Petunidin derivatives in black goji berries, their extraction method, and use.

Abstract

This invention discloses novel petunidin derivatives in black goji berries, as well as methods for extracting and using them. The extract of black goji berries can be separated by chromatography, and the novel petunidin derivatives can be obtained by several preparative separations. This invention further provides the use of novel compounds in the preparation of products for the prevention and / or treatment of inflammation, the use of novel compounds in the preparation of products for the prevention and / or treatment of glucose metabolism disorder-related disorders, and the use of novel compounds in the preparation of products for the prevention and / or treatment of lipid metabolism disorder-related disorders.

Description

Technical Field

[0001] <Cross - reference to Related Applications> This invention claims the priority of Chinese Patent Application CN2023106829845 filed on June 9, 2023. This invention incorporates the entire content of the above - mentioned Chinese patent application by reference.

[0002] This invention relates to the field of plant active ingredient separation technology, particularly to petunidin derivatives secopetanin, furanopetanin in black goji berries, their extraction methods and uses.

Background Art

[0003] Black goji berry (Lycium ruthenicum Murr) is a perennial thorny shrub of the Solanaceae family and genus Lycium. Its fruit is sweet, juicy and rich in nutrients, with extremely high research and development value, and is a desert medicinal plant variety unique to western China. Black goji berry contains various essential amino acids and rich mineral elements for the human body. In Tibetan medicine, it is used for treating psychosomatic fever, heart diseases, reducing cholesterol, stimulating the脑神经, enhancing immune function, preventing and treating cancer, anti - aging, beauty and skin care, irregular menstruation, amenorrhea, etc., and has remarkable medicinal effects.

[0004] Anthocyanins are water - soluble natural pigments widely present in plants in nature and belong to flavonoids. Anthocyanins mainly exist in the fruits, epidermis and flowers of plants in the form of glucosides, rhamnosides, etc. The anthocyanin content in black goji berries is very high, and more than 90% of them are petunidin derivatives, which have functions such as antioxidant, vision protection, tumor suppression, etc. Therefore, black goji berry anthocyanins have important biological activities and extraction values. With the increasing demand for natural - derived functional components without toxicity and side effects, as the most representative natural - derived functional component, anthocyanins have good coloring functions and excellent antioxidant activities, and are widely expected by consumers and the market.

[0005] Many of the active substances contained in black goji berries have not yet been isolated and identified. If we can conduct deeper and more detailed research on the novel chemical components in black goji berries and their pharmacological effects, and elucidate their mechanisms of activity, we can expect to develop safe and effective natural plant-derived drugs that can treat diseases. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The present invention provides an active ingredient extracted from black goji berries, a novel petunidin derivative, a method for extracting the same, and its use in the preparation of drugs for the prevention and / or treatment of inflammation-related diseases, wherein both the component and the compound have the effect of suppressing iNOS protein expression and reducing NO release.

[0007] The present invention provides black goji berry component Fr2-5, which has a retention time of 76 to 130 minutes and is prepared by the following method. (1) The black goji berry extract is prepared by preparative chromatography to obtain components Fr1, Fr2, and Fr3, respectively, with retention times of 12-39 min, 39-139 min, and 139-230 min; However, the conditions for the preparative chromatography described above include the following: Column: MCI medium-pressure column; preferred size is 49*460mm; Mobile phase: Water:A / Methanol:B / Dichloromethane:C; Gradient elution is performed according to 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) Separate component Fr2 by preparative chromatography to obtain component Fr2-5; However, the conditions for the preparative chromatography described above include the following: Column: MCI medium-pressure column; preferred size is 49*460mm; Mobile phase: A: Water / B: Methanol; Gradient elution is performed using the following procedure: 0-120 min, 0%-100% B; 20-140 min, 100% B.

[0008] In the present invention, the black goji berry extract refers to a concentrate obtained by extracting black goji berries with methanol, followed by filtration, blocking out light, and concentrating the product. The extraction method includes, but is not limited to, conventional extraction methods such as leaching, reflux, and ultrasonic extraction.

[0009] In a specific embodiment of the present invention, black goji berries are extracted using an infusion method, and the extraction conditions are: liquid sample ratio of 10-30 mL / g, preferably 20 mL / g; number of extractions of 2-5 times, 3-5 days each time, preferably 3 times, 4-5 days each time.

[0010] Furthermore, after extracting the black goji berries, the extract is filtered, concentrated under reduced pressure while blocking out light, and combined to obtain a methanol extract of black goji berry fruit.

[0011] In the present invention, polyamide is added to the methanol extract, and then the mixture is mixed, dried, pulverized, and sieved.

[0012] Furthermore, the methanol extract:polyamide ratio is 1:(0.5~2.5), preferably 1:1.

[0013] Furthermore, the drying method can be selected from options such as atmospheric pressure drying, reduced pressure drying, or freeze-drying.

[0014] Furthermore, the mesh size of the sieve is 20 to 50 mesh, preferably 20 mesh.

[0015] The present invention provides the black goji berry component Fr2-5-4, obtained by separating component Fr2-5 by preparative chromatography, wherein the retention time of component Fr2-5-4 is 21-27 min; However, the conditions for the preparative chromatography described above include the following:

[0016] Column: Kromasil C18 column; preferred size is 21.2 x 250 mm; Mobile phase: A: water / B: methanol; gradient elution is performed according to the following procedure: 0~60~65~90 min, 30%~42%~70%~95% B.

[0017] The present invention provides the black crow component Fr2-5-5, which is obtained by separating the component Fr2-5 by preparative chromatography, and the retention time of the component Fr2-5-5 is 27~36 min; However, the conditions of the preparative chromatography include the following.

[0018] Column: kromasil C18 column; the preferred specification is 21.2*250 mm; Mobile phase: A: water / B: methanol; gradient elution is performed according to the following procedure: 0~60~65~90 min, 30%~42%~70%~95% B.

[0019] The present invention provides a petunidin derivative IV having the structural formula of Formula IV, its salt, or an isomer. JPEG2026518247000002.jpg55170

[0020] The present invention extracts the fruits of black crow with methanol, separates them by preparative chromatography, and obtains a new petunidin derivative unexpectedly through one-dimensional and two-dimensional nuclear magnetic identification, which is named furanopetanin.

[0021] However, the petunidin derivative IV is obtained by separating the component Fr2-5-5 by preparative chromatography with a retention time of 92~95 min.

[0022] Furthermore, the conditions of the preparative chromatography include the following.

[0023] Column: kromasil C18 column; the preferred specification is 21.2*250 mm; Mobile phase: A: water / B: methanol; gradient elution is performed according to the following procedure: 0~30~120 min, 20%~28%~31% B.

[0024] The present invention provides petunidine derivative V having the structural formula of formula V, its salts, or isomers. JPEG2026518247000003.jpg69170

[0025] In this invention, we extracted the fruit of the black goji berry using methanol, separated it by preparative chromatography, and performed one-dimensional and two-dimensional nuclear magnetic identification to obtain a surprisingly novel petunidine derivative, which we named secopetanin.

[0026] However, petunidine derivative V was obtained by separating component Fr2-5-5 by preparative chromatography, and its retention time was 40-41 minutes.

[0027] Furthermore, the conditions for the preparative chromatography include the following: Column: Kromasil C18 column; preferred size is 21.2 x 250 mm; Mobile phase: A: Water / B: Methanol; Gradient elution is performed using the following procedure: 0-60 min, 30%-35% B.

[0028] In specific examples of the present invention, the preparative chromatographic separation includes at least one of the following conditions.

[0029] Detection wavelength: 210 nm; Column temperature: 25°C to 35°C; Flow rate: 15 to 60 mL / min; Sample injection volume: 0.1 mL to 8.0 mL.

[0030] The present invention provides for the use of petunidine derivatives IV and / or V, and their pharmaceutically acceptable salts, hydrates, or solvates, in the preparation of products for the treatment and / or prevention of inflammation-related diseases.

[0031] 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.

[0032] Furthermore, the two products mentioned above for the treatment and / or prevention of inflammation-related diseases are products that block and / or inhibit the NF-κB signaling pathway.

[0033] NF-κB is a protein complex that regulates transcribed DNA, cytokine production, and cell survival; it is involved in cellular responses to stimuli such as stress, cytokines, free radicals, heavy metals, UV irradiation, oxidized LDL, and bacterial or viral antigens; and it also plays a crucial role in regulating the immune response to infection.

[0034] Furthermore, the product is a product that reduces the release of at least one inflammatory factor, namely NO, PGE2, TNF-α, IL-β, IL-6, COX-2, and iNOS; and furthermore, the product is a product that reduces NO release.

[0035] Furthermore, the aforementioned product inhibits the expression of the iNOS protein.

[0036] The present invention further provides an anti-inflammatory product comprising one or more of the components Fr2-5, Fr2-5-4, Fr2-5-5, compound IV, and compound V.

[0037] The present invention provides for the use of petunidine derivatives IV and / or V, as well as pharmaceutically acceptable salts, hydrates, or solvates thereof, in the preparation of products for treating and / or preventing diseases related to glucose metabolism disorders.

[0038] 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 a product for treating and / or preventing glucose metabolism disorder-related diseases.

[0039] Furthermore, the products used to treat and / or prevent the two types of glucose metabolism disorders mentioned above all have the effect of lowering glucose levels, such as hypoglycemic agents.

[0040] In this invention, "glucose metabolism disorders" refer to abnormalities in the structure, function, or concentration of hormones or enzymes that regulate the metabolism of glucose, fructose, galactose, etc., or pathophysiological changes in tissues or organs, resulting in elevated blood glucose levels when blood glucose is monitored. Clinically important glucose metabolism disorders mainly involve blood glucose levels that are too high or too low.

[0041] Common diseases associated with impaired glucose metabolism include diabetes mellitus, hypoglycemia, fructose metabolism disorders, glycogen storage disorders, galactose metabolism disorders, and pyruvate metabolism disorders.

[0042] Diabetes mellitus is a metabolic disorder characterized by elevated blood glucose levels due to absolute insulin deficiency and / or reduced biological effectiveness of insulin, leading to chronic complications affecting blood vessels, nerves, and other tissues.

[0043] In this invention, "lowering blood sugar levels" means reducing the abnormally high blood sugar levels in patients. High blood sugar is often associated with diabetes.

[0044] In glycogen storage disease, a deficiency in a certain enzyme during the glycogen breakdown process leads to the accumulation of large amounts of glycogen in organs such as the liver, muscles, and kidneys, causing enlargement and dysfunction of these organs, and ultimately resulting in related diseases.

[0045] Products for treating and / or preventing the aforementioned glucose metabolism disorder-related diseases can lower blood glucose levels by inhibiting hepatic gluconeogenesis and hepatic glucose export, increasing the sensitivity of peripheral tissues to insulin, promoting glucose uptake and utilization, stimulating insulin secretion by insulin β-cells, enhancing insulin binding to receptors, and increasing the sensitivity of target cells to insulin.

[0046] Furthermore, the aforementioned product promotes the uptake of sugars and / or sugar analogs by adipocytes.

[0047] Glucose uptake by adipocytes in response to insulin stimulation is primarily carried out by insulin-sensitive GLUT4 (glucose transporter); under insulin stimulation, intracellular signaling of insulin receptor tyrosine phosphorylation phosphorylates insulin receptor substrate-1 (IRS-1), thereby activating phosphatidylinositol-3-kinase (PI3K), inducing the transfer of GLUT4 to the cell surface and increasing glucose uptake.

[0048] Furthermore, the aforementioned product increases the expression levels of p-AKT and p-PI3K proteins.

[0049] AKT is a serine / threonine protein kinase involved in a wide range of processes including glucose metabolism, apoptosis, cell proliferation, and cell transport. Activated AKT during glucose metabolism activates various downstream factors in the insulin signaling pathway, such as enzymes, kinases, and transcription factors, via the phosphorylation pathway, thereby regulating cellular function and insulin signaling. Normally, activated AKT promotes the translocation of glucose transporters from the cytoplasm to the cell membrane by activating its downstream phosphatidylinositol kinase 3 (PI3K), thereby facilitating glucose absorption and utilization, and playing a role in insulin signaling.

[0050] p-PI3K (mouse phosphorylated phosphoinositide 3-kinase) can activate or inhibit the activity of a series of downstream substrates and apoptosis-related proteins through phosphorylation, thereby regulating phenotypes such as cell proliferation, differentiation, apoptosis, and transitions.

[0051] The present invention provides for the use of petunidine derivatives IV and / or V, and their pharmaceutically acceptable salts, hydrates, or solvates, in the preparation of products for treating and / or preventing lipid metabolism disorders.

[0052] The present invention provides the use of at least one of the components Fr2-5, Fr2-5-4, and Fr2-5-5 in the preparation of a product for treating and / or preventing lipid metabolism disorder-related diseases.

[0053] The present invention further provides a method for treating and / or preventing diseases associated with inflammation, glucose metabolism disorders, or lipid metabolism disorders, the method of which is carried out by administering compounds IV and / or V of the present invention to a patient in a dose sufficient to treat and / or prevent inflammation, glucose metabolism disorders, or lipid metabolism disorders, the dose of which can be modified depending on the type of disease, the patient's age, weight, and general health condition, depending on the mode of administration.

[0054] Furthermore, the products used to treat and / or prevent the two types of lipid metabolism disorders mentioned above all have lipid-lowering effects, such as lipid-lowering drugs.

[0055] Furthermore, products for treating and / or preventing the two types of lipid metabolism disorders mentioned above include products that inhibit lipid droplet accumulation and / or cellular lipid synthesis; said products may be drugs, nutritional supplements, or other products that inhibit lipid droplet accumulation and / or cellular lipid synthesis.

[0056] If the product is a drug, the drug includes a drug that prevents and / or treats at least one of the following: obesity, hypertension, hyperlipidemia, cardiovascular disease, and metabolic syndrome-related disorders.

[0057] Obesity is a chronic metabolic disorder characterized by excessive accumulation of body fat and excessive body weight. Its diagnostic criteria vary and include body mass index, waist circumference, and body fat percentage. Obese individuals have a significantly higher probability of developing coronary heart disease and hypertension compared to non-obese individuals; obese individuals may experience decreased lung capacity, reduced lung compliance, and various lung function abnormalities; in obese individuals, excessive intake of sugars and lipids promotes triglyceride synthesis and catabolism, resulting in more active lipid metabolism and inhibited glucose metabolism. This metabolic change contributes to the formation of insulin resistance. Obesity often involves metabolic disorders despite active lipid metabolism, such as hypertriglyceridemia, hypercholesterolemia, and hypo-high-density lipoprotein cholesterolemia.

[0058] Hypertension is a condition in which the pressure exerted on the blood vessel walls while blood is flowing remains higher than normal. The causes of hypertension are often genetic factors and unhealthy lifestyles such as a high-salt diet, excessive alcohol consumption, long-term stress, and lack of physical activity.

[0059] Hyperlipidemia generally refers to elevated levels of triglycerides and / or total cholesterol in the plasma, elevated low-density lipoprotein cholesterol, and decreased high-density lipoprotein cholesterol. The causes of the disease include various environmental factors such as genetic mutations, poor dietary habits, lack of physical activity, and obesity, as well as diseases such as diabetes, nephrotic syndrome, and liver disease.

[0060] Cardiovascular disease, also known as circulatory system disease, is a group of diseases affecting the heart or blood vessels. It primarily includes ischemic heart disease, stroke, cardiomyopathy, rheumatic heart disease, hypertensive heart disease, endocarditis, arrhythmias, aortic disease, peripheral artery disease, and other cardiovascular and circulatory system diseases. Direct causes or high-risk factors for cardiovascular disease include smoking, lack of physical activity, obesity, hypertension, dyslipidemia, diabetes, infection, and genetic predisposition, as well as dietary imbalances and air pollution. Multiple factors do not exist independently but rather interact with each other, contributing to the development and mortality of cardiovascular disease. Obesity is associated with the development and outcomes of cardiovascular disease, increasing the risk of atherosclerosis in obese individuals, as well as increasing the risk of other conditions such as hypertension, diabetes, and dyslipidemia. However, the "obesity paradox" exists in patients with cardiovascular disease, with studies suggesting that groups with mild to moderately elevated body mass index (BMI) in patients with coronary heart disease, hypertension, and heart failure have lower overall mortality rates compared to groups with low BMI. Groups with severely oversized BMI have an increased overall mortality risk. Diabetes is a significant risk factor for cardiovascular diseases such as coronary heart disease and stroke. Long-term dyslipidemia can cause the formation of atherosclerotic plaques in the arterial walls, which can gradually narrow or block the arterial walls. Plaques can rupture, causing platelet aggregation and thrombosis, which can obstruct blood flow and lead to myocardial infarction, stroke, and even sudden death.

[0061] Metabolic syndrome refers to a condition in which substances such as proteins, fats, and carbohydrates in the human body undergo metabolic disorders. It is a complex collection of metabolic disorders and a risk factor for diabetic cardiovascular disease. It has the following characteristics: (1) Multiple metabolic disorders, including obesity, hyperglycemia, hypertension, dyslipidemia, high blood viscosity, high uric acid, high incidence of fatty liver, and hyperinsulinemia, are present together, and these metabolic disorders form the basis of the pathophysiology of the heart, cerebrovascular disease, and diabetes. Diabetes is not an isolated disease, but rather one of the components of metabolic syndrome. (2) They share a common pathological basis, and their common cause is thought to be insulin resistance and hyperinsulinemia due to obesity, especially central obesity. (3) It can lead to an increase in various diseases, including hypertension, coronary heart disease, stroke, and certain cancers, including breast cancer, endometrial cancer, prostate cancer, and gastrointestinal cancers such as pancreatic cancer, hepatobiliary cancer, and colon cancer, which are related to sex hormones.

[0062] Obesity, hypertension, hyperlipidemia, cardiovascular disease, metabolic syndrome, and other diseases are all closely related to the content of plasma lipids, such as triglycerides (TG), free cholesterol (FC), cholesterol esters (CE), and phospholipids. When plasma lipids in the body are reduced to a certain concentration range, these diseases can be effectively controlled or treated. Experiments in this invention have demonstrated that the active ingredients and / or novel compounds of this invention can effectively suppress the accumulation of lipid droplets, reduce TG content, and suppress cellular lipid differentiation and lipid synthesis by regulating the gene expression levels of adipocyte transcription factors and related protein expression levels via relevant signaling pathways. These compounds can be used in the preparation of products for the prevention and / or treatment of obesity, hypertension, hyperlipidemia, cardiovascular disease, and metabolic syndrome-related diseases.

[0063] The present invention provides the use of petunidine derivatives IV and / or V, and pharmaceutically acceptable salts, hydrates, or solvates thereof, in the preparation of at least one PPARγ antagonist, C / EBPα antagonist, FAS inhibitor, or ACC inhibitor.

[0064] The present invention provides the use of at least one of the components Fr2-5, Fr2-5-4, and Fr2-5-5 in the preparation of at least one PPARγ antagonist, C / EBPα antagonist, FAS inhibitor, and ACC inhibitor.

[0065] The aforementioned PPARγ antagonist, C / EBPα antagonist, FAS inhibitor, and ACC inhibitor are drugs that reduce the gene expression levels of the adipocyte transcription factors PPARγ and C / EBPα, and the protein expression levels of FAS and ACC.

[0066] The differentiation of undifferentiated cells into mature adipocytes requires the regulation of a complex and subtle series of transcription factors. PPARγ is an essential regulator for adipocyte formation and is highly expressed in the early stages of adipogenesis; C / EBPα, also known as CCAAT / enhancer-binding protein α, is highly expressed in the mid-stages of adipocyte differentiation.

[0067] Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor transcription factor superfamily that regulate the expression of target genes. Based on their structure, PPARs can be classified into three types: α, β (or δ), and γ. PPARγ is primarily expressed in adipose tissue and the immune system, and is closely related to adipocyte differentiation, somatic immunity, and insulin resistance. It is a target molecule for insulin sensitivity-improving drugs such as thiazolidinediones (troglitazone, TZDs). PPARγ is specific to adipose tissue, is activated by fatty acids and exogenous peroxisome proliferators, and can regulate the expression of enzymes involved in lipid metabolism. PPARγ is induced before the transcriptional activation of many adipocyte genes and plays a crucial role in cell differentiation. Insulin, glucocorticoids, and intracellular CAMP inducers can differentiate preadipocytes into adipocytes, epidermal growth factor (EGF) and transforming growth factors inhibit differentiation of primary cultures and preadipocyte lines, and mitogen-activated protein kinases can phosphorylate PPARγ, inhibiting the transcriptional activation function of ligands, suggesting that the transcriptional activation of PPARγ may be regulated by cytokine signaling pathways involved in adipocyte differentiation.

[0068] FAS is a fatty acid synthase that plays an important role in lipid synthesis; ACC is acetyl coenzyme A carboxylase and is the rate-limiting enzyme in the de novo synthesis of fatty acids.

[0069] The active ingredients and novel compounds according to the present invention can reduce the gene expression levels of the cellular transcription factors PPARγ and C / EBPα, as well as the protein expression levels of FAS and ACC. They are effective in suppressing lipid differentiation products and lipid synthesis in cells, and can be used in the preparation of products for the prevention and / or treatment of lipid metabolism disorders such as obesity, hypertension, hyperlipidemia, and cardiovascular disease.

[0070] An "antagonist" is a drug that, after binding to a receptor, does not produce any biological effect itself, but inhibits the action mediated by that receptor agonist.

[0071] "Inhibitors" primarily refer to enzyme inhibitors, which are substances that exert therapeutic effects by inhibiting the function of specific enzymes involved in certain diseases in the body.

[0072] The present invention also provides a glucose-lowering and / or lipid-lowering product comprising one or more of the components Fr2-5, Fr2-5-4, Fr2-5-5, compound IV, and compound V.

[0073] Products according to the present invention include, but are not limited to, pharmaceuticals, nutritional supplements, and food products.

[0074] The product of the present invention may contain excipients that are acceptable in pharmaceuticals, nutritional supplements, or foods.

[0075] The excipients in this invention are a general term for all additive materials other than the main component, and the excipients must have the following properties: (1) No toxic effects on the human body and virtually no side effects; (2) Chemically stable and not easily affected by temperature, pH, storage time, etc.; (3) No incompatibility with the main ingredient and not affecting the efficacy or quality inspection of the main ingredient; (4) No interaction with packaging materials. The excipients in this invention include, but are not limited to, fillers (diluents), lubricants (flow promoters or anti-adhesion agents), dispersants, wetting agents, binders, modifiers, solubilizers, antioxidants, bacteriostatic agents, emulsifiers, disintegrants, etc.The binders include syrup, gum arabic, gelatin, sorbitol, tragacanth, cellulose and its derivatives (e.g., microcrystalline cellulose, sodium carboxymethylcellulose, ethylcellulose or hydroxypropyl methylcellulose, etc.), gelatin, syrup, starch slurry or polyvinylpyrrolidone, etc.; the fillers include lactose, powdered sugar, dextrin, starch and its derivatives, cellulose and its derivatives, inorganic calcium salts (e.g., calcium sulfate, calcium phosphate, calcium hydrogen phosphate, precipitated calcium carbonate, etc.), sorbitol or glycine, etc.; the lubricants include silica gel powder, magnesium stearate, talc, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol, etc.; the disintegrants include starch and its derivatives (e.g., sodium carboxymethyl starch The ingredients include: starch (such as sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, corn starch, etc.), polyvinylpyrrolidone, or microcrystalline cellulose; humectants include sodium lauryl sulfate, water, alcohol, etc.; antioxidants include sodium sulfite, sodium bisulfite, sodium metabisulfite, dibutylbenzoic acid, etc.; bacteriostatic agents include 0.5% phenol, 0.3% cresol, 0.5% trichlorot-butanol, etc.; modifiers include hydrochloric acid, citric acid, potassium(sodium) hydroxide, sodium citrate, and buffers (including sodium dihydrogen phosphate and disodium hydrogen phosphate), etc.; emulsifiers include polysorbate-80, sorbitan oleate, Pluronic® F-68, lecithin, soy lecithin, etc.; solubilizers include Tween-80, bile, glycerin, etc.

[0076] The "salt" of the compound in this invention includes "pharmaceutically acceptable salts".

[0077] "Pharmacologically acceptable salt" refers to a salt suitable for medicinal use formed between the compound of the present invention and an acid or base. The acids and bases mentioned above are Lewis acids and bases in a broad sense. Acids suitable for forming salts include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, toluenesulfonic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.

[0078] The administration form of the compound or pharmaceutical composition of the present invention is not particularly limited, and typical administration forms include, but are not limited to, oral, extra-gastrointestinal (intravenous, intramuscular, subcutaneous), and topical administration.

[0079] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with any of the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, such as hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) dissolution and relaxation agents, such as paraffin; (f) absorption enhancers, such as quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, such as kaolin; (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include a buffering agent.

[0080] Solid dosage forms such as tablets, drops, capsules, pills, and granules can be prepared with coatings or shells, such as enteric coatings and other materials known in the art. These may contain opacifying agents, and the release of the active compound or compound in such compositions may be delayed in some part of the digestive tract. Examples of embedding components include polymers and waxes. If necessary, the active compound may form microcapsules with one or more of the above-mentioned excipients.

[0081] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, the liquid dosage form may also include inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, maize germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof.

[0082] In addition to these inert diluents, the composition may also contain auxiliary agents such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0083] In addition to the active compound, the suspension may also contain suspending agents, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures thereof.

[0084] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or aqueous solution, dispersion, suspension or emulsion, and sterile powder for redissolution in sterile injectable solution or dispersion. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0085] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required.

[0086] The compounds of the present invention can similarly be used in injectable formulations. Here, the injectable formulation is a liquid injectable formulation (water needle), a sterile powder for injection (powder needle), or an injectable tablet (referring to a molded tablet or mechanically compressed tablet in which the drug is manufactured using a sterile technique, which is dissolved in sterile water for injection at the time of use and administered by subcutaneous or intramuscular injection).

[0087] Here, the injectable powder contains, in addition to the above compound, at least an excipient. The excipient in the present invention is a component intentionally added to the drug and should not have pharmacological properties in the amount used, but the excipient may contribute to the processing, dissolution or dissolution, delivery via a targeted administration route, or stability of the drug.

[0088] Isomers of a functional group that result from the rapid movement of two atoms within a molecule are called tautomers.

[0089] Meso compounds contain asymmetric atoms within the molecule, but the presence of a symmetry factor results in zero total optical rotation within the molecule, meaning they do not exhibit optical activity.

[0090] A racemate is an equimolar mixture of a chiral molecule (see chirality) that exhibits optical activity (see optical isomers) and its enantiomer.

[0091] Stereoisomers that are mirror images of each other but do not overlap are called enantiomers. All enantiomers are optically active, one being levorotatory and the other dextrorotatory, and therefore enantiomers are also called optical isomers.

[0092] Diastereoisomers are stereoisomers of molecules that have two or more chiral centers and are not mirror images of each other.

[0093] The beneficial effect of this invention is that it provides three active ingredients and two novel compounds, furanopetanin and secopetanin, which have anti-inflammatory, carbohydrate-reducing, and lipid-reducing effects, thereby more comprehensively developing the active substances of black goji berry, while simultaneously more comprehensively exploring the pharmacological value of black goji berry, expanding its clinical applications, and providing more reference material for developing potential plant-derived drugs to treat diseases associated with inflammation, glucose metabolism disorders, and lipid metabolism disorders.

[0094] The present invention further provides a liquid chromatography method for analyzing the component Fr2-5-4, the chromatographic analysis conditions comprising the following: Column: C18, preferably with specifications of 4.6 × 250 mm and 5 μm; Mobile phase: A: Water / B: Methanol; Gradient elution procedure: 0-60 min, 30-35% methanol; Furthermore, the flow rate was 0.8-1.2 mL / min; the column temperature was 30±5℃; and the detection wavelength was 210±5 nm. Furthermore, Fr2-5-4-3 was used as a control group.

[0095] The present invention further provides a liquid chromatography method for analyzing the component Fr2-5-5, the chromatographic analysis conditions comprising the following: Column: C18, preferably with specifications of 4.6 × 250 mm and 5 μm; Mobile phase: A: Water / B: Methanol; Gradient elution procedure: 0-30-120 min, 20%-28%-31% methanol; Furthermore, the flow rate is 0.8-1.2 mL / min, and the detection wavelength is 210 ± 5 nm. Furthermore, Fr2-5-5-8 was used as a control group.

[0096] Based on the above method, the present invention further provides an analytical method for measuring the quality of black goji berries or their extracts, comprising the following procedure. (1) Pre-treat the black goji berry or its extract according to the preparation method of Fr2-5-4 or Fr2-5-5; (2) The pre-treated sample is detected using the chromatographic analysis conditions described above.

[0097] The above analytical method makes it possible to measure the quality of black goji berries or their extracts, which are useful in treating diseases related to inflammation, glucose metabolism disorders, and lipid metabolism disorders, and represents a new advance in establishing quality standards for related products. [Brief explanation of the drawing]

[0098] [Figure 1] This is an MCI preparative chromatogram of black goji berry fruit extract. [Figure 2] This is a preparative MCI chromatogram of the Fr2 fraction. [Figure 3] This is a preparative MCI chromatogram of the Fr2-5 fraction. [Figure 4] This is a preparative MCI chromatogram of the Fr2-5-5 fraction. [Figure 5] This is a purity analysis diagram of component Fr2-5-5-8 (furanopetanin). [Figure 6] This is the HSQC diagram for the novel compound Fr2-5-5-8. [Figure 7] This is the HMBC diagram of the novel compound Fr2-5-5-8. [Figure 8] This is the COSY diagram for the novel compound Fr2-5-5-8. [Figure 9] This is a preparative MCI chromatogram of the Fr2-5-4 fraction. [Figure 10] This is a purity analysis diagram of component Fr2-5-4-3 (secopetanin). [Figure 11] This is the HSQC diagram for the novel compound Fr2-5-4-3. [Figure 12] This is the HMBC diagram of the novel compound Fr2-5-4-3. [Figure 13] This is the COSY diagram for the novel compound Fr2-5-4-3. [Figure 14] This figure shows the effects of different concentrations of LPS on RAW264.7 cell viability and NO release. [Figure 15]This figure shows the effect of different concentrations of secopetanin on RAW264.7 cell viability. [Figure 16] This figure shows the effect of different concentrations of furanopetanin on RAW264.7 cell viability. [Figure 17] This figure shows the effects of furanopetanin and secopetanin on NO release in RAW264.7 cells. [Figure 18] This figure shows the effects of furanopetanin and secopetanin on iNOS protein expression in RAW264.7 cells. [Figure 19] This figure shows the effects of 2-NBDG uptake by 3T3-L1 adipocytes after treatment with furanopetanin and secopetanin. [Figure 20] This figure shows the effects of furanopetanin and secopetanin on the expression of p-AKT and p-PI3K in 3T3-L1 adipocytes. [Figure 21] This figure shows the effects of furanopetanin and secopetanin on lipid droplet accumulation in 3T3-L1 cells. [Figure 22] This figure shows the effects of furanopetanin and secopetanin on TG content in 3T3-L1 cells. [Figure 23] This figure shows the effects of furanopetanin and secopetanin on the expression of 3T3-L1 cell lipid synthesis transcription factors. [Figure 24] This figure shows the effects of furanopetanin and secopetanin on the expression of lipid metabolism-related proteins in 3T3-L1 cells. [Figure 25] This is a chromatographic analysis diagram of component Fr-5-4. [Figure 26] This is a chromatographic analysis diagram of component Fr-5-5. [Modes for carrying out the invention]

[0099] The preparation and use of the active substance and novel petunidin derivative of the present invention will be described in more detail below, with specific examples and tests, and their anti-inflammatory, glucose-lowering, and lipid-lowering effects will be verified and explained, and their mechanism of action will be investigated in a preliminary manner.

[0100] In the embodiments of the present invention, the method for detecting compound purity is obtained by area normalization using HPLC, and the calculation formula is: Target compound purity % = Target compound peak area / Total peak area * 100%.

[0101] <Example 1> Preparation of Black Goji Berry Component Fr2-5 (1) 10.0 kg of dried black goji berry fruit was weighed out and extracted by immersion in methanol at room temperature in the dark. Extraction conditions: liquid sample ratio 20 mL / g. Extraction was performed a total of 3 times for 4-5 days each time. After each extraction, the extract was filtered, concentrated under reduced pressure in the dark, and combined to obtain methanol extract of black goji berry fruit. (2) The methanol extract obtained in step (1) was added to the dried polyamide powder in a 1:1 ratio to prepare the sample. After drying in an oven at 40°C, it was polished and passed through a 20-mesh sieve. 50.00 g of the sieved powder was taken each time, packed into a small medium-pressure chromatography column (26*100 mm), and dry-packed by connecting it to a medium-pressure chromatography column (49*460 mm) containing MCI and a preparative liquid chromatograph. Elution was performed in a three-phase system of A:water / B:methanol / C:dichloromethane, with 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. As shown in Figure 1, components Fr1, Fr2, and Fr3 were obtained with retention times of 12–39 min, 39–139 min, and 139–230 min, respectively. (3) After dissolving the Fr2 obtained in step (2) with methanol, the preparation conditions were optimized, and finally the following settings were selected: mobile phase: A: water / B: methanol, gradient elution: 0-120 min, 0%-100% B; 120-140 min, 100% B; flow rate: 50 mL / min; detection wavelength: 254 nm; filler: MCI; column size: 49*460 mm; injection volume: 8 mL. Then, as shown in Figure 2, a total of five components, Fr2-1, Fr2-2, Fr2-3, Fr2-4, and Fr2-5, were obtained with retention times of 13-35 min, 35-55 min, 55-68 min, 68-76 min, and 76-130 min, respectively.

[0102] <Example 2> Preparation of Black Goji Berry Components Fr2-5-4 and Fr2-5-5 After dissolving the Fr2-5 obtained in step (3) of Example 1 with methanol, the preparation conditions were optimized, and finally a kromasil C18 preparative column (21.2*250 mm, 5 μm), mobile phase A: water / B: methanol was selected, gradient elution conditions: 0~60~65~90 min, 30%~42%~70%~95% B; flow rate: 19 mL / min; detection wavelength: 210 nm; injection volume: 300 μL. As shown in Figure 3, a total of 10 components, Fr2-5-1 to Fr2-5-10, were obtained with retention times of 2-9 min, 9-19 min, 19-21 min, 21-27 min, 27-36 min, 36-43 min, 43-50 min, 50-62 min, 62-72 min, and 72-85 min, respectively. Among these, Fr2-5-4 is component Fr2-5-4, and Fr2-5-5 is component Fr2-5-5.

[0103] <Example 3> Preparation of petunidine derivative IV (furanopetanin) (1) After dissolving the component Fr2-5-5 obtained in Example 2 with methanol, the preparation conditions were optimized, and finally, mobile phase: A: water / B: methanol, Kromasil C18 column (21.2*250 mm, 5 μm), gradient elution procedure: 0~30~120 min, 20%~28%~31% B, flow rate: 19 mL / min, detection wavelength: 210 nm. Then, as shown in Figure 4, a total of 10 components Fr2-5-5-1 to Fr2-5-5-10 were obtained, and the elemental compound Fr2-5-5-8 (furanopetanin) was obtained with a retention time of 92~95 min. (2) The purity of the sample Fr2-5-5-8 (furanopetanin) obtained in step (1) was measured by high-performance liquid chromatography detection; the chromatographic analysis conditions were as follows: kromasil C18 analytical column (4.6 × 250 mm, 5 μm); mobile phase: A: water / B: methanol; gradient elution procedure: 0~30~120 min, 20%~28%~31% methanol; flow rate: 1 mL / min; detection wavelength: 210 nm; injection volume: 10 μL. (3) As shown in Figure 5, the purity of compound Fr2-5-5-8(furanopetanin) prepared by the above method was 96.0% by HPLC. The structure of compound Fr2-5-5-8(furanopetanin) is as follows. Table 1 shows the results of the structural (nuclear magnetic data) verification of compound Fr2-5-5-8.

[0104] JPEG2026518247000005.jpg142170

[0105] <Example 4> Preparation of petunidine derivative V (secopetanin) (1) After dissolving the component Fr2-5-4 obtained in step (3) of Example 2 with methanol, the preparation conditions were optimized, and finally a kromasil C18 preparative column (21.2*250 mm, 5 μm), mobile phase: A: water / B: methanol was selected, gradient elution conditions: 0~60 min, 30~35% B; flow rate: 18 mL / min; detection wavelength: 210 nm; injection volume: 150 μL. As shown in Figure 9, a total of four components were obtained: Fr2-5-4-1, Fr2-5-4-2, Fr2-5-4-3, and Fr2-5-4-4, and a new elemental compound Fr2-5-4-3 (secopetanin) was obtained with a retention time of 40~41 min. (2) The purity of the sample Fr2-5-4-3 (secopetanin) obtained in step (1) was measured by high-performance liquid chromatography detection. The chromatographic analysis conditions were as follows: 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. (3) As shown in Figure 10, the purity of compound Fr2-5-4-3(secopetanin) prepared by the above method was 96.3% by HPLC. The structural formula of compound Fr2-5-4-3(secopetanin) is as follows. Table 2 shows the results of the structural (nuclear magnetic data) verification of the compound secopetanin.

[0106] JPEG2026518247000007.jpg142170

[0107] The beneficial effects of the petunidine derivative of the present invention are shown below through test examples. <Test Example 1> Effects of petunidin derivatives in black goji berries on inflammation Inflammation occurs in many tissues and organs of the human body and is deeply related to many diseases. Macrophages are specific cells that exert immune function in the body and play important physiological roles in inflammation, tumors, and autoimmune regulation. Numerous studies have shown that many inflammatory diseases in the body are closely related to macrophages, and lipopolysaccharides are components of the outer membrane of Gram-negative bacteria and are widely used to create inflammation models of macrophages. This invention constructs an inflammation model using LPS-induced RAW264.7 cells.

[0108] 1. Construction of an LPS-induced RAW264.7 cell inflammation model RAW264.7 macrophages at a density of 5 × 10⁻¹⁶ 4 Cells / mL were seeded in 150 μL portions per well in a 96-well plate and cultured in a cell incubator for 48 hours. After 24 hours of induction in media containing lipopolysaccharide at concentrations of 0.1, 1, 5, 10, 50, 100, and 200 μg / mL (containing 1% penicillin-streptomycin (biantibody) and 2% fetal bovine serum (FBS)), cell viability was measured by the MTT method, and the NO content in the cell supernatant was measured using an NO kit. Based on cell viability and NO content, the optimal effective concentration of lipopolysaccharide was determined.

[0109] As a result, the viability of RAW264.7 cells decreased with increasing LPS concentration, and cell viability was significantly suppressed when the LPS concentration was 50–200 μg / mL (Figure 14). When different concentrations of LPS were added, the amount of NO released by cells increased significantly in all cases, but NO release was high when the LPS concentration was 0.1–10 μg / mL. Based on both indicators, the LPS concentration selected for modeling in this experiment was 5 μg / mL.

[0110] 2. Experimental Method Based on the establishment of an LPS-induced RAW264.7 cell inflammation model, we conducted anti-inflammatory activity tests on petunidine derivatives. 2.1 Measurement of cell viability using the MTT method Logarithmically grown RAW264.7 cells, 5 × 10 4Cells were seeded at a cell density of cells / mL, with 150 μL per well in a 96-well plate. After 24 hours of incubation in a cell incubator, the medium was replaced with DMEM medium (containing 2% FBS) containing lipopolysaccharide or the test component, and incubation continued for another 24 hours. Subsequently, 10 μL of thiazole blue (MTT) solution was added to each well, and the cells were incubated in a cell incubator for 4 hours. Finally, the medium was discarded, 150 μL of DMSO-lysed cells were added to each well, and the absorbance value at a wavelength of 490 nm was read using a microplate reader. Cell viability was calculated from the formula. The formula for calculating cell viability is as follows: The MTT method measured the effect of each subject on the cell viability of RAW264.7, and the novel compounds Fr2-5-4-3 and Fr2-5-5-8 did not show significant inhibitory effects on RAW264.7 at concentrations of 0-100 μM. The concentration of the positive drug dexamethasone was 10 μM, and for consistency of experimental concentrations, all compounds were selected at 10 μM in subsequent experiments.

[0111] 2.2 Measurement of NO content by Griess method Griess reagents I and II were removed from the refrigerator and allowed to return to room temperature before the experiment. The standard was diluted with 10% FBS-containing DMEM to concentrations of 0, 1, 2, 5, 10, 20, 40, 60, 80, and 100 μM, respectively. 50 μL each of the standard and sample solution was added to a 96-well plate. Then, 50 μL each of Griess reagent I and Griess reagent II were added sequentially to each well. After mixing uniformly in a shaker, the absorbance at a wavelength of 540 nm was measured, and the NO content was calculated based on the calibration curve. The results showed that the novel compounds Fr2-5-4-3 and Fr2-5-5-8 had extremely significant effects in inhibiting NO release. In particular, compound Fr2-5-5-8 acted most significantly and was superior to the positive drug.

[0112] 2.3 Western blot analysis The experiment divided the participants into a control group, an LPS group, and an LPS + different drug group, and selected RAW264.7 cells in the logarithmic growth phase, 5*104 Cells were seeded at a density of cells / mL in 6-well plates and cultured in a cell incubator for 24 hours. After that, the medium was changed to DMEM medium containing 2% FBS. The model group was treated with LPS, and the test drug group was treated with LPS + drugs of different concentrations. Culture was continued for 24 hours, proteins were extracted, and electrophoresis was performed. The specific procedure is as follows. (1) Extraction of RAW264.7 cell proteins After processing the RAW264.7 cells, the cells were lysed and cellular proteins were extracted. The culture medium was aspirated using an aspirator, washed twice with PBS buffer, and then aspirated with PBS. Cell lysate was added, the 6-well plate was shaken to mix uniformly, and the cells were dissolved on ice for 10 minutes. The cells were scraped off with a cell scraper and collected in a centrifuge tube. The centrifuge tube was placed on ice and dissolved for 30 minutes. After dissolution was complete, the cells were centrifuged at 12000 r / min at 4°C for 15 minutes using a cryogenic centrifuge, and the supernatant containing proteins was collected in a new EP tube. (2) Measurement of cell protein concentration by BCA method Protein concentrations were measured using the BCA method. First, a protein standard was prepared to a concentration of 0.5 mg / mL in PBS buffer. The BCA working solution was prepared in a ratio of Solution A:B = 50:1 and uniformly mixed. Three parallel lines were set up, and a calibration curve was created by adding the protein standard solution and PBS buffer as shown in Table 3. 1 μL of cell-extracted protein solution sample was added to a 96-well plate, the volume was supplemented to 20 μL with PBS buffer, and then 200 μL of BCA working solution was added. The reaction was carried out at 37°C for 30 minutes, and the absorbance value at 562 nm was measured using a microplate reader. The protein concentration of each sample was calculated based on the calibration curve. JPEG2026518247000009.jpg55170(3) Protein denaturation Diluted protein samples were collected, mixed uniformly with protein loading buffer, and denatured in a metal bath at 100°C for 15 minutes. The denatured proteins were cooled to room temperature and then stored in a refrigerator at -20°C. (4) SDS-PAGE electrophoresis Two clean glass plates were taken, aligned, and clamped in a gel holder. Ultrapure water was added to check for leaks. After checking, the ultrapure water was poured out, and the remaining liquid between the two glass plates was absorbed with absorbent paper to prepare for gel injection. As shown in Table 4, separation gels (lower layer gels) of appropriate concentrations were prepared according to the molecular weight of the protein. Based on the molecular weight of the protein to be detected in the experiment, a 10% SDS-PAGE separation gel was prepared as shown in Table 5, and a 5% SDS-PAGE concentration gel (upper layer gel) was prepared as shown in Table 6. Approximately 4 mL of separation gel was placed between the two dry glass plates, isopropyl alcohol was added to seal the gel, and it was left for 1 hour to solidify. The isopropyl alcohol was discarded, the remaining isopropyl alcohol was absorbed with absorbent paper, and the concentration gel was poured in. A comb was inserted to prevent air bubbles from forming, and after the concentration gel solidified, the comb was gently removed. Two glass plates were placed in the electrophoresis tank, and electrophoresis solution was poured in so that the liquid surface covered the glass plates to load the protein sample. After loading was complete, electrophoresis was started. First, the sample was concentrated in the concentration gel at a constant voltage of 80V for about 30 minutes. Then, the voltage was adjusted to 120V and constant voltage electrophoresis was continued. Electrophoresis was stopped when the loading buffer had flowed to the bottom of the gel plate. JPEG2026518247000010.jpg39170JPEG2026518247000011.jpg45170JPEG2026518247000012.jpg45170(5) Transfer film The PVDF membrane was cut to the appropriate size, a corner was cut off as a mark to distinguish the front and back sides after gel cutting, and it was activated by immersion in methanol for 1 minute. The activated PVDF membrane, sponge, and transfer membrane filter paper were equilibrated in pre-cooled transfer membrane buffer. The glass plate was pried open with a gel cutter, the gel was cut according to the molecular weight of the target protein, and placed in electrophoresis solution. The black side of the transfer membrane sheet was placed down, and the sponge, filter paper, gel strip, PVDF membrane, filter paper, and sponge were placed in that order to sandwich the membrane. After standing, the sandwiched sheet was placed in the transfer membrane tank, the black sheet facing the black side of the tank, transfer membrane solution was added so that the sheet was submerged, an ice pack was placed to create a low temperature environment, and the constant current was set to 250 mA. The transfer time was set according to the molecular weight of the protein. (6) Blocking The blocking agent was 5% skim milk prepared with 1×TBST. After the membrane transfer was complete, the PVDF membrane was removed and placed in an incubator box. Skim milk was added until the PVDF membrane was submerged, and it was left on a sloshing bed at room temperature for 1 hour. The skim milk was then poured out, and the membrane was rinsed gently with 1×TBST for 10 minutes, and this process was repeated three times. (7) Incubation of the first antibody The primary antibodies COX-2 (CST, #12282), p-IκBα (CST, #2859), IκBα (CST, #4814), and β-actin (CST, #4970) were diluted in an antibody diluent at a ratio of 1:1000. A PVDF membrane was placed in the diluted primary antibody solution, and the primary antibodies were cultured overnight at a low speed in a 4°C shaker. The primary antibody solution was collected, washed with 1×TBST for 10 minutes, and washed three times. (8) Incubation of secondary antibodies The PVDF membrane was immersed in a 1:5000 diluted horseradish peroxidase-labeled secondary antibody and incubated at room temperature for 1 hour. After incubation, the secondary antibody solution was collected and washed with 1×TBST at room temperature for 10 minutes, repeating this process three times. (9) Developing Development was performed using the ECL chemical method. Developers A and B were uniformly mixed in a 1:1 ratio, 100 μL of which was dropped onto a PVDF film, developed using a developing machine, and then photographed. (10) Statistical analysis JPEG2026518247000013.jpg25170

[0113] 3.Result analysis When LPS activates RAW264.7 mouse macrophages, TLR-4 is stimulated to recognize and bind to LPS, activating the NF-κB signaling pathway and ultimately promoting the production of large amounts of inflammatory factors such as NO, PGE2, TNF-α, IL-β, IL-6, COX-2, and iNOS, inducing 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 can be used rudimentarily for screening the activity of individual compounds in anti-inflammatory models.

[0114] As shown in Figure 17, petunidine derivatives exhibit excellent anti-inflammatory activity in suppressing NO release in the inflammation model. After LPS stimulation, iNOS expression levels in RAW264.7 cells were upregulated, showing a very significant difference compared to the control group (P<0.01). At a petunidine derivative concentration of 10 μM, iNOS protein expression was significantly suppressed in both cases (P<0.01). As shown in Figure 18, it was demonstrated that the petunidine derivatives can exert anti-inflammatory effects by reducing NO release through suppression of iNOS protein expression.

[0115] <Test Example 2> Effect of petunidin derivatives in black goji berries on sugar metabolism 1. Experimental Method 1.1 Effect of petunidin derivatives on 2-NBDG uptake in 3T3-L1 cells 43T3-L1 preadipocytes 5×10 4 Cells were seeded at a density of cells / mL in a 12-well plate, and when the cell density reached over 80%, induced differentiation was performed. Eight days after induced differentiation and maturation, 1 μM Dex (dexamethasone) was added to the cell saturation to construct an IR model (insulin resistance model). A normal group, a model group, and a drug-treated group were set up. The normal group was cultured in complete medium, while the other groups were cultured in 1 μM Dex. 10 μM of a single compound was added to each drug-treated group, and they were cultured for 48 hours. The culture medium was then aspirated, washed once with DPBS, 500 μL of pancreatic enzyme was added, and digestion was carried out at 37°C for 1 minute. 2 mL of DPBS was added and blown in uniformly, and centrifugation was carried out at 1000 g for 6 minutes. The supernatant was discarded, and 1 mL of sugar-free medium containing 10 μM 2-NBDG was added to each well and cultured at 37°C for 30 minutes. Fluorescence intensity was detected by flow cytometry at a wavelength of 488 nm.

[0116] 1.2 Effects of petunidin derivatives on AKT phosphorylation in 3T3-L1 cells 3T3-L1 preadipocytes 5×10 4Cells were seeded in a 6-well plate at a density of cells / mL, and induced differentiation was performed when the cell density reached 80% or more. An IR model was constructed by adding 1 μM Dex to the cell culture medium on day 8 after induced differentiation and maturation. A normal group, a model group, and a drug-treated group were established. The normal group was cultured in complete medium, while the other groups were cultured in 1 μM Dex. 10 μM of a single compound was added to each drug-treated group, and after 48 hours of culture, the culture medium was aspirated and the cells were collected. The expression levels of PI3K and AKT proteins were measured using Western blot. The Western blot analysis method was the same as in Experimental Example 1.

[0117] 2.Result analysis 2.1 Effects of petunidin derivatives on 2-NBDG uptake in 3T3-L1 cells As shown in Figure 19, the uptake capacity of 2-NBDG in the normal group was higher than the glucose uptake capacity in the insulin-resistant model group. The glucose uptake capacity of cells in the model group was relatively weak, and the difference from the glucose uptake capacity of cells after insulin stimulation in the normal group was significant. After intervention with petunidine derivatives, 2-NBDG uptake by insulin-stimulated adipocytes was promoted to varying degrees, indicating that petunidine derivatives have the potential to improve insulin resistance.

[0118] 2.2 Effects of petunidine derivatives on the phosphorylation of PI3K and AKT in 3T3-L1 cells As a result, as shown in Figure 20, the expression levels of p-AKT and p-PI3K proteins in the model group were reduced compared to the normal group. After treatment with petunidine derivatives, the expression levels of p-AKT and p-PI3K in 3T3-L1 adipocytes were increased to varying degrees, thereby promoting glucose uptake and enhancing insulin sensitivity in 3T3-L1 adipocytes.

[0119] <Test Example 3> Effects of petrolatum derivatives in black goji berries on lipid metabolism 1. Experimental Method 1.1 Oil Red O-Dye 3T3-L1 cells in good cellular condition were placed in a 6-well plate at a density of 5 × 10⁴. 4 Cells were seeded at a rate of cells / mL and cultured in a high-sugar DMEM culture medium containing 10% FBS until the cell density reached approximately 85%-90%. The cell medium was then replaced, and after 2 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 cultured for 2 days (the day when the induction medium was added is considered day 0, labeled as Induction I). Subsequently, the culture medium was replaced with a 10 μg / mL Insulin-containing culture medium (Induction II) and cultured for another 2 days. Then, the culture medium was replaced with normal medium and 10 M single compounds and cultured together, with the medium being replaced every other day. After induction was complete, the cells were fixed with 4% neutral formaldehyde for 30 minutes. After cell fixation, the cell surface was stained with pre-prepared Oil Red O staining solution and allowed to stand in the dark for 60 minutes. After staining, the cells were washed with 70% ethanol, excess dye was discarded, and the cells were washed 3-4 times with ultrapure water. Finally, the cells were observed under a microscope and photographed.

[0120] 1.2 Measurement of TG content 3T3-L1 cells were placed in a 6-well plate in 5 × 10⁶ rows. 4 Cells were seeded at a density of cells / mL, and when the cell density reached approximately 85%-90%, induced differentiation was performed. TG content was measured on day 8 of induction, and the specific method was as follows: (1) Cell pretreatment: On day 8 of induction, the cell culture medium was aspirated, washed twice with cold PBS, and then the cells were digested with pancreatic enzyme digestate; (2) Cell collection: After cell digestion, the cells were resuspended with PBS and centrifuged at 1000g for 5 minutes to collect the cell pellet; (3) Ultrasonic disruption: An appropriate amount of PBS was added to the collected pellet, and then ultrasonic disruption (3 min) was performed; (4) Measurement: 2 μL of cell disruption suspension was added to each well of a 96-well plate, 2 μL of distilled water was added to the blank well, 2 μL of the standard was added to the standard well, then 200 μL of the measurement solution was added to each well and mixed uniformly. After incubation at 37°C for 10 minutes, the absorbance was read at 510 nm. The protein concentration in the sample was measured using the BCA method, corrected, and finally the TG content was calculated using the following formula. JPEG2026518247000014.jpg101701.3 Western blot analysis The Western blot analysis method is the same as in Example 1.

[0121] 2.Result analysis 2.1 Effects of petunidin derivatives on lipid droplet accumulation and TG content in 3T3-L1 cells As shown in Figure 21, there is no lipid droplet accumulation in undifferentiated cells, while differentiated cells contain a large amount of lipid droplets. After treatment with petunidin derivatives, intracellular lipid droplets were significantly reduced. As shown in Figure 22, the TG content in differentiated cells increased significantly compared to undifferentiated cells (P<0.01). Compared to differentiated cells, the TG content decreased after treatment with petunidine derivatives, and both differences were significant (P<0.01).

[0122] 2.2 Effects of petunidin derivatives on the expression of lipid metabolism proteins in 3T3-L1 cells (1) Effects of petunidin derivatives on the expression of lipid synthesis transcription factors in 3T3-L1 cells The effects of petunidine derivatives on the expression of lipid synthesis transcription factors in 3T3-L1 cells were analyzed by Western blot, and the results are shown in Figure 23. In uninducible differentiated 3T3-L1 cells, the expression levels of PPARγ and C / EBPα proteins were low. On the other hand, in differentiated cells, the expression levels of PPARγ and C / EBPα proteins were high. Compared to the differentiated group, treatment with petunidine derivatives could reduce the protein expression levels of PPARγ and C / EBPα transcription factors to some extent. (2) Effects of petunidin derivatives on the expression of lipid synthesis-related proteins in 3T3-L1 cells As shown in Figure 24, the expression levels of FAS and ACC proteins were low in uninducible differentiated 3T3-L1 cells. On the other hand, the expression levels of FAS and ACC proteins were significantly increased in induced differentiated cells. Compared to the differentiated group, the expression levels of FAS and ACC proteins can be significantly reduced after treatment with petunidin derivatives.

[0123] From these results, it can be seen that 3T3-L1 cells are regulated by transcription factors and lipid proteins during differentiation from pre-adipocytes to mature adipocytes, and their cell morphology also changes, for example, eventually resulting in the appearance of a "ring-like" shape. Petunidine derivatives can inhibit the accumulation of lipid droplets in 3T3-L1 cells and reduce the intracellular TG content; petunidine derivatives can also inhibit the differentiation of 3T3-L1 adipocytes and reduce the accumulation of intracellular lipid droplets by inhibiting the expression levels of transcription factors such as PPARγ and C / EBPα. At the same time, they suppress lipid synthesis by suppressing the expression levels of FAS and ACC proteins, thereby improving the lipid metabolism level of the cells.

[0124] <Example 5> Chromatographic analysis of component Fr-5-4 The component Fr2-5-4 obtained in Example 2 was chromatographically analyzed. The chromatographic analysis conditions were: kromasil C18 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 control. The results are shown in Figure 25. Component Fr2-5-4 was separated into four main components under the chromatographic analysis conditions of this experiment, and the separation of the four components was good. Of these, Fr-5-4-3 eluted at 40-50 min, which is the novel monomer compound of the present invention.

[0125] <Example 6> Chromatographic analysis of component Fr2-5-5 The component Fr2-5-5 obtained in Example 2 was chromatographically analyzed. The chromatographic analysis conditions were: kromasil C18 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 control. The results are shown in Figure 26. The component Fr2-5-5 was separated into 10 main components under the chromatographic analysis conditions of this experiment, and the elemental compound Fr2-5-5-8 was obtained.

[0126] The above description is merely an example of the present invention and does not limit the scope of the patent. Equivalent structures or process transformations created using the specification and drawings of the present invention, or their direct or indirect application to other related technical fields, are all within the scope of patent protection of the present invention.

Claims

1. A petunidine derivative or a salt or isomer thereof, characterized by having the structural formula of a compound of formula IV or formula V.

2. A method for preparing petunidine derivatives having the structural formula of a compound of formula IV or formula V, The preparation method for the compound of formula IV is as follows: (1) The extract of black goji berry is prepared by preparative chromatography to obtain components Fr1, Fr2, and Fr3, respectively, with retention times of 12-39 min, 39-139 min, and 139-230 min; However, the conditions for the preparative chromatography include the following: Column: MCI medium-pressure column, standard size 49 x 460 mm; Mobile phase: Water: A / Methanol: B / Dichloromethane: C; Gradient elution is performed according to 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) Separate component Fr2 by preparative chromatography to obtain component Fr2-5 with a retention time of 76 to 130 min; However, the conditions for the preparative chromatography include the following: Column: MCI medium-pressure column, standard size 49 x 460 mm; Mobile phase: A: Water / B: Methanol: Gradient elution is performed using the following procedure: 0–120 min, 0%–100% B; 20–140 min, 100% B; (3) Separate component Fr2-5 by preparative chromatography to obtain component Fr2-5-5 with a retention time of 27-36 min; However, the conditions for the preparative chromatography include the following: Column: kromasil C18 column; specification 21.2 x 250 mm; Mobile phase: A: Water / B: Methanol; Gradient elution is performed using the following procedure: 0–60–65–90 min, 30%–42%–70%–95% B; (4) Component Fr2-5-5 is separated by preparative chromatography to obtain petunidine derivative IV at a retention time of 92-95 min; However, the conditions for preparative chromatography are as follows: Column: kromasil C18 column; specification 21.2 x 250 mm; Mobile phase: A: Water / B: Methanol; Gradient elution is performed using the following procedure: 0–30–120 min, 20%–28%–31% B; Includes, The preparation method for the compound of formula V is as follows: (1) The extract of black goji berry is prepared by preparative chromatography to obtain components Fr1, Fr2, and Fr3, respectively, with retention times of 12-39 min, 39-139 min, and 139-230 min; However, the preparative chromatography conditions include the following: Column: MCI medium-pressure column, standard size 49 x 460 mm; Mobile phase: Water: A / Methanol: B / Dichloromethane: C; Gradient elution is performed according to 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) Separate component Fr2 by preparative chromatography to obtain component Fr2-5 with a retention time of 76 to 130 min; However, the conditions for the preparative chromatography include the following: Column: MCI medium-pressure column, standard size 49 x 460 mm; Mobile phase: A: Water / B: Methanol. Gradient elution is performed using the following procedure: 0–120 min, 0%–100% B; 20–140 min, 100% B; (3) Separate component Fr2-5 by preparative chromatography to obtain component Fr2-5-4 with a retention time of 21 to 27 minutes; However, the conditions for the preparative chromatography include the following: Column: kromasil C18 column; specification 21.2 x 250 mm; Mobile phase: A: Water / B: Methanol; Gradient elution is performed using the following procedure: 0–60–65–90 min, 30%–42%–70%–95% B; (4) Component Fr2-5-4 is separated by preparative chromatography to obtain petunidine derivative V with a retention time of 40-41 min; However, the conditions for the preparative chromatography include the following: Column: kromasil C18 column; specification 21.2 x 250 mm; Mobile phase: A: Water / B: Methanol; Gradient elution is performed using the following procedure: 0–60 min, 30%–35% B; A method for preparing a petunidine derivative according to claim 1, characterized by including the following.

3. Use of the petunidine derivative according to claim 1, and a pharmaceutically acceptable salt, hydrate, or solvate thereof, in the preparation of a product for treating and / or preventing inflammatory, glucose metabolism disorder, and lipid metabolism disorder-related diseases.

4. The use according to claim 3, characterized in that the product is a product that reduces the release of at least one inflammatory factor, namely NO, PGE2, TNF-α, IL-β, IL-6, COX-2, and iNOS.

5. The use according to claim 3, characterized in that the product is a product that enhances the expression levels of p-AKT and p-PI3K proteins.

6. The use according to claim 3, characterized in that the product is a product that inhibits the accumulation of cellular lipid droplets and / or the production of cellular lipids.

7. The use according to claim 3, characterized in that the product is at least one of a PPARγ antagonist, a C / EBPα antagonist, a FAS inhibitor, and an ACC inhibitor.

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