell Signaling Technology.

(II) Experimental method

1. Evaluation of Activity in animal in vitro treatment of obesity

1) Establishment of a lipid deposition model of HepG2 cells:

HepG2 cells were cultured in DMEM containing 10% fetal bovine serum, 10mg/mL penicillin and 10mg/mL streptomycin at 37 ℃ with 5% CO₂Culturing under the conditions, after inoculating HepG2 cells on a 6-well cell plate for 24 hours, HepG2 cells of a normal group are grown in a DMEM medium without 0.75mm inducer oleic acid, palmitic acid, linoleic acid and arachidonic acid, 29: 47: 18: 6(v/v)) or diosmin and a composition thereof, HepG2 cells of a model group are grown in a DMEM medium containing the inducer but not containing diosmin and a composition thereof, HepG2 cells of an administration group are grown in a DMEM medium containing the inducer and the diosmin and a composition thereof, before staining the cells with oil red O, the cells are washed with 1 × PBS three times, residual lipid components outside the cells are removed, the function of oil red O staining intracellular lipids alone is performed, 4% paraformaldehyde is fixed for 30min, the cells of a different group are washed again with 1 × times, the cells are treated with 60% isopropanol for 5min, then the cells are stained with oil red O solution at room temperature, 1h, the oil red O solution staining is performed with 1h, the distilled water is added to the dark room temperature, the content of the dark room temperature is measured, the content of the oil red O solution is measured, the dark room temperature is added, the dark room temperature is added with the dark room temperature, the dark room temperature is added withThe cells in a 6-well plate are fixed for 30min by 4% paraformaldehyde before staining, then a final concentration of 10 mu g/ml nile red and a final concentration of 1 mu g/ml DAPI solution are added, the cells are stained for 15min at room temperature in a dark condition, after the staining is finished, the nile red and DAPI staining solution are sucked out, the cells are washed for 3 times with 1 × PBS for 5min each time, and then the fluorescent imaging is carried out by inverting a fluorescent microscope to observe the state of captured lipid content.

2)3T3-L1 cell lipid deposition model establishment:

when the 3T3-L1 cells grow to 80% -90%, 1 × 10 is inoculated in a 6-well plate⁶3T3-L1 cells/well were cultured in a medium containing 10% newborn bovine serum. 3T3-L1 cells were cultured for 48h and replaced with medium containing 0.5mM 3-isobutyl-1-methylxanthine, 1. mu. mol/L dexamethasone and 10. mu.g/mL insulin. After 48h, the medium was changed to a medium containing 10. mu.g/mL insulin instead of the original medium. After 48h, the culture medium containing only fetal calf serum is changed to culture for 48h, and the differentiated cells can be used for subsequent experiments. The compound is added into the 3T3-L1 preadipocytes before the differentiation is not induced for action, and the compound is added into the 3T3-L1 mature adipocytes after the differentiation is successfully induced for action. The intracellular lipid content was determined after staining with HepG2 cell oil red O, 3T3-L1 cell oil red O as described above.

3) The evaluation method of the kit for intracellular TG content is as follows:

HepG2 and 3T3-L1 cells at 1 × 10 per well⁵Or 1 × 10⁶The method comprises the steps of inoculating the cells in a 6-well plate, collecting the cells according to the requirements of a kit after different culture and differentiation induction, washing the cells for three times by using precooled 1 × PBS, removing glycerol, adding TG lysate to crack the cells, uniformly mixing the cells and the lysate, cracking the cells at room temperature for 10min, quantifying the protein in the cells, heating the cells at 70 ℃ for 10min after the quantification is finished, separating the cells at the speed of 2000rpm for 5min, using supernatant for enzyme determination, and analyzing the content of TG in the cells according to a settlement formula in a reagent.

4) Western blot detection was as follows:

HepG2 cell and 3T3-L1 fat cell as 1 × 10⁵Or 1 × 10⁶The cell density is inoculated in a small dish, the cell density is placed in an incubator for culture, after the compound is processed, the cell density is continuously placed in the incubator for culture for sample collection time, the cells with different dosing concentrations are respectively collected and centrifuged for 5min at 2500rpm, the centrifugation is repeated twice, protein lysate is added, after the cell is lysed for 1h on ice, the centrifugation is carried out for 20min at 13500rpm, supernatant protein extract is taken and placed at-20 ℃ for storage, the protein concentration is measured, SDS-PAGE electrophoresis is carried out, each group of protein samples are transferred to a PVDF membrane, the PVDF membrane is sealed in 5% skimmed milk 1 × PBS solution, the PVDF membrane is washed with 1 × PBS for four times at room temperature by shaking for 1h, the primary antibody (1: 1000) and the PVDF membrane are incubated for one night at 4 ℃, the membrane is washed with 1 × PBS for four times by shaking for 2h at room temperature, the PVDF membrane is washed with 1 × for four times after the sealing is finished, each time for 10min, finally, the PVDF membrane is visually detected by a chemiluminescence blotting detection system, and the PVDF membrane is processed by Pro through Plus detection.

(III) evaluation of Activity

1. Effect on HepG2 cell model lipid deposition

Fat cells and liver cells are important regulation units of body glycolipid metabolism, and in order to verify the glycolipid metabolism activity of a compound on a cellular level, HepG2 liver cancer cells and 3T3-L1 fat cells are used as models to research the effect of diosmin and a composition thereof on HepG2 lipid accumulation so as to prove the action mechanism of the diosmin and the composition thereof for reducing fat and losing weight. HepG2 caused fat accumulation after treatment with oleic, palmitic, linoleic, and arachidonic acid mixed inducers, and intracellular lipid levels were significantly reduced after treatment with diosmin and its group 1 composition under conditions of high cell viability (fig. 3A). In addition, in the research of the kit for measuring the content of triglyceride in HepG2 cells, the content of triglyceride in the cells of the model group is obviously increased after induction, and the content of triglyceride in the diosmin composition 1 group is reduced by 36 percent compared with the content of triglyceride in the model group (figure 3B), so that the method is the best of the diosmin and the composition group thereof. The nile red and DAPI fluorescent staining can more intuitively reflect the influence of the compound on the lipid content (fig. 3C), and the experimental results show that diosmin and the composition thereof have good effect on the reduction of the lipid content of HepG2 cells.

2. Effect on lipid deposition in 3T3-L1 cell types

The activity of diosmin and the composition thereof on lipid reduction and weight loss in 3T3-L1 preadipocytes and 3T3-L1 mature adipocytes is researched, and as shown in FIGS. 4A-B, the fact that the lipid reduction content of diosmin and the composition of 1 group of diosmin is most obvious in the 3T3-L1 preadipocytes after the diosmin and the composition thereof are treated is found to be better than that of lovastatin at 50% under the same concentration. As shown in fig. 4C, in the 3T3-L1 mature adipocyte model, the content of triglyceride in the model group cells was significantly increased after induction, and after the treatment with diosmin and its composition, the diosmin composition 1 group was reduced by 68% compared with the model group, and the reduction effect was more significant compared with the diosmin group and lovastatin group at the same concentration. The experimental result shows that diosmin and the composition thereof have better lipid-lowering and weight-losing effects on 3T3-L1 fat cells.

3. Effect on the HepG2 cell AMPK pathway

Activation of the intracellular AMPK pathway inhibits fatty acid expression, reduces adipogenesis and produces anti-obesity activity. As shown in fig. 5, compared with the model group, the AMPK activator AICAR group can significantly enhance the phosphorylation levels of AMPK and ACC proteins, so that the p-AMPK/AMPK and p-ACC/ACC levels are increased, and the same results are also shown in the administration group treated by diosmin and the composition thereof, wherein the p-AMPK/AMPK is respectively increased by 443% and 509% and the p-ACC/ACC levels are respectively increased by 406% and 407% after the diosmin composition 1 group and the diosmin are administered; these results show that the diose and the composition thereof in the AMPK pathway of the HepG2 cell can obviously increase the phosphorylation levels of AMPK and ACC, play a role in regulating lipid metabolism and reducing weight, and have more obvious effect.

4. Effect on the 3T3-L1 cell AMPK pathway

After treatment with diosmin and compositions thereof, p-AMPK and p-ACC levels were significantly increased in 3T3-L1 preadipocytes and mature 3T3-L1 adipocytes and, in particular, the diosmin composition 1 group exceeded the positive control 250 μ M AICAR's activation of the AMPK pathway. Experimental results show that diosmin and the composition thereof in 3T3-L1 cells enhance the AMPK and ACC signal transduction activities, the phosphorylation level of the diosmin composition 1 group is strongest, and the effects of activating an AMPK pathway and recovering lipid metabolism disorder obesity are achieved. (see FIG. 6)

Application of penta-diosmin and diosmin

The medicine for treating obesity comprises diosmin serving as an active ingredient and pharmaceutically acceptable carriers or excipients, and the pharmaceutically acceptable carriers or excipients which are prepared into pharmaceutically acceptable dosage forms comprise one or more solid, semisolid or liquid auxiliary materials; the pharmaceutically acceptable dosage forms include tablets, capsules, granules, injections, pills, syrups, powders, ointments, liquid preparations and the like.

Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.

Claims

1. An application of natural plant diosmin in preparing anti-obesity product is disclosed.

2. Use according to claim 1, characterized in that: the structural formula of the natural plant molecule diosmin is as follows:

3. use according to claim 1, characterized in that: the product is functional food, health product or medicine.

4. A natural plant molecular composition containing diosmin for treating obesity is provided.

5. The natural plant molecule composition of claim 4, wherein: the composition comprises the following components in parts by weight: 0.5-5 parts of diosmin, and/or 1-15 parts of chrysanthemum aqueous extract, and/or 1-15 parts of chrysanthemum alcohol extract.

6. The natural plant molecule composition of claim 5, wherein: the preparation method of the chrysanthemum aqueous extract comprises the following steps:

weighing flos Chrysanthemi, pulverizing, adding 95% ethanol water solution and flos Chrysanthemi ethanol water solution at volume ratio of 1: 10-1: 40, reflux extracting for 3-5 hr, filtering, collecting ethanol layer, and evaporating to obtain flos Chrysanthemi ethanol extract.

7. The natural plant molecule composition of claim 5 or 6, wherein: the preparation method of the natural plant molecular composition comprises the following steps:

8. Use of a natural plant molecule composition as claimed in any one of claims 4 to 7 for the manufacture of a product for the treatment of obesity.

9. Use according to claim 8, characterized in that: the product is functional food, health product or medicine.