Furanopetanin, a petunidin derivative from lycium ruthenicum murr, its extraction method and use

Abstract

The application discloses a new petunidin derivative furanopetanin in Lycium ruthenicum, an extraction method and application thereof, and a preparation chromatographic separation of Lycium ruthenicum extract, and the new petunidin derivative furanopetanin can be obtained through multiple preparation separations. The application further provides application of the new compound furanopetanin in preparation of products for preventing and / or treating inflammation, in preparation of products for preventing and / or treating sugar metabolism disorder related diseases, and in preparation of products for preventing and / or treating lipid metabolism disorder related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant active component separation, in particular to a petunidin derivative furanopetanin in Lycium ruthenicum Murr and an extraction method and use thereof. BACKGROUND

[0002] Lycium ruthenicum Murr is a perennial shrub of Solanaceae Lycium with many spiny thorns, and its fruits are sweet and juicy, rich in nutrients, and have great research and development value. Lycium ruthenicum Murr is a unique desert medicinal plant species in the west of China. Lycium ruthenicum Murr contains various essential amino acids and rich mineral elements for the human body, and is used in Tibetan medicine to treat heart heat disease, heart disease, lower cholesterol, stimulate brain nerves, enhance immune function, prevent and treat cancer, anti-aging, beauty and skin care, irregular menstruation, menopause, etc., and has significant effects.

[0003] Anthocyanins are a class of water-soluble natural pigments widely existing in plants in nature, and belong to flavonoids. Anthocyanins mainly exist in the form of glucoside, rhamnoside, etc. in the fruits, epidermis and flowers of plants. The content of anthocyanins in Lycium ruthenicum Murr is extremely high, more than 90% of which are petunidin derivatives, which have functions of antioxidant, vision protection, tumor inhibition, etc., and have important biological activity and extraction value. With the increasing demand for functional components from natural sources and without toxic side effects, anthocyanins are the most representative functional components from natural sources. Due to their good coloring function and excellent antioxidant activity, they are favored by consumers and the market.

[0004] The rich active substances contained in Lycium ruthenicum Murr have not been separated and identified, and if the new chemical components in Lycium ruthenicum Murr and their pharmacological effects are further studied and the activity mechanism is explored, more safe and effective natural plant-derived drugs for treating diseases can be developed. SUMMARY

[0005] The present application provides an active component and a new petunidin derivative extracted from Lycium ruthenicum Murr, an extraction method thereof and a use thereof in preparing a drug for preventing and / or treating inflammation-related diseases, and the component and the compound both have the effect of inhibiting the expression of iNOS protein to reduce the amount of NO release.

[0006] The present application provides a Lycium ruthenicum Murr component Fr2-5, the retention time of which is 76-130 min, and the component Fr2-5 is prepared by the following method;

[0007] (1) Lycium ruthenicum Murr extract is prepared by preparative chromatography to obtain components Fr1, Fr2 and Fr3, and the retention times are 12-39 min, 39-139 min and 139-230 min, respectively;

[0008] The preparation chromatography conditions include:

[0009] The chromatographic column is MCI medium pressure chromatography tower, preferably with a specification of 49*460mm;

[0010] The mobile phase is water: A / methanol: B / dichloromethane: C; gradient elution is performed by using the following program: 0-120min, 100% A-100% B; 120-180min, 100% B-100% C; 180-210min, 100% B; 210-240min, 100% A;

[0011] (2) The component Fr2 is subjected to preparation chromatography separation to obtain the component Fr2-5;

[0012] The preparation chromatography conditions include:

[0013] The chromatographic column is MCI medium pressure chromatography tower, preferably with a specification of 49*460mm;

[0014] The mobile phase is A: water / B: methanol; gradient elution is performed by using the following program: 0-120min, 0%-100% B; 20-140min, 100% B.

[0015] In the present application, the Lycium ruthenicum Murr extract refers to a concentrate obtained by filtering and light-shielded concentration after methanol extraction of Lycium ruthenicum Murr, and the extraction method includes but is not limited to conventional extraction methods such as immersion extraction, hot reflux extraction, ultrasonic extraction, etc.

[0016] In the specific embodiment of the present application, Lycium ruthenicum Murr is extracted by using the immersion extraction method, and the extraction conditions are as follows: liquid-to-material ratio 10-30mL / g, preferably 20mL / g; extraction times: 2-5 times, 3-5 days each time, preferably 3 times, 4-5 days each time.

[0017] Further, the extract is filtered after extraction of Lycium ruthenicum Murr, and is subjected to light-shielded reduced-pressure concentration and combination to obtain Lycium ruthenicum Murr fruit methanol extract extract.

[0018] In the present application, polyamide is added to the methanol extract extract for sample mixing, drying, grinding and sieving.

[0019] Further, the methanol extract extract: polyamide is 1:(0.5-2.5), preferably 1:1.

[0020] Further, the drying method can be selected from normal-pressure drying, reduced-pressure drying and freeze-drying, etc.

[0021] Further, the sieving is 20-50 mesh, preferably 20 mesh.

[0022] The application provides a Lycium ruthenicum Murr component Fr2-5-4, and the component Fr2-5-4 is obtained by preparing chromatography separation on the component Fr2-5, and the retention time of the component Fr2-5-4 is 21-27 min.

[0023] The preparing chromatography comprises the following conditions:

[0024] The chromatographic column is a kromasil C18 chromatographic column, preferably with a specification of 21.2*250 mm.

[0025] The mobile phase is A: water / B: methanol, and gradient elution is performed by using the following program: 0-60-65-90 min, 30%-42%-70%-95% B.

[0026] The application provides a Lycium ruthenicum Murr component Fr2-5-5, and the component Fr2-5-5 is obtained by preparing chromatography separation on the component Fr2-5, and the retention time of the component Fr2-5-5 is 27-36 min.

[0027] The preparing chromatography comprises the following conditions:

[0028] The chromatographic column is a kromasil C18 chromatographic column, preferably with a specification of 21.2*250 mm.

[0029] The mobile phase is A: water / B: methanol, and gradient elution is performed by using the following program: 0-60-65-90 min, 30%-42%-70%-95% B.

[0030] The application provides a petunidin derivative IV, and a structural formula of the petunidin derivative IV is as shown in formula IV.

[0031]

[0032] The application extracts Lycium ruthenicum Murr fruits by using methanol, separates by preparing chromatography, and identifies by one-dimensional and two-dimensional nuclear magnetic resonance, so that a new petunidin derivative is unexpectedly obtained and is named as furanopetanin.

[0033] The petunidin derivative IV is obtained by preparing chromatography separation on the component Fr2-5-5, and the retention time is 92-95 min.

[0034] The preparing chromatography comprises the following conditions:

[0035] The chromatographic column is a kromasil C18 chromatographic column, preferably with a specification of 21.2*250 mm.

[0036] Mobile phase: A: water / B: methanol; gradient elution is carried out by using the following procedure: 0-60 min, 30%-35% B.

[0037] The present application provides a petunidin derivative V, and the structural formula is shown as formula V.

[0038]

[0039] The present application extracts the fruit of Lycium ruthenicum Murr by using methanol, separates by preparative chromatography, and identifies by one-dimensional and two-dimensional nuclear magnetic resonance, and unexpectedly obtains a new petunidin derivative, which is named secopetanin.

[0040] The petunidin derivative V is obtained by preparative chromatography separation of component Fr2-5-4, and the retention time is 40-41 min.

[0041] Further, the conditions of the preparative chromatography include:

[0042] The chromatographic column is a kromasil C18 chromatographic column, and the preferred specification is 21.2*250mm.

[0043] Mobile phase: A: water / B: methanol; gradient elution is carried out by using the following procedure: 0-60 min, 30%-35% B.

[0044] In the specific embodiments of the present application, the preparative chromatography separation further includes at least one of the following conditions:

[0045] Detection wavelength: 210nm;

[0046] Column temperature: 25-35℃;

[0047] Flow rate: 15-60mL / min;

[0048] Injection volume: 0.1-8.0mL.

[0049] The present application provides the above-mentioned petunidin derivatives IV and / or V, and pharmaceutically acceptable salts, hydrates or solvates thereof, and the application in the preparation of products for treating and / or preventing inflammation-related diseases.

[0050] The present application provides at least one of the above-mentioned components Fr2-5, component Fr2-5-4 and component Fr2-5-5, and the application in the preparation of products for treating and / or preventing inflammation-related diseases.

[0051] Further, the above-mentioned two products for treating and / or preventing inflammation-related diseases are products for blocking and / or inhibiting the signal pathway of NF-κB.

[0052] NF-κB is a protein complex that controls the transcription of DNA, cytokine production and cell survival; and is involved in the cellular response to stimuli, such as stress, cytokines, free radicals, heavy metals, ultraviolet irradiation, oxidized LDL and bacterial or viral antigens; and plays a key role in regulating the immune response to infection.

[0053] Further, the product is a product for reducing the release amount of at least one of NO, PGE2, TNF-α, IL-β, IL-6, COX-2 and iNOS; and more further, the product is a product for reducing the release amount of NO.

[0054] Further, the product is a product for inhibiting the expression of iNOS protein.

[0055] The present application also provides an anti-inflammatory product comprising one or more of component Fr2-5, component Fr2-5-4, component Fr2-5-5, compound IV and compound V.

[0056] The present application provides the use of the above-mentioned petunidin derivatives IV and / or V, and pharmaceutically acceptable salts, hydrates or solvates thereof, in the preparation of a product for treating and / or preventing a disease related to glucose metabolism disorder.

[0057] The present application provides the use of at least one of the above-mentioned component Fr2-5, component Fr2-5-4 and component Fr2-5-5, in the preparation of a product for treating and / or preventing a disease related to glucose metabolism disorder.

[0058] Further, both of the above-mentioned products for treating and / or preventing a disease related to glucose metabolism disorder are products with hypoglycemic effect, such as hypoglycemic drugs and the like.

[0059] The product for treating and / or preventing a disease related to glucose metabolism disorder can reduce blood glucose through the pathways of inhibiting hepatic gluconeogenesis and hepatic glucose output, increasing the sensitivity of peripheral tissues to insulin, promoting glucose uptake and utilization, stimulating the secretion of insulin by pancreatic beta cells, enhancing the binding of insulin to receptors, increasing the sensitivity of target cells to insulin and the like.

[0060] Further, the product is a product for promoting the uptake of glucose and / or sugar analogues by adipocytes.

[0061] The glucose uptake of adipocytes stimulated by insulin is mainly carried out through GLUT4 (glucose transporter) sensitive to insulin; under the stimulation of insulin, the intracellular transmission of insulin receptor tyrosine phosphorylation signal phosphorylates insulin receptor substrate-1 (IRS-1), thereby activating phosphatidylinositol-3-kinase (PI3K) and triggering the translocation of GLUT4 to the cell surface, increasing the uptake of glucose.

[0062] Further, the product is a product for increasing the expression level of p-AKT and p-PI3K proteins.

[0063] AKT is a serine / threonine protein kinase, which is involved in glucose metabolism, apoptosis, cell proliferation, cell transport, etc. In the process of glucose metabolism, activated AKT activates a variety of enzymes, kinases and transcription factors in the insulin signaling pathway through the phosphorylation pathway, and then regulates cell function and insulin signal transmission. Generally, activated AKT promotes the translocation of glucose transporter from cytoplasm to cell membrane by activating its downstream phosphatidylinositol kinase 3 (PI3K), accelerates the absorption and utilization of glucose, and thus plays a role in insulin signal transmission.

[0064] p-PI3K (mouse phosphorylated phosphoinositide 3 kinase) can activate or inhibit a series of downstream substrates through phosphorylation, and the activity of apoptosis-related proteins, thereby regulating cell proliferation, differentiation, apoptosis, migration and other phenotypes.

[0065] The application provides application of the above-mentioned petunidin derivatives IV and / or V, and pharmaceutically acceptable salts, hydrates or solvates thereof in preparation of products for treating and / or preventing diseases related to lipid metabolism disorders.

[0066] The application provides application of at least one of the above-mentioned components Fr2-5, component Fr2-5-4 and component Fr2-5-5 in preparation of products for treating and / or preventing diseases related to lipid metabolism disorders.

[0067] Further, the above-mentioned two types of products for treating and / or preventing diseases related to lipid metabolism disorders are products with lipid-lowering effects, such as lipid-lowering drugs.

[0068] Further, the above-mentioned two types of products for treating and / or preventing diseases related to lipid metabolism disorders include products for inhibiting lipid accumulation in adipocytes and / or products for inhibiting lipid synthesis in cells; the products can be drugs, health products or other products for inhibiting lipid accumulation in cells and / or products for inhibiting lipid synthesis in cells.

[0069] When the product is a drug, the drug includes at least one of the following: prevention and / or treatment of obesity, hypertension, hyperlipidemia, cardiovascular diseases and metabolic syndrome-related diseases.

[0070] Diseases such as obesity, hypertension, hyperlipidemia, cardiovascular disease, metabolic syndrome and the like are closely related to the content of plasma lipids such as triglyceride (TG), free cholesterol (FC), cholesterin lipids (CE) and phospholipids, and when the plasma lipids in the body are reduced to a certain concentration range, these diseases can be effectively controlled or treated. The present application proves through experiments that the active components and / or new compounds in the present application can effectively inhibit lipid droplet accumulation, reduce the content of TG, and regulate the gene expression level of adipocyte transcription factor and the expression level of related proteins through relevant signal pathways, thereby inhibiting the adipogenic differentiation and lipid generation of cells, and can be used for preparing products for preventing and / or treating obesity, hypertension, hyperlipidemia, cardiovascular disease, metabolic syndrome and the like.

[0071] The present application provides the use of the above-mentioned petunidin derivatives IV and / or V, and pharmaceutically acceptable salts, hydrates or solvates thereof, in the preparation of at least one of PPAR gamma antagonists, C / EBP alpha antagonists, FAS inhibitors, ACC inhibitors.

[0072] The present application provides the use of at least one of the above-mentioned components Fr2-5, component Fr2-5-4 and component Fr2-5-5 in the preparation of at least one of PPAR gamma antagonists, C / EBP alpha antagonists, FAS inhibitors, ACC inhibitors.

[0073] The PPAR gamma antagonists, C / EBP alpha antagonists, FAS inhibitors, ACC inhibitors are drugs that reduce the gene expression of adipocyte transcription factors PPAR gamma and C / EBP alpha and the protein expression level of FAS and ACC.

[0074] The differentiation of undifferentiated cells into mature adipocytes requires a series of complex and fine transcription factor regulation, and PPAR gamma is an indispensable regulatory factor for adipocyte formation and is highly expressed in the early stage of adipogenic process; C / EBP alpha, also known as CCAAT / enhancer binding protein alpha, is highly expressed in the middle stage of adipocyte differentiation.

[0075] FAS is a fatty acid synthase and plays an important role in lipid generation; ACC is acetyl-CoA carboxylase, which is a rate-limiting enzyme for de novo fatty acid synthesis.

[0076] The active components and new compounds in the present application can reduce the gene expression of cell transcription factors PPAR gamma and C / EBP alpha and the protein expression level of FAS and ACC, thereby being able to effectively inhibit the products of adipogenic differentiation of cells and inhibit the lipid generation of cells, and further being used for preparing products for preventing and / or treating diseases related to lipid metabolism disorders, such as obesity, hypertension, hyperlipidemia, cardiovascular disease and the like.

[0077] The application further provides a hypoglycemic and / or hypolipidemic product comprising one or more of the component Fr2-5, the component Fr2-5-4, the component Fr2-5-5, the compound IV, and the compound V.

[0078] The product described in the application includes but is not limited to drugs, health products, food, etc.

[0079] The application has the beneficial effects that the application provides three active components and two new compounds furanopetanin and secopetanin, the components and the compounds have the effects of anti-inflammation, hypoglycemia and hypolipidemia, the active substances of Lycium ruthenicum Murr are more comprehensively developed, the medicinal value of Lycium ruthenicum Murr is more comprehensively tapped, the clinical application thereof is expanded, and more reference bases are provided for developing potential plant sources of drugs for treating diseases related to inflammation, glucose metabolism disorder and lipid metabolism disorder.

[0080] The application further provides a liquid chromatography analysis method of the component Fr2-5-4, and the chromatography analysis conditions thereof include:

[0081] The chromatographic column is C18, preferably with a specification of 4.6*250mm, 5um;

[0082] The mobile phase is A: water / B: methanol, and the gradient elution program is 0-60min, 30%-35% methanol;

[0083] Further, the flow rate is 0.8-1.2mL / min, the column temperature is 30±5℃, and the detection wavelength is 210±5nm;

[0084] Further, Fr2-5-4-3 is used as the reference substance.

[0085] The application further provides a liquid chromatography analysis method of the component Fr2-5-5, and the chromatography analysis conditions thereof include:

[0086] The chromatographic column is C18, preferably with a specification of 4.6*250mm, 5um;

[0087] The mobile phase is A: water / B: methanol, and the gradient elution program is 0-30-120min, 20%-28%-31% methanol;

[0088] Further, the flow rate is 0.8-1.2mL / min, and the detection wavelength is 210±5nm;

[0089] Further, Fr2-5-5-8 is used as the reference substance.

[0090] Based on the above method, the application further provides an analysis method for measuring the quality of Lycium ruthenicum Murr or an extract thereof, including the following contents:

[0091] (1) The Lycium ruthenicum Miers or its extract is pretreated according to the preparation method of Fr2-5-4 or Fr2-5-5.

[0092] (2) The pretreated sample is detected using the above chromatographic analysis conditions.

[0093] The above analysis method provides a possibility for measuring the quality of Lycium ruthenicum Miers or its extract having a therapeutic effect on inflammation, glucose metabolism disorder and lipid metabolism disorder related diseases, and provides a new progress for establishing the quality standard of related products. BRIEF DESCRIPTION OF DRAWINGS

[0094] Figure 1 is the MCI isolation preparation chromatogram of Lycium ruthenicum Miers fruit extract;

[0095] Figure 2 is the MCI isolation preparation chromatogram of Fr2 fraction;

[0096] Figure 3 is the MCI isolation preparation chromatogram of Fr2-5 fraction;

[0097] Figure 4 is the MCI isolation preparation chromatogram of Fr2-5-5 fraction;

[0098] Figure 5 is the purity analysis chromatogram of component Fr2-5-5-8 (furanopetanin);

[0099] Figure 6 is the high resolution mass spectrum of new compound Fr2-5-5-8;

[0100] Figure 7 is the HSQC graph of new compound Fr2-5-5-8;

[0101] Figure 8 is the HMBC graph of new compound Fr2-5-5-8;

[0102] Figure 9 is the COSY graph of new compound Fr2-5-5-8;

[0103] Figure 10 is the MCI isolation preparation chromatogram of Fr2-5-4 fraction;

[0104] Figure 11 is the purity analysis chromatogram of component Fr2-5-4-3 (seco petanin);

[0105] Figure 12 is the high resolution mass spectrum of new compound Fr2-5-4-3;

[0106] Figure 13is the HSQC plot of new compound Fr2-5-4-3;

[0107] Figure 14 is the HMBC plot of new compound Fr2-5-4-3;

[0108] Figure 15 is the COSY plot of new compound Fr2-5-4-3;

[0109] Figure 16 is the effect of different concentrations of LPS on RAW264.7 cell viability and NO release amount;

[0110] Figure 17 is the effect of different concentrations of secopetanin on RAW264.7 cell viability;

[0111] Figure 18 is the effect of different concentrations of furanopetanin on RAW264.7 cell viability;

[0112] Figure 19 is the effect of furanopetanin and secopetanin on RAW264.7 cell NO release amount;

[0113] Figure 20 is the effect of furanopetanin and secopetanin on RAW264.7 cell iNOS protein expression;

[0114] Figure 21 is the effect of furanopetanin and secopetanin on 3T3-L1 adipocyte 2-NBDG uptake after treatment;

[0115] Figure 22 is the effect of furanopetanin and secopetanin on p-AKT and p-PI3K expression in 3T3-L1 adipocytes;

[0116] Figure 23 is the effect of furanopetanin and secopetanin on 3T3-L1 cell lipid droplet accumulation;

[0117] Figure 24 is the effect of furanopetanin and secopetanin on TG content in 3T3-L1 cells;

[0118] Figure 25 is the effect of furanopetanin and secopetanin on 3T3-L1 cell adipogenic transcription factor expression;

[0119] Figure 26 Figure is a graph showing the influence of furanopetanin and secopetanin on the expression of proteins related to lipid metabolism in 3T3-L1 cells;

[0120] Figure 27 Figure is a chromatogram of component Fr2-5-4;

[0121] Figure 28 Figure is a chromatogram of component Fr2-5-5. DETAILED DESCRIPTION

[0122] The active substance and the new petunidin derivative in the present application are further described below through specific examples and specific tests, and the anti-inflammatory, hypoglycemic and hypolipidemic effects thereof are verified and described, and the mechanism thereof is preliminarily discussed.

[0123] In the embodiment of the present application, the method for detecting the purity of the compound is obtained by using HPLC through area normalization method, and the calculation method is: target compound purity % = target compound peak area / total peak area*100%.

[0124] Preparation of Lycium ruthenicum Murr component Fr2-5

[0125] (1) 10.0 kg of dried Lycium ruthenicum Murr fruit was weighed, and methanol room temperature extraction was carried out under light protection, the extraction conditions were: liquid to material ratio 20 mL / g, a total of 3 times of extraction, 4-5 days each time, after each extraction, the extract was filtered and concentrated under light protection and reduced pressure, and then combined to obtain Lycium ruthenicum Murr fruit methanol extract extract;

[0126] (2) The methanol extract extract obtained in step (1) was added to dry polyamide powder 1:1 for sample mixing, dried in an oven at 40℃, and then ground, 50.00 g of sieved powder was taken each time, loaded into a small medium-pressure chromatography column (26*100 mm), and connected to a medium-pressure chromatography column (49*460 mm) loaded with MCI and a preparative liquid chromatograph for dry loading. A three-phase system of A: water / B: methanol / C: dichloromethane was used for elution, and the elution conditions were: 0-120 min, 100% A-100% B; 120-180 min, 100% B-100% C; 180-210 min, 100% B; 210-240 min, 100% A; flow rate: 50 mL / min; detection wavelength: 210 nm. Fr1, Fr2, Fr3 components were obtained, and the retention times were 12-39 min, 39-139 min, and 139-230 min, respectively, as shown in Figure 1

[0127] ​(3) The Fr2 obtained in step (2) was dissolved in methanol, and the preparation conditions were optimized, and finally the mobile phase A: water / B: methanol was selected, gradient elution: 0-120 min, 0%-100% B; 120-140 min, 100% B; flow rate: 50 mL / min; detection wavelength: 254 nm; filler: MCI, column size: 49*460 mm; sample size: 8 mL. Further, 5 components Fr2-1, Fr2-2, Fr2-3, Fr2-4, Fr2-5 were obtained, with retention times of 13-35 min, 35-55 min, 55-68 min, 68-76 min, and 76-130 min, respectively, as shown in Figure 2 .

[0128] Preparation of components Fr2-5-4 and Fr2-5-5 of Lycium ruthenicum Murr component Fr2-5

[0129] The Fr2-5 obtained in step (3) of Example 1 was dissolved in methanol, and the preparation conditions were optimized, and finally the kromasil C18 preparative column (21.2*250 mm, 5 μm) was selected, the mobile phase A: water / B: methanol was selected, gradient elution conditions: 0-60-65-90 min, 30%-42%-70%-95% B; flow rate: 19 mL / min; detection wavelength: 210 nm; sample size: 300 μL. 10 components Fr2-5-1-Fr2-5-10 were obtained, with retention times of 2-9 min, 9-19 min, 19-21 min, 21-27 min, 27-36 min, 36-43 min, 43-50 min, 50-62 min, 62-72 min, and 72-85 min, respectively, as shown in Figure 3 , wherein Fr2-5-4 is component Fr2-5-4, and Fr2-5-5 is component Fr2-5-5.

[0130] Preparation of furanopetanin derivative IV

[0131] (1) The component Fr2-5-5 obtained in Example 2 was dissolved in methanol, and the preparation conditions were optimized, and finally the mobile phase A: water / B: methanol was selected, Kromasil C18 column (21.2*250 mm, 5 μm), gradient elution program: 0-30-120 min, 20%-28%-31% B; flow rate: 19 mL / min; detection wavelength: 210 nm. Further, 10 components Fr2-5-5-1-Fr2-5-5-10 were obtained, and the monomeric compound Fr2-5-5-8 (furanopetanin) was obtained, with a retention time of 92-95 min, as shown in Figure 4 .

[0132] (2) The sample Fr2-5-5-8 (furanopetanin) obtained in step (1) was subjected to high performance liquid chromatography detection to determine the purity; the chromatographic analysis conditions were as follows: kromasil C18 analysis column (4.6*250 mm, 5 μm); mobile phase: A: water / B: methanol; gradient elution program: 0-30-120 min, 20%-28%-31% methanol; flow rate: 1 mL / min; detection wavelength: 210 nm; injection volume was 10 μL;

[0133] (3) The purity of the compound Fr2-5-5-8 (furanopetanin) prepared by the above method reached 96.0% as shown in the HPLC detection. Figure 5 The structure of the compound Fr2-5-5-8 (furanopetanin) is as follows:

[0134]

[0135] The structure (nuclear magnetic data) confirmation results of the compound Fr2-5-5-8 are shown in Table 1:

[0136] Table 1 DMSO-d6 / CF3COOD (9:1) furanopetanin 1 H and 13 C NMR spectral data

[0137]

[0138]

[0139] Example 4 Preparation of secopetanin derivative V

[0140] (1) The component Fr2-5-4 obtained in step (3) of Example 2 was dissolved with methanol, and then subjected to preparation condition optimization, and finally a kromasil C18 preparation column (21.2*250 mm, 5 μm) was selected, the mobile phase was A: water / B: methanol, and the gradient elution conditions were 0-60 min, 30-35% B; the flow rate was 18 mL / min; the detection wavelength was 210 nm, and the injection amount was 150 μL. Four components Fr2-5-4-1, Fr2-5-4-2, Fr2-5-4-3 and Fr2-5-4-4 were obtained, and a new monomer compound Fr2-5-4-3 (secopetanin) was obtained, and its retention time was 40-41 min as shown in Figure 10

[0141] ​(2) The sample Fr2-5-4-3 (secopetanin) obtained in step (1) is subjected to high performance liquid chromatography detection to determine the purity, and the chromatographic analysis conditions are as follows: kromasil C18 analysis chromatographic column (4.6x250mm, 5μm), mobile phase: A: water / B: methanol, gradient elution conditions: 0-60min, 30-35% methanol, flow rate: 1mL / min, column temperature: 30℃, detection wavelength: 210nm, injection volume is 10μL;

[0142] (3) The purity of the compound Fr2-5-4-3 (secopetanin) prepared by the above method is 96.3% as shown in the HPLC detection. Figure 11 The structural formula of the compound Fr2-5-4-3 (secopetanin) is as follows:

[0143]

[0144] The structure (nuclear magnetic data) confirmation results of the compound secopetanin are shown in Table 2:

[0145] Table 2 DMSO-d6 of secopetanin 1 H and 13 C NMR spectrum data

[0146]

[0147] The beneficial effects of the petunidin derivative of the present application are demonstrated by the following test examples:

[0148] Test Example 1 Influence of petunidin derivative in Lycium ruthenicum on inflammation

[0149] Inflammation can occur in many tissues and organs of the human body and is closely related to many diseases. Macrophages are a special cell in the body that plays an immune function, and plays an important physiological role in inflammation, tumors and autoimmune regulation systems. A large number of studies have shown that many inflammatory diseases of the body are closely related to macrophages, and lipopolysaccharide is a component of the outer membrane of gram-negative bacteria and is widely used to establish an inflammatory model of macrophages. The present application uses LPS to induce RAW264.7 cells to construct an inflammation model.

[0150] 1 Establishment of LPS-induced RAW264.7 cell inflammation model

[0151] RAW264.7 macrophages are seeded at a density of 5x10 4Cells were seeded at a concentration of 150 μL per well in a 96-well plate and cultured in a cell culture incubator for 48 h. After induction for 24 h, cells were incubated in medium containing 0.1, 1, 5, 10, 50, 100, and 200 μg / mL lipopolysaccharide (containing 1% penicillin-streptomycin (double antibiotic) and 2% fetal bovine serum (FBS)). Cell viability was detected by MTT assay, and NO content in cell supernatant was determined by NO kit. The optimal concentration of lipopolysaccharide was determined based on cell viability and NO content.

[0152] The results showed that the survival rate of RAW264.7 cells decreased with increasing LPS concentration, and cell viability was significantly inhibited when LPS concentration was 50–200 μg / mL. Figure 16 The addition of different concentrations of LPS significantly increased the NO release from cells. Figure 16 Among them, the NO release was relatively high when the LPS concentration was 0.1-10 μg / mL. Considering both indicators, the modeling concentration of LPS was selected as 5 μg / mL in this experiment.

[0153] 2 Experimental Methods

[0154] Based on the established LPS-induced inflammation model of RAW264.7 cells, the anti-inflammatory activity of petunia extract derivatives was studied.

[0155] 2.1 MTT assay for cell viability

[0156] Log-grown RAW264.7 cells were used at a ratio of 5*10-1 4 At a cell density of 150 μL / well, cells were seeded into each well of a 96-well plate. After incubation for 24 h, the medium was replaced with DMEM containing lipopolysaccharide or the test component (containing 2% FBS), and cultured for another 24 h. Then, 10 μL of thiazolyl blue (MTT) solution was added to each well, and the plate was incubated for 4 h. Finally, the medium was discarded, and 150 μL of DMSO was added to each well to lyse the cells. The absorbance at 490 nm was read using a microplate reader, and cell viability was calculated using the following formula: [Formula omitted for brevity].

[0157]

[0158] The effect of each test compound on the viability of RAW264.7 cells was detected by MTT assay. The new compounds Fr2-5-4-3 and Fr2-5-5-8 showed no significant inhibitory effect on RAW264.7 cells in the range of 0-100 μM. The positive control drug, dexamethasone, was used at a concentration of 10 μM. To ensure consistency of experimental concentrations, all compounds were used at 10 μM in subsequent experiments.

[0159] 2.2 Determination of NO content by Griess method

[0160] The refrigerator takes out Griess reagent I, II, and waits for it to recover to room temperature for experiment. The standard is diluted with DMEM containing 10% FBS to make its concentration 0, 1, 2, 5, 10, 20, 40, 60, 80, 100 μM in turn. In the 96-well plate, 50 μL of standard and sample solution is added to each well, and 50 μL of Griess reagent I and 50 μL of Griess reagent II are added to each well in turn. After mixing on the shaking bed, the absorbance value at 540 nm is measured, and the NO content is calculated according to the standard curve.

[0161] The results show that the new compounds Fr2-5-4-3 and Fr2-5-5-8 have a very significant effect on inhibiting the release of NO. Especially, the compound Fr2-5-5-8 has the most significant effect, and is better than the positive drug.

[0162] 2.3 Western blot analysis

[0163] The experiment is divided into control group, LPS group and LPS+different drug group. The logarithmic growth period RAW264.7 cells are inoculated in 6-well plates at a density of 5*10 4 The model group is treated with LPS, and the test drug group is treated with LPS+different concentrations of drugs. After 24h of incubation in the cell incubator, the medium is replaced with DMEM containing 2% FBS, and the protein is extracted for electrophoresis. The specific operation method is as follows:

[0164] (1) Extraction of RAW264.7 cell protein

[0165] After the treatment of RAW264.7 cells, the cells are lysed and the cell protein is extracted. The culture solution is pumped out with a liquid pump, and PBS buffer is washed twice. The PBS is pumped out. Add cell lysis solution, shake the 6-well plate, and place it on ice for 10 min. Scrape the cells with a cell scraper and collect them in a centrifuge tube. Place the centrifuge tube on ice for 30 min. After lysis, use a low-temperature centrifuge to centrifuge at 12000 r / min, 4°C for 15 min. Collect the supernatant containing protein in a new EP tube.

[0166] (2) BCA method for measuring cell protein concentration

[0167] The determination of protein concentration adopts BCA method, first, the protein standard is prepared into 0.5mg / mL with PBS buffer, BCA working solution is prepared according to the ratio of A liquid:B liquid=50:1, and mixed. Three parallels are set, according to Table 3, protein standard solution and PBS buffer are added, and the standard curve is drawn. 1μL of cell extract protein solution sample is taken and added to a 96-well plate, and the volume is made up to 20μL with PBS buffer, then 200μL of BCA working solution is added, 37℃ reaction for 30min, and the absorbance value at 562nm is determined by microplate reader, and the protein concentration of each sample is calculated according to the standard curve.

[0168] Table 3 BCA method protein quantification table

[0169]

[0170] (3) Protein denaturation

[0171] Take the diluted protein sample, add protein loading buffer and mix, and denature in a metal bath at 100℃ for 15min. After the denatured protein is cooled to room temperature, it is stored in a-20℃ refrigerator for standby.

[0172] (4) SDS-PAGE electrophoresis

[0173] Take two clean glass plates, align them on the gel holder and clamp them, add ultrapure water to check for leaks, after checking for leaks, pour out the ultrapure water and use absorbent paper to dry the residual liquid between the two glass plates, and prepare for pouring glue. According to the size of the protein molecular weight, prepare the appropriate concentration of separation gel (lower gel) as shown in Table 4, according to the protein molecular weight to be detected in the experiment, prepare 10% SDS-PAGE separation gel according to Table 5, and prepare 5% SDS-PAGE concentrated gel (upper gel) according to Table 6. Add about 4mL of separation gel between the two dry glass plates, add isopropyl alcohol to seal the glue, and let it stand for 1h to solidify the separation gel. Pour out the isopropyl alcohol, use absorbent paper to absorb the residual isopropyl alcohol, pour in the concentrated gel, insert the comb to avoid air bubbles, and gently pull out the comb after the concentrated gel solidifies. Clamp the two glass plates on the electrophoresis tank, pour in the electrophoresis liquid so that the liquid level is above the glass plate, load the protein sample, after loading is completed, start electrophoresis, first use constant voltage 80V to concentrate the sample in the concentrated gel for about 30min, adjust the voltage to 120V and continue constant voltage electrophoresis, until the loading buffer reaches the bottom of the gel plate, stop electrophoresis.

[0174] Table 4 SDS-PAGE gel optimal separation range

[0175]

[0176] Table 5 10% SDS-PAGE separation gel preparation table

[0177]

[0178] Table 6 SDS-PAGE concentrated gel preparation table

[0179]

[0180] (5) Transferring membrane

[0181] Cut the PVDF membrane to the appropriate size, cut off a corner as a marker to distinguish the front and back after cutting the gel, and soak it in methanol for 1 min. After activation, the PVDF membrane, foam, and transfer filter paper are soaked in pre-cooled transfer buffer. Use a gel cutter to pry open the glass plate, cut the gel according to the target protein molecular weight, and place it in the electrophoresis solution. Place the black side of the transfer clamp on the bottom, and then place the foam, filter paper, adhesive tape, PVDF membrane, filter paper, and foam in order. Tighten the clamp. Place the clamp in the transfer slot with the black clamp facing the black side of the slot. Pour the transfer solution to immerse the clamp plate. Place an ice bag in a low-temperature environment. Set the current to 250 mA. Set the transfer time according to the size of the protein molecular weight.

[0182] (6) Blocking

[0183] The blocking solution is 5% skimmed milk prepared in 1x TBST. After transferring the membrane, remove the PVDF membrane and place it in an incubation box. Add skimmed milk to cover the PVDF membrane. Place it in a shaking incubator at room temperature for 1 hour. Pour out the skimmed milk and wash it with 1x TBST for 10 minutes. Repeat the washing process three times.

[0184] (7) Incubating primary antibody

[0185] Dilute the primary antibody COX-2 (CST, #12282), p-IκBα (CST, #2859), IκBα (CST, #4814), and β-actin (CST, #4970) with the antibody diluent at a ratio of 1:1000. Place the PVDF membrane in the diluted primary antibody solution and incubate it at 4°C in a shaking incubator at low speed overnight. Recover the primary antibody solution and wash it with 1x TBST for 10 minutes. Repeat the washing process three times.

[0186] (8) Incubating secondary antibody

[0187] Place the PVDF membrane in a 1:5,000 dilution of horseradish peroxidase-labeled secondary antibody and incubate it at room temperature for 1 hour. After incubation, recover the secondary antibody solution and wash the membrane with 1x TBST at room temperature for 10 minutes. Repeat the washing process three times.

[0188] (9) Developing

[0189] Developing uses ECL chemical development method. Mix the developing solution A and B at a ratio of 1:1. Add 100 μL of the mixture to the PVDF membrane. Develop the membrane in a developing instrument and take a photo.

[0190] (10) Statistical analysis

[0191] Western blot data were collected and processed using Image J software, and the gray value of the protein band was quantitatively analyzed. β-actin was used as an internal reference for gray analysis of the protein band. Graphpad prism 8.0 statistical software was used for statistics, and the data were expressed as mean ± standard deviation The mean values between groups were analyzed by one-way ANOVA (One-way ANOVA), and P<0.05 was considered to have statistical significance.

[0192] 3 Result analysis

[0193] When LPS activates RAW264.7 mouse macrophages, TLR-4 is stimulated to recognize and bind with LPS, and then activates the signaling pathway of NF-κB, ultimately promotes the production of a large number of inflammatory factors such as NO, PGE2, TNF-α, IL-β, IL-6, COX-2 and iNOS, and triggers a cytokine storm. NO plays a core role in inflammation, and among the NOS family, iNOS is particularly involved in the pathological overproduction of NO. Therefore, the amount of NO release can be used as a preliminary screening of the activity of monomeric compounds in anti-inflammatory models.

[0194] Petroselinids derivatives showed good anti-inflammatory activity in inhibiting the release of NO in the inflammation model, as shown in Figure 19 After LPS stimulation, the expression level of iNOS in RAW264.7 cells was up-regulated, which had a very significant difference compared with the control group (P<0.01); at a concentration of 10 μM, petroselinids derivatives could significantly inhibit the expression of iNOS protein (P<0.01), as shown in Figure 20 , indicating that the above petroselinids derivatives can reduce the release of NO by inhibiting the expression of iNOS protein to play an anti-inflammatory role.

[0195] Test example 2 Effect of petroselinids derivatives in Lycium ruthenicum Murr on glucose metabolism

[0196] 1 Experimental method

[0197] 1.1 Effect of petroselinids derivatives on 2-NBDG uptake by 3T3-L1 cells

[0198] 3T3-L1 preadipocytes were seeded at 5×10 4Cells were seeded at a density of [number] cells / mL into 12-well plates. Once the cell density reached over 80%, differentiation was induced. On day 8 after induced differentiation and maturation, 1 μM Dex (dexamethasone) was added to the cell slurry to establish an insulin resistance (IR) model. Three groups were established: a normal group, a model group, and a drug-treated group. Except for the normal group, which was cultured in complete medium, the other groups were cultured in 1 μM Dex. In the drug-treated groups, 10 μM of the monomeric compound was added to each cell and cultured for 48 h. The culture medium was then aspirated, the cells were washed once with DPBS, and 500 μL of trypsin was added for digestion at 37°C for 1 min. 2 mL of DPBS was added, and the cells were mixed thoroughly and centrifuged at 1000g for 6 min. The supernatant was discarded, and 1 mL of sugar-free medium containing 10 μM 2-NBDG was added to each well. The cells were incubated at 37°C for 30 min, and the fluorescence intensity was detected by flow cytometry at a wavelength of 488 nm.

[0199] 1.2 Effects of petunia extract derivatives on AKT phosphorylation in 3T3-L1 cells

[0200] 3T3-L1 preadipocytes were divided into 5×10 4 Cells were seeded at a density of [number] cells / mL in 6-well plates. Once the cell density reached over 80%, differentiation was induced. On day 8 after induced differentiation and maturation, 1 μM Dex was added to the cell culture medium to establish an IR model. Normal, model, and drug-treated groups were set up. Except for the normal group, which was cultured in complete medium, the other groups were cultured in 1 μM Dex. The drug-treated groups were each cultured with 10 μM of a monomeric compound for 48 hours. Afterward, the culture medium was aspirated, cells were collected, and the expression levels of PI3K and AKT proteins were detected by Western blot. The Western blot analysis method was the same as in Example 1.

[0201] 2 Results Analysis

[0202] 2.1 Effects of petunia extract derivatives on 2-NBDG uptake in 3T3-L1 cells

[0203] like Figure 21 As shown, the uptake capacity of 2-NBDG in the normal group was higher than that in the insulin resistance model group. The glucose uptake capacity of cells in the model group was weaker, showing a significant difference compared to the glucose uptake capacity of cells in the normal group after insulin stimulation. Intervention with petunia extract derivatives promoted the uptake of 2-NBDG by adipocytes under insulin stimulation to varying degrees, indicating that petunia extract derivatives have the potential to improve insulin resistance.

[0204] 2.2 Effects of petunia extract derivatives on PI3K and AKT phosphorylation in 3T3-L1 cells

[0205] The results are as follows Figure 22As shown, compared with the normal group, the expression levels of p-AKT and p-PI3K proteins in the model group were reduced. After treatment with petunidin derivatives, the expression levels of p-AKT and p-PI3K in 3T3-L1 adipocytes were increased to different degrees, thereby promoting glucose uptake and enhancing insulin sensitivity of 3T3-L1 adipocytes.

[0206] Effect of petunidin derivatives in Lycium ruthenicum Murr. on lipid metabolism

[0207] 1 Experimental method

[0208] 1.1 Oil red O staining

[0209] The 3T3-L1 cells in good condition were inoculated on a 6-well plate at a plating density of 5×10 4 The cells were cultured in high-glucose DMEM medium containing 10% FBS until the cell density reached about 85%-90%, then the medium was replaced, and the cells were contacted for two days. Then the complete culture medium was discarded, and culture medium containing 10 μg / mL insulin, 0.5 mM IBMX, and 1 μM Dex was added and cultured for 2 d (the time when the inducer was added was recorded as day 0, induction I). Subsequently, the culture medium was replaced with culture medium containing 10 μg / mL insulin (induction II) and cultured for 2 d. Subsequently, the culture medium was replaced with normal culture medium and 10 μM monomer compound for co-incubation, and the medium was replaced every other day. After induction, the cells were fixed with 4% neutral formaldehyde for 30 min. After the cells were fixed, the oil red O working solution prepared in advance was added to the cell surface, and the cells were incubated in the dark for 60 min. After staining, the cells were washed with 70% ethanol, the excess dye was discarded, and the cells were washed with ultrapure water for 3-4 times. Finally, the cells were observed under a microscope and photographed.

[0210] 1.2 TG content determination

[0211] The 3T3-L1 cells were inoculated on a 6-well plate at a plating density of 5×10 4The cells were seeded in 6-well plates at a density of 1 x 104 / mL, and induced to differentiate when the cell density reached 85% to 90%. The TG content was determined on the 8th day of induction by the following method: (1) cell pretreatment: on the 8th day of induction, the cell culture solution was removed, and the cells were digested with trypsin after being washed twice with cold PBS; (2) cell collection: after the cells were digested, the cells were resuspended in PBS and centrifuged at 1000 g for 5 min to collect the cell pellet; (3) ultrasonic disruption: the collected cell pellet was added with an appropriate amount of PBS and ultrasonically disrupted (3 min); (4) determination: 2 μL of the cell disruption suspension was added to each well of a 96-well plate, 2 μL of distilled water was added to the blank wells, and 2 μL of the standard was added to the standard wells, followed by the addition of 200 μL of the determination solution to each well, mixing, incubation at 37°C for 10 min, and reading of the absorbance at 510 nm. The protein concentration in the sample was determined by the BCA method, and the TG content was calculated according to the following formula.

[0212]

[0213] 1.3 Western blot analysis

[0214] The Western blot analysis method was the same as in Test Example 1.

[0215] 2. Results analysis

[0216] 2.1 Effect of the petunidin derivatives on the lipid droplet accumulation and TG content in 3T3-L1 cells

[0217] As shown in Figure 23 , no lipid droplets accumulated in the undifferentiated cells, but a large number of lipid droplets were present in the differentiated cells. After treatment with the petunidin derivatives, the intracellular lipid droplets were significantly reduced.

[0218] As shown in Figure 24 , the TG content in the differentiated cells was significantly increased (P < 0.01) compared with that in the undifferentiated cells. Compared with the differentiated cells, the TG content was reduced after treatment with the petunidin derivatives, and the difference was extremely significant (P < 0.01).

[0219] 2.2 Effect of the petunidin derivatives on the lipid metabolism protein expression in 3T3-L1 cells

[0220] (1) Effect of the petunidin derivatives on the expression of the adipogenic transcription factor in 3T3-L1 cells

[0221] The effect of the petunidin derivatives on the expression of the adipogenic transcription factor in 3T3-L1 cells was analyzed by Western blot, and the results are shown in Figure 25As shown, the expression levels of PPARγ and C / EBPα proteins were low in undifferentiated 3T3-L1 cells, while the expression levels of PPARγ and C / EBPα proteins were high in differentiated cells. Compared with the differentiation group, treatment with petunia extract derivatives could reduce the protein expression levels of PPARγ and C / EBPα transcription factors to some extent.

[0222] (2) Effects of petunia extract derivatives on the expression of lipidogenesis-related proteins in 3T3-L1 cells

[0223] like Figure 26 As shown, the expression levels of FAS and ACC proteins were low in undifferentiated 3T3-L1 cells. However, the expression levels of FAS and ACC proteins were significantly increased in differentiated cells. Compared with the differentiation group, treatment with petunia extract derivatives significantly reduced the expression levels of FAS and ACC proteins.

[0224] Based on the above results, it can be concluded that the differentiation of 3T3-L1 cells from preadipocytes into mature adipocytes is regulated by transcription factors and lipoproteins, and the cell morphology also changes, eventually exhibiting a "ring-like" appearance. Petunia derivatives can inhibit the accumulation of lipid droplets in 3T3-L1 cells and reduce intracellular TG content; petunia derivatives can also inhibit the differentiation of 3T3-L1 adipocytes and reduce intracellular lipid droplet accumulation by inhibiting the expression levels of transcription factors such as PPARγ and C / EBPα. Simultaneously, they improve cellular lipid metabolism by inhibiting the expression levels of FAS and ACC proteins, thereby suppressing lipid production.

[0225] Chromatographic analysis of component Fr2-5-4 in Example 5

[0226] The fraction Fr2-5-4 prepared in Example 2 was subjected to chromatographic analysis under the following conditions: Kromasil C18 analytical column (4.6 × 250 mm, 5 μm); mobile phase: A: water / B: methanol; gradient elution conditions: 0–60 min, 30%–35% methanol; flow rate: 1 mL / min; column temperature: 30 °C; detection wavelength: 210 nm; injection volume: 10 μL; and Fr2-5-4-3 was used as a reference standard. The results are as follows: Figure 27 As shown, the component Fr2-5-4 was separated into four main components under the chromatographic analysis conditions of this experiment, and the separation degree of the four components was good. Among them, Fr2-5-4-3, which is a new monomer compound in this invention, eluted after 40-50 min.

[0227] Chromatographic analysis of component Fr2-5-5 in Example 6

[0228] The component Fr2-5-5 prepared in Example 2 was subjected to chromatographic analysis under the following conditions: kromasil C18 analytical column (4.6 x 250 mm, 5 μm), mobile phase: A: water / B: methanol, gradient elution conditions: 0-30-120 min, 20%-28%-31% methanol, flow rate: 1 mL / min; detection wavelength: 210 nm, injection volume: 10 μL, and Fr2-5-5-8 was used as a reference. The results are shown in Table 1. Figure 28 As shown in Table 1, component Fr2-5-5 was separated into 10 main components by the chromatographic analysis conditions of this experiment, and monomeric compound Fr2-5-5-8 was obtained.

[0229] The above description is merely an example of the present application, and does not limit the patent scope of the present application. Any equivalent structure or equivalent process transformation, or direct or indirect application in other related technical fields, made by using the content of the present application specification and drawings, are also included in the patent protection scope of the present application.

Claims

1. A petunia-derived derivative IV, characterized in that, The structural formula is as formula IV:

2. The method for preparing the dihydromatellin derivative IV according to claim 1, characterized by, Comprising the following steps: (1) 10.0 kg of dry Lycium ruthenicum Murr. fruits are soaked and extracted with methanol at room temperature in the dark, the extraction conditions are as follows: liquid to material ratio is 20 mL / g, a total of 3 times of extraction, 4-5 days each time, after each extraction, the extract is filtered, and concentrated under light-shielded reduced pressure, combined, to obtain Lycium ruthenicum Murr. fruit methanol extract extract; (2) the methanol extract extract obtained in step (1) is mixed with dry polyamide powder 1:1, dried in an oven at 40°C, ground, 50.00 g of powder is taken each time, loaded into a 26*100 mm small medium-pressure chromatography column, connected with a medium-pressure chromatography column loaded with MCI, specifications are 49*460 mm, and loaded onto the column by dry method, eluted with an A: water / B: methanol / C: dichloromethane three-phase system, elution conditions are as follows: 0-120 min, 100% A-100% B; 120-180 min, 100% B-100% C; 180-210 min, 100% B; 210-240 min, 100% A; flow rate: 50 mL / min, detection wavelength: 210 nm, to obtain Fr1, Fr2, Fr3 components, retention times are 12-39 min, 39-139 min, and 139-230 min, respectively; (3) the Fr2 component obtained in step (2) is dissolved with methanol, the mobile phase is selected as follows: A: water / B: methanol, gradient elution: 0-120 min, 0%-100% B; 120-140 min, 100% B; flow rate: 50 mL / min, detection wavelength: 254 nm, filler: MCI, column specifications: 49*460 mm, sample size: 8 mL, to further obtain Fr2-1, Fr2-2, Fr2-3, Fr2-4, Fr2-5, a total of 5 components, retention times are 13-35 min, 35-55 min, 55-68 min, 68-76 min, and 76-130 min, respectively; (4) the Fr2-5 component obtained in step (3) is dissolved with methanol, a 21.2*250 mm, 5 μm kromasil C18 preparative chromatography column is selected, the mobile phase is A: water / B: methanol, gradient elution conditions are as follows: 0-60-65-90 min, 30%-42%-70%-95% B; flow rate: 19 mL / min, detection wavelength: 210 nm, sample size is 300 μL, to obtain Fr2-5-1-Fr2-5-10, a total of 10 components, retention times are 2-9 min, 9-19 min, 19-21 min, 21-27 min, 27-36 min, 36-43 min, 43-50 min, 50-62 min, 62-72 min, and 72-85 min, respectively; (5) The obtained component Fr2-5-5 was dissolved with methanol, and then the mobile phase was selected: A: water / B: methanol, 21.2*250mm, 5μm kromasil C18 column, gradient elution procedure: 0~30~120min, 20%~28%~31%B; flow rate: 19mL / min; detection wavelength: 210nm, to further obtain 10 components Fr2-5-5-1~Fr2-5-5-10, and obtain monomer compound Fr2-5-5-8 with retention time of 92~95min; compound Fr2-5-5-8 is petunidin derivative IV.

3. Use of petunidin derivative IV according to claim 1, and pharmaceutically acceptable salts thereof, in the preparation of a product for treating and / or preventing inflammation.

4. Use according to claim 3, characterized in that, The product is a product for reducing the release of NO.

5. An anti-inflammatory product, characterized in that, The product comprises compound IV; 6. Use of petunidin derivative IV according to claim 1, and pharmaceutically acceptable salts thereof, in the preparation of a product for treating and / or preventing glucose metabolism and / or lipid metabolism.

7. Use according to claim 6, characterized in that, The product is a product for increasing the expression level of p-AKT and p-PI3K proteins.

8. Use according to claim 6, characterized in that, The product is a product for inhibiting the accumulation of cell lipid droplets and / or the generation of cell lipids.

9. A blood sugar and / or lipid lowering product, characterized in that, The product comprises compound IV;

Images