Tomato extract tomato saponin and its preparation method and application

Abstract

The present invention discloses a tomato extract, tomato saponin, and its preparation method and application, belonging to the field of biomedicine technology. The method comprises the following steps: S1. washing tomato fruits with clean water, squeezing to obtain tomato juice, and filtering for later use; S2. adding complex enzyme A and complex enzyme B to the filtered tomato juice in S1, performing continuous enzymolysis, and then inactivating the enzymes to obtain an enzyme-inactivated hydrolyzate; S3. subjecting the enzyme-inactivated hydrolyzate to membrane separation using an organic membrane coating to remove small molecular weight components, thereby obtaining an intercepted straight-through liquid; S4. heating the intercepted straight-through liquid after membrane separation at 50-100°C for 1-24 hours, and then concentrating and freeze-drying to obtain a dry powdered extract; and S5. subjecting the powdered extract obtained in S3 to gradient elution separation using an ODS C18 column chromatography. The method solves the problem in the prior art that the prevention and treatment of cardiovascular diseases and inflammatory-related diseases mainly rely on chemical drugs, which leads to a series of side effects and drug resistance. The method has the advantages of high extraction efficiency and good safety.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a tomato extract, tomato saponin, and a preparation method and application thereof. Background Art

[0002] Cardiovascular disease is one of the leading causes of death worldwide, primarily caused by atherosclerosis, of which hyperlipidemia is the primary culprit. Currently, statins and statins are commonly used lipid-lowering drugs in clinical practice, with Lipitor (atorvastatin) being the first blockbuster drug to surpass $100 billion in sales. Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, preventing hepatic cholesterol synthesis. Ezetimibe, a selective inhibitor of enterocyte cholesterol transporter 1 (NPC1L1), inhibits intestinal absorption of dietary cholesterol. In recent years, proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors have garnered attention for their long-lasting lipid-lowering effects. They lower LDL cholesterol levels by preventing PCSK9 from degrading the low-density lipoprotein receptor (LDLR). However, although statins are effective, long-term use may cause a series of side effects, such as muscle pain and abnormal liver function. Although ezetimibe and PCSK9 inhibitors have different mechanisms of action, they also have side effects or adverse reactions to varying degrees.

[0003] Tomato (Lycopersicon esculentum) is an important economic crop with a variety of varieties, among which the Millennium tomato (L. esculentum var. 'Millennium') is a common red cherry tomato. Tomatoes are rich in steroidal alkaloids and steroidal glycoalkaloids (SA / SGAs), compounds that contain nitrogen atoms and have plant defense functions. To date, at least 26 SA / SGAs are known to exist in tomatoes, most of which were discovered by the research team of Japanese scholar Toshihiro Nohara. These compounds can be divided into soladulcidine (22-αN), tomatidine (22-βN), solanocapsine, and pregnane types. Among them, esculeoside A (EscA), iso-esculeoside B, lycoperoside B (LycB), Lyc C, and Lyc G belong to the soladulcidine type; LycA, LycD, LycF, and α-tomatine belong to the tomatidine type; esculeoside B-1 (EscB-1), EscB-2, EscB-5, EscC, and LycH belong to the solanocapsine type; and EscD, tomato-pregnane, 5α-pregna-3β, 16β-diol-20-one3-O-β-lycotetraosyl belong to the pregnane type. Some SA / SGAs, such as α-tomatine, EscA, and LycH, have shown significant pharmacological activity. α-Tomatine is present in high concentrations in unripe green tomatoes, reaching up to 500 mg / kg. It has a bitter taste and anti-nutritional properties, and serves to protect the fruit during ripening. However, its content in ripe red tomatoes is lower, at approximately 5 mg / kg, due to its conversion to the non-bitter and less toxic EscA during the ripening process. Health-promoting components in tomatoes, such as LycH, have anti-inflammatory, antioxidant, and gut microbiota-regulating effects. They can prevent diabetes-related kidney disease, protect against 1,2-dimethylhydrazine (DMH)-induced colorectal cancer, improve epidermal dehydration, and alleviate symptoms of atopic dermatitis in IL-33 transgenic mice. EscA exhibits lipid-lowering, hypoglycemic, anti-inflammatory, and anti-dermatitis activities, and can reduce low-density lipoprotein cholesterol levels in Apoe-deficient mice and mice with experimental hyperlipidemia. EscA may be developed as a functional supplement for the treatment of diabetes. EscA can also protect against cardiac pathology in streptozotocin (STZ)-induced type 1 diabetic (T1DM) rats by activating the Nrf2 / antioxidant / NF-κB signaling axis to alleviate oxidative stress, inflammation, fibrosis, and apoptosis.Esculeogenin A (EsgA), the 23-deacetylated aglycone of EscA, also possesses immunotrophic properties for the treatment of atopic dermatitis. It significantly blocks hyaluronidase activity, improves experimental dermatitis in mice, and modulates Th2 / Th1 / Treg differentiation, reducing Th2 lymphocyte activity and CD4+ T lymphocyte activation. Furthermore, EsgA can ameliorate hyperlipidemia and atherosclerosis in Apoe-deficient mice by inhibiting acyl-CoA:cholesterol acyltransferase (ACAT). Given the significant pharmacological activities of various SA / SGAs found in tomatoes and their diverse health-promoting effects, the development of these natural ingredients as novel drugs or functional food supplements is of great significance. Scientifically extracting and purifying these components will not only enrich existing drug candidates but also provide new insights and strategies for the prevention and treatment of cardiovascular and inflammatory diseases.

[0004] Therefore, there is an urgent need for a tomato extract tomato saponin and a preparation method thereof, and its application in the preparation of anti-inflammatory and lipid-lowering drugs, in order to provide patients with safer and more effective treatment options. Summary of the Invention

[0005] To this end, the present invention provides a tomato extract tomato saponin and its preparation method and application, so as to solve the problem in the prior art that the prevention and treatment of cardiovascular diseases and inflammatory-related diseases rely on chemical drugs, resulting in a series of side effects and drug resistance.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] According to the first aspect of the present invention, a tomato extract tomato saponin is provided, the compound structural formula of which is:

[0008]

[0009] According to a second aspect of the present invention, a method for preparing tomato saponin from a tomato extract is provided, comprising the following steps:

[0010] S1. Wash the tomatoes with water, squeeze the tomato juice, and then filter it for later use;

[0011] S2. The tomato juice filtered by S1 was added with complex enzyme A and complex enzyme B for continuous enzymatic hydrolysis and then the enzyme was inactivated to obtain an enzyme hydrolyzate after enzyme inactivation;

[0012] S3. The enzyme-inactivated hydrolyzate is subjected to membrane separation using an organic membrane coating to remove small molecular weight components to obtain a retained straight-through solution;

[0013] S4. The retentate after membrane separation was heated at 50-100°C for 1-24h and then concentrated and freeze-dried to obtain a dry powdered extract;

[0014] S5. The powdered extract obtained in S3 was separated by ODS C18 column chromatography with gradient elution, and the collected elution fractions were separated by C18 column preparative liquid chromatography;

[0015] S6. The solution separated by the C18 column is concentrated and dried again to obtain a water-soluble tomato extract.

[0016] Furthermore, the S2 includes the following specific steps:

[0017] S21. Primary enzymatic hydrolysis: Add 8-10 times the amount of ultrapure water to the filtered tomato juice from S1, then stir to obtain a suspension. Adjust the pH to 4.5-5.5, then add 0.2-0.6% by weight of complex enzyme A to the suspension, and then add 1 / 10 by weight of stabilizer A to the complex enzyme A. Gradually raise the temperature to 45-55°C and perform enzymatic hydrolysis for 1-1.5 hours to obtain a primary enzymatic solution.

[0018] S22. Secondary enzymatic hydrolysis: The pH of the primary enzymatic solution was adjusted to 6.5-8.5, followed by addition of 0.1-0.3% by weight of the primary enzymatic solution of complex enzyme B for enzymatic hydrolysis, followed by addition of 1 / 10 by weight of the complex enzyme B of catalyst B, and the temperature was heated to 55-65 ° C and kept warm for 1.5-2h to obtain a secondary enzymatic solution;

[0019] S23. Inactivate enzymes: Heat the obtained secondary enzymatic hydrolysate to 90-95°C and keep it warm for 15-20 minutes to inactivate enzymes.

[0020] Furthermore, the complex enzyme A in S21 is composed of phytase, cellulase, pectinase, xylanase and β-glucosidase, and the mass ratio of phytase, cellulase, pectinase, xylanase and β-glucosidase in the complex enzyme A is (0.08-1.5): (0.05-3): (0.05-1): (0.05-1): (0.05-1);

[0021] The complex enzyme B in S22 is composed of papain, trypsin, Alcalase protease, endoprotease, exoprotease and flavor protease, the alkaline protease in the complex enzyme B is composed of papain and trypsin, and the mass ratio of papain, trypsin, Alcalase protease, endoprotease, exoprotease and flavor protease is (0.05-1.2): (0.05-1.2): (0.05-1.2): (0.08-1.5): (0.08-1.5).

[0022] Furthermore, the stabilizer A in S21 is one or more of CaCl2, MgCl2, and ZnSO4;

[0023] The catalyst B in S22 is one or more of N-acetylglucosamine, mannose, and trehalose.

[0024] Furthermore, the specific steps in S3 are:

[0025] The enzymatic hydrolysate after enzyme inactivation is passed through a membrane separation device loaded with a 500Da organic membrane coating for membrane separation. Components with a molecular weight less than 500 can pass through the organic coating and be discharged from the membrane separation device, while components with a molecular weight greater than 500 are retained in the membrane separation device to obtain retained straight-through liquid.

[0026] Furthermore, in S5, the eluent is acetonitrile and water in a volume ratio of 15:85-20:80, 50:50-100:0.

[0027] Furthermore, in S5, the elution fraction is collected from the elution fraction having a volume ratio of acetonitrile to water of 50:50 to 100:0.

[0028] Furthermore, the method also includes conducting structural characterization tests on the water-soluble tomato extract obtained by separation and preparation.

[0029] According to a third aspect of the present invention, a method for preparing tomato saponin from tomato extract is provided, and the use of the prepared tomato saponin in the preparation of anti-inflammatory drugs.

[0030] According to a fourth aspect of the present invention, a method for preparing tomato saponin from tomato extract is provided, and the use of the prepared tomato saponin in the preparation of lipid-lowering drugs.

[0031] The present invention has the following advantages:

[0032] 1. The present invention isolates a steroidal glycoalkaloid of the spirostane-type tomatidine subtype from millennium tomato fruit (i.e., a water-soluble tomato extract having the chemical structure of Formula I in the present application). The compound can inhibit LPS-induced NO production with a half-maximal inhibitory concentration of 4.2 μg / mL and a selectivity index greater than 4.76. It is applicable to the preparation of anti-inflammatory drugs; it is also applicable to the preparation of lipid-lowering drugs, and can inhibit triglyceride accumulation in a HepG2 cell steatosis model induced by sodium oleate and downregulate LDLR protein expression in HepG2 cells.

[0033] The present invention utilizes a specific enzymatic hydrolysis step combined with membrane separation technology to effectively remove small molecular weight impurities while retaining the target component. Compared to traditional extraction processes, this method can more accurately isolate the desired component and improve the purity of the target product. In particular, strict control of pH, temperature, and enzyme dosage during the enzymatic hydrolysis process maximizes enzyme activity, thereby increasing the yield of the target component. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0035] The structures, proportions, sizes, etc. illustrated in this specification are intended only to complement the contents disclosed herein and to facilitate understanding and reading by persons familiar with the art. They are not intended to limit the conditions under which the present invention may be implemented and therefore have no substantive technical significance. Any structural modifications, changes in proportions, or adjustments in sizes, without affecting the efficacy and objectives of the present invention, shall still fall within the scope of the technical contents disclosed herein.

[0036] Figure 1 The effects of different concentrations of XM-F170 provided by the present invention on the intracellular nitric oxide content;

[0037] Figure 2 The concentration-dependent curves of different concentrations of XM-F170 provided by the present invention inhibiting LPS-induced NO production in RAW 264.7 cells;

[0038] Figure 3 The effect of XM-F170 provided by the present invention on the viability of RAW264.7 cells in a concentration range of 0.125 to 2 μg / mL;

[0039] Figure 4 Effects of different concentrations of XM-F170 provided by the present invention on triglyceride content in HepG2 cells;

[0040] Figure 5 A schematic diagram of the protein immunoblotting test results provided by the present invention;

[0041] Figure 6 The present invention provides the effect of XM-F170 on the expression of LDLR protein in hepatocytes. DETAILED DESCRIPTION

[0042] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0043] The present invention provides a method for preparing tomato saponin from a tomato extract, comprising the following steps:

[0044] S1. Wash the tomatoes with water, squeeze the tomato juice, and then filter it for later use;

[0045] S2. The tomato juice filtered by S1 was added with complex enzyme A and complex enzyme B for continuous enzymatic hydrolysis and then the enzyme was inactivated to obtain an enzyme hydrolyzate after enzyme inactivation;

[0046] S3. The enzyme-inactivated hydrolyzate is subjected to membrane separation using an organic membrane coating to remove small molecular weight components to obtain a retained straight-through solution;

[0047] S4. The retentate after membrane separation was heated at 50-100°C for 1-24h and then concentrated and freeze-dried to obtain a dry powdered extract;

[0048] S5. The powdered extract obtained in S3 was separated by ODS C18 column chromatography with gradient elution, and the collected elution fractions were separated by C18 column preparative liquid chromatography;

[0049] S6. The solution separated by the C18 column was concentrated and dried again to obtain a water-soluble tomato extract having the structural formula of Formula I (laboratory code: XM-F170).

[0050] Formula I:

[0051]

[0052] Among them, S2 includes the following specific steps:

[0053] S21. Primary enzymatic hydrolysis: Add 8-10 times the amount of ultrapure water to the filtered tomato juice from S1, then stir to obtain a suspension. Adjust the pH to 4.5-5.5, then add 0.2-0.6% by weight of complex enzyme A to the suspension, and then add 1 / 10 by weight of stabilizer A to the complex enzyme A. Gradually raise the temperature to 45-55°C and perform enzymatic hydrolysis for 1-1.5 hours to obtain a primary enzymatic solution.

[0054] S22. Secondary enzymatic hydrolysis: The pH of the primary enzymatic solution was adjusted to 6.5-8.5, followed by addition of 0.1-0.3% by weight of the primary enzymatic solution of complex enzyme B for enzymatic hydrolysis, followed by addition of 1 / 10 by weight of the complex enzyme B of catalyst B, and the temperature was heated to 55-65 ° C and kept warm for 1.5-2h to obtain a secondary enzymatic solution;

[0055] S23. Inactivate enzymes: Heat the obtained secondary enzymatic hydrolysate to 90-95°C and keep it warm for 15-20 minutes to inactivate enzymes.

[0056] Among them, the complex enzyme A in S21 is composed of phytase, cellulase, pectinase, xylanase and β-glucosidase, and the mass ratio of phytase, cellulase, pectinase, xylanase and β-glucosidase in the complex enzyme A is (0.08-1.5): (0.05-3): (0.05-1): (0.05-1): (0.05-1);

[0057] The complex enzyme B in S22 is composed of papain, trypsin, Alcalase protease, endoprotease, exoprotease and flavor protease. The alkaline protease in the complex enzyme B is composed of papain and trypsin. The mass ratio of papain, trypsin, Alcalase protease, endoprotease, exoprotease and flavor protease is (0.05-1.2): (0.05-1.2): (0.05-1.2): (0.08-1.5): (0.08-1.5).

[0058] Specifically, the stabilizer A in S21 is one or more of CaCl2, MgCl2, and ZnSO4. Preferably, CaCl2 is selected in this application;

[0059] The catalyst B in S22 is one or more of N-acetylglucosamine, mannose, and trehalose. Preferably, N-acetylglucosamine is selected in this application.

[0060] The specific steps in S3 are:

[0061] The enzymatic hydrolysate after enzyme inactivation is passed through a membrane separation device loaded with a 500Da organic membrane coating for membrane separation. Components with a molecular weight less than 500 can pass through the organic coating and be discharged from the membrane separation device, while components with a molecular weight greater than 500 are retained in the membrane separation device to obtain retained straight-through liquid.

[0062] Specifically, the eluent in S5 is acetonitrile and water in a volume ratio of 15:85-20:80 and 50:50-100:0.

[0063] Specifically, the elution fraction in S5 is collected from the elution fraction having a volume ratio of acetonitrile to water of 50:50 to 100:0.

[0064] This also includes structural characterization testing of the water-soluble tomato extract obtained through separation and preparation.

[0065] The present invention also provides a method for preparing tomato saponin from tomato extract and application of the prepared tomato saponin in preparing anti-inflammatory drugs.

[0066] The present invention also provides a method for preparing tomato extract tomato saponin and application of the prepared tomato saponin in preparing lipid-lowering drugs.

[0067] Example 1

[0068] This embodiment provides a method for preparing tomato saponin from a tomato extract, comprising the following steps:

[0069] S1. Wash the tomatoes with water, squeeze the tomato juice, and then filter it for later use;

[0070] S2. Compound enzyme A and compound enzyme B are added to the tomato juice filtered by S1 for continuous enzymatic hydrolysis and then the enzyme is inactivated to obtain an enzyme hydrolyzate after enzyme inactivation; the specific steps are as follows:

[0071] S21. Primary enzymatic hydrolysis: The filtered tomato juice from S1 was added to 8 times the volume of ultrapure water, and then stirred to obtain a suspension. The pH was adjusted to 5, and then 0.4% by weight of complex enzyme A was added, followed by 1 / 10 by weight of the complex enzyme A at CaCl2. The temperature was gradually raised to 45-55°C, and the enzymatic hydrolysis time was 1-1.5 h to obtain a primary enzymatic solution.

[0072] S22. Secondary enzymatic hydrolysis: The pH of the primary hydrolyzate was adjusted to 7, followed by addition of 0.2% by weight of complex enzyme B to perform enzymatic hydrolysis, followed by addition of 1 / 10 by weight of N-acetylglucosamine of the complex enzyme B, and the temperature was heated to 55-65°C and kept warm for 2h to obtain a secondary enzymatic hydrolyzate.

[0073] S23. Inactivation of enzymes: The obtained secondary enzymatic hydrolysate was heated to 90°C and kept warm for 15 minutes to inactivate the enzymes.

[0074] Among them, the complex enzyme A in S21 is composed of phytase, cellulase, pectinase, xylanase and β-glucosidase, and the mass ratio of phytase, cellulase, pectinase, xylanase and β-glucosidase in the complex enzyme A is 0.5:1:0.5:0.5:0.5;

[0075] The complex enzyme B in S22 is composed of papain, trypsin, Alcalase protease, endoprotease, exoprotease and flavor protease. The alkaline protease in the complex enzyme B is composed of papain and trypsin. The mass ratio of papain, trypsin, Alcalase protease, endoprotease, exoprotease and flavor protease is 0.5:0.5:1:1:0.5.

[0076] S3. The enzyme-inactivated hydrolyzate is passed through a membrane separation device loaded with a 500Da organic membrane coating for membrane separation. Components with a molecular weight less than 500 can pass through the organic coating and be discharged from the membrane separation device, while components with a molecular weight greater than 500 are retained in the membrane separation device to obtain a retained straight-through liquid.

[0077] S4. The retentate after membrane separation was heated at 100°C for 12 h and then concentrated and freeze-dried to obtain a dry powder extract;

[0078] S5. The powdered extract obtained in S3 is subjected to gradient elution separation by ODS C18 column chromatography, and the collected elution fractions are then separated by C18 column preparative liquid chromatography; wherein, the eluent in S5 has a volume ratio starting from 20:80 and gradually transitioning to 100:0, and the elution fractions with a volume ratio of acetonitrile to water of 50:50-100:0 are collected.

[0079] S6. The solution separated by the C18 column is concentrated and dried again to obtain a water-soluble tomato extract having the structural formula I.

[0080] Example 2 Structural Evidence

[0081] 1H NMR (DMSO-d6, 600MHz): δ0.77 (3H, s, H3-19), 0.79 (3H, s, H3-18), 1.16 (3H, s, H3-21);

[0082] 13C NMR (DMSO-d6, 151MHz): δ12.3, 14.7, 18.9, 20.3, 25.8, 28.5, 29.1, 31.8, 32.4, 33.2, 34.1, 34.7, 35.5, 36.7, 36.9, 39.5, 42.3, 44.3, 47.0, 52.9, 54.5, 57.2, 57.9, 59.6, 60.9, 61.2, 6 1.5, 62.9, 63.1, 66.0, 68.0, 68.9, 69.5, 69.7, 70.1, 71.6, 72.6, 73.4, 73.6, 73.9, 74.4, 76.0, 76.1, 76.5, 76.7, 76.8, 77.1, 77.2, 77.2, 79.2, 79.6, 85.1, 92.8, 101.0, 102.7, 103.5;

[0083] HR-ESI-MS: m / z 1250.5181[M+Na] + ,(calcd for C 56 H 93 NO 28 Na + 1250.5776).

[0084] Example 3 Anti-inflammatory activity test

[0085] Mouse macrophage RAW 264.7 cell line was cultured to the logarithmic growth phase according to conventional methods. The desired cells were washed with preheated complete medium and gently pipetted to collect the desired cells. 100 μL of complete medium (complete medium is DMEM basal medium supplemented with 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin mixture, and L-glutamine) was added and resuspended into a single cell suspension. The cells were counted on a hemocytometer and the cell density was adjusted to 1×10 5 cells / mL, according to 100 μL / well, that is, 1×10 4 Cells were seeded in 96-well plates at 400 μg / well. After overnight seeding to ensure cell attachment, the drug stock solution to be tested was added to complete culture medium in a clean bench. A series of drug-containing medium concentrations was prepared by serial dilution. The highest concentration of drug-containing medium did not contain more than 1‰ DMSO by volume. The drug-containing medium was prepared and used immediately, and the complete culture medium in the 96-well plate was discarded. Five concentration groups (0.625, 1.25, 2.5, 5, and 10 μg / mL) were set up for XM-F170 administration, and 100 μL of the corresponding drug-containing medium was added to each well. For the positive drug group, 100 μL of 10 μM berberine-containing medium was added to each well. For the blank control group and the inflammatory model group, 100 μL of complete culture medium was added to each well. Three replicate wells were set up for each group. One hour after drug intervention, LPS was added to a final concentration of 1 μg / mL in all groups except the blank control group to induce macrophage differentiation into an inflammatory state. The cells were then cultured in an incubator for an additional 24 hours. After 24 hours of drug treatment, 50 μL of cell supernatant was collected and the NO content was determined using the Griess method according to the instructions of the nitric oxide detection kit. After normalizing the absorbance value of the blank control group, the NO production in each group was calculated.

[0086] Example 4 Cell Viability Test

[0087] Mouse macrophage RAW 264.7 cell line was cultured to the logarithmic growth phase according to conventional methods. The desired cells were washed with preheated complete medium and gently pipetted to collect the desired cells. 100 μL of complete medium (complete medium is DMEM basal medium supplemented with 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin mixture, and L-glutamine) was added and resuspended into a single cell suspension. The cells were counted on a hemocytometer and the cell density was adjusted to 5 × 10 4 cells / mL, according to 100 μL / well, that is, 5×10 3Cells were seeded in 96-well plates at 400 μg / well. After the cells were seeded overnight to ensure cell attachment, the stock solution of the drug to be tested was added to complete culture medium in a clean bench. A series of drug-containing culture medium concentrations were prepared by serial dilution. The highest concentration of drug-containing culture medium did not contain more than 1‰ DMSO by volume. The drug-containing culture medium was prepared and used immediately. The complete culture medium in the 96-well plate was discarded. Five concentration groups (0.625, 1.25, 2.5, 5, 10, and 20 μg / mL) were set up for the XM-F170 treatment group, and 100 μL of the corresponding drug-containing culture medium was added to each well. 100 μL of complete culture medium was added to each well of the negative control group. Eight replicate wells were set up for each group and cultured in an incubator for another 24 hours. After 24 hours of drug treatment, CCK8 working solution was added to the cell supernatant and incubated in the incubator for another 30 minutes. The absorbance value was measured according to the CCK8 kit instructions. After normalization to the absorbance value of the negative control group, the cell survival rate of each group was calculated.

[0088] Example 5 Sodium oleate-induced cell steatosis model

[0089] The human hepatoma cell line HepG2 was cultured to the logarithmic growth phase according to conventional methods. The desired cells were collected by washing with PBS, trypsinizing, and centrifuging. 100 μL of complete culture medium (complete culture medium is DMEM basal medium supplemented with 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin mixture, and L-glutamine) was added and resuspended into a single cell suspension. The cells were counted on a hemocytometer and the cell density was adjusted to 1×105 cells / mL. The cell density was adjusted to 1×105 cells / mL at 1 mL / well, i.e., 1×10 5 Cells were seeded at 400 μg / well in 24-well plates. After overnight seeding to ensure cell attachment, a 200 μM sodium oleate-containing medium was prepared in a clean bench. The stock solution of the drug to be tested was added to the sodium oleate-containing medium and serially diluted to prepare a series of drug-containing medium concentrations. The highest concentration of the drug-containing medium did not contain more than 1‰ of DMSO by volume. The drug-containing medium was prepared and used immediately, and the complete medium in the 24-well plate was discarded. Five concentration groups (1.25, 2.5, 5, and 10 μg / mL) of XM-F170 were set up in the treatment group, and 1 mL of the corresponding drug-containing medium was added to each well. 1 mL of complete medium was added to each well of the negative control group and the high-fat model group. 1 mL of sodium oleate-containing medium was added to each well of the positive drug group. 1 mL of the corresponding drug-containing medium was added to each well of the two drugs (berberine 10 μM and simvastatin 10 μM) in the positive drug group. Three replicate wells were set up in each group and cultured in an incubator for 24 hours. After 24 hours of drug treatment, the culture medium was discarded, and the cells were washed with pre-cooled PBS. Lysis buffer was added to lyse the cells and the total protein sample was collected. The protein concentration and triglyceride were tested according to the instructions of the triglyceride test kit, and the intracellular triglyceride content was calculated.

[0090] Example 6 Protein immunoblotting experiment

[0091] The human hepatoma cell line HepG2 was cultured to the logarithmic growth phase according to conventional methods. The desired cells were collected by washing with PBS, digesting with trypsin, and centrifuging. After adding an appropriate amount of complete culture medium to resuspend into a single cell suspension, the cells were counted on a hemocytometer and the cell density was adjusted to 4 × 10 5 Cells were seeded at 10 μg / mL in 5 mm medium-diameter culture dishes. After overnight seeding to ensure cell attachment, the drug stock solution was added to complete sodium chloride medium in a clean bench to prepare a 10 μg / mL drug-containing medium. 8 mL of drug-containing medium was added to each dish in the XM-F170-treated group, and 8 mL of complete medium was added to each dish in the normal control group. Three replicate wells were set up for each group and cultured in an incubator for an additional 24 hours. After 24 hours of drug exposure, the medium was discarded, and cells were washed with pre-chilled PBS. Lysis buffer was added to lyse the cells, and total protein samples were collected. Total protein samples were added to 1× loading buffer at a 4:1 volume ratio and heat-denatured. Western blotting was then performed. After gel separation and transfer to a membrane, the test protein samples were incubated with an anti-LDLR primary antibody. LDLR protein expression in HepG2 cells was semi-quantified using a secondary antibody and chemiluminescent solution under a microscope. The values ​​were analyzed using ImageJ software. Relative protein expression levels in the XM-F170 group were calculated after normalization to the grayscale values ​​of the normal control group.

[0092] Example 7 Experimental Results

[0093] (1) XM-F170 has anti-inflammatory activity

[0094] Anti-inflammatory activity tests found that XM-F170 has a concentration-dependent inhibitory effect on LPS-induced NO production in RAW 264.7 cells. The inhibitory effect at a concentration of 10 μg / mL is comparable to that of berberine at a concentration of 10 μg / mL. The calculated half-maximal inhibitory concentration of its anti-inflammatory activity is 4.2 μg / mL, indicating that XM-F170 has a significant effect in inhibiting inflammatory responses. Figure 1 and Figure 2 shown.

[0095] (2) XM-F170 has no killing effect on immune cells

[0096] Cell viability tests found that XM-F170 had no effect on the viability of RAW264.7 cells at concentrations of 1.25-20 μg / mL. Figure 3The median toxicity concentration for RAW 264.7 cells was calculated to be greater than 20 μg / mL. The selectivity index of XM-F170 was calculated to be greater than 4.76, as shown in Table 1. This indicates that XM-F170 has a good safety profile.

[0097] Table 1 Pharmacological parameters of XM-F170 on RAW 264.7 cells

[0098]

[0099] (3) XM-F170 inhibits triglyceride accumulation in cellular steatosis

[0100] Sodium oleate was used to induce HepG2 cells to establish a fatty degeneration model, and the intracellular triglyceride content test found that Figure 4 As shown in the results, XM-F170 inhibited the accumulation of triglycerides during cellular steatosis at a concentration of 1.25-10 μg / mL. The inhibitory effect at a concentration of 10 μg / mL was comparable to that of positive drugs (10 μM berberine, 10 μM simvastatin), indicating that XM-F170 has potential therapeutic effects in inhibiting steatosis.

[0101] (4) XM-F170 upregulates LDLR protein expression in hepatocytes

[0102] like Figure 5 and Figure 6 As shown, Western blotting revealed that XM-F170 treatment of hepatocytes upregulated the expression of the low-density lipoprotein receptor (LDLR). The LDLR recognizes circulating LDL cholesterol and internalizes it for degradation, thereby reducing cardiovascular risk. At a concentration of 10 μg / mL, LDLR protein was upregulated by 29±5%. This suggests that XM-F170 can promote the clearance of low-density lipoprotein cholesterol (LDL-C) by upregulating LDLR expression, thereby helping to reduce the risk of cardiovascular disease.

[0103] In summary, XM-F170 not only exhibits significant anti-inflammatory activity but also exhibits a favorable safety profile at the cellular level and has potential therapeutic efficacy in inhibiting steatosis and upregulating LDLR protein expression. These results suggest that XM-F170 has promising application prospects in the prevention and treatment of cardiovascular and inflammatory diseases.

[0104] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for preparing tomato saponin from tomato extract, characterized in that: The structural formula of the tomato extract tomato saponin compound is: The preparation method comprises the following steps: S1. Wash the tomatoes with water, squeeze the tomato juice, and then filter it for later use; S2. The tomato juice filtered by S1 was added with complex enzyme A and complex enzyme B for continuous enzymatic hydrolysis and then the enzyme was inactivated to obtain an enzyme hydrolyzate after enzyme inactivation; S3. The enzyme-inactivated hydrolyzate is subjected to membrane separation using an organic membrane coating to remove small molecular weight components to obtain a retained straight-through solution; S4. The retentate after membrane separation was heated at 50-100°C for 1-24h and then concentrated and freeze-dried to obtain a dry powdered extract; S5. The powdered extract obtained in S3 was separated by ODS C18 column chromatography with gradient elution, and the collected elution fractions were separated by C18 column preparative liquid chromatography; S6. The solution after separation by the C18 column is concentrated and dried again to obtain a water-soluble tomato extract; The S2 includes the following specific steps: S21. Primary enzymatic hydrolysis: Add 8-10 times the amount of ultrapure water to the filtered tomato juice from S1, then stir to obtain a suspension. Adjust the pH to 4.5-5.5, then add 0.2-0.6% by weight of complex enzyme A to the suspension, and then add 1 / 10 by weight of stabilizer A to the complex enzyme A. Gradually raise the temperature to 45-55°C and perform enzymatic hydrolysis for 1-1.5 hours to obtain a primary enzymatic solution. S22. Secondary enzymatic hydrolysis: The pH of the primary enzymatic solution was adjusted to 6.5-8.5, followed by addition of 0.1-0.3% by weight of the primary enzymatic solution of complex enzyme B for enzymatic hydrolysis, followed by addition of 1 / 10 by weight of the complex enzyme B of catalyst B, and the temperature was heated to 55-65 ° C and kept warm for 1.5-2h to obtain a secondary enzymatic solution; S23 enzyme inactivation: The resulting secondary enzymatic hydrolyzate was heated to 90-95 ° C, kept warm for 15-20min, and the enzyme was inactivated; The complex enzyme A in S21 is composed of phytase, cellulase, pectinase, xylanase and β-glucosidase, and the mass ratio of phytase, cellulase, pectinase, xylanase and β-glucosidase in the complex enzyme A is (0.08-1.5): (0.05-3): (0.05-1): (0.05-1): (0.05-1); The complex enzyme B in S22 is composed of papain, trypsin, Alcalase protease, endoprotease, exoprotease and flavor protease, the alkaline protease in the complex enzyme B is composed of papain and trypsin, and the mass ratio of papain, trypsin, Alcalase protease, endoprotease, exoprotease and flavor protease is (0.05-1.2): (0.05-1.2): (0.05-1.2): (0.08-1.5): (0.08-1.5); The stabilizer A in S21 is one or more of CaCl2, MgCl2, and ZnSO4; The catalyst B in S22 is one or more of N-acetylglucosamine, mannose, and trehalose.

2. The method for preparing tomato saponin from tomato extract according to claim 1, characterized in that: The specific steps in S3 are: The enzymatic hydrolysate after enzyme inactivation is passed through a membrane separation device loaded with a 500Da organic membrane coating for membrane separation. Components with a molecular weight less than 500 can pass through the organic coating and be discharged from the membrane separation device, while components with a molecular weight greater than 500 are retained in the membrane separation device to obtain retained straight-through liquid.

3. The method for preparing tomato saponin from tomato extract according to claim 1, characterized in that: In the S5, the eluent is acetonitrile and water in a volume ratio of 15:85-20:80, 50:50-100:

0.

4. The method for preparing tomato saponin from tomato extract according to claim 3, characterized in that: In S5, the elution fraction is collected from the elution fraction having a volume ratio of acetonitrile to water of 50:50 to 100:

0.

5. The method for preparing tomato saponin from tomato extract according to claim 1, characterized in that: It also includes structural characterization testing of the isolated and prepared water-soluble tomato extract.

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