CN116509918A

CN116509918A - Application of three natural product combinations in preparation of medicines for treating non-alcoholic fatty liver disease

Info

Publication number: CN116509918A
Application number: CN202310534990.6A
Authority: CN
Inventors: 向兰; 戚建华; 易美娟
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2023-05-12
Filing date: 2023-05-12
Publication date: 2023-08-01

Abstract

The invention provides application of three natural product combinations in preparing a medicine for treating non-alcoholic fatty liver, wherein the medicine is prepared from three natural products of peanut skin active components, geniposide and isoquercitrin according to a mass ratio of 16:10:1. According to the invention, through a non-alcoholic fatty liver disease animal model, the effects of reducing the weight and the liver fat content of the peanut coat active component, geniposide and isoquercitrin which are combined can be regulated by the microbial intestinal flora and TLR4-NF kB and AMPK signal paths are proved, and the non-alcoholic fatty liver disease animal model has obvious improvement effect on clinical symptoms of the non-alcoholic fatty liver disease. Can be applied to preparing medicines for treating non-alcoholic fatty liver disease, and also can be applied to preparing foods for preventing and treating non-alcoholic fatty liver disease and health foods.

Description

Application of three natural product combinations in preparation of medicines for treating non-alcoholic fatty liver disease

Technical Field

The invention belongs to the field of medical foods of compounds, and relates to application of three natural product combinations in preparation of medicines for treating non-alcoholic fatty liver, in particular to application of peanut coat active components, geniposide and isoquercetin combinations in preparation of medicines for treating non-alcoholic fatty liver.

Background

Nonalcoholic fatty liver disease refers to a clinical pathological syndrome characterized mainly by excessive deposition of intracellular fat in liver cells, except for alcohol and other definite liver-damaging factors, and is an acquired metabolic stress liver injury closely related to insulin resistance and genetic susceptibility. Including simple fatty liver, nonalcoholic steatohepatitis and liver cirrhosis related to the same, and serious liver cancer can also be caused.

Non-alcoholic fatty liver disease has progressed from a relatively unknown disease to the most common cause of chronic liver disease worldwide over the last 20 years. It is reported that 25% of the population worldwide is currently considered to suffer from non-alcoholic fatty liver disease, and recent studies predict that the incidence of non-alcoholic fatty liver disease will also be increasing, which will lead to a tremendous clinical and economic burden. Therefore, the development of drugs for non-alcoholic fatty liver disease is being advanced.

At present, steady progress is made in elucidating pathogenesis of non-alcoholic fatty liver disease, determining therapeutic targets and promoting drug development, but no drugs for treating the disease are available in the market. As a multifactorial disease, nonalcoholic fatty liver disease involves a complex series of metabolic changes, and many monotherapy effects against nonalcoholic fatty liver disease are clinically undesirable, with no more than 40% of patients benefiting from monotherapy. The natural products often act on multiple targets and have small toxic and side effects, and the combination of multiple natural products has the advantages of improving the curative effect, reducing the side effects and the like. Therefore, the treatment of nonalcoholic fatty liver disease has been increasingly developed after the combination of various natural products.

Peanut skin active ingredient (PSE), geniposide (PC) and Isoquercitrin (IQC) are three pharmaceutically and dietetically homologous active ingredients with anti-aging activity and a single compound. The active component of peanut coat is mainly composed of oligosaccharide and flavonoid, geniposide is an iridoid glucoside, which is the main medicinal component of fructus Gardeniae, and isoquercetin is flavonoid derived from herba Apocyni Veneti. We found that the mixture of peanut coat active ingredient, geniposide and isoquercitrin prepared in a mass ratio of 16:10:1 produced significant prophylactic effects on experimental non-alcoholic fatty liver disease mice by modulating the intestinal flora, AMPK and TLR4-NF kB signaling pathways.

Disclosure of Invention

The invention aims to provide application of three natural product combinations in preparing medicaments for treating non-alcoholic fatty liver diseases, which are prepared by combining active components (PSE) of peanut skin with anti-aging activity, flavonoid compound geniposide (PC) and flavonoid compound Isoquercetin (IQC), wherein the mass ratio of the components is PSE to the PC to the IQC=16 to 10 to 1. The preparation method of peanut coat active component has been published in patent (patent No. ZL 2019 1 0040231.8), mainly comprises oligosaccharide and flavonoid substances, and has geniposide (PC) structure shown in fig. 1 and Isoquercitrin (IQC) structure shown in fig. 2.

It is another object of the present invention to provide the use of the three natural product combinations in health products or foods for weight reduction and liver fat reduction. The three natural products are peanut skin active components (PSE), flavonoid geniposide (PC) and flavonoid Isoquercetin (IQC), and the mass ratio of the components is PSE to PC to IQC=16 to 10 to 1.

The peanut coat active ingredient, geniposide, and isoquercetin may be prepared in accordance with any conventional procedure in a pharmaceutically acceptable carrier or diluent. The pharmaceutically acceptable carrier as described herein refers to a pharmaceutical carrier conventional in the pharmaceutical field, such as diluents, excipients, etc., fillers such as starch, sucrose, microcrystalline cellulose, etc.; binders such as starch slurry, hydroxypropylcellulose, gelatin, polyethylene glycol, etc.; wetting agents such as magnesium stearate, silica gel micropowder, polyethylene glycols, etc.; absorption promoter such as polysorbate, lecithin, etc., surfactant such as poloxamer, sorbitan fatty acid, polysorbate, etc., and other adjuvants such as flavoring agent, sweetener, etc. can be added into the composition.

The dosage forms for administration may be tablets, pills, powders, dispersible tablets, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft capsules, hard capsules, sterile injectable solutions, liniments or suppositories. Can be made into conventional quick release, slow release or delayed release.

The mixture of the present invention may be administered by a variety of routes including oral, intramuscular, subcutaneous, intraperitoneal, intravenous injection, and the like.

The invention has the advantages that the raw materials adopted by the invention are peanut, gardenia and apocynum venetum which are easy to obtain and rich in resources. Peanut coats are used in traditional Chinese medicine for treating hemophilia, primary and secondary thrombocytopenic purpura, liver hemorrhage, etc. Fructus Gardeniae is used for treating icteric hepatitis, sprain and contusion, hypertension, diabetes, etc. in clinical practice. Herba Apocyni Veneti has effects of clearing heat, lowering blood pressure, tonifying heart, promoting urination, and treating heart disease. The three components are prepared into a medicine according to the mass ratio of 16:10:1, and the medicine has remarkable prevention effect on the high-fat-induced non-alcoholic fatty liver disease mice. And has no obvious toxic and side effects.

In the anti-aging activity assay, the above active ingredients and single compounds and mixtures thereof all extended the replicative life of K6001 yeast (FIG. 3).

In the anti-aging activity assay, the above active ingredients and single compounds and mixtures thereof all inhibited etoposide-induced PC12 cell senescence (FIGS. 4-5).

In the in vivo experiments for preventing the non-alcoholic fatty liver disease, the mixture can be found to remarkably reduce the food intake, the weight and the intestinal microbial flora change of a non-alcoholic fatty liver disease model mouse and reduce fat infiltration in the liver compared with a single component or a single compound (figures 6-11). The above mixture can produce the effect of preventing nonalcoholic fatty liver disease by modulating intestinal microbiota and AMPK and TLR4-NF kB signaling pathways (FIGS. 9-13).

Drawings

Fig. 1 is a view of geniposide (PC) structure.

FIG. 2 is a diagram of the structure of Isoquercetin (IQC).

FIG. 3 is the effect of peanut coat active ingredient (PSE), geniposide (PC), isoquercetin (IQC), and mixtures thereof (MIX) on replicative longevity of K6001 yeasts. In the figure: * P <0.05, < P <0.01, < P <0.001 represents the treatment group vs blank group.

FIG. 4 is the effect of PSE, PC, IQC and mixtures thereof (MIX) on etoposide-induced aging PC12 cells. In the figure, the numbers in brackets indicate the proportion of cells stained blue in the field of view (i.e., cells containing the cellular senescence β -galactosidase marker) to all cells.

FIG. 5 is a graph quantifying the effect of PSE, PC, IQC and mixtures thereof (MIX) on etoposide-induced senescent PC12 cells. In the figure, P<0.05，**P<0.01，***P<0.001 represents a model control vs blank, ^# P<0.05， ^## P<0.01， ^### P<0.001 represents the treatment group vs model control group.

FIG. 6 is a graph of the effect of MET, PSE, PC, IQC and MIX (MIX) on body weight, food intake and water intake of non-alcoholic fatty liver disease mice. In the figure: * P (P)<0.05，**P<0.01，***P<0.001 represents the administration group vs high fat diet group, ^# P<0.05， ^## P<0.01， ^### P<0.001 represents a high fat diet group vs normal diet group. Number of animals per group: n=10. ND is normal feed group, HFD is high fat feed group, MET is high fat feed group plus metformin group (positive control), PSE is high fat feed group plus peanut coat active group, PC is high fat feed group plus geniposide group, IQC is high fat feed group plus isoquercitrin group, MIX is high fat feed group plus mixture group (PSE+PC+IQC).

FIG. 7 is the result of hematoxylin & eosin staining of liver of non-alcoholic fatty liver disease mice with MET, PSE, PC, IQC and MIX (MIX).

FIG. 8 is a graph showing the results of oil red O staining of liver of non-alcoholic fatty liver disease mice by MET, PSE, PC, IQC and MIX (MIX).

FIG. 9 is the effect of MET, PSE and Mixture (MIX) on the diversity of intestinal flora α in non-alcoholic fatty liver disease mice.

FIG. 10 is the effect of MET, PSE and Mixture (MIX) on β diversity in intestinal flora of non-alcoholic fatty liver disease mice.

FIG. 11 is the effect of MET, PSE and Mixture (MIX) on the composition of non-alcoholic fatty liver disease mice intestinal flora at the portal level.

FIG. 12 is the effect of MET, PSE and Mixture (MIX) on the composition of the intestinal flora of non-alcoholic fatty liver mice at the genus level.

FIG. 13 is the effect of MET, PSE, PC, IQC and Mixture (MIX) on inflammatory factor levels in non-alcoholic fatty liver disease mice. In the figure: * P (P)<0.05，**P<0.01，***P<0.001 represents the administration group vs high fat diet group, ^# P<0.05， ^## P<0.01， ^### P<0.001 represents a high fat diet group vs normal diet group.

FIG. 14 is the effect of MET, PSE, PC, IQC and Mixture (MIX) on the levels of AMPK and TLR4-NF kB signaling pathway proteins in non-alcoholic fatty liver disease mice.

Detailed Description

The invention is further described with reference to the drawings and examples.

Example 1 peanut coat active ingredient extraction and ingredient analysis (see patent ZL 2019 1 0040231.8 for details)

The experimental method comprises the following steps:

extracting peanut skin with 60% ethanol for 2 times, 1 hr for the first time and 0.5 hr for the second time, vacuum filtering, concentrating to 1/50 of the original volume, separating with macroporous adsorbent resin HP-20, eluting with 40% ethanol, and collecting the fraction as active component of peanut skin. The peanut coat active component is separated into two parts by High Performance Liquid Chromatography (HPLC) with the following conditions: development ODS-UG-5 (. Phi.20X1250 mm), nomura Chemical column, flow rate of 6ml/min, isocratic elution with 18% methanol, and collection as saccharide component; 15-25min, 18-100% methanol gradient elution, 25-40min,100% methanol isocratic elution, and collecting as non-saccharide component. The saccharide and non-saccharide fraction fractions were then analyzed by spectroscopic analysis, high resolution mass spectrometry and chemical derivatization methods.

Experimental results:

analysis of these two fractions confirmed that the two fractions were a saccharide fraction and a non-saccharide fraction, respectively. The saccharide part mainly comprises arabinose, xylose, D- (+) -inositol, myo-inositol, mannose, glucose and galactose, and the molar ratio is 3:6.9:1:1.5:3.2:21.5:3.8. The non-sugar moiety consists of polyphenols, mainly including dimers, trimers and tetramers of procyanidins of type a or type B (table 1).

TABLE 1 Main chemical Components of peanut coat active ingredients

Example 2 Effect of peanut coat active ingredient (PSE), geniposide (PC), isoquercetin (IQC), and mixtures thereof (MIX) on replicative longevity of K6001 Yeast

The experimental method comprises the following steps:

the effect of PSE, PC, IQC alone or after mixing on replicative life of K6001 yeasts was determined using the K6001 yeast system (a system for evaluating anti-aging activity of compounds). K6001 yeast cells frozen at-30℃were first inoculated into 5 ml of galactose liquid medium (containing 3% galactose, 2% polypeptone and 1% yeast extract) and cultured with shaking at 28℃for 24 hours. The next day 5 ml of glucose solid medium (containing 2% glucose, 2% polypeptone, 2% agar and 1% yeast extract) was poured into small glass dishes, and 150 μl of absolute ethanol or different concentrations of sample solution were added to each dish after the medium solidified. After the solvent had evaporated, 1 ml of yeast cells incubated for 24 hours were taken in a centrifuge tube and washed three times with 9 ml of PBS. The washed yeast cells were counted by a blood cell counting plate, and about 4000 yeast cells were uniformly spread in a small dish and subjected to stationary culture at 28℃for 48 hours. Each dish was randomly picked under a microscope for 40 microcolonies and the number of subcellular (i.e., algebraic) of each microcolonie was calculated. The experiment uses resveratrol with the concentration of 2.282 mug/mL as a positive control.

Experimental results:

PSE significantly extended K6001 yeast replicative life at 3 and 10 μg/mL (FIG. 3A), PC significantly extended K6001 yeast replicative life at 3.884 μg/mL (FIG. 3B), IQC significantly extended K6001 yeast replicative life at 1.393 and 4.644 μg/mL (FIG. 3C), and mixtures thereof significantly extended K6001 yeast replicative life at 1 μg/mL PSE+0.0625 μg/mL PC+0.0625 μg/mL and 3 μg/mL PSE+1.875 μg/mL PC+0.1875 μg/mL IQC concentrations with average algebraic higher than these three components acting alone (FIG. 3A-FIG. 3D). The above results demonstrate that a better prolongation of the replicative life of K6001 yeast can be achieved by combining PSE, PC and IQC.

Example 3 Effect of PSE, PC, IQC and mixtures thereof (MIX) on Etoposide-induced aging PC12 cells

The experimental method comprises the following steps:

PC12 cells were inoculated into a 24-well plate containing CM culture solution, after culturing in a carbon dioxide incubator at 37℃for 1 day, CM culture solution was changed to EM culture solution containing no-use sample concentration, after culturing in an incubator at 37℃for another 1 day, culture solution was changed to EM culture solution containing etoposide at 1 micromolar concentration, and culturing was continued for another 1 day (the blank control group was replaced with EM culture solution containing no etoposide). After washing with PBS, 200. Mu.l of 4% paraformaldehyde was added, and after fixation for 15 minutes at room temperature, the cell fixative was aspirated, after washing 3 times with PBS, 200. Mu.l of staining working fluid (containing 10. Mu.l of beta-galactosidase staining fluid A, 10. Mu.l of beta-galactosidase staining fluid B, 930. Mu.l of beta-galactosidase staining fluid C and 50. Mu.l of X-Gal solution) was added, and the mixture was sealed with a preservative film to avoid evaporation of the solution and incubated overnight in a 37℃incubator without carbon dioxide. Cells were observed under a normal light microscope and the proportion of cells stained blue in the field of view (i.e., cells containing the cell senescence β -galactosidase marker) to all cells was calculated. The experiment uses rapamycin at a concentration of 0.457 μg/mL as a positive control.

Experimental results:

the negative control group had 14.95% of cells that were positive for age-related beta-galactosidase, while etoposide induced aging of PC12 cells, making 35.76% of cells positive for age-related beta-galactosidase (fig. 4). Etoposide-induced PC12 cells exhibited various degrees of reduction in senescence-associated beta-galactosidase positives following addition of various concentrations of PSE, PC, IQC and mixtures thereof, with 0.1, 0.3, 1, 3, 10. Mu.g/mL PSE,0.388, 1.165, 3.884. Mu.g/mL PC,0.464, 1.393, 4.644. Mu.g/mL IQC and mixtures thereof (mixed at 16:10:1) being most effective (FIGS. 4-5). These results indicate that combining PSE, PC and IQC inhibits etoposide-induced aging of PC12 cells.

Example 4 influence of mixture of PSE, PC, IQC (MIX) on weight, food intake and Water intake of non-alcoholic fatty liver disease mice

The experimental method comprises the following steps:

in this example, six week old male mice were divided into seven groups of ten mice. The normal feed group (ND) was drenched with water and normal diet. The High Fat Diet (HFD) control group was infused with the same amount of vehicle and fed the high fat diet ad libitum. The high fat diet plus metformin group (MET, positive control) was infused with metformin at a dose of 140 mg/kg body weight/day and fed ad libitum with high fat diet and drinking water. The high-fat feed and peanut coat active ingredient group (PSE) are infused with the peanut coat active ingredient according to the dosage of 80 mg/kg body weight/day and the high-fat feed and drinking water are freely taken. The high-fat feed plus geniposide group (PC) is infused with geniposide according to the dosage of 50 mg/kg body weight/day and can freely eat the high-fat feed and drink water. The high-fat feed plus isoquercitrin group (IQC) is infused with isoquercitrin at a dosage of 5 mg/kg body weight/day and fed with high-fat feed and drinking water freely. The high-fat diet plus MIX group (MIX, PC, IQC 80, 50, 5 mg/kg body weight/day) was infused with the MIX and was free to ingest the high-fat diet and drinking water. The experimental period was 12 weeks, and body weight, food intake, and water intake were recorded weekly.

Experimental results:

the mice in the HFD group had a significantly increased body weight and feed intake compared to the mice in the ND group (FIG. 6). The MET and PSE mice had significantly less body weight, no significant change in PC and IQC group body weight, and the MIX group body weight was most reduced compared to the HFD group (fig. 6A). The HFD group had significantly increased food intake and each treatment group had decreased food intake relative to the HFD group (FIG. 6B). However, the water intake of the HFD group was reduced relative to the ND group, as was the water intake of each treatment group (FIG. 6C). Thus, the MIX group had a remarkable weight-reducing effect.

EXAMPLE 5 Effect of mixture of PSE, PC, IQC (MIX) on non-alcoholic fatty liver disease mouse tissue and serum Triglycerides, total cholesterol and liver function index

The experimental method comprises the following steps:

after the last administration, the animals are fasted without water forbidding for 12 hours, blood is taken to detect indexes of TG (triglyceride), TC (total cholesterol), AST (aspartate aminotransferase) and ALT (alanine aminotransferase) in serum, and tissues such as liver, fat, spleen, kidney and the like are taken for weighing.

Experimental results:

comparing the tissue weights of the groups (table 2), it was found that the fat of MET, PSE and MIX groups was significantly reduced relative to the HFD group, the liver weights of the administered groups and HFD groups were significantly different, and the MIX group liver weight was most reduced. Therefore, MIX has a remarkable effect of reducing the weight of fat and liver tissue.

TABLE 2 influence of mixture of PSE, PC, IQC (MIX) on tissue weight of non-alcoholic fatty liver disease mice

In addition, the serum indices were compared (Table 3), and it was found that there was a significant difference between the serum indices of each administration group and the HFD group, indicating that MIX improved liver function index in mice.

TABLE 3 influence of mixture of PSE, PC, IQC (MIX) on serum index of non-alcoholic fatty liver disease mice

Group of	ALT(U/L)	AST(U/L)	TG(mmol/L)	TC(mmol/L)
					ND	34.67±4.96	87.74±6.79	1.58±0.18	2.96±0.30
HFD	51.85±7.89 ^##	142.33±26.34 ^###	1.96±0.21 ^##	4.16±0.37 ^###
					MET	24.14±3.79 ^***	96.23±14.56 ^**	1.11±0.17 ^***	3.50±0.35 ^*
PSE	26.91±5.80 ^***	95.69±16.07 ^**	1.03±0.11 ^***	3.68±0.37
					PC	24.27±4.15 ^***	92.34±13.08 ^**	0.81±0.14 ^***	3.44±0.18 ^**
IQC	28.04±2.32 ^***	87.49±10.77 ^***	0.83±0.12 ^***	3.64±0.37
					MIX	25.6±3.95 ^***	86.82±12.66 ^**	0.82±0.12 ^***	3.73±0.14 ^*

In this test, ALT stands for alanine aminotransferase, AST stands for aspartate aminotransferase, TG for triacylglycerol, and TC for total cholesterol; ^#，##，### shows that the high-fat feed group is compared with the normal feed group P<0.05,P<0.01,P<0.001； ^*，**，*** The expression of P in the administration group compared with the high-fat diet group<0.05,P<0.01,P<0.001; the number of animal samples was 8.

Example 6 Effect of mixture of PSE, PC, IQC (MIX) on liver pathology in non-alcoholic fatty liver disease mice

The experimental method comprises the following steps:

the livers of mice were taken and fixed in 4% paraformaldehyde solution for 48 hours, a portion of the livers after fixation was embedded with paraffin to prepare paraffin sections (section thickness 3 μm), another portion was embedded with OCT after gradient dehydration with 15% and 30% sucrose solution to prepare frozen sections (section thickness 10 μm), the paraffin sections were subjected to hematoxylin & eosin staining, and the frozen sections were subjected to oil red O staining.

Experimental results:

hematoxylin & eosin staining results showed that HFD group had severe hepatocyte damage, and administration group was able to reduce hepatocyte damage in mice, with MIX group having the best effect of reducing hepatocyte damage (fig. 7). Oil red O staining showed severe liver fat accumulation in HFD groups, with the MIX group reducing liver fat content the most (fig. 8). The results show that the mixture MIX of PSE, PC, IQC can reduce liver fat content in non-alcoholic fatty liver disease mice and alleviate liver cell damage in non-alcoholic fatty liver disease mice.

Example 7 effect of pse, pc, iqc Mixture (MIX) on intestinal microbiota of non-alcoholic fatty liver disease mice.

The experimental method comprises the following steps:

after 12 weeks of dosing, the cages were cleaned and alcohol sterilized and samples of approximately 100 mg (2-3 grains) of the faeces of the mice from group ND, HFD, MET, PSE and MIX were collected. DNA was extracted and the 16S rDNA V4 region of the sample was amplified according to the designated sequencing region. And PE250 sequencing analysis was performed using a Hiseq 2500 instrument. The experimental results were analyzed for intra-group species diversity and inter-group species differences.

Experimental results:

the results of the analysis of the α diversity of the intestinal microbiota of mice (fig. 9) showed that the HFD group significantly reduced the species richness of the intestinal microbiota of mice, while each dosing group restored it to the same level as the ND group. Beta diversity analysis (fig. 10) showed that HFD group significantly changed the mice intestinal flora composition, whereas each dosing group had an intestinal flora composition close to ND group, with MIX group intestinal flora composition closest to ND group. The species analysis results showed that at the portal level, the abundance of MIX group Bacteroidota (bacteroides) increased (fig. 11). This is mainly because at the genus level, MIX group significantly increases the abundance of lactobacillus salivarius (fig. 12), a beneficial bacterium having antibacterial, immune and intestinal microbial action, and studies have demonstrated that lactobacillus salivarius can improve nonalcoholic fatty liver disease.

The results show that the mixture MIX of PSE, PC and IQC can regulate the intestinal microbial environment homeostasis of non-alcoholic fatty liver disease mice.

Example 8 effect of pse, pc, iqc Mixture (MIX) on inflammatory factor levels in non-alcoholic fatty liver disease mice.

The experimental method comprises the following steps:

mouse liver 30-50 mg collected after dissection was taken, 500. Mu.l PBS and 5. Mu.l protease inhibitor were added, the tissues were ground, and supernatant was collected after centrifugation at 12000rpm and 4℃to obtain a mouse liver protein solution, and mouse liver and serum LPS (lipopolysaccharide), TNF-. Alpha. (tumor necrosis factor) and IL-6 (interleukin-6) levels were measured using an Elisa kit. In addition, 30-50 mg of the feces of the mice collected at 12 weeks of administration were taken, milled with PBS, centrifuged, the supernatant was taken, filtered with a 0.22 μm filter membrane, and inactivated in a water bath at 90℃for 15 minutes, and the LPS level was measured with an Elisa kit.

Experimental results:

inflammatory factor level measurements are shown in figure 13. The HFD group significantly increased the levels of mouse fecal LPS and serum and liver LPS, TNF- α and IL-6, while the levels of these inflammatory factors were significantly reduced in each of the dosing groups (FIGS. 13A-G). The results indicate that MIX is able to reduce the inflammatory level in mice.

Example 8 effect of PSE, PC, IQC Mixture (MIX) on protein expression levels in non-alcoholic fatty liver disease mice.

The experimental method comprises the following steps:

the method comprises the steps of taking 30-50 mg of mouse liver collected after dissection of the mouse, adding 500 microliter of RIPA lysate, 5 microliter of protease inhibitor and phosphorylation inhibitor, grinding tissues, centrifuging, collecting supernatant to obtain a mouse liver protein solution, and taking the same amount of protein solution from each group for immunoblotting after measuring protein concentration so as to measure the expression level of the mouse liver protein.

Experimental results:

the results of protein expression level measurement are shown in FIG. 14. The HFD group significantly increased the expression levels of inflammation-associated proteins such as mouse liver TLR4 (Toll-like receptor 4), phosphorylated IKKK beta (activating factor-stimulated enzyme inhibitor beta), phosphorylated IKB alpha (nuclear factor kappa B-inhibited protein alpha), etc., while the expression levels of these proteins were significantly decreased in each of the administration groups (FIG. 14A). While protein expression levels associated with fatty acid synthesis and metabolism were reduced in HFD groups, phosphorylated AMPK (adenylate activated protein kinase) and phosphorylated ACC (acetyl CoA carboxylase), etc., these protein expression levels were significantly increased in each of the administration groups (FIG. 14B). The result shows that MIX can regulate TLR4-NF kB and AMPK signal paths, thereby achieving the effect of preventing nonalcoholic fatty liver diseases.

In this test, 1,2 is ND, 3,4 is HFD, 5,6 is MET, 7,8 is PSE, 9,10 is PC, 11,12 is IQC, and 13,14 is MIX.

Claims

1. The application of the combination of three natural products in preparing the medicine for treating the non-alcoholic fatty liver disease is characterized in that the three natural products are peanut skin active components, geniposide and isoquercitrin, and the combination mass ratio is 16:10:1.

2. The use according to claim 1, wherein the medicament is in the form of a solid or liquid formulation and the route of administration is enteral or parenteral.

3. The application of the combination of three natural products in preparing health care products or foods for reducing weight and liver fat content is characterized in that the three natural products are peanut skin active components, geniposide and isoquercitrin, and the combination mass ratio is 16:10:1.

4. The use according to claim 3, wherein the formulation of the health product or food is a solid or liquid formulation.