CN118059172A

CN118059172A - Active component Fr2-5 in lycium ruthenicum, and extraction method and application thereof

Info

Publication number: CN118059172A
Application number: CN202410196137.2A
Authority: CN
Inventors: 胡娜; 姬治宇; 王洪伦; 董琦; 铁芳芳
Original assignee: Northwest Institute of Plateau Biology of CAS
Current assignee: Northwest Institute of Plateau Biology of CAS
Priority date: 2023-06-09
Filing date: 2024-02-22
Publication date: 2024-05-24
Also published as: CN118063528A; CN118059171A; CN118059170A; CN118063529A; CN118059169A

Abstract

The invention discloses an active component Fr2-5 in lycium ruthenicum and an extraction method thereof, relates to the technical field of plant active component separation, and also provides application of the active Fr2-5 in preparing products for preventing and/or treating inflammation, application in preparing products for preventing and/or treating diseases related to sugar metabolism disorder and application in preparing products for preventing and/or treating diseases related to lipid metabolism disorder.

Description

Active component Fr2-5 in lycium ruthenicum, and extraction method and application thereof

Technical Field

The invention relates to the technical field of separation of plant active components, in particular to an active component Fr2-5 in lycium ruthenicum, and an extraction method and application thereof.

Background

The black fruit matrimony vine (Lycium ruthenicum Murr) is perennial spiny shrub of the genus matrimony vine of the family Solanaceae, has sweet and succulent fruit taste, is rich in nutrition, has great research and development values, and is a special desert medicinal plant variety in the western part of China. The lycium ruthenicum contains various amino acids and rich mineral elements necessary for human bodies, and the Tibetan medicine is used for treating heart fever and heart disease, reducing cholesterol, exciting cerebral nerves, enhancing immune function, preventing and treating cancers, resisting aging, maintaining beauty and keeping young, irregular menstruation, stopping menstruation and the like, and has obvious efficacy.

Anthocyanin (Anthocyanins) is a water-soluble natural pigment widely existing in plants in nature, and belongs to flavonoid compounds. Anthocyanin exists mainly in the forms of glucoside, rhamnoside and the like and is contained in fruits, epidermis and flowers of plants. The anthocyanin content in the lycium ruthenicum is extremely high, wherein more than 90% of anthocyanin is petuniin derivatives, and the lycium ruthenicum has the functions of resisting oxidation, protecting eyesight, inhibiting tumors and the like, and has important biological activity and extraction value. With the higher the demand of people for functional components of natural origin and no toxic or side effects, anthocyanin is the most representative functional component of natural origin. It is popular with consumers and markets because of its good coloring function and excellent antioxidant activity.

The rich active substances contained in the lycium ruthenicum are still not separated and identified, and if the novel chemical components and the pharmacological actions of the novel chemical components in the lycium ruthenicum can be subjected to deeper and finer research and active mechanism discussion, more natural plant-derived medicaments which can treat diseases and are safe and effective are expected to be developed.

Disclosure of Invention

The invention provides an active component extracted from lycium ruthenicum, a novel petuniin derivative, an extraction method thereof and application thereof in preparing medicines for preventing and/or treating inflammation related diseases, wherein the components and the compounds have the effects of inhibiting expression of iNOS protein so as to reduce NO release amount.

The invention provides a lycium ruthenicum murr component Fr2-5, wherein the retention time of the component Fr2-5 prepared by the following method is 76-130 min;

(1) Preparing the lycium ruthenicum extract through preparation chromatography to obtain components Fr1, fr2 and Fr3, wherein the retention time is respectively 12-39 min, 39-139 min and 139-230 min;

Wherein the preparation chromatographic conditions include:

Chromatographic column: a MCI medium pressure chromatographic column, preferably 49 x 460mm;

Mobile phase: water, methanol, B, methylene dichloride and C; gradient elution was performed using the following procedure: 0-120 min,100% A-100% B; 120-180 min,100% B-100% C; 180-210 min,100% B; 210-240 min,100% A;

(2) Subjecting the component Fr2 to preparative chromatographic separation to obtain the component Fr2-5;

Wherein the conditions of the preparative chromatography include:

Chromatographic column: MCI medium pressure chromatographic column; the preferred specification is 49 x 460mm;

Mobile phase: water/methanol; gradient elution was performed using the following procedure: 0-120 min, 0-100% B; 20-140 min,100% B.

In the present invention, the lycium ruthenicum extract refers to a concentrate obtained by extracting lycium ruthenicum with methanol, filtering and concentrating in dark place, and the extraction method includes, but is not limited to, leaching, thermal reflux, ultrasonic extraction and other conventional extraction methods.

In the specific embodiment of the invention, the lycium ruthenicum is extracted by adopting an extraction method, and the extraction conditions are as follows: the liquid-material ratio is 10-30 mL/g, preferably 20mL/g; the extraction times are as follows: 2 to 5 times, 3 to 5 days each, preferably 3 times, 4 to 5 days each.

Further, filtering the extracting solution after the lycium ruthenicum is extracted, concentrating the extracting solution under reduced pressure in a dark place, and combining the extracting solution to obtain the lycium ruthenicum fruit methanol extract.

In the invention, polyamide is added into the methanol extract to be stirred, dried, ground and sieved.

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

Further, the drying method may be selected from normal pressure drying, reduced pressure drying, freeze drying, and the like.

Further, the screen is 20 mesh to 50 mesh, preferably 20 mesh.

The invention provides a lycium ruthenicum murr component Fr2-5-4, wherein the component Fr2-5 is subjected to preparative chromatographic separation to obtain the component Fr2-5-4, and the retention time of the component Fr2-5-4 is 21-27 min;

Wherein the conditions of the preparative chromatography include:

chromatographic column: kromasil C18 column; the preferred specification is 21.2 x 250mm;

mobile phase: water/methanol; gradient elution was performed using the following procedure: 0 to 60 to 65 to 90min,30 to 42 to 70 to 95 percent of B.

The invention provides a lycium ruthenicum murr component Fr2-5-5, wherein the component Fr2-5 is subjected to preparative chromatographic separation to obtain the component Fr2-5-5, and the retention time of the component Fr2-5-5 is 27-36 min;

Wherein the conditions of the preparative chromatography include:

The invention provides petuniin derivatives IV, which have a structural formula shown in the formula IV:

the invention adopts methanol to extract lycium ruthenicum fruit, separates through preparative chromatography, and obtains novel petuniin derivatives unexpectedly through one-dimensional and two-dimensional nuclear magnetic identification, and the petuniin derivatives are named furanopetanin.

Wherein, petuniin derivative IV is obtained by performing preparative chromatographic separation on the component Fr2-5-5, and the retention time is 92-95 min.

Further, the conditions of the preparative chromatography include:

mobile phase: water/methanol; gradient elution was performed using the following procedure: 0-30-120 min, 20-28-31% B.

The invention provides petuniin derivative V, which has a structural formula as shown in formula V:

The invention adopts methanol to extract lycium ruthenicum fruit, separates through preparative chromatography, and obtains novel petuniin derivatives unexpectedly through one-dimensional and two-dimensional nuclear magnetic identification, and the petuniin derivatives are named secopetanin.

Wherein, the kaladain compound V is obtained by carrying out preparation chromatographic separation on the component Fr2-5-5, and the retention time is 40-41 min.

Further, the conditions of the preparative chromatography include:

Mobile phase: water/methanol; gradient elution was performed using the following procedure: 0 to 60min,30 to 35 percent of B.

In a specific embodiment of the invention, the preparative chromatographic separation further comprises at least one of the following conditions:

Detection wavelength: 210nm;

column temperature: 25-35 ℃;

Flow rate: 15-60 mL/min;

Sample injection amount: 0.1-8.0 mL.

The invention provides application of petuniin derivatives IV and/or V and pharmaceutically acceptable salts, hydrates or solvates thereof in preparing products for treating and/or preventing inflammation-related diseases.

The invention provides the use of at least one of the above components Fr2-5, fr2-5-4 and Fr2-5-5 for the preparation of a product for the treatment and/or prophylaxis of diseases associated with inflammation.

Further, the two products for treating and/or preventing inflammation-related diseases are products for blocking and/or inhibiting NF- κB signaling pathway.

NF- κB, a protein complex, controls transcribed DNA, cytokine production and cell survival; and are involved in cellular responses to stimuli such as stress, cytokines, free radicals, heavy metals, ultraviolet radiation, oxidized LDL and bacterial or viral antigens; in addition, it plays a key role in regulating the immune response to infection.

Further, the product is a product that reduces the amount of release of at least one inflammatory factor of NO, PGE2, TNF- α, IL- β, IL-6, COX-2, and iNOS; further, the product is a product with reduced NO release.

Further, the product is a product that inhibits expression of iNOS protein.

The invention also provides an anti-inflammatory product which comprises one or more of the components Fr2-5, fr2-5-4, fr2-5-5, the compound IV and the compound V.

The invention provides application of petuniin derivatives IV and/or V and pharmaceutically acceptable salts, hydrates or solvates thereof in preparing products for treating and/or preventing diseases related to sugar metabolism disorder.

The invention provides the application of at least one of the components Fr2-5, fr2-5-4 and Fr2-5-5 in preparing products for treating and/or preventing diseases related to sugar metabolism disorder.

Further, both of the above-mentioned products for treating and/or preventing diseases related to the disorder of sugar metabolism are products having a hypoglycemic effect, such as hypoglycemic drugs and the like.

The product for treating and/or preventing the diseases related to the glucose metabolism disorder can reduce blood sugar by inhibiting hepatic gluconeogenesis and hepatic gluconeogenesis, increasing the sensitivity of peripheral tissues to insulin, promoting glucose uptake and utilization, stimulating islet beta cells to secrete insulin, enhancing the action of combining insulin with a receptor, increasing the sensitivity of target cells to insulin and the like.

Further, the product is a product that promotes uptake of sugar and/or sugar analogs by adipocytes.

Glucose uptake by adipocytes stimulated by insulin is mainly performed by GLUT4 (glucose transporter), which is sensitive to insulin; under insulin stimulation, the internalization of insulin receptor tyrosine phosphorylation signals phosphorylates insulin receptor substrate-1 (IRS-1), thereby activating phosphatidylinositol-3-kinase (PI 3K), triggering translocation of GLUT4 to the cell surface, and increasing glucose uptake.

Further, the product is a product that increases the expression levels of p-AKT and p-PI3K proteins.

AKT is a serine/threonine protein kinase, and is involved in glucose metabolism, apoptosis, cell proliferation, cell transportation and other aspects; AKT activated during glucose metabolism activates downstream factors such as various enzymes, kinases and transcription factors in the insulin signaling pathway through the phosphorylation pathway, thereby regulating cellular functions and transmission of insulin signals. Generally activated AKT acts as insulin signaling by activating phosphatidylinositol kinase 3 (PI 3K) downstream thereof to promote translocation of glucose transporter from the cytosol to the cell membrane, and by accelerating glucose uptake and utilization.

P-PI3K (mouse phosphoinositide 3 kinase) can phosphorylate, activate or inhibit a series of downstream substrates, apoptosis-related protein activities, thereby regulating cell proliferation, differentiation, apoptosis, migration and other phenotypes.

The invention provides application of petuniin derivatives IV and/or V and pharmaceutically acceptable salts, hydrates or solvates thereof in preparing products for treating and/or preventing diseases related to lipid metabolism disorder.

The invention provides the application of at least one of the components Fr2-5, fr2-5-4 and Fr2-5-5 in preparing products for treating and/or preventing diseases related to lipid metabolism disorder.

Further, both of the above-mentioned products for treating and/or preventing diseases related to lipid metabolism disorder are products having lipid-lowering effect, such as lipid-lowering drugs and the like.

Further, the above two types of products for treating and/or preventing diseases related to lipid metabolism disorders include products for inhibiting accumulation of lipid droplets of fat cells and/or products for inhibiting generation of lipid of cells; the product may be a pharmaceutical, nutraceutical or other product having the effect of inhibiting accumulation of cell lipid droplets and/or inhibiting production of cell lipids.

When the product is a medicament, the medicament comprises at least one of preventing and/or treating obesity, hypertension, hyperlipidemia, cardiovascular diseases and metabolic syndrome related diseases.

Obesity, hypertension, hyperlipidemia, cardiovascular diseases, metabolic syndrome, etc. are all closely related to the content of plasma lipids such as Triglyceride (TG), free Cholesterol (FC), cholesterol lipid (CE) and phospholipids, and when the plasma lipids are reduced to a certain concentration range in vivo, these diseases can be effectively controlled or treated. The experiment proves that the active components and/or the novel compounds in the invention can effectively inhibit lipid drop accumulation, reduce TG content, regulate the gene expression level of the adipocyte transcription factor and the expression level of related protein through related signal paths, inhibit the adipogenic differentiation and lipid generation of cells, and can be used for preparing products for preventing and/or treating obesity, hypertension, hyperlipidemia, cardiovascular diseases and metabolic syndrome related diseases.

The invention provides application of petuniin derivatives IV and/or V and pharmaceutically acceptable salts, hydrates or solvates thereof in preparing at least one of PPARgamma antagonists, C/EBP alpha antagonists, FAS inhibitors and ACC inhibitors.

The invention provides application of at least one of the components Fr2-5, fr2-5-4 and Fr2-5-5 in preparing at least one of PPARgamma antagonist, C/EBPalpha antagonist, FAS inhibitor and ACC inhibitor.

The PPARgamma antagonist, the C/EBP alpha antagonist, the FAS inhibitor and the ACC inhibitor are medicines for reducing the gene expression of the adipocyte transcription factors PPARgamma and C/EBP alpha and the protein expression level of FAS and ACC.

Differentiation of undifferentiated cells into mature adipocytes requires a series of sophisticated transcription factor regulation, pparγ is an essential regulator of adipocyte formation, highly expressed early in the adipogenic process; C/EBP alpha, the so-called CCAAT/enhancer binding protein alpha, is expressed in large amounts in the metaphase stage of adipocyte differentiation.

FAS is a fatty acid synthase that plays an important role in lipid production; ACC is an acetyl coa carboxylase, the rate limiting enzyme for fatty acid re-head synthesis.

The active component and the novel compound can reduce gene expression of cell transcription factors PPARgamma and C/EBPalpha and protein expression levels of FAS and ACC, so that products for effectively inhibiting cell adipogenic differentiation and inhibiting cell lipid generation can be effectively inhibited, and the novel compound can be used for preparing and preventing and/or treating related diseases with lipid metabolism disorder, such as obesity, hypertension, hyperlipidemia, cardiovascular diseases and the like.

The invention also provides a hypoglycemic and/or hypolipidemic product, which comprises one or more of components Fr2-5, fr2-5-4, fr2-5-5, compound IV and compound V.

Products of the present invention include, but are not limited to, pharmaceuticals, nutraceuticals, foods, and the like.

The beneficial effects of the invention are as follows: the invention provides three active components and two novel compounds furanopetanin and secopetanin, the components and the compounds of the invention have the effects of anti-inflammatory, blood sugar reducing and lipid lowering, the active substances of the lycium ruthenicum are more comprehensively developed, the medicinal value of the lycium ruthenicum is more comprehensively excavated, the clinical application of the lycium ruthenicum is expanded, and more reference bases are provided for developing potential plant-derived medicaments for treating diseases related to inflammation, sugar metabolism disorder and lipid metabolism disorder.

The invention also provides a liquid chromatography analysis method of the component Fr2-5-4, wherein the chromatographic analysis conditions comprise:

chromatographic column: c18, preferably 4.6X105 mm,5 μm;

Mobile phase: water/methanol gradient elution procedure: 0-60 min, 30-35% methanol;

Further, the flow rate is 0.8-1.2 mL/min; the column temperature is 30+/-5 ℃; the detection wavelength is 210+/-5 nm;

further, fr2-5-4-3 was used as a control.

The invention also provides a liquid chromatography analysis method of the component Fr2-5-5, wherein the chromatographic analysis conditions comprise:

Chromatographic column: c18, preferred specification is 4.6x250mm, 5 μm;

Mobile phase: water/methanol gradient elution procedure: 0-30-120 min, 20-28-31% methanol;

further, the flow rate: 0.8-1.2 mL/min; detection wavelength: 210+ -5 nm;

Further, fr2-5-5-8 was used as a control.

Based on the method, the invention also provides an analysis method for measuring the quality of the lycium ruthenicum or the extract thereof, which comprises the following steps:

(1) Referring to Fr2-5-4 or Fr2-5-5, pre-treating Lycium ruthenicum or its extract;

(2) The pretreatment samples were tested using the chromatographic conditions described above.

The analysis method provides possibility for measuring the quality of lycium ruthenicum mill or extracts thereof for treating diseases related to inflammation, sugar metabolism disorder and lipid metabolism disorder, and provides new progress for establishing quality standards of related products.

Drawings

FIG. 1 is a chromatogram of the MCI separation preparation of an extract of Lycium ruthenicum fruit;

FIG. 2 is a MCI separation preparative chromatogram of a Fr2 fraction;

FIG. 3 is a MCI separation preparative chromatogram of the Fr2-5 fraction;

FIG. 4 is a MCI separation preparative chromatogram of a Fr2-5-5 fraction;

FIG. 5 is a chart of purity analysis of component Fr2-5-5-8 (furanopetanin);

FIG. 6 is a high resolution mass spectrum of novel compounds Fr 2-5-5-8;

FIG. 7 is a HSQC diagram of novel compounds Fr 2-5-5-8;

FIG. 8 is a HMBC diagram of novel compounds Fr 2-5-5-8;

FIG. 9 is a COSY diagram of novel compounds Fr 2-5-5-8;

FIG. 10 is a MCI separation preparative chromatogram of a Fr2-5-4 fraction;

FIG. 11 is a chart of purity analysis of component Fr2-5-4-3 (secopetanin);

FIG. 12 is a high resolution mass spectrum of novel compound Fr 2-5-4-3;

FIG. 13 is a HSQC diagram of novel compound Fr 2-5-4-3;

FIG. 14 is a HMBC diagram of novel compound Fr 2-5-4-3;

FIG. 15 is a COSY diagram of novel compounds Fr 2-5-4-3;

FIG. 16 is a graph showing the effect of different concentrations of LPS on RAW264.7 cell viability and NO release;

FIG. 17 is a graph showing the effect of secopetanin at various concentrations on RAW264.7 cell viability;

FIG. 18 is a graph showing the effect of furanopetanin at various concentrations on RAW264.7 cell viability;

FIG. 19 is a graph showing the effect of furanopetanin and secopetanin on the amount of NO released by RAW264.7 cells;

FIG. 20 is a graph showing the effect of furanopetanin and secopetanin on expression of the iNOS protein in RAW264.7 cells;

FIG. 21 is a graph showing the effect of 3T3-L1 adipocyte 2-NBDG uptake after furanopetanin and secopetanin treatment;

FIG. 22 is a graph showing the effect of furanopetanin and secopetanin on p-AKT and p-PI3K expression in 3T3-L1 adipocytes;

FIG. 23 is a graph showing the effect of furanopetanin and secopetanin on lipid droplet accumulation in 3T3-L1 cells;

FIG. 24 is a graph showing the effect of furanopetanin and secopetanin on TG levels in 3T3-L1 cells;

FIG. 25 is a graph showing the effect of furanopetanin and secopetanin on expression of 3T3-L1 cell adipogenic transcription factors;

FIG. 26 is a graph showing the effect of furanopetanin and secopetanin on the expression of a 3T3-L1 cell lipid metabolism-related protein;

FIG. 27 is a chromatographic chart of component Fr 2-5-4;

FIG. 28 is a chromatographic chart of component Fr 2-5-5;

FIG. 29 is the effect of varying concentrations of Lycium ruthenicum components Fr2-5 on RAW264.7 cell viability;

FIG. 30 is a graph showing the effect of Lycium ruthenicum Murr component Fr2-5 on NO release from RAW264.7 cells;

FIG. 31 is the effect of Lycium ruthenicum component Fr2-5 on 1L-1β concentration of RAW264.7 cells.

Detailed Description

The preparation and application of the active substances in the invention are further illustrated by specific examples and specific experiments, and the anti-inflammatory, hypoglycemic and hypolipidemic effects are verified and illustrated, and the action mechanism is primarily discussed.

In the embodiment of the invention, the method for detecting the purity of the compound is obtained by an HPLC through an area normalization method, and the calculation mode is as follows: target compound purity% = target compound peak area/total peak area x 100%.

Example 1 preparation of Lycium ruthenicum Murr component Fr2-5

(1) Weighing 10.0kg of dried lycium ruthenicum fruit, and soaking and extracting the dried lycium ruthenicum fruit in methanol at room temperature in a dark place under the conditions of: extracting for 3 times, 4-5 d each time, filtering the extracting solution after each extraction, concentrating under reduced pressure in dark place, and combining to obtain the methanol extract of the lycium ruthenicum fruit;

(2) Adding the methanol extract obtained in the step (1) into dry polyamide powder 1:1 for sample mixing, drying at 40 ℃ in an oven, grinding, sieving with a 20-mesh sieve, taking 50.00g of sieved powder each time, loading into a small-sized medium-pressure chromatographic column (26 x 100 mm), and connecting a medium-pressure chromatographic column (49 x 460 mm) filled with MCI with a preparation liquid chromatograph for dry sample loading. Eluting with a three-phase system of A, water, B, methanol and C, and dichloromethane under the following elution conditions: 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: 50mL/min; detection wavelength: 210nm. The Fr1, fr2 and Fr3 components are obtained, the retention time is respectively 12-39 min, 39-139 min and 139-230 min, as shown in figure 1;

(3) After the Fr2 obtained in the step (2) is dissolved by methanol, the mobile phase is finally selected through the optimization of the preparation conditions: water/methanol, gradient elution: 0-120 min, 0-100% B; 120-140 min,100% B; flow rate: 50mL/min; detection wavelength: 254nm; and (3) filling: MCI, column specification: 49 x 460mm; sample injection amount: 8mL. Further, 5 components of Fr2-1, fr2-2, fr2-3, fr2-4 and Fr2-5 were obtained, and the retention times were 13 to 35min,35 to 55min,55 to 68min,68 to 76min and 76 to 130min, respectively, as shown in FIG. 2.

Example 2 preparation of Lycium ruthenicum Components Fr2-5-4 and Fr2-5-5

After dissolving Fr2-5 obtained in step (3) of example 1 with methanol, the kromasil C18 preparative chromatography column (21.2×250mm,5 μm) was finally selected by optimizing the preparation conditions, mobile phase: water/methanol, gradient elution condition: 0 to 60 to 65 to 90min,30 to 42 to 70 to 95 percent of B; flow rate: 19mL/min; detection wavelength: 210nm; the sample loading was 300. Mu.L. 10 components Fr2-5-1 to Fr2-5-10 are obtained, the retention time is respectively 2 to 9min,9 to 19min,19 to 21min,21 to 27min,27 to 36min,36 to 43min,43 to 50min,50 to 62min,62 to 72min and 72 to 85min, as shown in figure 3, wherein Fr2-5-4 is the component Fr2-5-4 and Fr2-5-5 is the component Fr2-5-5.

EXAMPLE 3 preparation of petuniin derivative IV (furanopetanin)

(1) After dissolving the components Fr2-5-5 obtained in example 2 with methanol, the mobile phase is finally selected by optimizing the preparation conditions: water/B: methanol, kromasil C18 column (21.2 x 250mm,5 μm), gradient elution procedure: 0-30-120 min, 20-28-31% B; flow rate: 19ml/min; detection wavelength: 210nm. Further, 10 components were obtained in total from Fr2-5-5-1 to Fr2-5-5-10 to obtain a monomer compound Fr2-5-5-8 (furanopetanin) having a retention time of 92 to 95 minutes as shown in FIG. 4;

(2) Performing high performance liquid chromatography detection on the sample Fr2-5-5-8 (furanopetanin) obtained in the step (1) to determine the purity; the chromatographic conditions were: kromasil C18 analytical column (4.6X250 mm,5 μm); mobile phase: a: water/B: methanol; gradient elution procedure: 0-30-120 min, 20-28-31% methanol; flow rate: 1mL/min; detection wavelength: 210nm; the sample injection volume is 10 mu L;

(3) The purity of the compound Fr2-5-5-8 (furanopetanin) prepared by the method reaches 96.0% through HPLC detection, and is shown in figure 5. The structure of compound Fr2-5-5-8 (furanopetanin) is as follows:

the structure (nuclear magnetic data) of the compound Fr2-5-5-8 was confirmed as shown in Table 1:

TABLE 1 ¹ H and ¹³ C NMR spectroscopic data of furanopetanin in DMSO-d6/CF ₃ COOD (9:1)

EXAMPLE 4 preparation of petuniin derivative V (secopetanin)

(1) After dissolving the component Fr2-5-4 obtained in step (3) of example 2 with methanol, the kromasil C18 preparative chromatography column (21.2×250mm,5 μm) was finally selected by optimization of the preparation conditions, mobile phase: a: water/B: methanol, gradient elution conditions were used: 0 to 60min,30 to 35 percent of B; flow rate: 18mL/min; detection wavelength: 210nm, and the sample injection amount is 150 mu L. Further, 4 components of Fr2-5-4-1, fr2-5-4-2, fr2-5-4-3, fr2-5-4-4 were obtained to obtain a novel monomer compound Fr2-5-4-3 (secopetanin) having a retention time of 40 to 41 minutes as shown in FIG. 10;

(2) Performing high performance liquid chromatography detection on the sample Fr2-5-4-3 (secopetanin) obtained in the step (1) to determine the purity, wherein the chromatographic analysis conditions are as follows: kromasil C18 analytical column (4.6x250 mm,5 μm), mobile phase: a: water/B: methanol, gradient elution conditions were used: 0 to 60min,30 to 35 percent of methanol, flow rate: 1mL/min, column temperature: 30 ℃, detection wavelength: 210nm, and the sample injection volume is 10 mu L;

(3) The purity of the compound Fr2-5-4-3 (secopetanin) prepared by the method reaches 96.3 percent through HPLC detection, and is shown in figure 11. The structural formula of the compound Fr2-5-4-3 (secopetanin) is as follows:

the structure (nuclear magnetic data) of compound secopetanin was confirmed as shown in table 2:

TABLE 2 ¹ H and ¹³ C NMR spectroscopic data of secopetanin in DMSO-d6

The following test examples prove that the petuniin derivatives have the beneficial effects:

Test example 1 Effect of petunidin derivatives in Lycium ruthenicum Murr on inflammation

Inflammation, which can occur in multiple tissues and organs of the human body, has a close relationship with many diseases. Macrophages are special cells in the body which play an immune function and play important physiological roles in inflammation, tumor, autoimmune regulation system and the like. A large number of researches show that many inflammatory diseases of the organism are closely related to macrophages, and lipopolysaccharide is a component part of an outer membrane of gram-negative bacteria and is widely used for establishing an inflammation model of the macrophages. The invention adopts LPS to induce RAW264.7 cells to construct an inflammation model.

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

RAW264.7 macrophages are inoculated into 96-well plates at a density of 5X 10 ⁴/mL and 150 mu L per well, cultured for 48 hours in a cell culture incubator, and then induced with medium (containing 1% penicillin-streptomycin (diabody) and 2% Fetal Bovine Serum (FBS)) containing 0.1, 1, 5, 10, 50, 100 and 200 mu g/mL lipopolysaccharide for 24 hours, respectively, and then cell viability is detected by MTT method, NO content in cell supernatant is measured by NO kit, and the optimal action concentration of lipopolysaccharide is determined according to cell viability and NO content.

As a result, it was found that the survival rate of RAW264.7 cells decreased with increasing LPS concentration, and that the cell viability was extremely significantly inhibited at LPS concentrations of 50 to 200. Mu.g/mL (FIG. 16). The NO release amount of cells is obviously increased by adding LPS with different concentrations (figure 16), wherein when the LPS concentration is 0.1-10 mug/mL, the NO release amount is higher, and the modeling concentration of LPS is 5 mug/mL by combining two indexes.

2 Experimental methods

Based on the established LPS-induced RAW264.7 cell inflammation model, anti-inflammatory activity research is carried out on petuniin derivatives.

2.1 Determination of cell viability by MTT method

Log-grown RAW264.7 cells were plated in 96-well plates at a cell density of 5×10 ⁴/mL, 150 μl per well, incubated in a cell incubator for 24h, changed to DMEM medium (containing 2% FBS) containing lipopolysaccharide or test components, and incubated for 24h, followed by addition of 10 μl of thiazole blue (MTT) solution per well, and incubated in the cell incubator for 4h. Finally, the culture medium is discarded, 150 mu L of DMSO is added into each well to dissolve the cells, an enzyme-labeled instrument is used for reading absorbance values at the wavelength of 490nm, and the cell viability is calculated according to a formula. The cell viability calculation formula is as follows:

The MTT method detects the influence of each compound on the activity of RAW264.7 cells, and the novel compounds Fr2-5-4-3 and Fr2-5-5-8 have no obvious inhibition effect on RAW264.7 at 0-100 mu M. The positive drug dexamethasone was 10. Mu.M in concentration, and for consistency of experimental concentration, in the following experiments, 10. Mu.M was chosen for each compound.

2.2Griess method for determining NO content

And taking out Griess reagents I and II in a refrigerator, and carrying out experiments after the Griess reagents I and II are restored to room temperature. Standards were diluted with DMEM containing 10% fbs to concentrations of 0, 1,2, 5, 10, 20, 40, 60, 80, 100 μm in this order. In a 96-well plate, 50. Mu.L of standard substance and sample solution are added into each well, 50. Mu.L of Griess reagent I and 50. Mu.L of Griess reagent II are sequentially added into each well, and after shaking table mixing, the absorbance at 540nm wavelength is measured, and the NO content is calculated according to a standard curve.

The results show that: the novel compounds Fr2-5-4-3 and Fr2-5-5-8 have very remarkable effect in inhibiting NO release. In particular, the compound Fr2-5-5-8 has the most remarkable action and is superior to positive medicines.

2.3Western blot analysis

The experiment is divided into a control group, an LPS group and an LPS+medicine group with different concentrations, RAW264.7 cells in the logarithmic growth phase are inoculated into a 6-hole plate at the density of 5 x 10 ⁴/mL, after incubation for 24 hours in a cell culture box, the culture medium is replaced by a DMEM culture medium containing 2% FBS, a model group is treated by LPS, a test medicine group is treated by the LPS+medicine group with different concentrations, the culture is continued for 24 hours, protein is extracted, and electrophoresis is carried out. The specific operation method is as follows:

(1) Extraction of RAW264.7 cell proteins

After RAW264.7 cells are treated, the cells are lysed to extract cellular proteins. The culture was aspirated with a suction pump, washed 2 times with PBS buffer, and the PBS was aspirated. Adding cell lysate, shaking 6-well plate, mixing, placing on ice for 10min, scraping with cell scraper, and collecting into centrifuge tube. The tube was placed on ice for further lysis for 30min. After completion of the lysis, the supernatant containing the protein was collected in a new EP tube by centrifugation at 12000r/min at 4℃for 15min using a cryocentrifuge.

(2) BCA method for measuring cell protein concentration

Protein concentration was determined by BCA method, first protein standards were formulated to a concentration of 0.5mg/mL using PBS buffer, BCA working solution according to solution a: solution B = 50:1, and mixing evenly. Three replicates were set and standard curves were drawn by adding protein standard solution and PBS buffer as shown in table 3. Taking 1 mu L of cell extraction protein solution sample, adding into 96-well plate, adding PBS buffer solution to make up the volume to 20 mu L, adding 200 mu L BCA working solution, reacting at 37 ℃ for 30min, measuring 562nm absorbance value by enzyme-labeling instrument, and calculating protein concentration of each sample according to standard curve.

TABLE 3 quantitative protein Table by BCA method

(3) Protein denaturation

Taking a diluted protein sample, adding a protein loading buffer solution, uniformly mixing, and carrying out denaturation in a metal bath at 100 ℃ for 15min. After the denatured protein is cooled to room temperature, the protein is stored in a refrigerator at the temperature of minus 20 ℃ for standby.

(4) SDS-PAGE electrophoresis

And (3) taking two clean glass plates, aligning and placing the two clean glass plates on a glue frame for clamping, adding ultrapure water for leak detection, pouring the ultrapure water after leak detection, sucking residual liquid between the two glass plates by using water-absorbing paper, and preparing glue filling. As shown in Table 4, a separation gel (lower gel) was prepared at a proper concentration according to the molecular weight of the protein, a 10% SDS-PAGE separation gel was prepared according to Table 5 according to the molecular weight of the protein to be detected in the experiment, and a 5% SDS-PAGE concentration gel (upper gel) was prepared according to Table 6. About 4mL of separating gel is added between the two drying glass plates, isopropanol sealing gel is added, and the separating gel is solidified after being placed for 1h. Pouring out isopropanol, sucking residual isopropanol with water absorbing paper, pouring concentrated glue, inserting a comb to avoid bubbles, and slightly pulling out the comb after the concentrated glue is solidified. And clamping the two glass plates on an electrophoresis tank, filling electrophoresis liquid to enable the liquid level to be beyond the glass plates, loading a protein sample, starting electrophoresis after loading, concentrating the sample in concentrated gel for about 30min by using constant voltage 80V, adjusting the voltage to 120V, continuing constant voltage electrophoresis, and stopping electrophoresis when loading buffer liquid runs to the bottom of the gel plate.

TABLE 4 optimal separation Range for SDS-PAGE gels

TABLE 5 preparation of 10% SDS-PAGE separating gel

TABLE 6 preparation of 5% SDS-PAGE gel

(5) Transfer film

Cutting PVDF film to proper size, cutting off one corner as mark to distinguish the front and back sides after cutting, soaking in methanol and activating for 1min. The activated PVDF membrane, foam and membrane transfer filter paper are soaked in a precooled membrane transfer buffer solution for balancing. And (3) prying the glass plate by using a glue cutting knife, cutting glue according to the molecular weight of target protein, putting the glass plate into electrophoresis liquid, placing the black side of the transfer film clamp below, sequentially putting foam, filter paper, adhesive tape, PVDF film, filter paper and foam, and clamping. Placing the clamp into a film transferring groove after placing, reversing film liquid to submerge the clamp plate with the black clamp facing the black surface of the groove, placing the ice bag in a low-temperature environment, and setting the current to be constant current of 250mA. The transfer time is set according to the molecular weight of the protein.

(6) Closure

The sealing liquid is 5% skimmed milk prepared by 1 XTBE, after the membrane transfer is finished, the PVDF membrane is taken out and put into an incubation box, the skimmed milk is added until the PVDF membrane is over, the mixture is placed on a shaking table at room temperature and slowly shaken for 1h, the skimmed milk is poured out, and the 1 XTBE is gently shaken and washed for 10min, and the mixture is washed for 3 times.

(7) Incubation of primary antibody

Primary anti-COX-2 (CST, # 12282), p-ikbα (CST, # 2859), ikbα (CST, # 4814), β -actin (CST, # 4970) were diluted with antibody according to 1: diluting in proportion of 1000, putting PVDF membrane into diluted primary antibody solution, incubating the primary antibody overnight at low speed by a shaking table at 4 ℃, recovering the primary antibody solution, washing for 10min by 1 XTBST, and washing for 3 times.

(8) Incubation of secondary antibody

PVDF membrane is put into horseradish peroxidase marked secondary antibody diluted by 1:5 000 for incubation for 1h at room temperature, secondary antibody solution is recovered after incubation, and membrane is washed for 3 times at room temperature by 1 XTBST for 10min each time.

(9) Development process

The development adopts ECL chemical development method, and the developing solutions A and B are prepared according to the following weight ratio of 1:1, 100 mu L of the mixture is dripped on a PVDF film, developed by a developing instrument and photographed.

(10) Statistical analysis

Western blot data is collected and processed by Image J software, protein band gray values are quantitatively analyzed, beta-actin is used as an internal reference for gray analysis of protein bands, GRAPHPAD PRISM 8.0.0 statistical software is used for statistics, and the data are obtained by means of average number +/-standard deviationAnd (5) expressing. One-way ANOVA (One-way ANOVA) was used between the averages of each group, and P <0.05 considered the difference to be statistically significant.

3 Analysis of results

When LPS activates RAW264.7 mouse macrophages, TLR-4 is stimulated to recognize and bind to LPS, subsequently activating NF- κb signaling pathways, ultimately promoting a number of inflammatory factors such as: production of NO, PGE2, TNF- α, IL- β, IL-6, COX-2 and iNOS triggers a cytokine storm. NO plays a central role in inflammatory actions, and iNOS is particularly involved in pathological overproduction of NO in the NOs family. Thus, the amount of NO released can be used initially for screening of the activity of monomeric compounds in anti-inflammatory models.

Petuniin derivatives showed good anti-inflammatory activity in inhibiting the amount of NO released in the inflammatory model, as shown in fig. 19. After LPS stimulation, iNOS expression levels in RAW264.7 cells were up-regulated with very significant differences compared to the control group (P < 0.01); when the concentration of petuniin derivatives is 10 mu M, the expression of iNOS protein can be obviously inhibited (P < 0.01), and as shown in figure 20, the petuniin derivatives can play an anti-inflammatory role by inhibiting the expression of iNOS protein and reducing the release amount of NO.

Test example 2 Effect of petunidin derivatives in Lycium ruthenicum on sugar metabolism

1 Experimental method

1.1 Effect of petunidin derivatives on uptake of 2-NBDG by 3T3-L1 cells

3T3-L1 preadipocytes were seeded into 12-well plates at a density of 5X 10 ⁴/mL, induced differentiation was performed when the cell density was 80% or more, and an IR model (insulin resistance model) was established by adding 1. Mu.M Dex (dexamethasone) to the cell sap on day 8 after maturation of the induced differentiation. Setting a normal group, a model group and a drug treatment group respectively, wherein each group is cultured by 1 mu M Dex except the normal group which is cultured by a complete culture medium; after 10 mu M of monomer compound is added to each of the drug treatment groups for culturing for 48 hours, the culture solution is sucked away, the mixture is washed 1 time by DPBS, 500 mu L of pancreatin is added for digestion for 1min at 37 ℃, 2mL of DPBS is added for blowing and beating uniformly, and 1000g is centrifuged for 6min. The supernatant was discarded, 1mL of sugar-free medium containing 10. Mu.M 2-NBDG was added to each well, and incubated at 37℃for 30 minutes, and the fluorescence intensity was measured at a wavelength of 488nm by flow-through.

1.2 Effect of petunidin derivatives on AKT phosphorylation in 3T3-L1 cells

3T3-L1 preadipocytes were seeded into 6 well plates at a density of 5X 10 ⁴/mL, induced differentiation was performed when the cell density was 80% or more, and 1. Mu.M Dex was added to the cell culture broth on day 8 after maturation of the induced differentiation to establish an IR model. The normal group, the model group and the drug treatment group are respectively arranged, the other groups are cultivated by 1 mu M Dex except the normal group which is cultivated by a complete culture medium, after 10 mu M monomer compound is added into the drug treatment group for cultivation for 48 hours, the culture solution is sucked away, cells are collected, and the expression level of PI3K and AKT proteins is detected by Western blot. The Western blot analysis method is the same as that of test example 1.

2 Analysis of results

2.1 Effect of petunidin derivatives on uptake of 2-NBDG by 3T3-L1 cells

As shown in FIG. 21, the uptake capacity of 2-NBDG in the normal group was higher than that in the insulin resistance model group, and the uptake capacity of glucose in the model group was weaker than that in the cells after insulin stimulation in the normal group. After stem prognosis of petunidin derivatives, the uptake of 2-NBDG by adipocytes under insulin stimulation can be promoted to different degrees, and the petunidin derivatives can be seen to have the potential of improving insulin resistance.

2.2 Effect of petunidin derivatives on phosphorylation of PI3K and AKT in 3T3-L1 cells

The results are shown in FIG. 22, where the model group had reduced p-AKT and p-PI3K protein expression levels compared to the normal group. After petuniin derivatives are treated, the expression level of p-AKT and p-PI3K in 3T3-L1 adipocytes can be improved to different degrees, so that the glucose uptake of the 3T3-L1 adipocytes is promoted, and the insulin sensitivity is enhanced.

Test example 3 Effect of petunidin derivatives in Lycium ruthenicum on lipid metabolism

1 Experimental method

1.1 Oil Red O staining

3T3-L1 cells with good cell state are inoculated on a 6-well plate, the plating density is 5 multiplied by 10 ⁴/mL, the cells are changed when the cell density reaches about 85% -90% by using high sugar DMEM culture solution containing 10% FBS, the complete culture solution is discarded after two days of contact inhibition, the culture solution containing 10 mu g/mL of Insulin, 0.5mM IBMX and 1 mu M Dex is added for 2d (the added induction solution is marked as 0d, the induction I) and then the culture solution is changed to 10 mu g/mL of Insulin (the induction II) for continuous culture for 2d, then the normal culture solution is changed to 10 mu M of monomer compound for co-incubation, and the culture solution is changed every other day. After the induction is finished, fixing the cells for 30min by using 4% neutral formaldehyde, adding the oil red O working solution prepared in advance to dye the surfaces of the cells after the cell fixation is finished, and standing for 60min in a dark place. After the dyeing is finished, washing the cells with 70% ethanol, discarding redundant dye, washing with ultrapure water for 3-4 times, and finally observing and photographing under a microscope.

1.2TG content determination

3T3-L1 cells are inoculated into a 6-hole plate at the density of 5 multiplied by 10 ⁴/mL, induced differentiation is carried out when the cell density reaches about 85% -90%, and the TG content is measured at the 8d of induction, and the specific method is as follows: (1) cell pretreatment: at the 8d of induction, sucking away the cell culture solution, washing twice with cold PBS, and adding pancreatin digestive juice to digest cells; (2) cell collection: after cell digestion, adding PBS to resuspend cells, centrifuging for 5min under the condition of 1000g, and collecting cell pellet; (3) ultrasonic disruption: adding a proper amount of PBS into the collected precipitate, and then performing ultrasonic crushing (3 min); (4) measurement: mu.L of cell disruption suspension was added to each well of a 96-well plate, 2. Mu.L of distilled water was added to a blank well, 2. Mu.L of standard substance was added to a standard well, 200. Mu.L of assay solution was added to each well, and after incubation at 37℃for 10min, absorbance was read at 510 nm. And measuring the protein concentration in the sample to be measured by means of the BCA method, correcting, and finally calculating the content of TG according to the following formula.

1.3Western blot analysis

Western blot analysis was performed in the same manner as in test example 1.

2 Analysis of results

2.1 Effect of petunidin derivatives on accumulation of lipid droplets and TG content in 3T3-L1 cells

As shown in fig. 23, there was no accumulation of lipid droplets in the undifferentiated cells, and the differentiated cells contained a large amount of lipid droplets. After petuniin derivatives are treated, intracellular lipid droplets are obviously reduced.

As shown in fig. 24, the TG content in the differentiated cells was significantly increased (P < 0.01) compared to the undifferentiated cells. TG content was reduced after petuniin derivative treatment compared to differentiated cells and all had very significant differences P < 0.01).

2.2 Effect of petunidin derivatives on expression of 3T3-L1 cell lipodystrophin

(1) Effect of petunidin derivatives on expression of 3T3-L1 cell lipogenic transcription factor

The effect of petuniin derivatives on expression of 3T3-L1 cell adipogenic transcription factor was analyzed by Western blot, and the results are shown in FIG. 25. The expression level of PPARgamma and C/EBPalpha proteins in the 3T3-L1 cells which are not induced to differentiate is lower. Whereas the expression level of PPARgamma, C/EBPalpha protein is higher in the cells induced to differentiate. Compared with the differentiated group, the petuniin derivative treatment can reduce the protein expression level of PPARgamma and C/EBPalpha transcription factors to a certain extent.

(2) Effect of petunidin derivatives on expression of 3T3-L1 cell lipid production-related proteins

As shown in FIG. 26, FAS and ACC protein expression levels were low in the 3T3-L1 cells that did not induce differentiation. While FAS, ACC protein expression levels were significantly elevated in induced differentiated cells. Compared with the differentiated group, the petuniin derivative can obviously reduce the expression level of FAS and ACC proteins after being treated.

Based on the above results, it is found that 3T3-L1 cells are regulated by transcription factors and lipoproteins when they differentiate from preadipocytes into mature adipocytes, and that the cell morphology is also altered such as the eventual appearance of "arming loops". The petuniin derivative can inhibit accumulation of 3T3-L1 intracellular lipid drops and reduce intracellular TG content; petuniin derivatives can also inhibit the differentiation of 3T3-L1 adipocytes and reduce the accumulation of intracellular lipid droplets by inhibiting the expression level of transcription factors such as PPARgamma, C/EBPalpha and the like. And simultaneously, the expression level of FAS and ACC proteins is inhibited, so that the lipid generation is inhibited, and the lipid metabolism level of cells is improved.

EXAMPLE 5 chromatographic analysis of Components Fr2-5-4

The component Fr2-5-4 prepared in example 2 was subjected to chromatography under the following conditions: kromasil C18 analytical column (4.6x250 mm,5 μm), mobile phase: a: water/B: methanol, gradient elution conditions were used: 0 to 60min,30 to 35 percent of methanol, flow rate: 1mL/min, column temperature: 30 ℃, detection wavelength: 210nm, the sample volume was 10. Mu.L, and Fr2-5-4-3 was used as a control. As a result, as shown in FIG. 27, the fraction Fr2-5-4 was separated into four main fractions by the chromatographic conditions of the experiment, and the four fractions were separated to a good degree, wherein the effluent of 40 to 50min was Fr2-5-4-3, which was the novel monomer compound in the present invention.

EXAMPLE 6 chromatographic analysis of component Fr2-5-5

The component Fr2-5-5 prepared in example 2 was subjected to chromatography under the following conditions: kromasil C18 analytical column (4.6x250 mm,5 μm), mobile phase: a: water/B: methanol, gradient elution conditions were used: 0-30-120 min, 20-28-31% methanol, flow rate: 1mL/min; detection wavelength: 210nm, the sample volume was 10. Mu.L, and Fr2-5-5-8 was used as a control. As a result, as shown in FIG. 28, the fraction Fr2-5-5 was separated into 10 main fractions by the chromatographic conditions of the experiment, to obtain the monomer compound Fr2-5-5-8.

EXAMPLE 7 Effect of Lycium ruthenicum active ingredient Fr2-5 on inflammation

1 Experimental method

Based on the established LPS-induced RAW264.7 cell inflammation model, anti-inflammatory activity research is performed on the Lycium ruthenicum component Fr 2-5.

1.1 Determination of cell viability by MTT method

The measurement method was as described in test example 1. The MTT assay detects the effect of components on RAW264.7 cell viability, and components Fr2-5 have no significant inhibitory effect on RAW264.7 at 0-100. Mu.g/mL (FIG. 29). The concentration of the component Fr2-5 in the present invention was selected to be 10. Mu.g/mL.

1.2 Determination of inflammatory factors

(1) Determination of NO content by Griess method

The results are shown in FIG. 30: after LPS treatment, the NO release amount of an inflammation model is obviously increased (P < 0.01), and the Lycium ruthenicum component Fr2-5 can inhibit the NO release.

(2) Determination of IL-1 beta

Dividing a 96-well plate into a blank hole, a sample hole and a standard hole, adding a sample with proper concentration and a standard substance with 100 mu L/hole, sealing the sample with a sealing plate film, incubating for 2 hours at room temperature, washing the plate for 5 times, and placing the plate on absorbent paper for drying. 100 mu L of horseradish peroxidase-labeled strepitavidine is added to each well, the sealing plate is carried out again by using a sealing plate membrane, the mixture is incubated at room temperature for 20min in dark, 50 mu L of stop solution is added to each well, the absorbance value is measured at 450nm after uniform mixing, and the concentration of the sample is calculated through a standard curve.

The results are shown in FIG. 31: after LPS treatment, the IL-1 beta of the inflammation model is obviously increased (P < 0.01), and the Lycium ruthenicum component Fr2-5 can obviously reduce the expression of the IL-1 beta of the inflammation factor.

2 Analysis of results

When LPS activates RAW264.7 mouse macrophages, TLR-4 is stimulated to recognize and bind to LPS, subsequently activating NF- κb signaling pathways, ultimately promoting a number of inflammatory factors such as: production of NO, PGE2, TNF- α, IL- β, IL-6, COX-2 and iNOS triggers a cytokine storm. NO plays a central role in inflammatory actions, and iNOS is particularly involved in pathological overproduction of NO in the NOs family. Thus, the amount of NO released can be used initially for screening of active ingredients in anti-inflammatory models.

Lycium ruthenicum Murr component Fr2-5 showed good anti-inflammatory activity, as shown in FIGS. 30 and 31. NO release and IL-1β levels in RAW264.7 cells were significantly elevated following LPS stimulation, with significant differences compared to control (P < 0.01); at a concentration of 10 μg/mL for Lycium ruthenicum Murr component Fr2-5, the release of NO (P < 0.05) and the expression of inflammatory factor IL-1 beta (P < 0.05) can be significantly inhibited. In conclusion, lycium ruthenicum Murr component Fr2-5 can exert anti-inflammatory effect by reducing NO release amount and inhibiting expression of inflammatory factor IL-1β.

The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related technical fields are included in the scope of the present invention.

Claims

1. A lycium ruthenicum component Fr2-5, characterized in that the component Fr2-5 is prepared by the following method:

Wherein the preparation chromatographic conditions include:

chromatographic column: the preferred specification of the MCI medium pressure chromatographic column is 49 x 460mm;

(2) Separating the component Fr2 by preparative chromatography to obtain a component Fr2-5, wherein the retention time is 76-130 min;

Wherein the conditions of the preparative chromatography include:

chromatographic column: an MCI medium pressure chromatographic column; the preferred specification is 49 x 460mm;

2. Use of a component according to claim 1 for the preparation of a product for the treatment and/or prophylaxis of diseases which are associated with inflammation.

3. The use according to claim 2, wherein the product is a product that reduces the release of at least one inflammatory factor of NO, PGE2, TNF- α, IL-1β, IL-6, COX-2 and iNOS; further, the inflammatory factor is selected from the group consisting of NO, IL-1β.

4. An anti-inflammatory product comprising components Fr2-5.

5. Use of a component according to claim 1 for the preparation of a product for the treatment and/or prophylaxis of diseases which are associated with disturbed carbohydrate metabolism and/or lipid metabolism.

6. The use according to claim 5, wherein the product promotes the uptake of sugar and/or sugar analogues by adipocytes.

7. The use according to claim 5, wherein the product is a product that increases the expression level of p-AKT and p-PI3K proteins.

8. The use according to claim 5, wherein the product is a product that inhibits accumulation of cellular lipid droplets and/or production of cellular lipids.

9. Use of a component according to claim 1 for the preparation of a medicament for the treatment of at least one of ppary antagonists, C/ebpa antagonists, FAS inhibitors, ACC inhibitors.

10. A hypoglycemic and/or hypolipidemic product, characterized by comprising components Fr2-5.