Preparation method and application of white oak essential oil

Abstract

The invention belongs to the technical field of oak functional development, and particularly relates to a preparation method and application of white oak essential oil. The white oak essential oil provided by the invention can up-regulate the expression level of genes participating in lipid hydrolysis, such as atgl-1 and hosl-1, and genes participating in fatty acid beta oxidation, such as acs-2, and down-regulate the expression level of genes participating in lipid synthesis, such as fat-5, fat-6, fat-7 and sbp-1, so that lipid hydrolysis is promoted, fatty acid beta oxidation is promoted, and lipid synthesis is inhibited; in addition, the oak can be combined with pancreatic lipase through reversible chemical bonds, the activity of pancreatic lipase, cholesterol esterase and alpha-glucosidase is inhibited, synthesis of blood fat components such as triglyceride is inhibited, the blood fat reducing effect is achieved, and therefore the technical problem that existing oak is low in functional development degree is solved.

Description

Technical Field

[0001] The present application belongs to the technical field of oak functionalization development, and in particular relates to a preparation method and application of white oak essential oil. Background Art

[0002] Unbalanced diet, excessive intake of unhealthy foods such as high-fat and high-sugar foods, and lack of exercise may cause abnormal increases in blood lipid components such as triglycerides and cholesterol, and the body's fat metabolism will be disordered. Excessive lipids will accumulate in fat cells, leading to fat cell hypertrophy and hyperplasia, causing weight gain and obesity. Obese people often have excess energy intake, and excess energy will be stored in the body in the form of fat. Adipose tissue will release a large amount of free fatty acids into the blood, increasing the raw materials for the liver to synthesize triglycerides, which in turn leads to increased triglyceride levels in the blood. The increase in blood lipid components such as triglycerides will affect the body's normal metabolic function and lead to the occurrence of hyperlipidemia. Hyperlipidemia can be diagnosed when the fasting plasma triglyceride level exceeds a certain level.

[0003] Obesity and hyperlipidemia may cause a series of diseases. For example, excessive lipids in the blood of people with obesity and hyperlipidemia will be deposited under the endothelial cells of blood vessels, triggering an inflammatory response and gradually forming atherosclerotic plaques. As the plaques grow and increase in number, they can lead to narrowing or even blockage of blood vessels, affecting the blood supply to important organs such as the heart and brain, and causing cardiovascular diseases such as coronary heart disease. In addition, obesity and hyperlipidemia may also lead to insulin resistance. Excessive adipose tissue will secrete large amounts of adipocytokines and inflammatory mediators, which reduce the sensitivity of cells to insulin, leading to insulin resistance. To maintain normal blood sugar levels, the pancreas will compensatory secretion of more insulin. Long-term excessive secretion may lead to islet cell failure and insufficient insulin secretion, thus causing diabetes. At the same time, obesity and hyperlipidemia lead to a large accumulation of fat in the body, which can cause sclerosis of the arterioles and left ventricular hypertrophy, leading to increased vascular resistance, increasing the burden on the heart and blood vessels, and raising blood pressure, resulting in hypertension.

[0004] Orlistat is a potent, specific, long-acting gastrointestinal lipase inhibitor used for weight loss. It inhibits gastrointestinal pancreatic lipase, reduces fat absorption, improves insulin resistance, achieves weight loss effects, and improves dyslipidemia such as triglycerides. Orlistat is a chemically synthesized drug. In addition to chemical synthesis, natural plant extraction is also considered an important way to obtain active ingredients. The essential oils extracted by steam distillation of the fruits, rhizomes, leaves, and wood of natural aromatic plants are rich in active ingredients such as terpenes, polyphenols, and aromatic compounds, and have antibacterial, antioxidant, and anti-inflammatory effects. Natural aromatic plant essential oils have been used in skin beauty, health care massage, food flavor regulation, and other aspects. The safety of natural aromatic plant essential oils has been widely recognized.

[0005] Oak contains some volatile organic compounds such as phenols, aldehydes, and ketones. These substances evaporate to produce a unique aroma. For example, in the winemaking process, oak barrels are used to age wine to enhance its flavor. However, there is currently a lack of in-depth research on the active ingredients in oak, and the functional development of oak is insufficient. Summary of the Invention

[0006] In view of this, the present application provides a preparation method and application of white oak essential oil, which is used to solve the technical problem of low level of functional development of existing oak.

[0007] A first aspect of the present application provides a method for preparing white oak essential oil, comprising the steps of placing white oak chips in a steam distillation apparatus, heating deionized water in the steam distillation apparatus to boiling, and distilling to obtain a white oak essential oil fraction.

[0008] Preferably, the white oak chips are obtained by passing through a 100-200 mesh sieve;

[0009] The mass volume ratio of white oak chips and deionized water in the steam distillation apparatus is 1 g:10-30 mL;

[0010] The distillation time is 1 to 3 hours;

[0011] After the distillation obtains the white oak essential oil fraction, the method further includes drying with anhydrous sodium sulfate to remove moisture from the white oak essential oil fraction.

[0012] The second aspect of the present application provides the use of the white oak essential oil described in the first aspect in preparing a lipid-lowering composition.

[0013] Preferably, the use of the white oak essential oil in preparing a lipid-lowering composition is specifically: use of the white oak essential oil in preparing a lipid-lowering composition for preventing or treating obesity.

[0014] Preferably, the use of the white oak essential oil in preparing a lipid-lowering composition is specifically: use of the white oak essential oil in preparing a lipid-lowering composition for preventing or treating hyperlipidemia.

[0015] Preferably, the use of the white oak essential oil in preparing a lipid-lowering composition is specifically: use of the white oak essential oil in preparing a lipid-lowering composition for inhibiting the activity of at least one of pancreatic lipase, cholesterol esterase, and α-glucosidase.

[0016] Preferably, the use of the white oak essential oil in the preparation of a lipid-lowering composition is specifically: use of the white oak essential oil in the preparation of a lipid-lowering composition that upregulates the gene expression level of genes involved in lipid hydrolysis, upregulates the gene expression level of genes involved in fatty acid β-oxidation, or downregulates the gene expression level of genes involved in lipid synthesis.

[0017] Preferably, the gene involved in lipid hydrolysis is selected from atgl-1 and / or hosl-1;

[0018] The gene involved in fatty acid β-oxidation is selected from acs-2;

[0019] The gene involved in lipid synthesis is selected from at least one of fat-5, fat-6, fat-7, and sbp-1.

[0020] Preferably, the use of the white oak essential oil in preparing a lipid-lowering composition is specifically: use of the white oak essential oil in preparing a lipid-lowering composition for reducing the content of at least one of cholesterol, triglycerides, phospholipids, free fatty acids, and lipoproteins in the blood.

[0021] The third aspect of the present application provides a lipid-lowering composition, comprising the white oak essential oil described in the first aspect and a pharmaceutically acceptable carrier or excipient.

[0022] Preferably, the lipid-lowering composition is selected from at least one of granular powder, tablet, capsule, solution, emulsion, injection, inhalant, and suppository.

[0023] The compositions described in the first, second and third aspects of the present application may be food, health products or medicines.

[0024] Compared with the prior art, the white oak essential oil provided by this application has at least the following technical effects:

[0025] 1. The white oak essential oil provided in this application can bind to pancreatic lipase with a reversible chemical bond and inhibit the activities of pancreatic lipase, cholesterol esterase and α-glucosidase.

[0026] 2. The white oak essential oil provided in this application can inhibit the synthesis of blood lipid components such as triglycerides, thereby exerting a lipid-lowering effect.

[0027] 3. The white oak essential oil provided in this application can upregulate the gene expression levels of genes involved in lipid hydrolysis and genes involved in fatty acid β-oxidation, and downregulate the gene expression levels of genes involved in lipid synthesis, thereby promoting lipid hydrolysis, promoting fatty acid β-oxidation and inhibiting lipid synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific implementation of this application or the technical solutions in the prior art,

[0029] A brief introduction will be given to the drawings required for use in specific implementation methods or descriptions of prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0030] Figure 1 This is a graph showing the results of an experimental inhibition experiment on pancreatic lipase activity, cholesterol esterase activity, and α-glucosidase activity by white oak essential oil OEO, orlistat, and acarbose in Example 1 of the present application;

[0031] Figure 2 This is a graph showing the inhibition type of white oak essential oil OEO on pancreatic lipase PL in Example 2 of the present application;

[0032] Figure 3 These are the experimental results of Red O staining of the blank group (B), high-fat nematode model group (A), orlistat group (C), 10 μg / mL white oak essential oil group (D), and 20 μg / mL white oak essential oil group (E) in Example 3 of the present application;

[0033] Figure 4 This is a graph showing the experimental results of relative fat content and triglyceride content in the blank group, high-fat nematode model group, orlistat group, 10 μg / mL white oak essential oil group, and 20 μg / mL white oak essential oil group in Example 3 of the present application;

[0034] Figure 5 This is a graph showing the gene expression levels of genes involved in lipid hydrolysis, genes involved in fatty acid β-oxidation, and genes involved in lipid synthesis before and after treatment of Caenorhabditis elegans with 20 μg / mL white oak essential oil in Example 4 of the present application. DETAILED DESCRIPTION

[0035] The present application provides a preparation method and application of white oak essential oil, which is used to solve the technical problem of low level of functional development of existing oak.

[0036] The following will clearly and completely describe the technical solution of this application in conjunction with the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of this application.

[0037] Given that oak is currently mainly used to make furniture, sports equipment, wine barrels, etc., and oak contains a variety of chemical components, such as some volatile organic compounds such as phenols, aldehydes, ketones, etc., which may have special effects, the current research on the active ingredients in oak is not in-depth enough, and there is a lack of functional development of oak; this application provides a preparation method and application of white oak essential oil.

[0038] The present application provides a method for preparing white oak essential oil using a steam distillation apparatus. The process comprises: passing white oak chips through a 160-mesh sieve and placing them at the bottom of a 1L round-bottom flask in the steam distillation apparatus. The mouth of the round-bottom flask is connected to a condenser. Deionized water is added to the steam distillation apparatus at a material-liquid weight-to-volume ratio of 1:20 (solid in g, liquid in mL). The round-bottom flask is heated with an electric heating mantle to keep the liquid in the flask boiling. The oil is distilled for 2 hours, and the condensate is collected and dried over anhydrous sodium sulfate to obtain the white oak essential oil. The white oak essential oil extracted by this method is a crude extract with a light yellow color and a density of 0.97 g / mL. Dozens of different compounds were identified using gas chromatography-mass spectrometry (GC-MS), but the efficacy of these compounds is still poorly understood.

[0039] The present application conducts a performance test on the prepared white oak essential oil, and the performance test includes a test on the inhibition of pancreatic lipase, cholesterol esterase and α-glucosidase activity by white oak essential oil. The results show that white oak essential oil and pancreatic lipase can be combined with reversible chemical bonds and inhibit pancreatic lipase. At the same time, it can also inhibit cholesterol esterase and α-glucosidase, thereby reducing the activity of pancreatic lipase, cholesterol esterase and α-glucosidase. Pancreatic lipase is an important enzyme for hydrolyzing dietary fat. Inhibiting the activity of pancreatic lipase can effectively reduce the absorption efficiency of fat in the small intestine, thereby achieving the purpose of lipid reduction. At the same time, cholesterol esterase can also inhibit pancreatic lipase and α-glucosidase. Enzymes play an important role in the conversion of cholesterol esters to free cholesterol. By inhibiting the activity of cholesterol esterase, the conversion of cholesterol esters to free cholesterol can be slowed down, thereby reducing its absorption rate by the human body, helping to reduce the cholesterol content in plasma and exerting a lipid-lowering effect. In addition, α-glucosidase can hydrolyze polysaccharides into polysaccharides such as glucose, causing blood sugar to rise. Excessive blood sugar can lead to insulin resistance. The white oak essential oil provided in this application is expected to reduce blood lipid components such as triglycerides and cholesterol by inhibiting the activity of pancreatic lipase, cholesterol esterase, and α-glucosidase, thereby achieving the effect of lowering blood lipids.

[0040] Based on the discovery that white oak essential oil can inhibit the activity of pancreatic lipase, cholesterol esterase and α-glucosidase, this application takes into account that the fat metabolism process of Caenorhabditis elegans has certain similarities with that of humans, and that it has genes involved in lipid hydrolysis, fatty acid β-oxidation or lipid synthesis, and contains blood lipid components such as triglycerides in the body. This application also studies the effects of white oak essential oil on fat deposition in Caenorhabditis elegans and the expression levels of genes related to fat metabolism in Caenorhabditis elegans. The results showed that white oak essential oil can reduce the fat content and triglyceride content of Caenorhabditis elegans, and can upregulate the gene expression levels of genes involved in lipid hydrolysis, upregulate the gene expression levels of genes involved in fatty acid β-oxidation, or downregulate the gene expression levels of genes involved in lipid synthesis. This shows that the white oak essential oil provided by this application can reduce blood lipid components such as triglycerides and cholesterol, and is used to prepare drugs, health products or foods for treating or preventing obesity and hyperlipidemia.

[0041] Preferably, when the white oak essential oil provided in the present application is used to prepare lipid-lowering drugs, health products or foods for treating or preventing obesity and hyperlipidemia, the lipid-lowering target can be pancreatic lipase, cholesterol esterase or α-glucosidase, or genes involved in lipid hydrolysis such as atgl-1 and hosl-1, or genes involved in fatty acid β-oxidation such as acs-2, or genes involved in lipid synthesis such as fat-5, fat-6, fat-7, sbp-1.

[0042] Preferably, when the white oak essential oil provided in the present application is used to prepare lipid-lowering drugs, health products or foods for treating or preventing obesity and hyperlipidemia, it can be prepared into dosage forms for gastrointestinal administration such as granular powders, tablets, capsules, solutions, emulsions, or dosage forms for non-gastrointestinal administration such as injections, inhalants, and suppositories.

[0043] Example 1

[0044] In this example, the white oak essential oil extracted by the steam distillation device was subjected to an experiment to test the effect of the white oak essential oil on pancreatic lipase activity, cholesterol esterase activity, and α-glucosidase activity.

[0045] The experimental principle is as follows: pancreatic lipase, cholesterol esterase, and α-glucosidase can hydrolyze p-nitrophenol ester (PNPP), hydrolyze 4-nitrophenyl butyrate (PNPB), and p-nitrophenyl-α-D-pyranoglucopyranoside (PNPG), respectively, to produce 4-nitrophenol; 4-nitrophenol has a characteristic absorbance value at a wavelength of 405nm. By measuring the absorbance changes before and after treatment with white oak essential oil, the effect of white oak essential oil on the activities of pancreatic lipase, cholesterol esterase, and α-glucosidase can be indirectly reflected.

[0046] The experimental materials included white oak essential oil, pancreatic lipase, cholesterol esterase, α-glucosidase, PNPP (p-nitrophenol ester), 4-nitrophenyl butyrate (PNPB), p-nitrophenyl-β-D-galactopyranoside (PNPG), acarbose, orlistat, and DMSO (dimethyl sulfoxide). 6.06 g of Tris was dissolved in 1000 mL of deionized water, followed by the addition of 500 μL of Triton X-100 and 100 mg of gum arabic powder. After stirring, the pH was adjusted to 8.0 with hydrochloric acid to prepare Tris-HCl buffer, which was then prepared using PBS buffer (0.1 M, pH = 7.0, containing 5.16 mM sodium taurocholate and 0.1 M sodium chloride). The instruments included a multifunctional microplate reader, a water bath, an electronic balance, a micropipette, and a constant temperature incubator.

[0047] The experimental groups included: white oak essential oil was dissolved in 10% DMSO to prepare a group of white oak essential oil solution sample groups with concentrations of 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 mg / mL, respectively; at the same time, a group of orlistat (McLean, purity 98%) solution positive sample groups with concentrations of 2, 4, 6, 8, and 10 μg / mL were prepared accordingly; a group of acarbose solution positive sample groups with concentrations of 2, 5, 10, 20, and 40 μg / mL were prepared; and 3% / 4% / 10% DMSO was used to replace white oak essential oil to prepare blank groups.

[0048] 1.1. The test process of the experiment on the effect of white oak essential oil on pancreatic lipase activity includes:

[0049] First, weigh 10 mg of p-nitrophenol ester (PNPP) and dissolve it in 5 mL of isopropanol. Appropriate sonication and heating can be used to promote dissolution. Then, the volume is adjusted to 100 mL with Tris-HCl buffer to prepare a substrate solution. Dissolve pancreatic lipase in Tris-HCl buffer at a concentration of 1 mg / mL. Centrifuge at 10,000 rpm for 5 minutes and collect the supernatant to prepare a pancreatic lipase solution.

[0050] To a 5 mL centrifuge tube, 480 μL of Tris-HCl buffer, 240 μL of white oak essential oil solution, and 720 μL of pancreatic lipase solution were added in sequence. After incubation at 37°C for 10 min, 960 μL of substrate solution was added. After incubation at 37°C for 15 min, the absorbance at 405 nm was measured using a microplate reader. At the same time, Tris-HCl buffer was used instead of pancreatic lipase solution as a sample control group. After incubation at the same temperature and time, the absorbance at 405 nm was tested.

[0051] Orlistat solution was used instead of white oak essential oil solution as the positive sample group. 480 μL of Tris-HCl buffer, 240 μL of orlistat solution and 720 μL of pancreatic lipase solution were added to a 5 mL centrifuge tube in sequence. After incubation at 37°C for 10 min, 960 μL of substrate solution was added. After incubation at 37°C for 15 min, the absorbance at 405 nm was measured using a microplate reader. At the same time, Tris-HCl buffer was used instead of pancreatic lipase solution as the positive sample control group. After incubation at the same temperature and time, the absorbance at 405 nm was tested.

[0052] 10% DMSO was used instead of white oak essential oil solution as the blank sample group. 480 μL of Tris-HCl buffer, 240 μL of 10% DMSO and 720 μL of pancreatic lipase solution were added to a 5 mL centrifuge tube in sequence. After incubation at 37°C for 10 min, 960 μL of substrate solution was added. After incubation at 37°C for 15 min, the absorbance at 405 nm was measured using a microplate reader. At the same time, Tris-HCl buffer was used instead of pancreatic lipase solution as the blank control group. After incubation at the same temperature and time, the absorbance at 405 nm was tested.

[0053] 1.2. The test process of the effect of white oak essential oil on cholesterol esterase activity includes:

[0054] First, weigh 10 mg of 4-nitrophenylbutyrate (PNPB) and dissolve it in acetonitrile to prepare a substrate stock solution with a concentration of 10 mg / mL. The stock solution must be stored in the dark. The substrate stock solution and PBS buffer are mixed in a ratio of 1:9 to prepare a 1 mg / mL substrate solution. Cholesterol esterase is dissolved in PBS buffer to obtain a cholesterol esterase solution with a concentration of 0.163 U / mL.

[0055] To a 5 mL centrifuge tube, add 100 μL PBS buffer, 50 μL white oak essential oil solution and 50 μL cholesterol esterase solution in sequence. After incubation at 37°C for 10 min, add 100 μL substrate solution. After incubation at 37°C for 15 min, measure the absorbance at 405 nm using a microplate reader. At the same time, use PBS buffer instead of cholesterol esterase solution as a sample control group, and after incubation at the same temperature and time, test the absorbance at 405 nm.

[0056] Orlistat solution was used instead of white oak essential oil solution as the positive sample group. 100 μL PBS buffer, 50 μL orlistat solution and 50 μL cholesterol esterase solution were added to a 5 mL centrifuge tube in sequence. After incubation at 37°C for 10 min, 100 μL substrate solution was added. After incubation at 37°C for 15 min, the absorbance at 405 nm was measured using a microplate reader. At the same time, PBS buffer was used instead of cholesterol esterase solution as the positive sample control group. After incubation at the same temperature and time, the absorbance at 405 nm was tested.

[0057] 4% DMSO was used instead of white oak essential oil solution as the blank group. 100 μL PBS buffer, 50 μL 4% DMSO solution and 50 μL cholesterol esterase solution were added to a 5 mL centrifuge tube in sequence. After incubation at 37°C for 10 min, 100 μL substrate solution was added. After incubation at 37°C for 15 min, the absorbance at 405 nm was measured using a microplate reader. At the same time, PBS buffer was used instead of cholesterol esterase solution as the blank control group. After incubation at the same temperature and time, the absorbance at 405 nm was tested.

[0058] 1.3. The test process of the effect of white oak essential oil on α-glucosidase activity includes:

[0059] p-Nitrophenyl-β-D-galactopyranoside (PNPG) was dissolved in Tris-HCl buffer to obtain a substrate solution with a concentration of 0.5 mM, and α-glucosidase was dissolved in Tris-HCl buffer to obtain an α-glucosidase solution with a concentration of 0.5 U / mL.

[0060] To a 5 mL centrifuge tube, 240 μL of Tris-HCl buffer, 100 μL of white oak essential oil solution, and 100 μL of α-glucosidase solution were added in sequence. After incubation at 37°C for 10 min, 100 μL of substrate solution was added. After incubation at 37°C for 20 min, the absorbance at 405 nm was measured using a microplate reader. At the same time, Tris-HCl buffer was used instead of α-glucosidase solution as a sample control group. After incubation at the same temperature and time, the absorbance at 405 nm was tested.

[0061] Acarbose solution was used instead of white oak essential oil solution as the positive sample group. 240 μL of Tris-HCl buffer, 100 μL of acarbose solution and 100 μL of α-glucosidase solution were added to a 5 mL centrifuge tube in sequence. After incubation at 37°C for 10 min, 100 μL of substrate solution was added. After incubation at 37°C for 20 min, the absorbance at 405 nm was measured using a microplate reader. At the same time, Tris-HCl buffer was used instead of α-glucosidase solution as the positive sample control group. After incubation at the same temperature and time, the absorbance at 405 nm was tested.

[0062] 3% DMSO was used instead of white oak essential oil solution as the blank group. 240 μL of Tris-HCl buffer, 100 μL of 3% DMSO and 100 μL of α-glucosidase solution were added to a 5 mL centrifuge tube in sequence. After incubation at 37°C for 10 min, 100 μL of substrate solution was added. After incubation at 37°C for 20 min, the absorbance at 405 nm was measured using a microplate reader. At the same time, Tris-HCl buffer was used instead of α-glucosidase solution as the blank control group. After incubation at the same temperature and time, the absorbance at 405 nm was tested.

[0063] The calculation of the experimental results includes: calculating the inhibition rate of pancreatic lipase, cholesterol esterase and α-glucosidase activities according to the formula: Inhibition rate (%) = (1-(A3-A4) / (A1-A2))×100; where: A1 is the absorbance of the sample group; A2 is the absorbance of the sample control group; A3 is the absorbance of the blank group; A4 is the absorbance of the blank control group; or A1 is the absorbance of the positive sample group; A2 is the absorbance of the positive sample control group; A3 is the absorbance of the blank group; A4 is the absorbance of the blank control group.

[0064] The calculation results are as follows Figure 1 As shown, from Figure 1 As can be seen from Figures A and B, the IC of orlistat to pancreatic lipase PL 50 was 1.76±0.15μg / mL, while the IC value of white oak essential oil for pancreatic lipase PL was 50 The inhibitory effect of white oak essential oil on pancreatic lipase PL was 0.62±0.05mg / mL, and within a certain concentration range, as the concentration increased, the inhibitory effect of white oak essential oil on pancreatic lipase PL became stronger and stronger; Figure 1 As shown in Figures C and D, the IC of orlistat to cholesterol esterase 50 was 1.03±0.02μg / mL, while the IC 50 The inhibitory effect of white oak essential oil on cholesterol esterase activity was 0.20±0.04 mg / mL, and within a certain concentration range, as the concentration increased, the inhibitory effect of white oak essential oil on cholesterol esterase activity became stronger and stronger; at the same time, Figure 1As shown in Figures E and F, the IC of acarbose on α-glucosidase 50 was 6.16±0.43μg / mL, while the IC of white oak essential oil for α-glucosidase was 50 It is 0.63±0.01 mg / mL, and within a certain concentration range, as the concentration increases, the white oak essential oil inhibits the activity of α-glucosidase more and more strongly; the experiment shows that the white oak essential oil provided in this application can inhibit the activity of pancreatic lipase, cholesterol esterase, and α-glucosidase.

[0065] Example 2

[0066] In this example, white oak essential oil extracted by the steam distillation apparatus was used for experiments. Considering that pancreatic lipase is the most important enzyme in the digestion and absorption of dietary lipids, responsible for the hydrolysis of 50-70% of dietary lipids, we only studied the type of inhibition of pancreatic lipase by white oak essential oil. The experiments in this example analyzed the type of inhibition of pancreatic lipase activity by white oak essential oil.

[0067] The experimental principle is as follows: different concentrations of white oak essential oil react with the enzyme, and the specific substrate p-nitrophenol ester (PNPP) is used to catalyze the reaction. The substrate is hydrolyzed to generate a product with absorbance. The optical density (OD) value per minute is measured at a wavelength of 405nm, and the linear slope of "time-OD value" represents the reaction rate of the enzyme. By plotting the reaction rate graph of different concentrations of white oak essential oil and enzyme, its reversible inhibition type is analyzed. By plotting the relationship between 1 / V and 1 / [S], the Lineweaver-Burk plot provides a straight line, where the slope is K m / V max , the Y intercept is 1 / V max By analyzing the changes in these parameters, the type of competitive inhibition can be determined.

[0068] The experimental raw materials include: pancreatic lipase, p-nitrophenol ester (PNPP), sodium acetate, Triton X-100, orlistat (McLean, purity 98%); and the instruments include: multifunctional microplate reader, water bath, electronic balance, micropipette gun, constant temperature incubator.

[0069] The experimental testing process includes: referring to Example 1, fixing the essential oil concentrations at 0, 0.4, and 0.8 mg / mL, and the p-nitrophenolate (PNPP) concentration at 0.1 mg / mL, measuring the pancreatic lipase activity at 0.4, 0.8, 1.2, 1.6, and 2.0 mg / mL, plotting the reaction velocity v as the ordinate and the enzyme mass concentration [S] as the abscissa, and determining the type of reversible inhibition based on the slope and intersection of the curve; fixing the essential oil concentrations at 0, 0.4, and 0.8 mg / mL, and the pancreatic lipase concentration at 1.0 mg / mL, and measuring the pancreatic lipase activity using 0.06, 0.08, 0.10, 0.12, and 0.14 mg / mL of p-nitrophenolate (PNPP). The calculation of experimental results includes: using the inverse of the reaction rate (1 / V) and the inverse of the substrate concentration (1 / [S]) to draw a Lineweaver-Burk plot, according to the Michaelis-Menten equation: 1 / V=K m / V max ×1 / [S]+1 / V max Determine the type of competitive inhibition, where K m For Michaelisconstant, V max is the maximal velocity, and V is the reaction velocity.

[0070] The experimental results are as follows Figure 2 As shown, from Figure 2 As can be seen from Figure A in the figure, the relationship between pancreatic lipase concentration and reaction rate is a straight line, and when the concentration of white oak essential oil is changed, the graph passes through the origin, and the slope of the fitting equation decreases with the increase of white oak essential oil. These results show that white oak essential oil does not completely inactivate pancreatic lipase, but only reduces the catalytic rate of the enzyme. In addition, white oak essential oil binds to pancreatic lipase through non-covalent bonds and reversibly inhibits pancreatic lipase. The Lineweaver-Burk curves under different concentrations of white oak essential oil are shown in Figure 1. Figure 3 As shown in Figure B, all the curves intersect at the negative half of the x-axis, and the intercepts on the x-axis remain basically unchanged. Moreover, after adding white oak essential oil, the slope of the curve increases, indicating that the inhibition type of white oak essential oil on pancreatic lipase is non-competitive inhibition.

[0071] The experimental results show that the inhibition between white oak essential oil and pancreatic lipase is a reversible chemical bond binding type; reversible binding inhibition does not lead to permanent inactivation of pancreatic lipase, and under certain conditions, the enzyme can dissociate and restore its activity. The white oak essential oil and pancreatic lipase form a temporary complex; white oak essential oil can bind to the inactive site of pancreatic lipase to form an enzyme-substrate-inhibitor complex to inhibit the activity of the enzyme.

[0072] Example 3

[0073] In this example, the white oak essential oil extracted by the steam distillation device was used to conduct an experiment to test the effect of the white oak essential oil on fat deposition in Caenorhabditis elegans.

[0074] The experimental principle is as follows: Oil Red O is a fat-soluble dye that can specifically bind to lipids. The fat in the body of C. elegans mainly exists in the form of triglycerides. Oil Red O penetrates the cell membrane of the nematode and enters the cell, combining with lipid components such as triglycerides in fat droplets. After binding, Oil Red O maintains its red properties in the lipids, thereby dyeing the fat-containing areas red, so that the distribution of fat in the body of C. elegans can be clearly observed under a microscope.

[0075] The experimental materials include: white oak essential oil, Caenorhabditis elegans (wild type N2), Escherichia coli (E. coli OP50), Oil Red O, NGM medium, orlistat (McLean, purity 98%), M9 buffer, DMSO (dimethyl sulfoxide), glucose; the experimental instruments include: fluorescence inverted microscope, multi-function microplate reader.

[0076] The experimental testing process includes:

[0077] 100 μL of Escherichia coli (E. coli OP50) and 10 mM glucose were added to the surface of NGM culture medium as food for C. elegans to establish a high-fat nematode model group, while the culture medium of the high-fat nematode blank group did not contain glucose; white oak essential oil was dissolved in DMSO to prepare white oak essential oil solution sample groups with concentrations of 10 and 20 μg / mL, respectively, which were added to the surface of NGM culture medium with 100 μL of Escherichia coli (E. coli OP50) and 10 mM glucose as food for C. elegans to establish a 10 / 20 μg / mL white oak essential oil group; a positive sample group of orlistat solution with a concentration of 10 μg / mL was prepared, which was added to the surface of NGM culture medium with 100 μL of E. coli (E. coli OP50) and 10 mM glucose as food for C. elegans to establish an orlistat group.

[0078] The Oil Red O stock solution (5 mg / mL dissolved in isopropanol) was diluted to 60% with distilled water, filtered with a 0.22 μm filter membrane, and allowed to stand for 12 h. The nematodes (about 500) in the model group, blank group, 10 / 20 μg / mL white oak essential oil group, and orlistat group were collected with M9 buffer and washed three times. The nematodes were then fixed with 60% isopropanol for 15 min and stained with Oil Red O solution for 2 h. The excess stain was then washed away with M9 buffer, and the images were taken with a fluorescence inverted microscope. Representative photos of the Oil Red O staining of the nematodes in the model group, blank group, 10 / 20 μg / mL white oak essential oil group, and orlistat group are shown in Figure 2. Figure 3As shown; Image J software was used to analyze the Oil Red O staining area. At least 15 nematodes were measured in each group, and the relative fat content was calculated based on the Oil Red O staining area of C. elegans. The results are shown in Figure 4 As shown in Figure A; nematodes (about 1000) in the model group, blank group, 10 / 20 μg / mL white oak essential oil group, and orlistat group were collected and washed with M9 buffer, and then ultrasonically disrupted and centrifuged to obtain the supernatant. The total TG content was detected using the Amplex Red triglyceride assay kit, and the protein content was measured using the BCA protein content assay kit. The TG content was normalized according to the protein content. Three biological replicates were performed for each group of experiments, and the relative content of triglycerides in the nematodes was calculated. The results are shown in Figure 3. Figure 4 As shown in Figure B.

[0079] The experimental results of red O staining are as follows Figure 3 As shown, from Figure 3 As shown in Figure A of the representative photos of Oil Red O staining of Caenorhabditis elegans, the high-fat nematode model group has a high lipid accumulation level due to the addition of glucose as food for Caenorhabditis elegans, and the Oil Red O staining area is large, continuous and dense. As can be seen from Figure B, the blank group did not add glucose as food for Caenorhabditis elegans, and the lipid accumulation level was low, and the Oil Red O staining area was small and more dispersed. Comparison of Figures A and B shows that the addition of glucose to the NGM culture medium established a high-fat model; at the same time, comparison of Figures A and C to E shows that the lipid accumulation of Caenorhabditis elegans in the white oak essential oil group and the orlistat group decreased, and the Oil Red O staining area became smaller and more dispersed.

[0080] The experimental results of relative fat content and triglyceride are as follows Figure 4 As shown, from Figure 4 As can be seen from Figure A, the fat accumulation in the blank group of Caenorhabditis elegans was low, and the relative fat content was only 43.94% of that in the high-fat nematode model group, indicating that the glucose added to the NGM medium established a high-fat model; compared with the high-fat nematode model group, the fat content of the Caenorhabditis elegans in the white oak essential oil group and the orlistat group decreased, among which, the relative fat content of the Caenorhabditis elegans after taking 10 μg / mL and 20 μg / mL of white oak essential oil decreased by 14.29% and 26.46%, respectively; at the same time, it can also be seen from Figure B that compared with the high-fat nematode model group, the triglyceride content of the Caenorhabditis elegans after taking 10 μg / mL and 20 μg / mL of white oak essential oil decreased by 10.67% and 21.35%, respectively; the experimental results show that the white oak essential oil provided in the present application has an inhibitory effect on the accumulation of fat and triglycerides in Caenorhabditis elegans.

[0081] Example 4

[0082] In this example, the white oak essential oil extracted by the steam distillation apparatus was used to conduct an experiment to investigate the effect of white oak essential oil on the expression levels of genes related to fat metabolism in Caenorhabditis elegans. The C. elegans were treated with 20 μg / mL of white oak essential oil, and then the experiment was conducted using real-time fluorescence quantitative qPCR.

[0083] The real-time fluorescence quantitative qPCR testing process includes: total RNA extraction according to the instructions of the SteadyPureQuick RNA Extraction Kit, removal of residual genomic DNA in the extracted RNA using the PrimeScript RT reagent Kit, configuration of the reverse transcription system according to the instructions of the TB Green Premix Ex Taq II Kit, cDNA amplification using the CFX Connect Real-Time System, and signal intensity analysis using the 2-ΔΔCt method. Each experiment was performed with four biological replicates. Primers were designed using the NCBI Primer-BLAST tool, using the β-actin gene as the primer. The primer sequences are shown in Appendix Table 1.

[0084] The experiments were conducted using real-time fluorescence quantitative qPCR to test the expression levels of genes involved in lipid hydrolysis, such as atgl-1 (encoding adipose triglyceride lipase) and hosl-1 (encoding hormone-sensitive lipase); the expression levels of genes involved in fatty acid β-oxidation, such as acs-2 (encoding acyl-CoA synthetase short-chain family member 2) and aak-2 (encoding AMP-activated protein kinase); and the expression levels of genes involved in lipid synthesis, such as fat-5, fat-6, fat-7 (encoding Δ9 desaturase) and sbp-1 (encoding sterol regulatory element binding protein-1).

[0085] The results of gene expression levels of genes involved in lipid hydrolysis, fatty acid β-oxidation, and lipid synthesis before and after treatment of Caenorhabditis elegans with 20 μg / mL white oak essential oil are shown in Figure 2. Figure 5 shown; from Figure 5It can be seen that white oak essential oil can increase the relative expression levels of genes involved in lipid hydrolysis such as atgl-1 and hosl-1 by 44% and 69% respectively, indicating that white oak essential oil can promote lipid hydrolysis; white oak essential oil can increase the expression of the gene acs-2 involved in fatty acid β-oxidation by 22%, thereby promoting the conversion of long-chain fatty acids into a series of short-chain acyl-CoA and then β-oxidation; white oak essential oil can reduce the expression levels of genes involved in lipid synthesis such as fat-5, fat-6, fat-7, and sbp-1 by 71.79%, 10.70%, 19.45% and 33.17% respectively, indicating that white oak essential oil can inhibit the synthesis of lipids such as cholesterol and fatty acids; the experimental results show that the white oak essential oil provided in this application can promote lipid hydrolysis, promote fatty acid β-oxidation and inhibit lipid synthesis.

[0086] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing white oak essential oil, characterized in that: The method comprises the following steps: placing white oak chips in a steam distillation device, heating deionized water in the steam distillation device to boiling, and distilling to obtain white oak essential oil.

2. Use of a white oak essential oil prepared by the preparation method according to claim 1 in preparing a lipid-lowering composition.

3. The use according to claim 2, characterized in that The specific application is: use of white oak essential oil in preparing a composition for lowering blood lipids to prevent or treat obesity.

4. The use according to claim 2, characterized in that The specific application is: use of white oak essential oil in preparing a composition for lowering blood lipids to prevent or treat hyperlipidemia.

5. The use according to claim 2, characterized in that The specific application is: use of white oak essential oil in preparing a composition for inhibiting the activity of at least one of pancreatic lipase, cholesterol esterase, and α-glucosidase to lower blood lipids.

6. The use according to claim 2, characterized in that The specific application is: the use of white oak essential oil in preparing a composition for lowering blood lipids by upregulating the gene expression level of genes involved in lipid hydrolysis, upregulating the gene expression level of genes involved in fatty acid β-oxidation, or downregulating the gene expression level of genes involved in lipid synthesis.

7. The use according to claim 6, characterized in that The application is specifically as follows: the gene involved in lipid hydrolysis is selected from atgl-1 and / or hosl-1; The gene involved in fatty acid β-oxidation is selected from acs-2; The gene involved in lipid synthesis is selected from at least one of fat-5, fat-6, fat-7, and sbp-1.

8. The use according to claim 2, characterized in that The specific application is: use of white oak essential oil in preparing a lipid-lowering composition for reducing the content of at least one of cholesterol, triglycerides, phospholipids, free fatty acids, and lipoproteins in the blood.

9. A lipid-lowering composition, characterized in that: The invention comprises the white oak essential oil described in the first aspect and a pharmaceutically acceptable carrier or auxiliary material.

10. The lipid-lowering composition according to claim 9, characterized in that: The lipid-lowering composition is selected from at least one of granular powder, tablet, capsule, solution, emulsion, injection, inhalant, and suppository.

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