阅读说明：本技术 一种具有抑制肥胖和脂肪肝作用的罗汉果提取物及其制备方法 (fructus Siraitiae Grosvenorii extract with obesity and fatty liver inhibiting effect, and its preparation method ) 是由 谢伟东 宋云飞 张晓冰 丁艺佩 廖玲 孙鹏博 张雅鸥 于 2018-06-05 设计创作，主要内容包括：本发明公开了一种罗汉果提取物及其制备方法与应用。所述罗汉果提取物的制备方法如下：1)取鲜罗汉果洗净,加入水进行打浆,得到浆液；2)向所述浆液中加入复合果胶酶进行酶解1；然后再加入活性酵母菌进行酶解2；酶解2结束后,离心收集上清液；再向所述上清液中加入活性炭进行吸附处理,过滤,收集滤液,即得罗汉果提取物。该提取物可作为脂肪代谢的促进剂,用于抑制肥胖和脂肪肝的形成。该组分作用机制可能涉及激活AMPK,促进ECH1表达,以促进能量及脂肪代谢,降低肥胖小鼠腹部脂肪重量和体重。并且该组分能够减少肝损伤,使血液中ALT、AST酶活下降,达到保肝的目的。(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.)

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.

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.