CN110559331A - fructus Siraitiae Grosvenorii extract with obesity and fatty liver inhibiting effect, and its preparation method - Google Patents

Abstract

The invention discloses a momordica grosvenori extract and a preparation method and application thereof. The preparation method of the momordica grosvenori extract comprises the following steps: 1) cleaning fresh fructus Siraitiae Grosvenorii, adding water, and pulping to obtain pulp; 2) adding compound pectinase into the pulp for enzymolysis 1; then adding active yeast for enzymolysis 2; after the enzymolysis 2 is finished, centrifuging and collecting supernatant; and adding active carbon into the supernatant for adsorption treatment, filtering, and collecting filtrate to obtain the fructus momordicae extract. The extract can be used as fat metabolism promoter for inhibiting obesity and fatty liver. The action mechanism of the component may relate to the activation of AMPK, the promotion of ECH1 expression, the promotion of energy and fat metabolism, and the reduction of abdominal fat weight and body weight of obese mice. The component can reduce liver injury, lower ALT and AST enzyme activity in blood, and protect liver.

Description

fructus Siraitiae Grosvenorii extract with obesity and fatty liver inhibiting effect, and its preparation method

Technical Field

the invention belongs to the technical field of biological medicines, and particularly relates to a momordica grosvenori extract with the function of inhibiting obesity and fatty liver and a preparation method thereof.

Background

obesity is caused by a long-term imbalance in energy intake and metabolism, including nutrient excess and lifestyle changes. With the increase in the living standard of people, obesity has been widely prevalent worldwide, and the obesity rate in developed countries such as the united states has rapidly increased after 1980 but now has become smooth, but the obesity rate in developing countries is rapidly increasing. Worldwide, the number of obese people now doubles in 1980, and has exceeded 5 hundred million people. Besides the problems of body shape, metabolism, psychology and the like caused by the obesity, the complications caused by the obesity become the 'invisible killer' of the human beings. Complications such as hypertension, diabetes, global chronic kidney disease, cardiovascular disease, fatty liver, etc. can be caused by obesity.

currently, the over-the-counter drug most used in the obesity treatment drugs is the lipase inhibitor Orlistat (Orlistat)^[1]. Orlistat was the first gastrointestinal lipase inhibitor approved by the FDA^[2]it can reduce fat absorption by inhibiting lipase activity to reduce weight loss, and has certain regulation effect on blood pressure, blood fat and insulin resistance^[3]. However, orlistat not only causes flatulence, diarrhea, dyspepsia and other side effects, but also is reported to cause acute liver and kidney injury^[4]. Other weight-reducing medicines have serious side effects, and an ideal prevention and treatment medicine is still lacking at present.

research shows that obesity is often complicated by the occurrence of fatty liver^[5]. Fatty liver has become the most common cause of chronic liver disease and is one of the major global health problems^[6]. Insulin resistance, chronic systemic inflammation and dyslipidemia due to fatty liver disease, which has led to fatty liver being associated with many chronic diseases, such as diabetes and cardiovascular diseases^[7]. Wherein, the liver inflammation caused by the serious fatty liver can increase AST and ALT enzyme activity in blood, and in fact, most liver cell injuries can cause the enzyme activity of two enzymes in the blood to increase^[8]. Obesity and fatty liver formation are associated with high-fat or high-calorie food intake, and slow metabolism^[9]. At present, ideal medicines are also lacking in prevention and treatment of fatty liver.

the liver is one of the major organs of fat oxidative metabolism. Adenylate kinase (AMPK) is a key factor in the regulation of energy metabolism, and AMPK activation such as phosphorylation of AMPK can promote lipid or energy metabolism. Mitochondria and peroxisomes are the major organelles responsible for the oxidative metabolism of fat. enoyl-CoA hydratase 1(ECH1) is a key enzyme of lipid metabolism, and dominates the oxidative metabolism of fat in peroxisomes^[10]The up-regulation of ECH1 means the increase of fat oxidation metabolism in peroxisome in liver, thereby reducing the accumulation of liver fat and achieving the effects of losing weight and treating fatty liver.

Fructus Siraitiae Grosvenorii is fruit of perennial vine of Cucurbitaceae, and has effects of moistening lung, relieving cough, promoting fluid production, quenching thirst, and loosening bowel to relieve constipation^[11]. Mogroside V is one of the effective components, but there is no report on the application of obesity and fatty liver.

[1]Narayanaswami V,Dwoskin L P.Obesity:Current and potential pharmacotherapeutics and targets[J].Pharmacology&Therapeutics,2016,170:116.

[2]Mcneely W,Benfield P.Orlistat.[J].Drugs,1998,56(2):241-249.

[3]Siebenhofer A,Jeitler K,Berghold A,et al.Long-term effects of weight-reducing diets in hypertensive patients.[M]//The Cochrane Library.John Wiley&Sons,Ltd,2009:CD008274.

[4]And C F D E.Postmarket Drug Safety Information for Patients and Providers-FDA Drug Safety Communication:Completed safety review of Xenical/Alli(orlistat)and severe liver injury[J].

[5]Maher J J.New Insights from Rodent Models of Fatty Liver Disease[J].Antioxidants&Redox Signaling,2011,15(2):535-50.

[6]Mikolasevic I,Milic S,Turk W T,et al.Nonalcoholic fatty liver disease-A multisystem disease？[J].World Journal of Gastroenterology,2016,22(43):9488-9505.

[7]Bang K B,Kyun C Y.Comorbidities and Metabolic Derangement of NAFLD[J].Journal of Lifestyle Medicine,2015,5(1):7-13.

[8]Giannini E,Botta F,Fasoli A,et al.Progressive Liver Functional Impairment Is Associated with an Increase in AST/ALT Ratio[J].Digestive Diseases&Sciences,1999,44(6):1249-1253.

[9]Gortmaker S L,Swinburn B A,Levy D,et al.Changing the future of obesity:science,policy,and action[J].Lancet,2011,378(9793):838-847.

[10]Xie W,Zhang S,Lei F,et al.Ananas comosus L.Leaf Phenols and p-Coumaric Acid Regulate Liver Fat Metabolism by Upregulating CPT-1Expression[J].Evidence-Based Complementray and Alternative Medicine,2014,(2014-8-12),2014,2014(15):903258.

[11]Liu D D,Ji X W,Li R W.Effects of Siraitia grosvenorii Fruits Extracts on Physical Fatigue in Mice[J].Iranian Journal of Pharmaceutical Research,2013,12(1):115-121.

Disclosure of Invention

An object of the present invention is to provide a momordica grosvenori extract.

The fructus momordicae extract provided by the invention is prepared by the method comprising the following steps:

1) Cleaning fresh fructus Siraitiae Grosvenorii, placing in a fruit crusher, adding water, and pulping to obtain pulp;

2) Adding compound pectinase into the pulp for enzymolysis 1; then adding active yeast for enzymolysis 2; after the enzymolysis 2 is finished, centrifuging and collecting supernatant; and adding active carbon into the supernatant for adsorption treatment, filtering, and collecting filtrate to obtain fructus Siraitiae Grosvenorii extract solution.

In the method, the water in the step 1) is hot water at the temperature of 50-100 ℃. The mass ratio of the fresh momordica grosvenori to the added water is 1: 0.5-5, preferably 1:1-2, such as 1: 1.5.

The fruit crusher which can ensure the full crushing of the fruits and the integrity of the momordica grosvenori seeds is preferably selected in the step 1).

In the method, the compound pectinase in the step 2) consists of pectinase, cellulase and compound plant hydrolase; the mass ratio of the pectinase to the cellulase to the composite plant hydrolase is 1-7: 0.1-4: 0.1-4, specifically 7: 2: 1.

The enzyme activity of the pectinase is 14000-20000U/g; the enzyme activity of the cellulase is 700-1000U/g, and the enzyme activity of the composite plant hydrolase is 800-1000U/g.

The pectinase, cellulase and complex plant hydrolase are all available from Novozymes (Novozymes) or Japan Wildlife corporation (Amano Enzyme).

Pectinase, such as Novozymes, Catalogue number Pectinex Ultra SP-L; novozymes, a cellulase available under the catalog number Celluclast; novozymes, Inc. (Novozymes), Catalogue number Viscozyme L complex plant hydrolases.

The addition amount of the compound pectinase is 0.5-15 Kg/per ton of fresh fructus Siraitiae Grosvenorii, preferably 5-12 Kg/per ton of fresh fructus Siraitiae Grosvenorii, specifically 10 Kg/per ton of fresh fructus Siraitiae Grosvenorii.

the conditions of the enzymolysis 1 are as follows: enzymolysis is carried out for 1-2 hours at 20-55 ℃ (preferably 50-55 ℃).

The active yeast can be edible active dry yeast or fresh yeast used for brewing wine, food fermentation and the like. The active yeast is specifically available from Angel yeast.

The adding amount of the active yeast is 0.5-20 Kg/ton of fresh fructus momordicae, preferably 5-15 Kg/ton of fresh fructus momordicae.

The conditions of the enzymolysis 2 are as follows: enzymolysis is carried out for 1-10 hours (preferably 3 hours) at 20-55 ℃ (preferably 40-50 ℃).

the using amount of the activated carbon is 0.5-5% of the mass of the supernatant, and the preferable using amount is 1%.

The conditions of the adsorption treatment are as follows: stirring and preserving heat for 10-60 minutes (preferably preserving heat for 30 minutes) at 30-100 ℃ (preferably 80-90 ℃).

The method further comprises the following purification steps: passing the fructus Siraitiae Grosvenorii extract solution through plate-and-frame filtration equipment, passing through ion exchange resin LX-T5 when the medicinal liquid is cooled to normal temperature, collecting effluent, concentrating, and drying to obtain fructus Siraitiae Grosvenorii extract product (GO-Luo).

Another purpose of the invention is to protect the application of the fructus momordicae extract.

the momordica grosvenori extract provided by the invention has at least one application as follows:

1) Preparing a product for preventing and/or treating obesity;

2) preparing a product for preventing and/or treating fatty liver;

3) Preparing a fat metabolism promoter;

4) Preparing a liver protection product;

5) preparing a product for reducing the weight of the liver;

6) Preparing a product for reducing abdominal fat weight;

7) Preparing a product for promoting the expression of p-AMPK (phosphorylated adenylate activated protein kinase);

8) A product is prepared which promotes the expression of the ECH1 (enoyl hydratase coenzyme 1) protein.

The invention mainly provides a natural medicine active component fructus momordicae extract (Go-Luo), the extract contains 45-55% of fructus momordicae saponin V, and the extract can be used as a fat metabolism promoter and used for inhibiting obesity and fatty liver formation. The action mechanism of the component may relate to the activation of AMPK, the promotion of ECH1 expression, the promotion of energy and fat metabolism, and the reduction of abdominal fat weight and body weight of obese mice. The component can reduce liver injury, lower ALT and AST enzyme activity in blood, and protect liver.

In addition, the invention also protects various products prepared by taking the momordica grosvenori extract as an active ingredient; the product can be specifically a medicine or a health-care product.

the administration route of the momordica grosvenori extract provided by the invention is oral administration. Optionally, one or more pharmaceutically acceptable carriers can be added into the fructus Siraitiae Grosvenorii extract. The carrier comprises a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant and a synergist which are conventional in the pharmaceutical field. The medicine can be in the form of liquid, dry powder or extract, and can be made into various dosage forms with health promotion or medicinal effects such as capsule, tablet, injection, pill, granule, powder, etc. with various conventional adjuvants, and can be used alone or in combination with other medicines for preventing obesity and protecting liver.

the invention has the following beneficial effects: the action mechanism of the momordica grosvenori extract provided by the invention is mainly characterized in that ECH1 is up-regulated by activating liver AMPK, and energy and fat metabolism are promoted, so that the purpose of preventing obesity and fatty liver is achieved; and has effects of reducing serum ALT and AST enzyme activity, and protecting liver. The extract is a natural active component of medicine, and has low side effect and high safety.

Drawings

FIG. 1 is an HPLC chart of Go-Luo (LH); 1. 11-oxo-mogroside V, 2, mogroside V, 3 and mogroside IV.

Figure 2 is the mean body weight of mice in each group and data are presented as mean ± standard deviation (n 10); P <0.05, # P <0.01vs Model, # P <0.01vs Normal. Normal, Normal control group; model, high fat Model group; LH200, Lo Han Guo extract low dose group (200 mg/kg); LH400, Lo Han Guo extract medium dose group (400 mg/kg); LH800, Lo Han Guo extract bulk group (800 mg/kg); OL60, orlistat (60 mg/kg).

Figure 3 is the mean liver weight of mice in each group and data are presented as mean ± standard deviation (n 10); P <0.05, # P <0.01vs Model, # P <0.01vs Normal. Normal, Normal control group; model, high fat Model group; LH200, Lo Han Guo extract low dose group (200 mg/kg); LH400, Lo Han Guo extract medium dose group (400 mg/kg); LH800, Lo Han Guo extract bulk group (800 mg/kg); OL60, orlistat (60 mg/kg).

Figure 4 is the mean weight of abdominal fat in each group of mice, and data are presented as mean ± standard deviation (n 10); P <0.05, # P <0.01vs Model, # P <0.01vs Normal. Normal, Normal control group; model, high fat Model group; LH200, Lo Han Guo extract low dose group (200 mg/kg); LH400, Lo Han Guo extract medium dose group (400 mg/kg); LH800, Lo Han Guo extract bulk group (800 mg/kg); OL60, orlistat (60 mg/kg). .

Figure 5 is the mean food intake of mice in each group and data are presented as mean ± standard deviation (n-10) — P <0.05 vsModel. Normal, Normal control group; model, high fat Model group; LH200, Lo Han Guo extract low dose group (200 mg/kg); LH400, Lo Han Guo extract medium dose group (400 mg/kg); LH800, Lo Han Guo extract bulk group (800 mg/kg); OL60, orlistat (60 mg/kg); NCD, normal food; HFD, high fat food.

Fig. 6 shows blood glucose values for each group of mice, and data are presented as mean ± standard deviation (n ═ 10), # # P <0.01 vsNormal. Normal, Normal control group; model, high fat Model group; LH200, Lo Han Guo extract low dose group (200 mg/kg); LH400, Lo Han Guo extract medium dose group (400 mg/kg); LH800, Lo Han Guo extract bulk group (800 mg/kg); OL60, orlistat (60 mg/kg).

Figure 7 is the serum total cholesterol level for each group of mice, and the data are presented as mean ± standard deviation (n 10), # # P <0.01vs Normal. Normal, Normal control group; model, high fat Model group; LH200, Lo Han Guo extract low dose group (200 mg/kg); LH400, Lo Han Guo extract medium dose group (400 mg/kg); LH800, Lo Han Guo extract bulk group (800 mg/kg); OL60, orlistat (60 mg/kg).

Figure 8 is a graph of serum triglyceride content for each group of mice, with data presented as mean ± standard deviation (n-10). Normal, Normal control group; model, high fat Model group; LH200, Lo Han Guo extract low dose group (200 mg/kg); LH400, Lo Han Guo extract medium dose group (400 mg/kg); LH800, Lo Han Guo extract bulk group (800 mg/kg); OL60, orlistat (60 mg/kg).

Figure 9 is the triglyceride content per mg of feces in the mice in each group and the data are presented as mean ± standard deviation (n 10), # # P <0.01vs Model. Normal, Normal control group; model, high fat Model group; LH200, Lo Han Guo extract low dose group (200 mg/kg); LH400, Lo Han Guo extract medium dose group (400 mg/kg); LH800, Lo Han Guo extract bulk group (800 mg/kg); OL60, orlistat (60 mg/kg).

FIG. 10 shows the fatty liver condition of each group. Normal, Normal control group; model, high fat Model group; LH200, Lo Han Guo extract low dose group (200 mg/kg); LH400, Lo Han Guo extract medium dose group (400 mg/kg); LH800, Lo Han Guo extract bulk group (800 mg/kg); OL60, orlistat (60 mg/kg).

FIG. 11 shows pathological liver sections of mice in each group. Normal, Normal control group; model, high fat Model group; LH200, Lo Han Guo extract low dose group (200 mg/kg); LH400, Lo Han Guo extract medium dose group (400 mg/kg); LH800, Lo Han Guo extract bulk group (800 mg/kg); OL60, orlistat (60 mg/kg).

FIG. 12 shows pathological abdominal fat sections of mice in each group. Normal, Normal control group; model, high fat Model group; LH200, Lo Han Guo extract low dose group (200 mg/kg); LH400, Lo Han Guo extract medium dose group (400 mg/kg); LH800, Lo Han Guo extract bulk group (800 mg/kg); OL60, orlistat (60 mg/kg).

FIG. 13 shows the expression of AMPK and ECH1 proteins in liver of each group. Normal, Normal control group; model, high fat Model group; LH200, Lo Han Guo extract low dose group (200 mg/kg); LH400, Lo Han Guo extract medium dose group (400 mg/kg); LH800, Lo Han Guo extract bulk group (800 mg/kg); OL60, orlistat (60 mg/kg).

Detailed Description

The present invention is described below with reference to specific embodiments, but the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

The pectinase used in the following examples is purchased from Novozymes corporation (Novozymes), the catalog number is Pectinex Ultra SP-L, and the enzyme activity is 14000-20000U/g; the cellulase used was purchased from Novozymes corporation (Novozymes), catalog number Celluclast, enzyme activity 700U/g; the compound plant hydrolase is purchased from Novozymes corporation (Novozymes), the catalog number is Viscozyme L, and the enzyme activity is 10000-20000U/g; the active yeast used was purchased from Angel Yeast, catalog number BV 818.

Example 1 preparation of Momordica grosvenori extract (named Go-Luo, hereinafter substituted for LH)

1.1 preparation steps:

5000g of fresh momordica grosvenori is taken and washed clean by clear water, and then put into a fruit crusher and added with 1.5 times of hot water with the temperature of 90 ℃ for pulping. Then 50g of composite pectinase (pectinase: cellulase: composite plant hydrolase: 7: 2:1, mass ratio) is added, enzymolysis is carried out for 1-2 hours at 50 ℃, 60g of active yeast is added when the mixture is naturally placed to about 40 ℃, and heat preservation is carried out for 3 hours at 40 ℃. Centrifuging the liquid medicine after enzymolysis by a centrifugal machine to remove solid insoluble substances, adding active carbon (the dosage is 1 percent of the mass of the filtrate) into the filtrate, stirring at 80 ℃, and preserving heat for 30 minutes. And (3) passing the liquid medicine through a plate-and-frame filter device while the liquid medicine is hot, passing the liquid medicine through macroporous acrylic acid type ion exchange resin LX-T5 when the liquid medicine is cooled to the normal temperature, collecting effluent, concentrating and drying to obtain 45g of a fructus momordicae extract product (GO-Luo).

1.2 content detection

the content of mogrosides in LH is determined by High Performance Liquid Chromatography (HPLC), which is mainly determined according to 0512 high performance liquid chromatography of the four-part general rules of 2015 pharmacopoeia of the people's republic of China. Through detection, the content of mogroside V in the extract is 46%, the content of mogroside IV is 3.2%, and the content of 11-O-mogroside V is 6.8%. The HPLC results are shown in FIG. 1.

Example 2, Go-Luo (LH) anti-obesity and fatty liver Activity test

1. Experimental Material

1.1 Experimental animals and food:

NIH male mice (about 20 g) were purchased from Guangdong province medical laboratory animal center (SPF grade, permit number: Yue Jue character, SCXK 2013-; SPF-grade food was purchased from the center of medical laboratory animals in Guangdong province; high fat food (H10141) was purchased from beijing huafukang biotech inc.

1.2 drugs for experiments

The Go-Luo (LH) extract was prepared according to example one and provided by Guilin Rhine Biotech Co., Ltd; orlistat (Orlistat, hereinafter, OL substituted) tablets were purchased from zhejiang haizheng medicinal company, ltd, and manufactured under a lot number of 21607031.

1.3 Experimental reagents:

absolute ethanol was purchased from west longa science ltd; sodium citrate and anhydrous methanol were purchased from Baishi chemical Co., Ltd, Tianjin; triton X-100 was purchased from Biotechnology engineering (Shanghai) Inc.

1.4 reagent kit for experiments

1.5Western Blot antibody reagent:

GAPDH antibody (ABS118936A), rabbit antibody, purchased from abin; AMPK antibody (2532S), P-AMPK antibody (2535S) rabbit antibody, purchased from CST corporation; ECH1 antibody (ARP43561), rabbit antibody, purchased from AVIVASYSBIO.

rabbit secondary antibody (074-1506) was purchased from KPL.

1.6 Experimental consumables:

96-well plates, pipettes, and centrifuge tubes were purchased from Biofil, Inc.; 1.5ml EP tubes were purchased from axygen.

2 method of experiment

2.1 high fat animal model fabrication and in vivo drug Activity testing

selecting 60 male SPF (specific pathogen free) NIH mice with the age of four weeks, randomly numbering the animals, randomly dividing the animals into 6 groups, ensuring that the average weight of each group is equal, wherein the ten groups are shown in the table, dividing the groups into groups, dissolving the medicines in distilled water, feeding the medicines G1 to normal food, feeding the medicines G2 to G5 to high-fat food, feeding the medicines for four weeks, and performing regular and quantitative intragastric administration once a day, wherein the administration amount is shown in the table.

The main evaluation indexes are as follows: the main evaluation indexes include body weight, fat content in vivo, volume of fat cells, blood lipid level, fatty liver, food and water intake, etc.

2.2.1 collecting the samples to be tested

And 3, collecting feces and freezing and storing the feces in-80 for standby within 2 hours after 2-4 weeks of the gavage. After the stomach is irrigated for four weeks, after the mice are fasted for 6 hours, the experimental animals are weighed and anesthetized by 10 percent of black sugar, blood is taken from eye sockets, and serum is centrifugally taken and frozen at the temperature of minus 20 ℃ for biochemical detection; taking liver tissues and abdominal fat of animals for weighing, taking about 100mg of the tissues to be placed in 10% formalin solution for fixation, and then carrying out conventional pathological section analysis; and (3) instantly freezing the rest tissues by using liquid nitrogen, putting the frozen tissues into a 24-hole plate, preserving the tissues at-80 ℃ after plastic sealing, and then performing a Western-Blot experiment.

2.2.2 Biochemical experiments

2.2.2.1 measurement of blood glucose

1) Taking 2 μ L of serum, using distilled water as blank, adding standard hole, adding 200 μ L of working solution, mixing, incubating at 37 deg.C for 10min, and measuring absorbance at 505 nm;

2) Calculating the formula: the glucose content (mmol/L) × (sample OD value-blank OD value)/(standard OD value-blank OD value) × standard concentration (5 mmol/L).

2.2.2.2 measurement of blood Cholesterol

1) Taking 2 μ L of serum, using distilled water as blank, adding standard hole, adding 200 μ L of working solution, mixing, incubating at 37 deg.C for 10min, and measuring absorbance at 505 nm;

2) The formula cholesterol content (mmol/L) ═ sample OD-blank OD)/(standard OD-blank OD) × standard concentration (5.17mmol/L) was calculated.

2.2.2.3 determination of triglyceride content in blood

1) Taking 2 μ L of serum, using distilled water as blank, adding standard hole, adding 200 μ L of working solution, mixing, incubating at 37 deg.C for 10min, and detecting its absorbance value under the light of 510nm wavelength of enzyme-labeling instrument;

2) Calculating the formula: triglyceride content (mmol/L) × (sample OD value-blank OD value)/(standard OD value-blank OD value) × standard concentration (2.26 mmol/L).

2.2.2.4 determination of ALT content in blood

1) making a standard curve: respectively adding 0 μ L, 2 μ L, 4 μ L, 6 μ L, 8 μ L and 10 μ L of 2 μmol/L sodium pyruvate standard solution, adding 20 μ L matrix solution, mixing, incubating at 37 deg.C for 30min, repeating steps 3) and 4), and obtaining standard curve;

2) Adding 5 μ L of serum into the measurement hole, adding 5 μ L of double distilled water into the blank hole, adding 20 μ L of matrix solution preheated to 20mi at 37 deg.C, mixing, and incubating at 37 deg.C for 30 min;

3) Adding 20 μ L of 2, 4-dinitrophenylhydrazine solution into the measurement holes and the blank holes, mixing uniformly, and incubating for 20min at 37 ℃;

4) adding 0.4mol/L sodium hydroxide solution to terminate the reaction, standing at room temperature for 15min, and detecting the absorbance value of the solution under the light with the wavelength of 510nm of an enzyme-labeling instrument;

5) calculating the formula: and (4) determining the OD value of the hole-the OD value of a blank hole, and checking a standard curve to obtain a corresponding ALT activity value.

2.2.2.5 determination of AST content in blood

1) Making a standard curve: respectively adding 0 mu L, 2 mu L, 4 mu L, 6 mu L and 8 mu L of 2 mu mol/L sodium pyruvate standard solution, adding 20 mu L of matrix solution, uniformly mixing, incubating at 37 ℃ for 30min, and repeating the steps 3) and 4) to obtain a standard curve;

2) Adding 5 μ L of serum into the measurement hole, adding 5 μ L of double distilled water into the blank hole, adding 20 μ L of matrix solution preheated to 20mi at 37 deg.C, mixing, and incubating at 37 deg.C for 30 min;

3) Adding 20 μ L of 2, 4-dinitrophenylhydrazine solution into the measurement holes and the blank holes, mixing uniformly, and incubating for 20min at 37 ℃;

4) Adding 0.4mol/L sodium hydroxide solution to terminate the reaction, standing at room temperature for 15min, and detecting the absorbance value of the solution under the light with the wavelength of 510nm of an enzyme-labeling instrument;

5) Calculating the formula: and (4) determining the OD value of the hole-OD value of a blank hole, and checking a standard curve to obtain the corresponding AST activity value.

2.2.2.6 determination of triglyceride content in feces

Collecting a small amount of feces, drying in an oven at 60 deg.C for 30min, determining weight, adding 100 μ L PBS, standing for a period of time to wet thoroughly, adding 800 μ L chloroform-methanol mixed solution (volume ratio 2:1) for homogenizing, shaking up and down, standing at room temperature for 5min, centrifuging at 4 deg.C and 12000rpm, taking 200 μ L into an EP tube, drying in an oven at 40 deg.C for 1h, attaching triglyceride to the bottom wall of the EP tube, adding 250 μ L triglyceride determination working solution, blowing thoroughly to dissolve triglyceride at the bottom of the tube into the working solution, incubating at 37 deg.C for 30min, sucking 200 μ L, determining absorbance at 505nm with an enzyme reader, and calculating with the same formula as 2.2.2.3.

2.2.3Western Blotting protein expression experiment

1) Protein sample preparation: selecting 3 samples in each group, taking 60mg liver, washing twice by PBS, adding 800ul cell lysate, homogenizing, centrifuging at 4 ℃, 12000rpm for 10min, carefully selecting tube bottom DNA, and obtaining supernatant as the collected protein sample.

2) protein concentration determination: coomassie brilliant blue G250200 mu L is added into each hole of a 96-hole plate, then 2 mu L of protein samples are respectively added and mixed evenly, and the absorbance is detected at 595nm by using an enzyme-linked immunosorbent assay. Similarly, the absorbance of a series of BSA standards with known concentrations was measured, a standard curve of concentration versus absorbance was plotted, and the concentration of the unknown protein sample was calculated.

3) Preparing an electrophoresis sample: according to the result obtained by the concentration measurement in the previous step, electrophoresis samples with the same protein content and the same volume are prepared, 5 Xloading buffer is added, and the protein is denatured by metal bath at 100 ℃ for 10 min.

4) Gel electrophoresis: and (3) placing the prepared rubber plate in an electrophoresis tank, adding electrophoresis liquid, spotting, adding protein marker, and then starting rubber running. Running at constant voltage of 80V for 30min, and after running the upper layer concentrated gel, performing electrophoresis at constant voltage of 120V until the electrophoresis is finished.

5) Film transfer: sequentially assembling membrane transferring clamps from a negative electrode to a positive electrode according to a sandwich structure of sponge, filter paper, gel, membrane, filter paper and sponge, placing the membrane transferring clamps in a membrane transferring groove, pouring membrane transferring liquid, and transferring the membrane for 1.5h at constant pressure of 69V, or transferring the membrane for 1.5h at constant pressure of 80V if protein with the molecular weight of more than 90kd is run.

6) And (3) sealing: taking out the transferred membrane, washing with TBST at 120rpm for 5min, adding into 5% skimmed milk/TBST sealing solution, shaking at room temperature at 80rpm, and sealing for 1 h; incubating the primary antibody: the blocking solution was decanted, the desired band of interest was cut off according to the size of the protein of interest, primary antibody diluted with 3% BSA/TBST was added, shaken at 80rpm for 30min at room temperature, and then incubated overnight at 4 ℃ G.

7) Washing the membrane: the primary antibody was recovered and TBST was added to wash the membrane for 10min at 120rpm, and repeated 4 times.

8) Incubation of secondary antibody: secondary antibodies of mice, rabbits and goats were incubated according to the source of the primary antibody, and the secondary antibodies were diluted with 5% milk/TBST and shaken at 80rpm for 1h at room temperature.

9) Washing the membrane: the secondary antibody was recovered and TBST was added to wash the membrane for 10min at 120rpm, and this was repeated 5 times.

10) Incubation substrate: carefully clamping the membrane from TBST, gently washing in ultrapure water, slightly draining on absorbent paper by siphoning, uniformly dripping the prepared ECL substrate onto the protein membrane, reacting for 2min, and placing the membrane in an exposure clamp to enter a dark room for development.

11) And (3) developing: and (3) placing a film into the exposure clamp, exposing for a plurality of minutes, taking out the film, placing the film into the developing solution and the fixing solution in sequence, washing with clear water, and drying.

12) And (3) analysis results: and marking the developed film, scanning the film into a color mode picture, and analyzing the result.

3 results of the experiment

3.1 Effect of Go-Luo (LH) and Positive drugs on mouse body weight, liver weight, abdominal fat weight, and food intake

As shown in fig. 2, we found that mice in the model group were obese and had significantly higher body weight than the normal group (P < 0.01); the weight of the LH group (LH200) which is administered 200mg/kg per day is reduced compared with that of the model group, but no significant difference is generated; the weight of a 400mg/kg LH group (LH400) and a 800mg/kg LH group (LH800) which are given daily is remarkably reduced compared with that of a high-fat model group of a negative control; the group administered 60mg/kg daily OL (OL60) showed a significant decrease in body weight.

As shown in fig. 3, the liver weight of the model mice is significantly increased compared with the normal group (P < 0.01); the liver weights of LH200, LH400 and LH800 groups are reduced remarkably (P is less than 0.05) compared with the liver weights of the model group; the liver weight of the OL60 group was significantly reduced compared to the model group (P < 0.01).

As shown in fig. 4, the abdominal fat weight of the model mice is significantly increased compared with that of the normal group (P < 0.01); the abdominal fat weight of the LH200 and LH400 groups is reduced compared with that of the model group, but no significant change is generated; the weight of the abdominal fat of the LH800 group is remarkably reduced compared with that of the model group (P < 0.05); the OL60 group showed a significant decrease in abdominal fat weight (P <0.01) compared to the model group.

As shown in fig. 5, the food intake of LH group mice with 3 doses in 24h is not significantly different from that of the model group; the food intake in the OL60 group mice within 24h is obviously increased compared with the model group (P < 0.05).

3.2 Effect of Go-Luo (LH) and Positive drugs on glycolipid metabolism in mice

After 4 weeks of induction of hyperlipidemia, as shown in FIGS. 6-8, model mice developed hyperglycemia, hypercholesterolemia, and no hypertriglyceridemia. The blood sugar and cholesterol of mice in each administration group are not significantly different from those in the model group.

FIG. 9 shows the triglyceride content in the mouse feces, and no significant increase in the oil content in the feces of the model group was observed compared with the normal group; compared with the model group, the LH group did not affect the level of oil in feces, but the OL60 group showed a very significant increase in oil in feces, which is consistent with its mechanism of action of inhibiting gastrointestinal lipase.

3.3 Effect of Go-Luo (LH) and Positive drugs on fatty liver and hepatoprotective effect in mice

The liver color of the LH group and OL60 group in fig. 10 were more normal. FIG. 11 is a HE section of liver of each group of mice, the HE section of liver of control group of mice shows no significant fatty liver change, the liver of model group of mice shows significant steatosis, vacuole generated by lipid occupies most of the space, liver cells are squeezed to one side of the cells, and the lipid vacuole of liver of 3 doses of LH group and OL60 group of mice is significantly reduced.

3.4 Effect of Go-Luo (LH) and Positive drugs on Abdominal adipocyte volume in mice

As shown in fig. 12, the abdominal adipocytes in the model group had increased volume compared to the normal group, and the adipocytes in the LH and OL groups had slightly decreased volume compared to the model group.

3.5 Effect of Go-Luo (LH) and Positive drugs on AMPK and ECH1 metabolism-related proteins in the liver

as shown in fig. 13, LH activates AMPK in the liver, promotes the expression of p-AMPK, and promotes the expression of ECH1 protein.

LH can reduce the body weight of high-fat food-induced obese mice and inhibit the formation of fatty liver, and has similar effect to positive drugs; combining liver weight, abdominal fat HE slices, food intake and mouse fecal triglyceride data we can see that LH produces weight loss and inhibits fatty liver formation, and may be associated with promoting energy metabolism, not by reducing mouse food intake, nor inhibiting oil absorption in the gastrointestinal tract of mice.

The liver is an important energy metabolism site of the body, and when the synthesis, transportation, decomposition and other pathways of fatty acid and triglyceride in the liver are hindered, lipid is likely to be deposited in the liver to gradually form fatty liver. The liver weight, the photograph, the liver HE slice, the blood ALT and the AST data show that LH can reduce the liver weight of a high fat induced mouse, so that the liver of the mouse becomes smaller, and the formation of fatty liver is inhibited by reducing the formation of fat in the liver; and can reduce liver injury, reduce ALT and AST enzyme activity in blood, and protect liver.

LH activates phosphorylation of AMPK and also increases ECH-1 expression. APMK can promote energy metabolism after being activated, and ECH-1 is a key enzyme for beta oxidation of fatty acid. These results indicate that LH promotes energy metabolism and fatty acid beta oxidation in the liver to reduce liver fat deposition, inhibit fatty liver formation, and may therefore inhibit obesity.

Claims (10)

1. A method for preparing a Luo Han Guo extract, comprising the steps of:

1) Cleaning fresh fructus Siraitiae Grosvenorii, placing in a fruit crusher, adding water, and pulping to obtain pulp;

2) adding compound pectinase into the pulp for enzymolysis 1; then adding active yeast for enzymolysis 2; after the enzymolysis 2 is finished, centrifuging and collecting supernatant; and adding active carbon into the supernatant for adsorption treatment, filtering, and collecting filtrate to obtain fructus Siraitiae Grosvenorii extract solution.

2. the method of claim 1, wherein: in the step 1), the water is water with the temperature of 50-100 ℃; the mass ratio of the fresh momordica grosvenori to the added water is 1: 0.5-5, preferably 1: 1-2.

3. The method according to claim 1 or 2, characterized in that: in the step 2), the compound pectinase consists of pectinase, cellulase and compound plant hydrolase; wherein the mass ratio of the pectinase to the cellulase to the composite plant hydrolase is 1-7: 0.1-4: 0.1 to 4;

the enzyme activity of the pectinase is 14000-20000U/g; the enzyme activity of the cellulase is 700-1000U/g, and the enzyme activity of the composite plant hydrolase is 800-1000U/g;

the adding amount of the compound pectinase is 0.5-15Kg per ton of fresh fructus momordicae, and preferably 5-12Kg per ton of fresh fructus momordicae is added;

The conditions of the enzymolysis 1 are as follows: carrying out enzymolysis for 1-2 hours at 20-55 ℃.

4. The method according to any one of claims 1-3, wherein: in the step 2) of the said step,

The adding amount of the active yeast is 0.5-20 Kg/ton of fresh fructus momordicae, preferably 5-15 Kg/ton of fresh fructus momordicae;

the conditions of the enzymolysis 2 are as follows: carrying out enzymolysis for 1-10 hours at 20-55 ℃.

5. the method according to any one of claims 1-3, wherein: in the step 2), the using amount of the activated carbon is 0.5-5% of the mass of the supernatant;

the conditions of the adsorption treatment are as follows: stirring and preserving heat for 10-60 minutes at 30-100 ℃.

6. The method according to any one of claims 1-5, wherein: the method further comprises the following purification steps: and (3) passing the momordica grosvenori extract solution through a plate-and-frame filtering device while the momordica grosvenori extract solution is hot, passing through ion exchange resin when the liquid medicine is cooled to the normal temperature, collecting effluent liquid, concentrating and drying to obtain the momordica grosvenori extract.

7. A Lo Han Guo extract prepared by the method of any one of claims 1 to 6.

8. use of a lo han guo extract of claim 7 in the preparation of at least one of:

1) preparing a product for preventing and/or treating obesity;

2) Preparing a product for preventing and/or treating fatty liver;

3) Preparing a fat metabolism promoter;

4) preparing a liver protection product;

5) preparing a product for reducing the weight of the liver;

6) Preparing a product for reducing abdominal fat weight;

7) Preparing a product for promoting the expression of p-AMPK;

8) Preparing a product for promoting the expression of ECH1 protein;

9) Inhibiting obesity and/or fatty liver formation.

9. use according to claim 8, characterized in that: the product is a medicine or a health product.

10. A product whose active ingredient is the momordica grosvenori extract of claim 7;

The product has at least one of the following uses:

1) Prevention and/or treatment of obesity;

2) preventing and/or treating fatty liver;

3) a fat metabolism promoter;

4) Protecting the liver;

5) Reducing liver weight;

6) Reducing abdominal fat weight;

7) Products that promote p-AMPK expression;

8) Products that promote the expression of ECH1 protein;

9) Inhibiting obesity and/or fatty liver formation.