Lipase inhibitor with specific structure as well as preparation method and application of lipase inhibitor

Abstract

The invention belongs to the technical field of natural medicines, and particularly discloses a lipase inhibitor with a specific structure as well as a preparation method and application thereof, the inhibitor is derived from grape leaves and contains C6-C3-C6, 1, 2-benzopyrone, diphenyl ethylene and a phenolic hydroxyl structure, and the lipase inhibitor is a lipase inhibitor with a specific structure. Comprising the following components in percentage by mass: 72.1 to 97.5 percent of flavonoids, 2.5 to 5.1 percent of coumarins, 2.0 to 3.2 percent of stilbenes and 5.1 to 8.3 percent of phenolic acids. According to the lipase inhibitor with the specific structure as well as the preparation method and the application of the lipase inhibitor, the inhibitor can inhibit secretion of lipase, prevent fat from being decomposed into absorbable free fatty acid and monoacylglycerol, and reduce fat absorption; the total cholesterol and the low-density lipoprotein cholesterol are reduced, the blood fat level is improved, and the composition can be applied to preparation of health-care products, functional foods, sports nutritious foods and formula foods for special medical purposes.

Description

Technical Field

[0001] The present invention relates to the technical field of natural medicines, and in particular to a lipase inhibitor with a specific structure, its preparation method and application. Background Art

[0002] Obesity and hyperlipidemia have increased rapidly worldwide in the past two decades. Obesity was classified as a chronic metabolic disease in the 1990s and was listed as a disease code in the International Classification of Diseases (ICD). At the annual meeting in 2013, the American Medical Association officially defined obesity as a chronic disease. Obesity is not just a weight problem but a disease that requires medical intervention. The health hazards of obesity are extensive and severe, involving multiple aspects such as cardiovascular, metabolic, respiratory, osteoarticular, psychological, reproductive, and digestive systems. Preventing and controlling obesity and hyperlipidemia is crucial for maintaining health.

[0003] There are many causes of obesity and hyperlipidemia, and abnormal fat metabolism is a very important one. For example, long-term high-fat diet, lack of exercise, etc. can lead to changes in the activity of enzymes related to fat metabolism or disorders in metabolic pathways. Lipase inhibitors are substances that can inhibit the activity of lipase. Lipase inhibitors bind to lipase, inhibiting its activity, thereby reducing the digestion and absorption of fat in food, reducing energy intake, and controlling weight from the source. Orlistat is a chemically synthesized lipase inhibitor and the only weight-loss drug approved for marketing in China at present, which gives it a unique position in the field of weight-loss drugs. However, it has the risk of liver and kidney function damage. Therefore, the development of natural and risk-free lipase inhibitors for application in controlling weight, preventing and treating hyperlipidemia, overweight or obesity is of great significance.

[0004] Currently, the patent with the patent number CN114805483B discloses five pancreatic lipase inhibitors derived from adzuki bean protein and their applications, mainly regarding peptides with pancreatic lipase inhibitor effects. The patent with the patent number CN113087751B discloses a flavone with lipase inhibitory activity, its preparation method and application, mainly involving a new flavonoid compound naringenin-4′-O-(6-O-sinapoyl)-β-D-glucosyl-7-O-α-L-rhamnosyl-(1→2)-[6″-O-(3-hydroxy-3-methylglutaryl)]-β-D-glucopyranoside. However, currently in the field of controlling weight, preventing and treating overweight and obesity, there is no research on lipase inhibitors containing structures such as C6-C3-C6, 1,2-benzopyrone, stilbene, and phenolic hydroxyl groups. Summary of the Invention

[0005] The object of the present invention is to provide a lipase inhibitor with a specific structure, its preparation method and application, to overcome the disadvantages in the prior art. By using organic solvent extraction and aqueous extraction of grape leaves, compounds containing structures such as C6-C3-C6, 1,2-benzopyranone, stilbene, and phenolic hydroxyl groups can be obtained, which can inhibit the secretion of lipase, prevent the decomposition of fat into absorbable free fatty acids and monoacylglycerols, reduce fat absorption, lower total cholesterol and low-density lipoprotein cholesterol, improve blood lipid levels, and can be applied in the preparation of health products, functional foods, sports nutrition foods, and foods for special medical purposes formula foods.

[0006] To achieve the above object, the present invention provides a lipase inhibitor with a specific structure. The inhibitor is derived from grape leaves and contains structures such as C6-C3-C6, 1,2-benzopyranone, stilbene, and phenolic hydroxyl groups, and includes the following components by mass percentage: flavonoids 72.1-97.5%, coumarins 2.5-5.1%, stilbenes 2.0-3.2%, phenolic acids 5.1-8.3%.

[0007] Preferably, the flavonoids include the following components by mass fraction: baicalin 40.5-58.2%, kaempferol 38.4-53.5%, genistein 0.4-0.8%, naringenin 0.2-0.6%, luteolin 0.1-0.3%.

[0008] Preferably, the coumarins include the following components by mass fraction: esculetin 50.7-70.1%, 7-hydroxycoumarin 24.0-33.9%, scopoletin 5.2-10.0%.

[0009] Preferably, the stilbenes include resveratrol.

[0010] Preferably, the phenolic acids include the following components by mass fraction: sinapic acid 24.1-34.2%, gallic acid 20.1-29.3%, caffeic acid 18.0-25.5%, vanillic acid 10.3-16.4%, ferulic acid 1.5-2.7%, cinnamic acid 1.2-2.6%, 4-hydroxybenzoic acid 0.9-1.9%, p-coumaric acid 0.7-1.1%, syringic acid 0.7-1.1%, 2-hydroxycinnamic acid 0.4-0.8%, 3,4-dihydroxybenzoic acid 0.1-0.4%, 2,5-dihydroxybenzoic acid 0.1-0.3%.

[0011] The present invention also provides a preparation method of a lipase inhibitor with a specific structure, including organic solvent extraction and aqueous extraction. The steps are as follows:

[0012] Select grape leaves, soak them in tap water, and rinse them repeatedly to remove impurities. After washing, dry them in a cool place at a controlled temperature of 25 ± 2 °C and a humidity of 55 ± 5 RH%. Grind the dried samples with an ultrafine grinder, sieve the ground powder through a 60-mesh sieve, and store it sealed and protected from light for later use;

[0013] Organic solvent extraction includes the following steps:

[0014] S1: Mix the grape leaf powder with 97% n-hexane or petroleum ether or ethyl acetate, and then add 50% ethanol. The mass-volume ratio of grape leaf powder to n-hexane is 1:3 - 1:6, and the mass-volume ratio of grape leaf powder to ethanol is 1:4 - 1:10;

[0015] S2: Then shake the mixture at a speed of 150 - 300 revolutions per minute at room temperature for 24 - 48 hours. Every 2 hours, perform ultrasonic treatment for 10 - 30 min;

[0016] S4: Filter the mixture using a Buchner funnel;

[0017] S5: Rotate and evaporate the filtrate at 25 °C, then lyophilize it, weigh it, and store it;

[0018] Aqueous agent extraction includes the following steps:

[0019] A: Soak the grape leaf powder in warm water at 50 °C - 60 °C. The mass-volume ratio of grape leaf powder to warm water is 1:6 - 1:10;

[0020] B: Perform ultrasonic treatment for 10 - 30 min, extract 3 times, each time for 30 - 60 min;

[0021] C: Combine the extraction solutions, evaporate and concentrate to 1000 mL, and then precipitate impurities with ethanol at a final concentration of 50%;

[0022] D: Filter, remove ethanol by rotary evaporation, and freeze-dry the concentrated solution to obtain a powdery extract, weigh it, and store it.

[0023] Preferably, step S2 of the organic solvent extraction can also be to shake the mixture at a speed of 150 - 300 revolutions per minute at room temperature for 24 - 48 hours or perform ultrasonic treatment 3 times at 50 °C, each time for 45 min;

[0024] The present invention also provides an application of a lipase inhibitor with a specific structure in the preparation of products for controlling body weight, preventing and treating hyperlipidemia, and preventing and treating obesity. The products include health care products, functional foods, sports nutrition foods, and foods for special medical purposes.

[0025] The advantages and beneficial effects of the present invention adopting the above lipase inhibitor with a specific structure, its preparation method, and application are:

[0026] In this application, a lipase inhibitor compound with a specific structure is obtained by extracting grape leaves using organic solvents and aqueous solutions, resulting in compounds containing structures such as C6-C3-C6, 1,2-benzopyrone, stilbene, and phenolic hydroxyl groups. These compounds can inhibit the secretion of lipase, prevent the breakdown of fat into absorbable free fatty acids and monoacylglycerols, reduce fat absorption, lower total cholesterol and low-density lipoprotein cholesterol, and improve blood lipid levels. They can be applied in the field of preparing products for controlling body weight, preventing and treating hyperlipidemia, overweight, or obesity.

[0027] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and examples. Brief Description of the Drawings

[0028] Figure 1 It is a bar graph of the serum pancreatic lipase amount of the lipase inhibitor compound with a specific structure of the present invention;

[0029] Figure 2 It is a curve graph of the reduction of the body weight gain of rats by the lipase inhibitor compound with a specific structure of the present invention;

[0030] Figure 3 It is a bar graph of the reduction of serum triglycerides in rats by the lipase inhibitor compound with a specific structure of the present invention;

[0031] Figure 4 It is a bar graph of the reduction of serum total cholesterol in rats by the lipase inhibitor compound with a specific structure of the present invention;

[0032] Figure 5 It is a bar graph of the reduction of serum low-density lipoprotein cholesterol in rats by the lipase inhibitor compound with a specific structure of the present invention;

[0033] Figure 6 It is a bar graph of the reduction of liver triglycerides in rats by the lipase inhibitor compound with a specific structure of the present invention;

[0034] Figure 7 It is a bar graph of the reduction of liver total cholesterol in rats by the lipase inhibitor compound of the present invention. Detailed Embodiments

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings and examples.

[0036] Unless otherwise defined, the technical terms or scientific terms used in the present invention should have the ordinary meaning understood by those of ordinary skill in the field to which the present invention belongs.

[0037] Unless otherwise defined, the reagents and equipment used in the present invention are all obtained from conventional commercial sources.

[0038] The lipase inhibitor of the present invention is not used for the diagnosis and treatment of diseases.

[0039] Example 1

[0040] A preparation method of a lipase inhibitor with a specific structure, including organic solvent extraction and aqueous extraction, the steps are as follows:

[0041] Select grape leaves, soak them in tap water, and rinse repeatedly to remove impurities. After washing, dry them in the shade at a controlled temperature of 25 ± 2 °C and a humidity of 55 ± 5 RH%. The dried sample is pulverized with an ultrafine pulverizer, and the pulverized powder is sieved through a 60-mesh sieve and stored in a sealed and light-proof manner for later use;

[0042] The organic solvent extraction includes the following steps:

[0043] S1: Mix the grape leaf powder with 97% n-hexane or petroleum ether or ethyl acetate, and then add 50% ethanol. The mass-volume ratio of the grape leaf powder to n-hexane is 1:3 - 1:6, and the mass-volume ratio of the grape leaf powder to ethanol is 1:4 - 1:10;

[0044] S2: Then shake the mixture at a speed of 150 - 300 revolutions per minute at room temperature for 24 - 48 hours, and ultrasonicate for 10 - 30 min every 2 hours;

[0045] S3: Filter the mixture using a Buchner funnel;

[0046] S4: Rotate and evaporate the filtrate at 25 °C, then lyophilize it, weigh and store it;

[0047] The aqueous extraction includes the following steps:

[0048] A. Soak the grape leaf powder in warm water at 50 °C - 60 °C, and the mass-volume ratio of the grape leaf powder to the warm water is 1:6 - 1:10;

[0049] B. Ultrasonicate for 10 - 30 min, extract 3 times, each time for 30 - 60 min;

[0050] C. Combine the extracts, evaporate and concentrate, and then precipitate impurities with ethanol with a final concentration of 50%;

[0051] D. Filter, remove ethanol by rotary evaporation, and the concentrated solution is freeze-dried to obtain a powdery extract, weigh and store it.

[0052] Use a liquid chromatography (LC) system (Agilent Technologies 1200 series) coupled with a triple quadrupole mass spectrometer (MS) with an electrospray ionization source (Agilent Technologies 6410A), and a Zorbax Eclipse XDB-C18 chromatographic column (50 mm × 4.6 mm, 1.8 μm) to analyze the components of the grape leaf extract.

[0053] A lipase inhibitor with a specific structure, the inhibitor is derived from grape leaves and contains C6-C3-C6, 1,2-benzopyrone, stilbene, and phenolic hydroxyl structures, and includes the following components by mass percentage: flavonoids 72.1-97.5%, coumarins 2.5-5.1%, stilbenes 2.0-3.2%, phenolic acids 5.1-8.3%.

[0054] The flavonoids include the following components by mass fraction (i.e., the percentage of each component in the mass of flavonoids): baicalein 40.5-58.2%, kaempferol 38.4-53.5%, genistein 0.4-0.8%, naringenin 0.2-0.6%, luteolin 0.1-0.3%.

[0055] The coumarins include the following components by mass fraction (i.e., the percentage of each component in the mass of coumarins): aesculetin 50.7-70.1%, 7-hydroxycoumarin 24.0-33.9%, scopoletin 5.2-10.0%.

[0056] The stilbenes include resveratrol.

[0057] The phenolic acids include the following components by mass fraction (i.e., the percentage of each component in the mass of phenolic acids): sinapic acid 24.1-34.2%, gallic acid 20.1-29.3%, caffeic acid 18.0-25.5%, vanillic acid 10.3-16.4%, ferulic acid 1.5-2.7%, cinnamic acid 1.2-2.6%, 4-hydroxybenzoic acid 0.9-1.9%, p-coumaric acid 0.7-1.1%, syringic acid 0.7-1.1%, 2-hydroxycinnamic acid 0.4-0.8%, 3,4-dihydroxybenzoic acid 0.1-0.4%, 2,5-dihydroxybenzoic acid 0.1-0.3%.

[0058] The structures of the compounds contained in the lipase inhibitor are shown in Table 1 - Table 4.

[0059] Table 1 C6-C3-C6 structure

[0060]

[0061] Table 2 1,2-benzopyrone structure

[0062]

[0063]

[0064] Table 3 Stilbene structure

[0065]

[0066] Table 4 Phenolic hydroxyl structure

[0067]

[0068]

[0069] Example 2

[0070] A preparation method of a lipase inhibitor with a specific structure, wherein the organic solvent extraction comprises the following steps:

[0071] S1: Mix 50 grams of grape leaf powder with 200 mL of n-hexane (97%) and 300 mL of 50% ethanol;

[0072] S2: Then shake the mixture at a speed of 250 revolutions per minute at room temperature for 48 hours, and perform ultrasonic treatment for 20 minutes every 2 hours;

[0073] S3: Filter, and filter with a Buchner funnel until there are no solid particles;

[0074] S4: Rotate and evaporate the filtrate at 25°C, then freeze-dry it, weigh it and store it.

[0075] A liquid chromatography (LC) system (Agilent Technologies 1200 series) is used in combination with a triple tandem mass spectrometry (MS) with an electrospray ionization source (Agilent Technologies 6410A), and a Zorbax Eclipse XDB-C18 chromatographic column (50 mm × 4.6 mm, 1.8 μm) is used to analyze the components of the grape leaf extract.

[0076] The lipase inhibitor compound contains a C6-C3-C6, 1,2-benzopyranone, stilbene, and phenolic hydroxyl structure. This compound contains 85.0% flavonoids, 5.1% coumarins, 2.5% stilbenes, and 7.4% phenolic acids.

[0077] The flavonoids include 51.3% baicalein, 47.3% kaempferol, 0.7% genistein, 0.5% naringenin, and 0.2% luteolin.

[0078] The coumarins include 61.8% esculetin, 30.0% 7-hydroxycoumarin, and 8.2% scopoletin.

[0079] The phenolic acids include 29.7% sinapic acid, 25.6% gallic acid, 22.1% caffeic acid, 14.3% vanillic acid, 2.1% ferulic acid, 1.9% cinnamic acid, 1.3% 4-hydroxybenzoic acid, 0.9% p-coumaric acid, 1.0% syringic acid, 0.6% 2-hydroxycinnamic acid, 0.3% 3,4-dihydroxybenzoic acid, and 0.2% 2,5-dihydroxybenzoic acid.

[0080] Lipase inhibitor compound, the extraction rate of lipase inhibitor active ingredient is 13.4%, the total flavonoid content is 3.9%, and the total polyphenol content is 9.8%.

[0081] Determination of lipase activity inhibition rate:

[0082] S1: Preparation of enzyme solution and tri-Hcl buffer solution.

[0083] Preparation of enzyme solution: 20mg / ml, prepared with distilled water.

[0084] Preparation of Tris-Hcl buffer solution: Dissolve 1.2114g of Tris in distilled water to a volume of 200ml, and adjust the pH to 7.0 with hydrochloric acid.

[0085] S2: Preparation of p-nitrophenol standard curve.

[0086] p-NP standard solution: Accurately weigh 10mg of p-NP and dissolve it in 1000mL of distilled water to prepare a 10ug / mL solution.

[0087] Prepare standard solutions with different p-NP concentrations, measure the absorbance at 410nm, and plot the standard curve.

[0088] S3: Reaction

[0089] Solution A: Weigh 30mg of p-NPP powder and dissolve it in 10ml of isopropanol. Appropriate heating and shaking can be used to accelerate dissolution, and it should be stored at 4°C. Generally, it should be used up within two days, as the substrate will hydrolyze spontaneously after a long time.

[0090] Solution B: Prepare a 0.5% concentration solution of Tritox-X-100 with 0.05mol / L pH8.0 Tris-HCl buffer solution, and heating can be used to promote dissolution.

[0091] After mixing solution A and solution B evenly, add 1ml of Tris-HCl solution, mix evenly, and incubate at 37°C for 2 minutes. Add the extract and enzyme solution and mix, and react in a 37°C water bath for 15 minutes. Do not terminate the reaction. Since the extract itself has color, the value measured for the control group is the absorbance of the extract.

[0092] The amount of reaction = experimental group - control group, and calculate the enzyme activity by referring to the standard curve.

[0093] Lipase inhibitor compound, lipase inhibitor activity is 65.4%.

[0094] Grape leaves are traditional medicinal and edible plants in China. Grape leaves are sweet, astringent, neutral in nature, and non-toxic. They have the effects of stopping bleeding, clearing heat and relieving summer heat, and are used to treat edema, dysuria, red eyes, and carbuncles. In Turkey, Greece, and the Middle East, they are mostly used as cooking materials for making dishes with corn and minced meat. Fresh grape leaves are good materials for making tea and cooking.

[0095] Example 3

[0096] A preparation method of a lipase inhibitor with a specific structure. The organic solvent extraction includes the following steps:

[0097] S1: Mix 50 grams of grape leaf powder with 200 mL of n-hexane (97%) and 300 mL of 50% ethanol;

[0098] S2: Then shake the mixture at a speed of 250 revolutions per minute at room temperature for 48 hours;

[0099] S3: Filter with a Buchner funnel until there are no solid particles;

[0100] S4: Rotate and evaporate the filtrate at 25 °C, then freeze-dry, weigh and store.

[0101] Lipase inhibitor compound, the extraction rate of lipase inhibitor active ingredient is 10.2%, the total flavonoid content is 3.0%, and the total polyphenol content is 6.7%.

[0102] Lipase inhibitor compound, the lipase inhibitor activity is 57%.

[0103] Example 4

[0104] The preparation of the organic solvent extraction process includes the following steps:

[0105] S1: Mix 50 grams of grape leaf powder with 200 mL of n-hexane (97%) and 300 mL of 50% ethanol;

[0106] S2: Then ultrasonicate 3 times at 50 °C, each time for 45 min;

[0107] S3: Filter with a Buchner funnel until there are no solid particles;

[0108] S4: Rotate and evaporate the filtrate at 25 °C, then freeze-dry, weigh and store.

[0109] Lipase inhibitor compound, the extraction rate of lipase inhibitor active ingredient is 8.1%, the total flavonoid content is 2.5%, and the total polyphenol content is 5.4%.

[0110] Lipase inhibitor compound, the lipase inhibitor activity is 44.6%.

[0111] As can be seen from Examples 2-4, changing the reaction conditions of the preparation method of the organic solvent of the lipase inhibitor affects the extraction rate of the active ingredient of the lipase inhibitor, the total flavonoid content, the total polyphenol content and the activity of the lipase inhibitor.

[0112] Example 5

[0113] The preparation process of the aqueous extract includes the following steps:

[0114] S1: Mix 50 grams of grape leaf powder with 450 mL of pure water;

[0115] S2: Ultrasonic for 15 min, extract 3 times, 30 min each time;

[0116] S3: Combine the extracts, evaporate and concentrate to 1000 mL, and then precipitate impurities with ethanol with a final concentration of 50%;

[0117] S4: Filter, remove ethanol by rotary evaporation, and the concentrated solution is freeze-dried to obtain a powdery extract, weigh and store.

[0118] For the lipase inhibitor compound, the extraction rate of the active ingredient of the lipase inhibitor is 7.4%, the total flavonoid content is 2.2%, and the total polyphenol content is 4.8%.

[0119] For the lipase inhibitor compound, the activity of the lipase inhibitor is 25.3%.

[0120] Test Example 1

[0121] Animal test:

[0122] The experimental test sample is a chewable tablet candy made of the lipase inhibitor compound with a specific structure obtained in Example 2.

[0123] After the modeling period using the obesity prevention model method, 50 obesity-sensitive rats were randomly divided into 5 groups according to body weight, namely the blank control group, the model control group, and three dose groups (high dose group, medium dose group, low dose group). The model control group and the sample group were given a high-calorie model diet, and the blank control group was given a maintenance diet. Each dose group was intragastrically administered different doses of the test sample, the model control group and the blank control group were given an equal amount of the corresponding solvent, and the test sample was administered for 6 weeks. The grouping situation is shown in Table 5.

[0124] Table 5 Grouping design of animal experiments

[0125]

[0126] After the experiment, the body weight was measured. The rats were anesthetized with 1% sodium pentobarbital (0.5 ml / 100 g BW), and the perirenal fat and peritesticular fat were dissected and weighed to calculate the fat / body ratio. Indexes such as body weight gain, triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) in rat serum, pancreatic lipase secretion, liver triglyceride, and liver total cholesterol were measured.

[0127] As Figure 1 shown, the amount of serum pancreatic lipase in the sample group was significantly lower than that in the model control group, and with the increase of the dose of the sample group, the content of serum pancreatic lipase was less. As Figure 2 shown, the body weight gain in the sample group was significantly lower than that in the model control group, and it was found that the body weight gain in the high-dose group was lower than that in the blank control group at the 5th and 6th weeks. As Figure 3 shown, the content of serum triglyceride in the sample group was significantly lower than that in the model control group, and with the increase of the dose of the sample group, the content of serum triglyceride was less. As Figure 4 shown, the content of serum total cholesterol in the sample group was significantly lower than that in the model control group, and with the increase of the dose of the sample group, the content of serum total cholesterol was less. As Figure 5 shown, the content of serum low-density lipoprotein cholesterol in the sample group was significantly lower than that in the model control group, and with the increase of the dose of the sample group, the content of serum low-density lipoprotein cholesterol was less. As Figure 6 shown, the content of liver triglyceride in the sample group was significantly lower than that in the model control group, and the liver triglyceride in the medium-dose and high-dose groups of the sample group was lower than that in the blank control group. As Figure 7 shown, the content of liver total cholesterol in the sample group was significantly lower than that in the model control group, and with the increase of the dose of the sample group, the liver total cholesterol was less.

[0128] From Figures 1-7 the analysis, the fat content in rats was lower than that in the model control group, and the difference was significant (P < 0.05). The sample group (specific structural lipase inhibitor compound) was helpful for regulating the body fat function and maintaining the healthy level of blood lipids (cholesterol / triglyceride).

[0129] Human trial test:

[0130] The test sample for the human trial test was a special dietary (endurance type) beverage prepared with the specific structural lipase inhibitor compound obtained in Example 5.

[0131] People with a body mass index (BMI) greater than 30 were selected as the test population. Thirty people were selected and evenly divided into 2 groups, an experimental group and a control group. The experimental group consumed 150 mL of the special dietary (endurance type) beverage 20 minutes before lunch and dinner, and ate foods containing fat for lunch and dinner. The control group consumed 150 mL of drinking water 20 minutes before lunch and dinner, and ate foods containing fat for lunch and dinner.

[0132] During the subsequent 4-week weight maintenance follow-up period, body weight and body fat were tracked, and the data are shown in Table 6.

[0133] Table 6 Data table of body weight and body fat

[0134] Index Experimental group Control group Significance (p-value) Average weight loss (kg) 2.9 -2.1 p<0.01 Reduction in body fat percentage (%) 1.5 -0.8 p<0.05

[0135] In summary, it can be seen that the specific structural lipase inhibitor compound in the present invention uses organic solvent extraction and aqueous extraction of grape leaves to obtain compounds containing structures such as C6-C3-C6, 1,2-benzopyrone, stilbene, and phenolic hydroxyl groups, which can inhibit the secretion of lipase, reduce triglycerides, reduce total cholesterol, reduce low-density lipoprotein cholesterol, and improve blood lipid levels; reduce free fatty acids, reduce weight gain, and can be applied in the fields of weight control, prevention and treatment of hyperlipidemia, overweight or obesity.

[0136] Therefore, the present invention adopts the above-mentioned lipase inhibitor with a specific structure, its preparation method and application. The specific structural lipase inhibitor compound uses organic solvent extraction and aqueous extraction of grape leaves to obtain compounds containing structures such as C6-C3-C6, 1,2-benzopyrone, stilbene, and phenolic hydroxyl groups, which can inhibit the secretion of lipase, prevent the decomposition of fat into absorbable free fatty acids and monoacylglycerols, and reduce fat absorption; reduce total cholesterol and low-density lipoprotein cholesterol, and improve blood lipid levels. It can be applied in the field of preparing products for weight control, prevention and treatment of hyperlipidemia, overweight or obesity.

[0137] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A lipase inhibitor of a specific structure, characterized in that: The inhibitor is derived from grape leaves, contains C6-C3-C6, 1,2-benzopyrone, diphenylethylene, and phenolic hydroxyl structures, and includes the following components in mass percentages: flavonoids 72.1-97.5%, coumarins 2.5-5.1%, stilbenes 2.0-3.2%, and phenolic acids 5.1-8.3%.

2. The lipase inhibitor of a specific structure according to claim 1, characterized in that: The flavonoids include the following components by mass fraction: 40.5-58.2% of scutellaria baicalensis, 38.4-53.5% of kaempferol, 0.4-0.8% of genistein, 0.2-0.6% of naringenin, and 0.1-0.3% of luteolin.

3. The lipase inhibitor of a specific structure according to claim 1, characterized in that: The coumarins include the following components by mass fraction: 50.7-70.1% of esculin, 24.0-33.9% of 7-hydroxycoumarin and 5.2-10.0% of scopoletin.

4. The lipase inhibitor of a specific structure according to claim 1, characterized in that: The stilbenes include resveratrol.

5. The lipase inhibitor of a specific structure according to claim 1, characterized in that: The phenolic acids include the following components by mass fraction: 24.1-34.2% of sinapinic acid, 20.1-29.3% of gallic acid, 18.0-25.5% of caffeic acid, 10.3-16.4% of vanillic acid, 1.5-2.7% of ferulic acid, 1.2-2.6% of cinnamic acid, 0.9-1.9% of 4-hydroxybenzoic acid, 0.7-1.1% of p-hydroxycinnamic acid, 0.7-1.1% of syringic acid, 0.4-0.8% of 2-hydroxycinnamic acid, 0.1-0.4% of 3,4-dihydroxybenzoic acid and 0.1-0.3% of 2,5-dihydroxybenzoic acid.

6. The method for preparing a lipase inhibitor of a specific structure according to any one of claims 1 to 5, characterized in that: Including organic solvent extraction and aqueous extraction, the steps are as follows: Select grape leaves, soak them in tap water, rinse them repeatedly to remove impurities, and then dry them in a cool place at a controlled temperature of 25±2°C and a humidity of 55±5RH%. The dried samples are crushed with an ultrafine grinder, and the crushed powder is passed through a 60-mesh sieve, sealed and kept away from light for later use; Organic solvent extraction includes the following steps: S1: Mix grape leaf powder with 97% n-hexane or petroleum ether or ethyl acetate, and then add 50% ethanol to mix, the mass volume ratio of grape leaf powder to n-hexane is 1:3-1:6, and the mass volume ratio of grape leaf powder to ethanol is 1:4-1:10; S2: The mixture was then shaken at room temperature at 150-300 rpm for 24-48 hours, and sonicated for 10-30 min every 2 hours; S3: Filter the mixture using a Buchner funnel; S4: The filtrate was rotary evaporated at 25°C, then freeze-dried, weighed and stored; Aqueous extraction includes the following steps: A. Soak the grape leaf powder in 50℃-60℃ warm water, with the mass volume ratio of grape leaf powder to warm water being 1:6-1:10; B. Ultrasonication for 10-30 minutes, extraction 3 times, 30-60 minutes each time; C. Combine the extracts, evaporate and concentrate, and then use ethanol with a final concentration of 50% to precipitate impurities; D. Filter and remove ethanol by rotary evaporation. The concentrated solution is freeze-dried to obtain a powdered extract, which is weighed and stored.

7. The method for preparing a lipase inhibitor of a specific structure according to claim 6, characterized in that: The organic solvent extraction step S2 can also be performed by shaking the mixture at room temperature at a speed of 150-300 rpm for 24-48 hours or by ultrasonicating at 50° C. for 3 times, each time for 45 minutes.

8. Use of a lipase inhibitor of a specific structure as claimed in any one of claims 1 to 5, characterized in that: It is used to prepare products for controlling weight, preventing and treating hyperlipidemia, and preventing and treating obesity. The products include health products, functional foods, sports nutrition foods, and special medical purpose formula foods.

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