The active component Fr2-5 in black goji berries, its extraction method and uses

Abstract

This invention discloses the active component Fr2-5 in black goji berries and its extraction method, relating to the field of plant active component separation technology. This invention also provides the application of active Fr2-5 in the preparation of products for the prevention and / or treatment of inflammation, the preparation of products for the prevention and / or treatment of disorders related to glucose metabolism, and the preparation of products for the prevention and / or treatment of disorders related to lipid metabolism.

Description

Technical Field

[0001] This invention relates to the field of plant active component separation technology, and in particular to the active component Fr2-5 in black goji berries, its extraction method and uses. Background Technology

[0002] Black goji berry (Lycium ruthenicum Murr) is a perennial, thorny shrub belonging to the genus Lycium in the Solanaceae family. Its fruit is sweet, juicy, and rich in nutrients, making it highly valuable for research and development. It is a unique desert medicinal plant species native to western my country. Black goji berries contain various essential amino acids and abundant minerals. In Tibetan medicine, it is used to treat heart disease, lower cholesterol, stimulate brain nerves, enhance immune function, prevent and treat cancer, anti-aging, beautify the skin, regulate menstruation, and treat amenorrhea, with significant efficacy.

[0003] Anthocyanins are a class of water-soluble natural pigments widely found in plants, belonging to the flavonoid family. Anthocyanins mainly exist in the forms of glucosides and rhamnosides in the fruits, epidermis, and flowers of plants. Black goji berries have extremely high anthocyanin content, with over 90% of the anthocyanins being petunia derivatives. These anthocyanins possess antioxidant properties, protect vision, and inhibit tumors, exhibiting significant biological activity and extraction value. As the demand for naturally derived and non-toxic functional components increases, anthocyanins, as one of the most representative naturally derived functional components, are gaining widespread popularity among consumers and in the market due to their excellent coloring properties and superior antioxidant activity.

[0004] Many of the abundant active substances in black goji berries have yet to be isolated and identified. If we can conduct more in-depth and detailed research and explore the active mechanisms of new chemical components and their pharmacological effects in black goji berries, we can hope to develop more safe and effective natural plant-derived drugs that can treat diseases. Summary of the Invention

[0005] This invention provides active components and novel petunia derivatives extracted from black goji berries, their extraction methods, and their use in the preparation of drugs for the prevention and / or treatment of inflammation-related diseases. The components and compounds have the effect of inhibiting the expression of iNOS protein, thereby reducing NO release.

[0006] This invention provides a black goji berry component Fr2-5, which is prepared by the following method and has a retention time of 76-130 min;

[0007] (1) Black wolfberry extract was prepared by preparative chromatography to obtain components Fr1, Fr2 and Fr3, with retention times of 12-39 min, 39-139 min and 139-230 min, respectively.

[0008] The preparative chromatographic conditions include:

[0009] Chromatographic column: MCI medium-pressure chromatographic column, preferably 49*460mm;

[0010] Mobile phase: Water:A / Methanol:B / Dichloromethane:C; Gradient elution was performed using the following program: 0–120 min, 100% A–100% B; 120–180 min, 100% B–100% C; 180–210 min, 100% B; 210–240 min, 100% A;

[0011] (2) The component Fr2 was separated by preparative chromatography to obtain the component Fr2-5;

[0012] The conditions for preparing the chromatogram include:

[0013] Chromatographic column: MCI medium-pressure chromatographic column; preferred size is 49*460mm;

[0014] Mobile phase: A: water / B: methanol; gradient elution was performed using the following program: 0–120 min, 0%–100% B; 20–140 min, 100% B.

[0015] In this invention, the black goji berry extract refers to the concentrate obtained by extracting black goji berries with methanol, filtering, and concentrating in the dark. The extraction method includes, but is not limited to, conventional extraction methods such as immersion extraction, hot reflux extraction, and ultrasonic extraction.

[0016] In a specific embodiment of the present invention, black goji berries are extracted by immersion. The extraction conditions are: liquid-to-solid ratio of 10-30 mL / g, preferably 20 mL / g; extraction times: 2-5 times, each time for 3-5 days, preferably 3 times, each time for 4-5 days.

[0017] Furthermore, after extracting the black goji berries, the extract is filtered and concentrated under reduced pressure in the dark, then combined to obtain a methanol extract of black goji berries.

[0018] In this invention, polyamide is added to the methanol extract paste for mixing, drying, grinding, and sieving.

[0019] Furthermore, the ratio of methanol extract to polyamide is 1:(0.5-2.5), preferably 1:1.

[0020] Furthermore, the drying method can be selected from atmospheric pressure drying, reduced pressure drying, and freeze drying, etc.

[0021] Furthermore, the sieving is of 20 mesh to 50 mesh, preferably 20 mesh.

[0022] This invention provides a black goji berry component Fr2-5-4, which is obtained by preparative chromatographic separation of component Fr2-5, and the retention time of component Fr2-5-4 is 21-27 min;

[0023] The conditions for preparing the chromatogram include:

[0024] Chromatographic column: Kromasil C18 column; preferred size is 21.2*250mm;

[0025] Mobile phase: A: water / B: methanol; gradient elution was performed using the following program: 0–60–65–90 min, 30%–42%–70%–95% B.

[0026] This invention provides a black goji berry component Fr2-5-5, which is obtained by preparative chromatographic separation of component Fr2-5, and the retention time of component Fr2-5-5 is 27-36 min;

[0027] The conditions for preparing the chromatogram include:

[0028] Chromatographic column: Kromasil C18 column; preferred size is 21.2*250mm;

[0029] Mobile phase: A: water / B: methanol; gradient elution was performed using the following program: 0–60–65–90 min, 30%–42%–70%–95% B.

[0030] This invention provides a petunia-derived derivative IV, the structural formula of which is shown in Formula IV:

[0031]

[0032] This invention uses methanol to extract black wolfberry fruit, separates it by preparative chromatography, and unexpectedly obtains a new petunia derivative after one-dimensional and two-dimensional nuclear magnetic resonance identification, which is named furanopetanin.

[0033] Among them, petunia derivative IV was obtained by preparative chromatographic separation of component Fr2-5-5, with a retention time of 92-95 min.

[0034] Furthermore, the conditions for preparing the chromatogram include:

[0035] Chromatographic column: Kromasil C18 column; preferred size is 21.2*250mm;

[0036] Mobile phase: A: water / B: methanol; gradient elution was performed using the following program: 0–30–120 min, 20%–28%–31% B.

[0037] This invention provides a petunia-derived derivative V, the structural formula of which is shown in Formula V:

[0038]

[0039] This invention uses methanol to extract black wolfberry fruit, separates it by preparative chromatography, and unexpectedly obtains a new petunia-like derivative after one-dimensional and two-dimensional nuclear magnetic resonance identification, which is named secopetinin.

[0040] Among them, the phenanthrene compound V was obtained by preparative chromatographic separation of component Fr2-5-5, with a retention time of 40-41 min.

[0041] Furthermore, the conditions for preparing the chromatogram include:

[0042] Chromatographic column: Kromasil C18 column; preferred size is 21.2*250mm;

[0043] Mobile phase: A: water / B: methanol; gradient elution was performed using the following program: 0–60 min, 30%–35% B.

[0044] In a specific embodiment of the present invention, the preparative chromatographic separation further includes at least one of the following conditions:

[0045] Detection wavelength: 210nm;

[0046] Column temperature: 25℃~35℃;

[0047] Flow rate: 15–60 mL / min;

[0048] Injection volume: 0.1 mL to 8.0 mL.

[0049] The present invention provides the use of the above-mentioned petunia derivatives IV and / or V, as well as their pharmaceutically acceptable salts, hydrates or solvates, in the preparation of products for the treatment and / or prevention of inflammation-related diseases.

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

[0051] Furthermore, the two products mentioned above for treating and / or preventing inflammation-related diseases are products that block and / or inhibit the NF-κB signaling pathway.

[0052] NF-κB is a protein complex that controls the transcription of DNA, the production of cytokines, and cell survival; it also participates 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.

[0053] Furthermore, the product is a product that reduces the release of at least one inflammatory factor, including NO, PGE2, TNF-α, IL-β, IL-6, COX-2, and iNOS; even further, the product is a product that reduces the release of NO.

[0054] Furthermore, the product is a product that inhibits the expression of iNOS protein.

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

[0056] The present invention provides the use of the above-mentioned petunia derivatives IV and / or V, as well as their pharmaceutically acceptable salts, hydrates or solvates, in the preparation of products for the treatment and / or prevention of diseases related to glucose metabolism disorders.

[0057] The present invention provides at least one of the above-mentioned components Fr2-5, Fr2-5-4 and Fr2-5-5 for use in the preparation of products for treating and / or preventing diseases related to glucose metabolism disorders.

[0058] Furthermore, both of the above-mentioned products for treating and / or preventing diseases related to glucose metabolism disorders are products with hypoglycemic effects, such as hypoglycemic drugs.

[0059] The products for treating and / or preventing disorders of glucose metabolism can lower blood glucose through various pathways, including inhibiting hepatic gluconeogenesis and glucose output, increasing peripheral tissue sensitivity to insulin, promoting glucose uptake and utilization, stimulating pancreatic β-cells to secrete insulin, enhancing the binding of insulin to receptors, and increasing the sensitivity of target cells to insulin.

[0060] Furthermore, the product is one that promotes the uptake of sugars and / or sugar analogues by fat cells.

[0061] Adipocytes primarily utilize insulin-sensitive GLUT4 (glucose transporter) for glucose uptake. Under insulin stimulation, the inward transmission of insulin receptor tyrosine phosphorylation signals phosphorylates insulin receptor substrate-1 (IRS-1), thereby activating phosphatidylinositol-3-kinase (PI3K), triggering GLUT4 to translocate to the cell surface, and increasing glucose uptake.

[0062] Furthermore, the product is a product that enhances the expression levels of p-AKT and p-PI3K proteins.

[0063] AKT is a serine / threonine protein kinase involved in various aspects of glucose metabolism, apoptosis, cell proliferation, and cell transport. During glucose metabolism, activated AKT activates various downstream factors in the insulin signaling pathway, including enzymes, kinases, and transcription factors, through phosphorylation, thereby regulating cellular function and insulin signal transduction. Typically, activated AKT activates its downstream phosphatidylinositol kinase 3 (PI3K), prompting glucose transporters to translocate from the cytoplasm to the cell membrane, accelerating glucose uptake and utilization, and thus playing a role in insulin signal transduction.

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

[0065] The present invention provides the use of the above-mentioned petunia derivatives IV and / or V, as well as their pharmaceutically acceptable salts, hydrates or solvates, in the preparation of products for the treatment and / or prevention of lipid metabolism disorder-related diseases.

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

[0067] Furthermore, both of the above-mentioned products for treating and / or preventing lipid metabolism disorders are products with lipid-lowering effects, such as lipid-lowering drugs.

[0068] Furthermore, the above two categories of products for treating and / or preventing lipid metabolism disorders include products that inhibit the accumulation of lipid droplets in adipocytes and / or products that inhibit the production of lipids in cells; the products may be drugs, health products, or other products that inhibit the accumulation of lipid droplets in cells and / or inhibit the production of lipids 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 disease, and metabolic syndrome-related diseases.

[0070] Obesity, hypertension, hyperlipidemia, cardiovascular disease, and metabolic syndrome are all closely related to the levels of plasma lipids such as triglycerides (TG), free cholesterol (FC), cholesterol esters (CE), and phospholipids. Reducing plasma lipid levels to a certain concentration range can effectively control or treat these diseases. This invention demonstrates through experiments that the active components and / or novel compounds in this invention can effectively inhibit lipid droplet accumulation, reduce TG levels, and regulate the gene expression levels of adipocyte transcription factors and related protein expression levels through relevant signaling pathways, thereby inhibiting adipogenic differentiation and lipid production. This invention can be used to prepare products for the prevention and / or treatment of obesity, hypertension, hyperlipidemia, cardiovascular disease, and metabolic syndrome.

[0071] The present invention provides the use of the above-mentioned petunia derivatives IV and / or V, and their pharmaceutically acceptable salts, hydrates or solvates, in the preparation of at least one of PPARγ antagonists, C / EBPα antagonists, FAS inhibitors and ACC inhibitors.

[0072] The present invention provides at least one of the above-mentioned components Fr2-5, Fr2-5-4 and Fr2-5-5 for use in the preparation of at least one of PPARγ antagonists, C / EBPα antagonists, FAS inhibitors and ACC inhibitors.

[0073] The PPARγ antagonists, C / EBPα antagonists, FAS inhibitors, and ACC inhibitors are drugs that reduce the gene expression of adipocyte transcription factors PPARγ and C / EBPα and the protein expression levels of FAS and ACC.

[0074] The differentiation of undifferentiated cells into mature adipocytes requires a series of complex and precise transcription factors. PPARγ is an essential regulatory factor for adipocyte formation and is highly expressed in the early stages of adipogenesis. C / EBPα, also known as CCAAT / enhancer-binding protein α, is expressed in large quantities in the middle stages of adipocyte differentiation.

[0075] FAS is fatty acid synthase, which plays an important role in lipid synthesis; ACC is acetyl-CoA carboxylase, which is the rate-limiting enzyme in the heavy head synthesis of fatty acids.

[0076] The active components and novel compounds described in this invention can reduce the gene expression of cellular transcription factors PPARγ and C / EBPα and the protein expression levels of FAS and ACC, thereby effectively inhibiting adipogenic differentiation and lipid production. They can then be used to prepare products for the prevention and / or treatment of diseases related to lipid metabolism disorders, such as obesity, hypertension, hyperlipidemia, and cardiovascular diseases.

[0077] The present invention also provides a blood sugar and / or lipid-lowering product, comprising one or more of component Fr2-5, component Fr2-5-4, component Fr2-5-5, compound IV, and compound V.

[0078] The products described in this invention include, but are not limited to, pharmaceuticals, health products, and food.

[0079] The beneficial effects of this invention are as follows: This invention provides three active components and two new compounds, furanopetanin and sepoetanin, whose components and compounds have anti-inflammatory, hypoglycemic, and lipid-lowering effects, thus more comprehensively developing the active substances of black goji berries. At the same time, it further explores the medicinal value of black goji berries and expands their clinical applications, providing more reference for the development of potential plant-derived drugs for the treatment of diseases related to inflammation, glucose metabolism disorders, and lipid metabolism disorders.

[0080] This invention also provides a liquid chromatography analysis method for the component Fr2-5-4, the chromatographic analysis conditions of which include:

[0081] Chromatographic column: C18, preferably 4.6×250mm, 5μm;

[0082] Mobile phase: A: water / B: methanol, gradient elution program: 0–60 min, 30%–35% methanol;

[0083] Furthermore, the flow rate was 0.8–1.2 mL / min; the column temperature was 30 ± 5 °C; and the detection wavelength was 210 ± 5 nm.

[0084] Furthermore, Fr2-5-4-3 was used as a control.

[0085] This invention also provides a liquid chromatography analysis method for the component Fr2-5-5, the chromatographic analysis conditions of which include:

[0086] Chromatographic column: C18, preferably 4.6*250mm, 5μm;

[0087] Mobile phase: A: water / B: methanol, gradient elution program: 0–30–120 min, 20%–28%–31% methanol;

[0088] Furthermore, the flow rate was 0.8–1.2 mL / min; the detection wavelength was 210 ± 5 nm.

[0089] Furthermore, Fr2-5-5-8 was used as a control.

[0090] Based on the above method, the present invention also provides an analytical method for measuring the quality of black goji berries or their extracts, comprising the following:

[0091] (1) Pretreatment of black goji berries or their extracts was carried out in accordance with the preparation methods of Fr2-5-4 or Fr2-5-5;

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

[0093] The above analytical methods provide a means to measure the quality of black goji berries or their extracts, which are used to treat diseases related to inflammation, glucose metabolism disorders, and lipid metabolism disorders, and offer new progress in establishing quality standards for related products. Attached Figure Description

[0094] Figure 1 This is the MCI separation and preparation chromatogram of black goji berry fruit extract;

[0095] Figure 2 This is a chromatogram of the MCI separation and preparation of the Fr2 fraction;

[0096] Figure 3 This is a chromatogram of the MCI separation and preparation of the Fr2-5 fraction;

[0097] Figure 4 This is a chromatogram of the MCI separation and preparation of the Fr2-5-5 fraction;

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

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

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

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

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

[0103] Figure 10 This is a chromatogram of the MCI separation and preparation of the Fr2-5-4 fraction;

[0104] Figure 11 This is a purity analysis chromatogram of component Fr2-5-4-3 (secopetanin);

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

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

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

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

[0109] Figure 16 The graph shows the effect of different concentrations of LPS on the viability and NO release of RAW264.7 cells.

[0110] Figure 17 This is a graph showing the effect of different concentrations of sepoetanin on the viability of RAW264.7 cells;

[0111] Figure 18 This is a graph showing the effect of different concentrations of furanophenin on the viability of RAW264.7 cells;

[0112] Figure 19 This is a graph showing the effects of furanopetanin and secopetanin on NO release in RAW264.7 cells;

[0113] Figure 20 This is a graph showing the effects of furanopetanin and sepopetanin on iNOS protein expression in RAW264.7 cells;

[0114] Figure 21 This is a graph showing the effect of furanopetanin and secopetanin treatment on 2-NBDG uptake in 3T3-L1 adipocytes;

[0115] Figure 22 This is a diagram showing the effects of furanopetanin and sepopetanin on the expression of p-AKT and p-PI3K in 3T3-L1 adipocytes;

[0116] Figure 23 This is a diagram showing the effects of furanopetanin and sepopetanin on lipid droplet accumulation in 3T3-L1 cells;

[0117] Figure 24 This is a graph showing the effects of furanopetanin and sepoetanin on TG levels in 3T3-L1 cells;

[0118] Figure 25 This is a graph showing the effects of furanopetanin and sepoetanin on the expression of adipogenic transcription factors in 3T3-L1 cells;

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

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

[0121] Figure 28 This is the chromatogram of component Fr2-5-5;

[0122] Figure 29 The effect of different concentrations of black goji berry component Fr2-5 on the viability of RAW264.7 cells;

[0123] Figure 30 The effect of black goji berry component Fr2-5 on NO release in RAW264.7 cells;

[0124] Figure 31 The effect of black goji berry component Fr2-5 on 1L-1β concentration in RAW264.7 cells. Detailed Implementation

[0125] The preparation and application of the active substances in this invention will be further explained through specific embodiments and experiments, and their anti-inflammatory, hypoglycemic and lipid-lowering effects will be verified and explained, and their mechanism of action will be preliminarily explored.

[0126] In this embodiment of the invention, the method used to detect the purity of the compound is to obtain it by area normalization using HPLC. The calculation method is: target compound purity % = target compound peak area / total peak area * 100%.

[0127] Example 1: Preparation of Fr2-5 component from black goji berries

[0128] (1) Weigh 10.0 kg of dried black wolfberry fruit and extract it by soaking in methanol at room temperature in the dark. Extraction conditions: liquid-to-solid ratio 20 mL / g, extract a total of 3 times, each time for 4-5 days. After each extraction, filter the extract and concentrate it under reduced pressure in the dark. Combine the extracts to obtain black wolfberry fruit methanol extract.

[0129] (2) The methanol extract obtained in step (1) was mixed with dry polyamide powder at a 1:1 ratio, dried in an oven at 40°C, ground, and passed through a 20-mesh sieve. 50.00 g of the sieved powder was loaded into a small medium-pressure chromatographic column (26*100 mm) and connected to a medium-pressure chromatographic column (49*460 mm) equipped with MCI for dry loading into preparative liquid chromatography. Elution was performed using a three-phase system of A:water / B:methanol / C:dichloromethane. 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: 50 mL / min; detection wavelength: 210 nm. Fr1, Fr2, and Fr3 fractions were obtained with retention times of 12–39 min, 39–139 min, and 139–230 min, respectively. Figure 1 As shown;

[0130] (3) After dissolving the Fr2 obtained in step (2) in methanol, the mobile phase was finally selected through optimization of the preparation conditions: 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; packing material: MCI, column size: 49*460 mm; injection volume: 8 mL. This yielded five components: Fr2-1, Fr2-2, Fr2-3, Fr2-4, and Fr2-5, with retention times of 13-35 min, 35-55 min, 55-68 min, 68-76 min, and 76-130 min, respectively. Figure 2 As shown.

[0131] Example 2: Preparation of black goji berry components Fr2-5-4 and Fr2-5-5

[0132] After dissolving Fr2-5 obtained in step (3) of Example 1 in methanol, the preparation conditions were optimized, and a Kromasil C18 column (21.2*250mm, 5μm) was finally selected for preparation. The mobile phase was A: water / B: methanol, and the gradient elution conditions were: 0~60~65~90min, 30%~42%~70%~95%B; the flow rate was 19mL / min; the detection wavelength was 210nm; and the injection volume was 300μL. Ten components, Fr2-5-1 to Fr2-5-10, were obtained, with retention times of 2~9min, 9~19min, 19~21min, 21~27min, 27~36min, 36~43min, 43~50min, 50~62min, 62~72min, and 72~85min, respectively. Figure 3As shown, Fr2-5-4 is component Fr2-5-4 and Fr2-5-5 is component Fr2-5-5.

[0133] Example 3: Preparation of Furanopetanin (a derivative of petunia)

[0134] (1) After dissolving the component Fr2-5-5 obtained in Example 2 in methanol, the mobile phase was finally selected through optimization of the preparation conditions: A: water / B: methanol, Kromasil C18 column (21.2*250mm, 5μm), gradient elution program: 0~30~120min, 20%~28%~31% B; flow rate: 19ml / min; detection wavelength: 210nm. Ten components, Fr2-5-5-1~Fr2-5-5-10, were obtained, yielding the monomer compound Fr2-5-5-8 (furanopetanin), with a retention time of 92~95min. Figure 4 As shown;

[0135] (2) The purity of the sample Fr2-5-5-8 (furanopetanin) obtained in step (1) was determined by high performance liquid chromatography. The chromatographic conditions were as follows: Kromasil C18 analytical column (4.6×250mm, 5μm); mobile phase: A: water / B: methanol; gradient elution program: 0~30~120min, 20%~28%~31% methanol; flow rate: 1mL / min; detection wavelength: 210nm; injection volume: 10μL.

[0136] (3) HPLC analysis showed that the purity of the compound Fr2-5-5-8 (furanopetanin) prepared by the above method reached 96.0%. Figure 5 As shown. The structure of compound Fr2-5-5-8 (furanopetanin) is as follows:

[0137]

[0138] The structural confirmation results (NMR data) of compound Fr2-5-5-8 are shown in Table 1:

[0139] Table 1. Furanopetanin in DMSO-d6 / CF3COOD (9:1) 1 H and 13 C NMR spectral data

[0140]

[0141] Example 4: Preparation of sepoetanin derivative V

[0142] (1) After dissolving the component Fr2-5-4 obtained in step (3) of Example 2 in methanol, the preparation conditions were optimized, and a Kromasil C18 column (21.2*250mm, 5μm) was finally selected. The mobile phase was A: water / B: methanol, and the gradient elution conditions were 0-60min, 30-35% B; the flow rate was 18mL / min; the detection wavelength was 210nm, and the injection volume was 150μL. This yielded four components: Fr2-5-4-1, Fr2-5-4-2, Fr2-5-4-3, and Fr2-5-4-4, resulting in a new monomeric compound Fr2-5-4-3 (secopetanin) with a retention time of 40-41min. Figure 10 As shown;

[0143] (2) The purity of the sample Fr2-5-4-3 (secopetanin) obtained in step (1) was determined by high performance liquid chromatography. Chromatographic analysis conditions: Kromasil C18 analytical column (4.6×250mm, 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: 10μL;

[0144] (3) HPLC analysis showed that the purity of the compound Fr2-5-4-3 (secopetanin) prepared by the above method reached 96.3%. Figure 11 As shown. The structural formula of compound Fr2-5-4-3 (secopetanin) is as follows:

[0145]

[0146] The structural confirmation results (NMR data) of the compound sepoetanin are shown in Table 2:

[0147] Table 2 shows the sepoetanin content in DMSO-d6. 1 H and 13 C NMR spectral data

[0148]

[0149] The following experimental examples demonstrate the beneficial effects of the petuniae derivatives of this invention:

[0150] Experimental Example 1: Effects of petuniain derivatives in black wolfberry on inflammation

[0151] Inflammation can occur in multiple tissues and organs of the human body and is closely related to many diseases. Macrophages are special cells in the body that play an important physiological role in inflammation, tumors, and the autoimmune regulatory system. Numerous studies have shown that many inflammatory diseases in the body are closely related to macrophages. Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria and is widely used to establish macrophage-based inflammation models. This invention uses LPS-induced RAW264.7 cells to construct an inflammation model.

[0152] 1. Establishment of an LPS-induced inflammation model in RAW264.7 cells

[0153] RAW264.7 macrophages were stored at a density of 5 × 10⁻⁶ cells. 4 Cells 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.

[0154] 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.

[0155] 2 Experimental Methods

[0156] Based on the established LPS-induced inflammation model of RAW264.7 cells, the anti-inflammatory activity of petunia extract derivatives was studied.

[0157] 2.1 MTT assay for cell viability

[0158] Log-grown RAW264.7 cells were used at a ratio of 5*10-1 4At 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].

[0159]

[0160] The effects of the MTT assay on the viability of RAW264.7 cells were examined. 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 in experimental concentrations, all compounds were used at 10 μM in subsequent experiments.

[0161] 2.2 Determination of NO content by Griess method

[0162] Remove Griess reagents I and II from the refrigerator and allow them to return to room temperature before conducting the experiment. Dilute the standards with DMEM containing 10% FBS to obtain concentrations of 0, 1, 2, 5, 10, 20, 40, 60, 80, and 100 μM, respectively. Add 50 μL of the standard and sample solution to each well of a 96-well plate, followed by 50 μL of Griess reagent I and 50 μL of Griess reagent II. Mix thoroughly on a shaker and measure the absorbance at 540 nm. Calculate the NO content based on the standard curve.

[0163] The results showed that the new compounds Fr2-5-4-3 and Fr2-5-5-8 had a highly significant effect in inhibiting NO release. In particular, compound Fr2-5-5-8 showed the most significant effect and was superior to the positive control drug.

[0164] 2.3 Western blot analysis

[0165] The experiment was divided into a control group, an LPS group, and LPS + different concentrations of drugs groups. RAW264.7 cells in logarithmic growth phase were used, and the cells were divided into groups of 5*10-1. 4 Cells were seeded at a density of [number] cells / mL in 6-well plates and incubated in a cell culture incubator for 24 hours. The medium was then replaced with DMEM containing 2% FBS. The model group was treated with LPS, and the test drug group was treated with LPS plus different concentrations of the drug. The cells were cultured for another 24 hours, and proteins were extracted and subjected to electrophoresis. The specific procedures are as follows:

[0166] (1) Extraction of proteins from RAW264.7 cells

[0167] After treatment, RAW264.7 cells were lysed to extract cellular proteins. The culture medium was aspirated using a suction pump, and the cells were washed twice with PBS buffer, after which the PBS was removed. Cell lysis buffer was added, and the 6-well plate was agitated to mix. The plate was then placed on ice for 10 min to lyse. Cells were scraped off using a cell scraper and collected into centrifuge tubes. The centrifuge tubes were placed on ice for another 30 min to continue lysis. After lysis, the cells were centrifuged at 12000 rpm at 4°C for 15 min using a low-temperature centrifuge, and the supernatant containing proteins was collected into new EP tubes.

[0168] (2) BCA method for measuring cell protein concentration

[0169] Protein concentration was determined using the BCA method. First, protein standards were prepared to a concentration of 0.5 mg / mL using PBS buffer. BCA working solution was prepared at a ratio of solution A:solution B = 50:1 and mixed thoroughly. Three replicates were set up, and protein standard solutions and PBS buffer were added as shown in Table 3 to plot a standard curve. 1 μL of cell-extracted protein solution was added to a 96-well plate, and the volume was brought up to 20 μL with PBS buffer. Then, 200 μL of BCA working solution was added, and the plate was incubated at 37°C for 30 min. The absorbance at 562 nm was measured using a microplate reader, and the protein concentration of each sample was calculated based on the standard curve.

[0170] Table 3. Protein Quantification by BCA Method

[0171]

[0172] (3) Protein denaturation

[0173] Take the diluted protein sample, add protein loading buffer and mix well. Denature the protein in a metal bath at 100°C for 15 min. After cooling the denatured protein to room temperature, store it at -20°C for later use.

[0174] (4) SDS-PAGE electrophoresis

[0175] Take two clean glass plates, align them, and clamp them on the gel holder. Add ultrapure water to check for leaks. After leak testing, pour out the ultrapure water and use absorbent paper to dry any remaining liquid between the two glass plates, preparing for gel pouring. As shown in Table 4, prepare a suitable concentration of separating gel (lower layer gel) according to the protein molecular weight. Prepare a 10% SDS-PAGE separating gel according to Table 5 based on the protein molecular weight to be detected in the experiment, and prepare a 5% SDS-PAGE stacking gel (upper layer gel) according to Table 6. Add about 4 mL of separating gel between the two dry glass plates, add isopropanol to seal the gel, and let it stand for 1 hour to allow the separating gel to solidify. Pour off the isopropanol, absorb any remaining isopropanol with absorbent paper, pour in the stacking gel, insert a comb (avoiding air bubbles), and wait for the stacking gel to solidify before gently removing the comb. Place two glass plates between the electrophoresis tank and pour in electrophoresis buffer until the liquid level covers the glass plates. Load the protein sample. After loading, start electrophoresis. First, use a constant voltage of 80V to concentrate the sample in the stacking gel for about 30 minutes. Then, adjust the voltage to 120V and continue constant voltage electrophoresis. Stop electrophoresis when the loading buffer reaches the bottom of the gel.

[0176] Table 4 Optimal Separation Range of SDS-PAGE Gel

[0177]

[0178] Table 5. Preparation of 10% SDS-PAGE separating gel

[0179]

[0180] Table 6. Preparation of 5% SDS-PAGE Stacking Gel

[0181]

[0182] (5) Transfer membrane

[0183] Cut the PVDF membrane to the appropriate size, trimming off a corner as a marker to distinguish the front and back sides after gel cutting. Activate by soaking in methanol for 1 minute. Equilibrate the activated PVDF membrane, foam, and transfer filter paper in pre-cooled transfer buffer. Pry open the glass plate with a gel cutter, cut the gel according to the target protein molecular weight, and place it in the electrophoresis buffer. Place the transfer clamp with the black side facing down, and then place the foam, filter paper, gel strip, PVDF membrane, filter paper, and foam in sequence, clamping tightly. After placement, place the clamp in the transfer tank with the black clamp facing the black side of the tank. Pour the transfer buffer to submerge the clamp, place an ice pack in a low-temperature environment, and set the current to a constant 250mA. Set the transfer time according to the protein molecular weight.

[0184] (6) Closed

[0185] The blocking solution was 5% skim milk prepared with 1×TBST. After the transfer was completed, the PVDF membrane was taken out and placed in an incubation box. Skim milk was added until it covered the PVDF membrane. The membrane was placed on a shaker at room temperature and shaken slowly for 1 hour. The skim milk was poured out and the membrane was gently shaken and washed with 1×TBST for 10 minutes. The washing was repeated 3 times.

[0186] (7) Incubation of primary antibody

[0187] The primary antibodies COX-2 (CST, #12282), p-IκBα (CST, #2859), IκBα (CST, #4814), and β-actin (CST, #4970) were diluted at a ratio of 1:1000 using antibody dilution buffer. The PVDF membrane was placed in the diluted primary antibody solution and incubated overnight at low speed on a shaker at 4°C. The primary antibody solution was then recovered, and the membrane was washed with 1×TBST for 10 min, and the washing was repeated 3 times.

[0188] (8) Incubation of secondary antibodies

[0189] The PVDF membrane was incubated with horseradish peroxidase-labeled secondary antibody diluted 1:5000 at room temperature for 1 hour. After incubation, the secondary antibody solution was recovered, and the membrane was washed 3 times with 1×TBST at room temperature for 10 minutes each time.

[0190] (9) Development

[0191] The development was performed using the ECL chemical oxidative method. Developer A and developer B were mixed in a 1:1 ratio, and 100 μL was dropped onto the PVDF membrane. The membrane was then developed and photographed using a developer.

[0192] (10) Statistical Analysis

[0193] Western blot data were acquired and processed using ImageJ software. Protein band grayscale values ​​were quantitatively analyzed, with β-actin used as an internal control for grayscale analysis. Statistical analysis was performed using Graphpad Prism 8.0 software. Data are presented as mean ± standard deviation. Statement. One-way ANOVA was used to compare the means among the groups, and a p-value < 0.05 was considered statistically significant.

[0194] 3 Results Analysis

[0195] When LPS activates RAW264.7 mouse macrophages, TLR-4 is stimulated to recognize and bind to LPS, subsequently activating the NF-κB signaling pathway. This ultimately promotes the production of numerous inflammatory factors such as NO, PGE2, TNF-α, IL-β, IL-6, COX-2, and iNOS, triggering a cytokine storm. NO plays a central role in inflammation, and within the NOS family, iNOS is particularly involved in the pathological overproduction of NO. Therefore, NO release levels can be used as a preliminary indicator for screening the activity of monomeric compounds in anti-inflammatory models.

[0196] Petunia extract derivatives exhibited good anti-inflammatory activity in inhibiting NO release in an inflammatory model, such as... Figure 19 As shown, LPS stimulation upregulated iNOS expression in RAW264.7 cells, showing a highly significant difference compared to the control group (P<0.01); petunia extract derivatives at a concentration of 10 μM significantly inhibited iNOS protein expression (P<0.01). Figure 20 As shown, the above petunia derivatives can exert anti-inflammatory effects by inhibiting the expression of iNOS protein, thereby reducing NO release.

[0197] Experimental Example 2: Effects of petuniain derivatives in black wolfberry on glucose metabolism

[0198] 1 Experimental Methods

[0199] 1.1 Effects of petunia extract derivatives on 2-NBDG uptake in 3T3-L1 cells

[0200] 3T3-L1 preadipocytes were divided into 5×10 4 Cells 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.

[0201] 1.2 Effects of petunia extract derivatives on AKT phosphorylation in 3T3-L1 cells

[0202] 3T3-L1 preadipocytes were divided into 5×104 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.

[0203] 2 Results Analysis

[0204] 2.1 Effects of petunia extract derivatives on 2-NBDG uptake in 3T3-L1 cells

[0205] 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.

[0206] 2.2 Effects of petunia extract derivatives on PI3K and AKT phosphorylation in 3T3-L1 cells

[0207] The results are as follows Figure 22 As shown, compared with the normal group, the expression levels of p-AKT and p-PI3K proteins were decreased in the model group. Treatment with petunia extract derivatives could increase the expression levels of p-AKT and p-PI3K in 3T3-L1 adipocytes to varying degrees, thereby promoting glucose uptake and enhancing insulin sensitivity in 3T3-L1 adipocytes.

[0208] Experimental Example 3: Effects of petuniain derivatives on lipid metabolism in black wolfberry

[0209] 1 Experimental Methods

[0210] 1.1 Oil Red O staining

[0211] Healthy 3T3-L1 cells were seeded into 6-well plates at a density of 5 × 10⁶ cells / well. 4Cells were cultured in high-glucose DMEM medium containing 10% FBS until the cell density reached approximately 85%–90%. The medium was then changed, and after two days of contact inhibition, the complete culture medium was discarded. A culture medium containing 10 μg / mL Insulin, 0.5 mM IBMX, and 1 μM Dex was added, and the cells were cultured for 2 days (recorded as day 0, Induction I). The medium was then changed to contain 10 μg / mL Insulin (Induction II) and cultured for another 2 days. Afterward, the medium was changed to normal medium with 10 μM of the monomeric compound for co-incubation, with the medium changed every other day. After induction, cells were fixed with 4% neutral formaldehyde for 30 min. After fixation, pre-prepared Oil Red O working solution was added to stain the cell surface, and the cells were incubated in the dark for 60 min. After staining, the cells were washed with 70% ethanol to remove excess dye, and then washed 3–4 times with ultrapure water. Finally, the cells were observed and photographed under a microscope.

[0212] 1.2 Determination of TG content

[0213] 3T3-L1 cells were used at a rate of 5 × 10⁻⁶ 4 Cells were seeded at a density of 10 cells / mL in 6-well plates. When the cell density reached about 85% to 90%, differentiation was induced. The TG content was measured on the 8th day of induction. The specific method is as follows: (1) Cell pretreatment: On the 8th day of induction, the cell culture medium was aspirated, the cells were washed twice with cold PBS, and then trypsin digestion solution was added to digest the cells; (2) Cell collection: After cell digestion, the cells were resuspended in PBS and centrifuged at 1000g for 5min to collect the cell pellet; (3) Ultrasonic disruption: An appropriate amount of PBS was added to the collected pellet and ultrasonic disruption was performed (3min); (4) Measurement: 2μL of cell disruption suspension was added to each well of a 96-well plate, 2μL of distilled water was added to the blank well, 2μL of standard was added to the standard well, and then 200μL of the assay solution was added to each well. After mixing, the absorbance was read at 510nm after incubation at 37℃ for 10min. The protein concentration in the sample was determined by BCA method, corrected, and the TG content was calculated according to the following formula.

[0214]

[0215] 1.3 Western blot analysis

[0216] The Western blot analysis method is the same as in Experiment 1.

[0217] 2 Results Analysis

[0218] 2.1 Effects of petunia extract derivatives on lipid droplet accumulation and TG content in 3T3-L1 cells

[0219] like Figure 23As shown, undifferentiated cells show no accumulation of lipid droplets, while differentiated cells contain a large number of lipid droplets. Treatment with petunia extract derivatives significantly reduced the number of intracellular lipid droplets.

[0220] like Figure 24 As shown, compared with undifferentiated cells, the intracellular TG content of differentiated cells was significantly increased (P<0.01). Compared with differentiated cells, the TG content of petunia extract derivatives decreased after treatment, and the differences were highly significant (P<0.01).

[0221] 2.2 Effects of petunia extract derivatives on the expression of lipid metabolism proteins in 3T3-L1 cells

[0222] (1) Effects of petunia extract derivatives on the expression of adipogenic transcription factors in 3T3-L1 cells

[0223] The effects of petunia extract derivatives on the expression of adipogenic transcription factors in 3T3-L1 cells were analyzed by Western blot, and the results are as follows: Figure 25 As 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.

[0224] (2) Effects of petunia extract derivatives on the expression of lipidogenesis-related proteins in 3T3-L1 cells

[0225] 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.

[0226] 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.

[0227] Chromatographic analysis of component Fr2-5-4 in Example 5

[0228] 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.

[0229] Chromatographic analysis of component Fr2-5-5 in Example 6

[0230] The fraction Fr2-5-5 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: 0–30–120 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 standard. The results are as follows: Figure 28 As shown, component Fr2-5-5 was separated into 10 major components under the chromatographic analysis conditions of this experiment, yielding the monomer compound Fr2-5-5-8.

[0231] Example 7: Effects of the active component Fr2-5 from black goji berries on inflammation.

[0232] 1 Experimental Methods

[0233] Based on the established LPS-induced RAW264.7 cell inflammation model, the anti-inflammatory activity of the black goji berry component Fr2-5 was studied.

[0234] 1.1 MTT assay for cell viability

[0235] The assay method was the same as that shown in Example 1. The MTT assay was used to detect the effect of the component on RAW264.7 cell viability. Component Fr2-5 showed no significant inhibitory effect on RAW264.7 cells in the range of 0-100 μg / mL. Figure 29 In this invention, the concentration of component Fr2-5 is selected as 10 μg / mL.

[0236] 1.2 Measurement of inflammatory factors

[0237] (1) Determination of NO content by Griess method

[0238] Remove Griess reagents I and II from the refrigerator and allow them to return to room temperature before conducting the experiment. Dilute the standards with DMEM containing 10% FBS to obtain concentrations of 0, 1, 2, 5, 10, 20, 40, 60, 80, and 100 μM, respectively. Add 50 μL of the standard and sample solution to each well of a 96-well plate, followed by 50 μL of Griess reagent I and 50 μL of Griess reagent II. Mix thoroughly on a shaker and measure the absorbance at 540 nm. Calculate the NO content based on the standard curve.

[0239] The results are as follows Figure 30 As shown, after LPS treatment, the NO release in the inflammatory model increased significantly (P<0.01), and the black goji berry component Fr2-5 was able to inhibit NO release.

[0240] (2) Measurement of IL-1β

[0241] The 96-well plate was divided into blank wells, sample wells, and standard wells. Appropriate concentrations of sample and standard were added at 100 μL / well. The plates were sealed with sealing film and incubated at room temperature for 2 hours. The plates were then washed 5 times and patted dry on absorbent paper. 100 μL of horseradish peroxidase-labeled streptavidin was added to each well, and the plates were sealed again with sealing film. The plates were incubated at room temperature in the dark for 20 minutes. 50 μL of stop solution was added to each well, and the absorbance was measured at 450 nm. The sample concentration was calculated using a standard curve.

[0242] The results are as follows Figure 31 As shown, after LPS treatment, the expression of IL-1β in the inflammatory model was significantly increased (P<0.01), and the black goji berry component Fr2-5 could significantly reduce the expression of the inflammatory factor IL-1β.

[0243] 2 Results Analysis

[0244] When LPS activates RAW264.7 mouse macrophages, TLR-4 is stimulated to recognize and bind to LPS, subsequently activating the NF-κB signaling pathway. This ultimately promotes the production of numerous inflammatory factors such as NO, PGE2, TNF-α, IL-β, IL-6, COX-2, and iNOS, triggering a cytokine storm. NO plays a central role in inflammation, and within the NOS family, iNOS is particularly involved in the pathological overproduction of NO. Therefore, NO release levels can be used as a preliminary indicator for screening active components in anti-inflammatory models.

[0245] The black goji berry component Fr2-5 exhibited good anti-inflammatory activity, such as... Figure 30 and 31As shown, LPS stimulation significantly increased NO release and IL-1β levels in RAW264.7 cells compared to the control group (P<0.01). At a concentration of 10 μg / mL, the black goji berry component Fr2-5 significantly inhibited NO release (P<0.05) and significantly suppressed the expression of the inflammatory cytokine IL-1β (P<0.05). In conclusion, the black goji berry component Fr2-5 exerts its anti-inflammatory effect by reducing NO release and inhibiting the expression of the inflammatory cytokine IL-1β.

[0246] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. An anti-inflammatory black goji berry component Fr2-5, characterized in that, The component Fr2-5 was prepared by the following method: (1) Weigh 10.0 kg of dried black wolfberry fruit and extract it by soaking in methanol at room temperature in the dark. Extraction conditions: liquid-to-solid ratio 20 mL / g, extract a total of 3 times, each time for 4-5 days. After each extraction, filter the extract and concentrate it under reduced pressure in the dark. Combine the extracts to obtain black wolfberry fruit methanol extract. (2) The methanol extract obtained in step (1) was mixed with dry polyamide powder at a 1:1 ratio, dried in an oven at 40°C, ground, and passed through a 20-mesh sieve. 50.00 g of the sieved powder was loaded into a 26*100 mm small medium-pressure chromatographic column and connected to a 49*460 mm medium-pressure chromatographic column equipped with MCI. Dry loading was performed with preparative liquid chromatography, using a three-phase system of A: water / B: methanol / C: dichloromethane for 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: 50 mL / min; detection wavelength: 210 nm; obtained Fr1, Fr2, and Fr3 components with retention times of 12–39 min, 39–139 min, and 139–230 min, respectively. (3) After dissolving the Fr2 component obtained in step (2) 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, packing material: MCI, column size: 49*460 mm, injection volume: 8 mL, thereby obtaining 5 components Fr2-1, Fr2-2, Fr2-3, Fr2-4, and Fr2-5, with retention times of 13-35 min, 35-55 min, 55-68 min, 68-76 min, and 76-130 min, respectively.

2. The use of the black goji berry Fr2-5 component as described in claim 1 in the preparation of products for treating and / or preventing inflammation.

3. The application according to claim 2, characterized in that, The product is designed to reduce the release of at least one inflammatory factor, including NO, PGE2, TNF-α, IL-1β, IL-6, COX-2, and iNOS.

4. An anti-inflammatory product, characterized in that, The active ingredient is the black goji berry component Fr2-5 as described in claim 1.

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