A flavonoid glycoside of golden chrysanthemum and its application in preparing medicine for treating hyperlipidemia and fatty liver

Abstract

The present invention discloses a golden chrysanthemum flavonoid glycoside derived from golden chrysanthemum, wherein the golden chrysanthemum flavonoid glycoside is apigenin 7-O-(6"-O-malonyl)-β-D-glucoside derived from golden chrysanthemum. The present invention separates and purifies apigenin 7-O-(6"-O-malonyl)-β-D-glucoside from golden chrysanthemum for the first time, completes the first synthesis through a 5-step synthesis process with a total yield of 2.7%, and discovers for the first time that the compound has activity in improving hyperlipidemia and fatty liver in vitro and in vivo. Therefore, the compound can be used in the preparation of products for treating related diseases such as hyperlipidemia and fatty liver.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural product preparation and medicine, in particular to a flavonoid glycoside derived from golden chrysanthemum and its application in preparing medicines for treating hyperlipidemia and fatty liver. Background Art

[0002] Golden Chrysanthemum is a type of chrysanthemum. As an ancient large chrysanthemum variety, it has a delicate and sweet fragrance and has been produced and sold as chrysanthemum tea in recent years. Originally cultivated in Wuyuan, Jiangxi Province, Golden Chrysanthemum is now also grown in large quantities in Anhui, Jiangsu, Qinghai, and other regions. Research reports indicate that Golden Chrysanthemum has antibacterial, antioxidant, antihypertensive, and anti-inflammatory properties. The unique pharmacological effects of its active ingredients, polysaccharides, caffeic acid derivatives, and flavonoids, have also been extensively studied.

[0003] Due to the unique physiological activities of flavonoids, research on their activity and chemical structure modification is ongoing. Isolation from natural medicinal and edible resources is also an important avenue for flavonoid discovery. Currently, chemical modification of the C2, C3, C7, C8, C3' (or C5'), and C4' residues of flavonoids is the main focus of structural modification. By introducing functional groups such as halogens, aryls, alkyls, sulfonic acids, phosphates, amino groups, and carboxyl groups, the chemical structure is altered, thereby improving physicochemical properties such as lipid solubility, water solubility, and stability, thereby enhancing bioactivity and bioavailability.

[0004] Apigenin-7-O-(6”-O-malonylglucoside)-β-D-glucoside (structural formula below, hereinafter referred to as AMG), also known as Apigenin-7-O-6”-malonylglucoside, CAS: 86546-87-4, has been isolated from chamomile, Huangshan tribute chrysanthemum, small yellow chamomile, lupine, salt-loving grass, Antarctic moss, and tenuifolia. It has been reported to exhibit antioxidant, liver damage relief, and anti-tumor cell proliferation activities.

[0005] The structural formula of apigenin 7-O-(6"-O-malonyl)-β-D-glucoside is as follows:

[0006]

[0007] Through searching, we found the following publications related to the patent application of this invention:

[0008] 1. Journal article: Isolation, identification and stability of acylated derivatives of apigenin7-O-glucoside from chamomile (Chamomilla recutita[L.]Rauschert). Phytochemistry, 2004, 65(16), 2323-2332. It was isolated and extracted from chamomile using a polyamide column combined with high performance liquid chromatography, but there is no research on its activity.

[0009] 2. Journal article: Identification of major flavonoids inpetals of edible chrysanthemum flowers and their suppressive effect on carbon tetrachloride-induced liver injury in mice. Food Science and Technology Research (2009), 15(5), 499-506. The compound was isolated and extracted from a Japanese chrysanthemum (Chrysanthemum morifolium Ramat.forma esculentum Makino, cv Kotobuki) and has antioxidant activity and activity in alleviating carbon tetrachloride-induced liver injury.

[0010] 3. Identification of the phenolic components of chrysanthemum flower (Chrysanthemum morifolium Ramat). Food Chemistry (2010), 120(1), 319-326. A series of compounds, including this compound, were identified by liquid chromatography-mass spectrometry. No information on the efficacy of the study was available.

[0011] In the prior art, apigenin 7-O-(6"-O-malonyl)-β-D-glucoside has antioxidant, liver damage alleviation, and anti-tumor cell proliferation activities, but does not have lipid-lowering and fatty liver alleviation activities. The present invention provides a new application of this compound for the treatment of lipid-lowering and fatty liver. Fatty liver disease is essentially different from liver damage. Fatty liver disease generally requires a "second hit," which affects metabolism and promotes the deposition of triglycerides in the liver, such as a high-fat diet, obesity, and insulin resistance, which is the "first hit" in the pathogenesis process; signal transduction processes, such as extracellular cytokines, adipokines, bacterial endotoxins, mitochondrial dysfunction and / or endoplasmic reticulum stress, or further progression to fatty liver disease, activation of inflammatory cascades and fibrosis are called the "second hit." To date, there are no effective methods or drugs for curing fatty liver disease on the market.

[0012] In addition, in the prior art, apigenin 7-O-(6"-O-malonyl)-β-D-glucoside has been isolated from chamomile, Huangshan tribute chrysanthemum, small yellow chamomile, lupine, salt-loving grass, Antarctic moss, and fine-leaved true moss, but there is no report on its isolation from golden chrysanthemum, and there is no report on its chemical synthesis. Summary of the Invention

[0013] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing flavonoid glycosides from Jinsihuangchrysanthemum and its application in preparing drugs for treating hyperlipidemia and fatty liver.

[0014] The technical solution adopted by the present invention to solve its technical problem is:

[0015] A flavonoid glycoside derived from golden chrysanthemum, wherein the flavonoid glycoside is apigenin 7-O-(6"-O-malonyl)-β-D-glucoside derived from golden chrysanthemum.

[0016] Furthermore, the structural formula of the apigenin 7-O-(6″-O-malonyl)-β-D-glucoside derived from Chrysanthemum morifolium is:

[0017]

[0018] Furthermore, the apigenin 7-O-(6"-O-malonyl)-β-D-glucoside can inhibit pancreatic lipase activity in vitro and reduce the accumulation of intracellular lipids; in vivo, it can significantly reduce the increase in TG, TC, LDL-C, ALT, and AST biochemical indicators in serum and liver, alleviate the decrease in HDL-C, and improve hyperlipidemia and fatty liver to a certain extent; it can also increase the serum SOD activity of mice, improve the increase in the activity of liver inflammatory factors IL-6 and MPO caused by fat accumulation; and can restore the liver morphology, fatty degeneration, inflammation, and oxidative damage of mice; and can also improve the enlargement of epididymal adipose white tissue.

[0019] Furthermore, the preparation method of the apigenin 7-O-(6″-O-malonyl)-β-D-glucoside comprises:

[0020] Method 1: The preparation method is to separate and purify Chrysanthemum officinale by combining multiple chromatographic separation methods;

[0021] Alternatively, method 2: the preparation method is to use apigenin as the starting material, synthesize it through a chemical synthesis process with a total yield of 2.7% through 5 steps of reaction.

[0022] Furthermore, the preparation method of apigenin 7-O-(6″-O-malonyl)-β-D-glucoside comprises the following steps:

[0023] Crush the golden chrysanthemum, weigh the golden chrysanthemum powder in a container, add water (1-500g / L), heat and stir for 0.5-24h; cool and filter, extract the filtrate with an organic solvent, combine the organic phases, and concentrate under reduced pressure to obtain a crude product, which is weighed;

[0024] The crude product was separated and purified using a high-performance liquid phase preparative column with a mobile phase consisting of water and acetonitrile containing 0.1% trifluoroacetic acid at a mass concentration, with a volume ratio of the aqueous phase to the organic phase of 6:4 to 1:9, and a retention time of 6-15 minutes. After drying, pure flavonoid glycosides as active monomers were obtained. After the pure compound was prepared and isolated, characterization methods such as mass spectrometry and nuclear magnetic resonance spectroscopy were used to infer the structure of the compound.

[0025] Furthermore, the organic solvent is ethyl acetate, dichloromethane, chloroform or n-butanol.

[0026] Furthermore, the synthetic route of the preparation method of apigenin 7-O-(6″-O-malonyl)-β-D-glucoside is:

[0027] Furthermore, the preparation method comprises the following steps:

[0028] (1) Synthesis of Compound 1: Apigenin and silver carbonate were dissolved in quinoline, acetyl bromide-α, D-glucose was added at room temperature, and the mixture was reacted at 50°C for 0.5 h. The reaction was completed when the starting material disappeared after TLC monitoring. The reaction solution was poured into 6M hydrochloric acid solution and extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Compound 1 was obtained after purification by silica gel column chromatography as a yellow solid with a yield of 40.3%.

[0029] The ratio of apigenin: silver carbonate: quinoline: acetyl bromide-α,D-glucose: hydrochloric acid solution is 2.7:5.5:100:4.5:200 in g:g:mL:g:mL;

[0030] (2) Synthesis of Compound 2: Compound 1 and potassium carbonate were dissolved in N,N-dimethylformamide, benzyl bromide was added, and the mixture was reacted at 50°C for 3 h. The reaction was monitored by TLC. The reaction solution was poured into a 2M dilute hydrochloric acid solution and extracted with ethyl acetate. The organic phases were combined and washed three times with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Compound 2 was purified by silica gel column chromatography to obtain a yellow solid with a yield of 72.5%.

[0031] The ratio of compound 1: potassium carbonate: N, N-dimethylformamide: benzyl bromide: dilute hydrochloric acid solution (g:g:mL:g:mL) is 1.4:1.3:20:0.8:200;

[0032] (3) Synthesis of Compound 3: Compound 2 was dissolved in methanol, sodium methoxide was added at room temperature, and the reaction was allowed to proceed at room temperature for 1 hour. The reaction was monitored by TLC. The reaction solution was poured into a 2M dilute hydrochloric acid solution and extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Compound 3 was obtained after column chromatography separation and purification as a light yellow solid with a yield of 85.4%.

[0033] Among them, the ratio of compound 2: methanol: sodium methoxide: dilute hydrochloric acid solution is 1:20:277:20 in g:mL:mg:mL;

[0034] (4) Synthesis of Compound 4: Compound 3 was dissolved in acetonitrile and N,N-dimethylformamide, malonic acid and tert-butyl isocyanide were added to the solution, and the mixture was reacted at 80°C for 8 h. The reaction was monitored by TLC. The reaction solution was poured into water and extracted with ethyl acetate. The organic phases were combined and washed three times with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Compound 4 was obtained after column chromatography purification as a light yellow solid with a yield of 40.6%.

[0035] Among them, the ratio of compound 3: acetonitrile: N,N-dimethylformamide: malonic acid: tert-butyl isocyanide: water in mg:mL:mL:mg:mg:mL is 670:5:5:208:249:20;

[0036] (5) Synthesis of apigenin 7-O-(6"-O-malonic acid monoyl)-β-D-glucoside: Compound 4 was dissolved in ethanol, and 5% to 15% palladium carbon was added. The mixture was reacted at room temperature under a pressure of 15 psi in a hydrogen atmosphere for 16 h. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed three times, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was then separated and purified by a high-performance liquid phase preparative column to obtain the compound apigenin 7-O-(6"-O-malonic acid monoyl)-β-D-glucoside as a yellow solid with a yield of 27.0%, which is the flavonoid glycoside of chrysanthemum strychnosyltransferase.

[0037] The ratio of compound 4: ethanol: palladium carbon in mg: mL: mg is 310:10:20.

[0038] Use of the chrysanthemum flavonoid glycosides as described above in the preparation of drugs for treating hyperlipidemia and / or fatty liver.

[0039] Furthermore, the flavonoid glycosides of Chrysanthemum morifolium can inhibit pancreatic lipase activity in vitro and reduce the accumulation of intracellular lipids; in vivo, it can significantly reduce the increase of TG, TC, LDL-C, ALT, and AST biochemical indicators in serum and liver, alleviate the decrease of HDL-C, and improve hyperlipidemia and fatty liver to a certain extent; it can also increase the serum SOD activity of mice, improve the increase of liver inflammatory factors IL-6 and MPO activity caused by fat accumulation; and can restore the liver morphology, fatty degeneration, inflammation, and oxidative damage of mice; it can also improve the enlargement of epididymal fat white tissue.

[0040] The advantages and positive effects achieved by the present invention are:

[0041] 1. The chrysanthemum flavonoid glycoside AMG of the present invention can inhibit pancreatic lipase activity in vitro and reduce intracellular lipid accumulation. In vivo, it can significantly reduce the elevation of biochemical indicators such as TG, TC, LDL-C, ALT, and AST in serum and liver, alleviate the decrease of HDL-C, and improve hyperlipidemia and fatty liver to a certain extent. It can also increase serum SOD activity in mice and improve the elevated activity of liver inflammatory factors IL-6 and MPO caused by fat accumulation. It can also restore liver morphology, steatosis, inflammation, and oxidative damage in mice. In addition, it can also improve the enlargement of epididymal adipose white tissue. Therefore, through in vitro and in vivo studies, it was found that AMG has a positive effect on the treatment of hyperlipidemia and fatty liver, and is worthy of further development and application.

[0042] 2. The present invention is the first to separate and purify AMG from Chrysanthemum morifolium, and completes the first synthesis through a 5-step synthesis process with a total yield of 2.7%. It is also the first to discover that the compound has activity in improving hyperlipidemia and fatty liver in vitro and in vivo. Therefore, the compound can be used in the preparation of products for treating hyperlipidemia, fatty liver and other related diseases.

[0043] 3. This invention provides a new naturally derived plant and achieves its first chemical synthesis, providing a compound of sufficient quality for further drug development. Therefore, AMG can be used as a drug for treating hyperlipidemia and / or fatty liver disease. Its preparation method is simple and feasible, and it exhibits minimal food-borne toxicity and side effects, thus possessing significant market potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 AMG nuclear magnetic resonance hydrogen spectrum of the present invention;

[0045] Figure 2 AMG carbon nuclear magnetic resonance spectrum of the present invention;

[0046] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of the synthetic intermediate 1 in the present invention;

[0047] Figure 4 is the hydrogen nuclear magnetic resonance spectrum of the synthetic intermediate 2 in the present invention;

[0048] Figure 5 is the hydrogen nuclear magnetic resonance spectrum of the synthetic intermediate 3 in the present invention;

[0049] Figure 6 is the hydrogen nuclear magnetic resonance spectrum of the synthetic intermediate 4 in the present invention;

[0050] Figure 7 This is a staining diagram of the lipid content of HepG2 cells by AMG in the present invention;

[0051] Figure 8 The figure shows the effect of AMG on the activity of SOD in serum; compared with the model group, ***P<0.01, **P<0.05;

[0052] Figure 9 This is a diagram showing the effect of AMG on liver morphology in the present invention;

[0053] Figure 10 The figure shows the effect of AMG on liver coefficient in the present invention; compared with the model group, ***P<0.01, **P<0.05;

[0054] Figure 11 This is a slice analysis diagram of the effect of AMG on liver lesions in the present invention;

[0055] Figure 12 The figure shows the effect of AMG on liver myeloperoxidase (MPO) in the present invention; compared with the model group, ***P<0.01, **P<0.05;

[0056] Figure 13 The figure shows the effect of AMG on liver interleukin-6 (IL-6) in the present invention; compared with the model group, ***P<0.01, **P<0.05;

[0057] Figure 14 This is a diagram showing the effect of AMG on epididymal white adipose tissue morphology in the present invention;

[0058] Figure 15 The figure shows the effect of AMG on epididymal white adipose tissue index in the present invention; compared with the model group,

[0059] **P<0.05, *P<0.01. DETAILED DESCRIPTION

[0060] The present invention will be further described below with reference to the following examples. The following examples are descriptive rather than restrictive, and the scope of protection of the present invention cannot be limited by the following examples.

[0061] The various experimental operations involved in the specific embodiments are all routine techniques in the field. For parts not specifically annotated in this document, ordinary technicians in this field can refer to various commonly used reference books, scientific literature or related instructions, manuals, etc. before the filing date of this invention to implement them.

[0062] A flavonoid glycoside derived from golden chrysanthemum, wherein the flavonoid glycoside is apigenin 7-O-(6"-O-malonyl)-β-D-glucoside derived from golden chrysanthemum.

[0063] Preferably, the structural formula of the apigenin 7-O-(6″-O-malonyl)-β-D-glucoside derived from Chrysanthemum morifolium is:

[0064]

[0065] Preferably, the apigenin 7-O-(6"-O-malonyl)-β-D-glucoside can inhibit pancreatic lipase activity in vitro and reduce the accumulation of intracellular lipids; in vivo, it can significantly reduce the increase in TG, TC, LDL-C, ALT, and AST biochemical indicators in serum and liver, alleviate the decrease in HDL-C, and improve hyperlipidemia and fatty liver to a certain extent; it can also increase the serum SOD activity of mice, improve the increase in the activity of liver inflammatory factors IL-6 and MPO caused by fat accumulation; and can restore the liver morphology, fatty degeneration, inflammation, and oxidative damage in mice; and can also improve the enlargement of epididymal adipose white tissue.

[0066] Preferably, the preparation method of apigenin 7-O-(6″-O-malonyl)-β-D-glucoside comprises:

[0067] Method 1: The preparation method is to separate and purify Chrysanthemum officinale by combining multiple chromatographic separation methods;

[0068] Alternatively, method 2: the preparation method is to use apigenin as the starting material, synthesize it through a chemical synthesis process with a total yield of 2.7% through 5 steps of reaction.

[0069] Preferably, the preparation method of apigenin 7-O-(6″-O-malonyl)-β-D-glucoside comprises the following steps:

[0070] Crush the golden chrysanthemum, weigh the golden chrysanthemum powder in a container, add water (1-500g / L), heat and stir for 0.5-24h; cool and filter, extract the filtrate with an organic solvent, combine the organic phases, and concentrate under reduced pressure to obtain a crude product, which is weighed;

[0071] The crude product was separated and purified using a high performance liquid phase reverse preparative column. The mobile phase was water and acetonitrile containing 0.1% trifluoroacetic acid at a mass concentration. The volume ratio of the aqueous phase to the organic phase was 6:4 to 1:9. The retention time was 6-15 minutes. After drying, the pure active monomer chrysanthemum flavonoid glycoside was obtained. After the pure compound was prepared and isolated, it was characterized by mass spectrometry, nuclear magnetic resonance spectroscopy and other means to infer the structure of the compound.

[0072] Preferably, the organic solvent is ethyl acetate, dichloromethane, chloroform or n-butanol.

[0073] Preferably, the synthetic route of the preparation method of apigenin 7-O-(6″-O-malonyl)-β-D-glucoside is:

[0074]

[0075] Preferably, the preparation method comprises the following steps:

[0076] (1) Synthesis of Compound 1: Apigenin and silver carbonate were dissolved in quinoline, acetyl bromide-α, D-glucose was added at room temperature, and the mixture was reacted at 50°C for 0.5 h. The reaction was completed when the starting material disappeared after TLC monitoring. The reaction solution was poured into 6M hydrochloric acid solution and extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Compound 1 was obtained after purification by silica gel column chromatography as a yellow solid with a yield of 40.3%.

[0077] The ratio of apigenin: silver carbonate: quinoline: acetyl bromide-α,D-glucose: hydrochloric acid solution is 2.7:5.5:100:4.5:200 in g:g:mL:g:mL;

[0078] (2) Synthesis of Compound 2: Compound 1 and potassium carbonate were dissolved in N,N-dimethylformamide, benzyl bromide was added, and the mixture was reacted at 50°C for 3 h. The reaction was monitored by TLC. The reaction solution was poured into a 2M dilute hydrochloric acid solution and extracted with ethyl acetate. The organic phases were combined and washed three times with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Compound 2 was purified by silica gel column chromatography to obtain a yellow solid with a yield of 72.5%.

[0079] The ratio of compound 1: potassium carbonate: N, N-dimethylformamide: benzyl bromide: dilute hydrochloric acid solution (g:g:mL:g:mL) is 1.4:1.3:20:0.8:200;

[0080] (3) Synthesis of Compound 3: Compound 2 was dissolved in methanol, sodium methoxide was added at room temperature, and the reaction was allowed to proceed at room temperature for 1 hour. The reaction was monitored by TLC. The reaction solution was poured into a 2M dilute hydrochloric acid solution and extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Compound 3 was obtained after column chromatography separation and purification as a light yellow solid with a yield of 85.4%.

[0081] Among them, the ratio of compound 2: methanol: sodium methoxide: dilute hydrochloric acid solution is 1:20:277:20 in g:mL:mg:mL;

[0082] (4) Synthesis of Compound 4: Compound 3 was dissolved in acetonitrile and N,N-dimethylformamide, malonic acid and tert-butyl isocyanide were added to the solution, and the mixture was reacted at 80°C for 8 h. The reaction was monitored by TLC. The reaction solution was poured into water and extracted with ethyl acetate. The organic phases were combined and washed three times with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Compound 4 was obtained after column chromatography purification as a light yellow solid with a yield of 40.6%.

[0083] Among them, the ratio of compound 3: acetonitrile: N,N-dimethylformamide: malonic acid: tert-butyl isocyanide: water in mg:mL:mL:mg:mg:mL is 670:5:5:208:249:20;

[0084] (5) Synthesis of AMG: Compound 4 was dissolved in ethanol, and 5% to 15% palladium on carbon was added. The reaction was carried out under a hydrogen atmosphere at 15 psi for 16 h at room temperature. The reaction solution was filtered through diatomaceous earth, and the filter cake was rinsed three times. The filtrate was concentrated under reduced pressure to obtain a crude product, which was then separated and purified by a high-performance liquid phase preparative column to obtain compound 'AMG as a yellow solid with a yield of 27.0%.

[0085] The ratio of compound 4: ethanol: palladium carbon in mg: mL: mg is 310:10:20.

[0086] Use of the chrysanthemum flavonoid glycosides as described above in the preparation of drugs for treating hyperlipidemia and / or fatty liver.

[0087] Preferably, the apigenin 7-O-(6"-O-malonyl)-β-D-glucoside can inhibit pancreatic lipase activity in vitro and reduce the accumulation of intracellular lipids; in vivo, it can significantly reduce the increase of TG, TC, LDL-C, ALT, and AST biochemical indicators in serum and liver, alleviate the decrease of HDL-C, and improve hyperlipidemia and fatty liver to a certain extent; it can also increase the serum SOD activity of mice, improve the increase of liver inflammatory factors IL-6 and MPO activity caused by fat accumulation; and can restore the liver morphology, fatty degeneration, inflammation, and oxidative damage of mice; and can also improve the enlargement of epididymal adipose white tissue.

[0088] Specifically, the relevant preparation and detection are as follows:

[0089] Example 1 Extraction, separation, purification and structural identification of the active monomer AMG from Chrysanthemum morifolium

[0090] First, crush the golden chrysanthemum, weigh 80g of golden chrysanthemum powder into a round-bottom flask, add 1L of deionized water, heat to 80°C, and stir for 30min. After cooling, filter the suspension, and extract the filtrate three times with ethyl acetate. The extraction volume of the organic solvent is 400mL each time. The organic phases after the three extractions are combined and concentrated under reduced pressure to obtain the first crude extraction product, weighing 1.2g. The remaining aqueous phase after the extraction is then extracted again with n-butanol three times. The extraction volume of the organic solvent is 400mL each time. The organic phases are combined and collected again and concentrated under reduced pressure to remove the solvent to obtain the second crude extraction product, weighing 0.8g.

[0091] The crude product was isolated and purified using a high-performance liquid phase preparative column to obtain pure AMG, the active monomer. The preparative purification was performed using a Phenomenex Luna 80*30mm*3um column, with a mobile phase of: A: water (0.1% trifluoroacetic acid) - B: acetonitrile, with a B percentage of 10%-35%, and a retention time of 8 minutes. After the pure compound was isolated, it was characterized by mass spectrometry and proton nuclear magnetic resonance spectroscopy, and its structure was inferred based on literature reports on the isolation, purification, and structural identification of chrysanthemum flavonoids.

[0092] The H NMR, C NMR and MS data of AMG are as follows: 1 H NMR (400MHz, MeOD) δ7.88(br d,J=8.6Hz,2H),6.94(d,J=8.6Hz,2H),6.78(s,1H),6.65(s,1H),6.50(d,J=1.7Hz,1H),5.10-5.01(m,1H),4.55(br d,J=10.9Hz,1H),4.31(dd,J=7.0,12.0Hz,1H),3.86-3.73(m,1H),3.54-3.47(m,2H),3.46-3.36(m,3H). 13 C NMR(400MHz,DMSO-d6)δ182.5(C-4),168.2(COOH),167.3(COOR),164.8(C-2),163. 1(C-7),161.8 / 161.5(C-5,C-4'),157.4(C-9),129.1(C-2',6'),121.5(C-1'),116. 5(C-3',C-5'),105.9(C-10),103.6(C-3),100.1 / 100.0(C-6,G-1),95.2(C-8),76.6 (G-3),74.3(G-5),73.5(G-2),70.1(G-4),64.5(G-6),41.8(CH2).MS(ESI):m / z[M+H + ]=519.3. H NMR spectrum is shown in Figure 1 , the C NMR spectrum is shown in Figure 2 .

[0093] Example 2 First Chemical Total Synthesis and Process Study of AMG

[0094] The present invention uses apigenin as the starting material and, through process optimization, fully synthesizes AMG with a total yield of 2.7%. The synthesis is obtained through the following route:

[0095]

[0096] The preparation of the above-mentioned AMG specifically includes the following steps:

[0097] (1) Synthesis of Compound 1: Apigenin (2.7 g, 10.0 mmol, 1.0 equivalent) and silver carbonate (5.5 g, 20.0 mmol, 2.0 equivalent) were dissolved in quinoline (100 mL). Acetyl bromide-α, D-glucose (4.5 g, 11.0 mmol, 1.1 equivalent) was added at room temperature. The mixture was microwaved at 50°C for 0.5 h. TLC (thin layer chromatography) monitored the disappearance of the starting material and the reaction was complete. The reaction solution was poured into 6 M hydrochloric acid solution (200 mL) and extracted with ethyl acetate (100 mL × 2). The organic phases were combined and washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. After purification by silica gel column chromatography, 1.4 g of compound 1 was obtained as a yellow solid with a yield of 40.3%.

[0098] The spectral data of compound 1 are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 13.05 (br s, 1H), 10.44 (br s, 1H), 7.97 (d, J = 8.9 Hz, 2H), 6.99-6.87 (m, 3H), 6.81 (d, J = 2.3 Hz, 1H), 6.46 (d, J = 2.3 Hz, 1H), 5.77 (d, J = 8.0 Hz, 1H), 5.48-5.35 (m, 1H), 5.12 (dd, J = 7.9, 9.7 Hz, 1H), 5.03 (t, J = 9.7 Hz, 1H), 4.41-4.31 (m, 1H), 4.26-4.09 (m, 2H), 2.08-2.02 (m, 9H), 1.99 (s, 3H). Figure 3 .

[0099] (2) Synthesis of Compound 2: Compound 1 (1.4 g, 2.3 mmol, 1.0 equivalent) and potassium carbonate (1.3 g, 9.2 mmol, 4.0 equivalent) were dissolved in N,N-dimethylformamide (20 mL), and benzyl bromide (0.8 g, 4.8 mmol, 2.1 equivalent) was added. The mixture was reacted at 50°C for 3 h. The reaction was monitored by TLC. The reaction solution was poured into 2M dilute hydrochloric acid solution (200 mL) and extracted with ethyl acetate (100 mL × 2). The organic phases were combined and washed three times with saturated brine (200 mL). The mixture was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure. After purification by silica gel column chromatography, 1 g of compound 2 was obtained as a yellow solid with a yield of 72.5%.

[0100] The spectral data of compound 2 are as follows: 1H NMR (400 MHz, CDCl3) δ7.81 (d, J = 8.8 Hz, 2H), 7.62 (d, J = 6.4 Hz, 2H), 7.45–7.38 (m, 8H), 7.08 (d, J = 8.8 Hz, 2H), 6.67 (d, J = 2.2 Hz, 1H), 6.60 (s, 1H), 6.48 (d, J = 2.4 Hz, 1H), 5.34–5.30 (m, 2H), 5.26 (s, 2H), 5.19–5.16 (m, 4H), 4.30–4.26 (m, 1H), 4.21–4.17 (m, 1H), 3.95–3.93 (m, 1H), 2.08–2.05 (m, 12H). Figure 4 .

[0101] (3) Synthesis of Compound 3: Compound 2 (1 g, 1.3 mmol, 1.0 equivalent) was dissolved in 20 mL of methanol. Sodium methoxide (277 mg, 5.1 mmol, 4.0 equivalent) was added in batches at room temperature. The reaction was allowed to react at room temperature for 1 hr. The reaction was monitored by TLC. The reaction solution was poured into 2 M dilute hydrochloric acid solution (20 mL) and extracted with ethyl acetate (20 mL × 2). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. After column chromatography, 670 mg of compound 3 was obtained as a light yellow solid with a yield of 85.4%.

[0102] The spectral data of compound 3 are as follows: 1 HNMR(400MHz,MeOD)δ8.02-7.92(m,2H),7.59(d,J=7.4Hz,2H),7.50-7.46(m,2H),7. 44-7.28(m,6H),7.20-7.15(m,2H),6.94(d,J=2.1Hz,1H),6.74(d,J=2.1Hz,1H),6.66 (s, 1H), 5.38-5.26 (m, 2H), 5.21 (s, 2H), 5.06-4.99 (m, 1H), 4.60 (s, 2H), 3.94 (dd, J = 2.1, 12.0 Hz, 1H), 3.71 (dd, J = 6.3, 12.0 Hz, 1H), 3.59-3.47 (m, 3H), 3.44-3.38 (m, 1H). Figure 5 .

[0103] (4) Synthesis of Compound 4: Compound 3 (670 mg, 1.1 mmol, 1.0 equivalent) was dissolved in 5 mL of acetonitrile and 5 mL of N,N-dimethylformamide. Malonic acid (208 mg, 2.2 mmol, 2.0 equivalent) and tert-butyl isocyanide (249 mg, 3.4 mmol, 3.0 equivalent) were added to the solution. The mixture was reacted at 80°C for 8 h. The reaction was monitored by TLC. The reaction solution was poured into 20 mL of water and extracted with ethyl acetate (10 mL × 2). The organic phases were combined and washed three times with saturated brine (20 mL). The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. After purification by column chromatography, 310 mg of compound 4 was obtained as a light yellow solid with a yield of 40.6%.

[0104] The spectral data of compound 4 are as follows: 1 H NMR(400MHz,DMSO-d6)δ7.80(d,J=1.6Hz,1H),7.73(br dd,J=1.8,8.3Hz,1H),7.58-7.53(m,5H),7.50-7.42(m,7H),7.28(d,J=8.4Hz,1H),6.93-6.89(m,1H),6.79( d,J=2.0Hz,1H),6.48(d,J=2.0Hz,1H),5.62-5.42(m,1H),5.40-5.23(m,1H),5.02(d,J=7.1Hz,1H),4.29(br d,J=11.1Hz,1H),4.07(br dd, J = 6.8, 12.0 Hz, 1H), 3.73-3.65 (m, 1H), 3.30-3.25 (m, 2H), 3.23-3.14 (m, 2H), 3.03-2.95 (m, 2H). H NMR spectrum is shown in Figure 6 .

[0105] (5) Synthesis of Compound AMG: Compound 4 (310 mg, 0.4 mmol, 1.0 equivalent) was dissolved in 10 mL of ethanol, and 20 mg of 15% palladium on carbon was added. The mixture was reacted under a hydrogen atmosphere at 15 psi for 16 h at room temperature. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed three times, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was then separated and purified by high-performance liquid phase preparative column to obtain 62 mg of compound AMG as a yellow solid, with a yield of 27.0%.

[0106] Example 4 Evaluation of Pancreatic Lipase Inhibitory Activity of AMG

[0107] Pancreatic lipase allows the breakdown products of dietary fat to be reabsorbed by the body, thereby inhibiting fat production. Most marketed pancreatic lipase inhibitors can cause side effects, such as diarrhea and stomach cramps, leading to gastrointestinal discomfort. Therefore, identifying new pancreatic lipase inhibitors with strong inhibitory activity and high safety from natural food-derived plant species is of great significance.

[0108] The inhibitory activity of active monomers at different concentrations on pancreatic lipase was detected by microplate assay, using orlistat as a positive control and 4-methylumbelliferyl oleate as a substrate. The method is as follows:

[0109] (1) Preparation of buffer solution: Dissolve Tris powder in ultrapure water, adjust the solution pH to 8.0 after constant volume, and obtain a Tris-HCl buffer solution with a system concentration of 13 mM.

[0110] (2) Preparation of compounds: Weigh the compound to be tested and dissolve it in DMSO reagent to obtain a test solution with a final concentration of 20 mM.

[0111] (3) Preparation of pancreatic lipase test solution: The reaction system uses 1 mg / mL pancreatic lipase working solution, that is, after weighing pancreatic lipase powder, add Tris-HCl buffer and place on ice until use.

[0112] (4) Preparation of substrate: 4-Methylumbelliferyl oleate was dissolved in DMSO to form a 5 mM solution, and then diluted with Tris-HCl to form a 0.1 mM substrate solution.

[0113] (5) Preparation of stop solution: Weigh citric acid and sodium citrate, add distilled water to dissolve them respectively, and mix the two solutions to prepare a reaction stop solution with a pH of 4.2 and a concentration of 0.1 mM.

[0114] (6) Pancreatic lipase inhibitory activity test system: Compound test group (A): 50 μL substrate solution + 10 μL test solution + 25 μL enzyme solution + 100 μL sodium citrate + 15 μL Tris-HCl; Compound blank group (B): 50 μL substrate solution + 10 μL test solution + 100 μL sodium citrate + 40 μL Tris-HCl; Control group (C): 50 μL substrate solution + 25 μL enzyme solution + 100 μL sodium citrate + 25 μL Tris-HCl; Blank group (D): 50 μL substrate solution + 100 μL sodium citrate + 50 μL Tris-HCl.

[0115] Tris-HCl buffer, substrate solution, and test solution of varying concentrations were sequentially added to a 96-well microtiter plate and thoroughly mixed. The enzyme solution was then added to initiate the reaction. After incubation at 25°C for 30 minutes, 100 μL of sodium citrate was added to each well to terminate the reaction. The absorbance (OD) was then immediately measured using a microplate reader at 340 nm and 460 nm emission wavelengths. The Tris-HCl buffer was pH 8.0. The results are from three independent experiments, each with two replicates.

[0116] The inhibition rate of pancreatic lipase activity was calculated as follows: inhibition rate / % = 1-(OD A -OD B ) / (OD C -OD D )]×100%.

[0117] Table 1 Effects on inhibition of pancreatic lipase activity

[0118]

[0119] Note: a The data are the mean of three independent experiments. b Orlistat was used as a positive control and the test concentration was 0.08 μM.

[0120] As can be seen from Table 1, the flavonoid AMG has pancreatic lipase inhibitory activity, and the inhibition rate can reach more than 80% at 250 μM, but the inhibitory activity is weaker than that of the positive control orlistat at 0.08 μM.

[0121] Example 5 Effect of AMG on lipid content in HepG2 cells

[0122] First, the cytotoxicity of 250μM and 50μM AMG on HepG2 cells was evaluated. No significant cytotoxicity was observed at these concentrations. Oil Red O staining was then used to evaluate the lipid-lowering activity of AMG at different concentrations. Lovastatin was used as a positive control. The specific method is as follows:

[0123] HepG2 cells in the logarithmic growth phase were taken and cultured in DMEM low-glucose medium at a cell density of 1×10 5The cells were seeded with 2 mL / mL in a 6-well plate, 2 mL per well, and cultured in a 37°C incubator overnight. After the cells adhered, the supernatant was discarded, the cells were washed once with 1×PBS, starved with serum-free DMEM high-glucose medium for 24 hours, washed once with 1×PBS, and HepG2 cells were induced with the prepared long-chain fatty acid (FFAS) inducer to establish a fat accumulation model for 24 hours. A drug control group (inducer + test substance + cells), a negative control group (inducer + DMSO + cells), and a blank control group (DMEM low-glucose medium solution containing only 1% BSA) + a positive control group (inducer + lovastatin + cells) were set up. Three replicate wells were set up for each concentration, and 10 μL of dilution containing different concentrations of the test substance was added to each well. The cells were placed in a carbon dioxide incubator and cultured for another 24 h, then washed three times with 1× PBS, fixed with 2 mL per well of 4% paraformaldehyde for 30 min, washed three times with 1× PBS, and treated with 2 mL per well of 60% isopropanol for 5 min to increase cell permeability. The cells were stained with 2 mL per well of Oil Red O for 1 h in the dark at room temperature. The cells were washed four times with distilled water, and then 1 mL of isopropanol was added to each well for binding for 10 min. The cells were washed out by shaking, and the absorbance was measured at 492 nm using a microplate reader.

[0124] Table 2 Effects on lipid content in HepG2 cells

[0125]

[0126] Note: a The data are the mean of three independent experiments; bLovastatin was used as a positive control.

[0127] As shown in Table 2, flavonoid AMG has the ability to significantly reduce the cell fat content induced by sodium oleate and sodium palmitate at the cellular level, and its effect in reducing fat content is more obvious than that of the positive control lovastatin at the same concentration.

[0128] Example 6 AMG staining experiment on lipid content of HepG2 cells

[0129] The test method is the same as in Example 5, and the results are as follows Figure 7 As shown in the results, AMG still has good lipid-lowering activity at a concentration of 0.2 μM, and the activity at this concentration is comparable to that of 10 μM lovastatin.

[0130] Example 7 Study on the activity of AMG in lowering blood lipids and treating non-alcoholic fatty liver disease in mice

[0131] (1) The in vivo activity evaluation method for hyperlipidemia and non-alcoholic fatty liver disease model mice is as follows:

[0132] (a) Establishment of mouse model

[0133] The experimental animals were three-week-old male Kunming mice purchased from Beijing Weitonglihua Animal Experiment Technology Co., Ltd. Tyloxapol is a non-ionic surfactant and oligomerization inducer that can cause dyslipidemia and liver damage. Tyloxapol can increase TC, TG, and LDL-C levels, reduce HDL-C levels, and increase ALT and AST activities, indicating the formation of fatty liver. In addition, tyloxapol can also inhibit the activity of lipase, thereby affecting the clearance of TG, and can be used to induce hyperlipidemia in mice. Therefore, when mice are injected with tyloxapol for 24 hours, the blood lipid level in the body rises to the highest level, which can cause a large amount of fat to accumulate in the liver in a short period of time, causing a significant increase in blood lipid levels. It can be used to screen drugs for the treatment of fatty liver.

[0134] In the experiment, AMPK (Adenosine 5'-monophosphate (AMP)-activated protein kinase), an AMP-dependent protein kinase, is a key molecule in regulating biological energy metabolism; AICAR (5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside), an AMPK activator, is used to study the mechanism of action of this model.

[0135] (b) Drug preparation

[0136] The compound was first dissolved in 5% Tween 80 solvent volume, and then physiological saline was added to make up to the various concentrations. The compound was shaken until it was dissolved without precipitation.

[0137] (c) Animal grouping

[0138] After one week of adaptive feeding with a normal diet, 42 mice were randomly divided into seven groups, with six mice in each group. These groups included a normal mouse group, a model mouse group (tyloxapol + an equal volume of diluent), a high-dose group (tyloxapol + AMG 200 mg / kg), a medium-dose group (tyloxapol + AMG 100 mg / kg), a low-dose group (tyloxapol + AMG 50 mg / kg), a positive control group (tyloxapol + fenofibrate 100 mg / kg), and an AMPK-positive control group (tyloxapol + AICAR 100 mg / kg).

[0139] (d) Mouse experimental process

[0140] Both the model and treatment groups were induced with intraperitoneal injection of 500 mg / kg tyloxapol (0.2 mL) per mouse. Approximately 30 minutes later, the model group mice were administered an equal volume of tyloxapol solvent. The treatment group mice were gavaged with an oral volume of 0.1 mL / 10 g (0.2 mL per mouse). The high-, medium-, and low-dose groups received AMG 200 mg / kg, 100 mg / kg, and 50 mg / kg, respectively. The positive control group received fenofibrate 100 mg / kg and AICAR 100 mg / kg, respectively. Twenty-four hours later, the mice were bled by eye removal. Plasma was allowed to rest on ice for a period of time and then centrifuged at 3500 rpm for 10 minutes to collect serum. Tissues, including the liver, spleen, and epididymal fat, were rapidly dissected and cleaned with saline until no blood was visible on the surface of the organs. The organs were then blotted dry with filter paper and weighed. A portion of the liver from each group of mice was taken from the hepatic lobule and immersed in 4% paraformaldehyde fixative for subsequent liver section staining with H&E and Oil Red O. The remaining liver, spleen, epididymal white fat, and other tissues were stored in a -80°C freezer until use.

[0141] (2) Effects of AMG on various indicators in mouse serum

[0142] Blood lipid levels are closely related to cardiovascular metabolic diseases. Increased levels of TG, TC, and LDL-C, and decreased HDL-C often indicate the presence of dyslipidemia. As shown in Table 3, the high, medium, and low dose groups of AMG administration were 200, 100, and 50 mg / kg, respectively, fenofibrate was 100 mg / kg, and AICAR was 100 mg / kg. Compared with the normal group of mice, the model group mice had significantly increased levels of TG, TC, and LDL-C in the serum, and significantly decreased levels of HDL-C, showing the distinct characteristics of hyperlipidemia. After administration, serum levels of TC, TG, and LDL-C decreased, while HDL-C levels increased, and related indicators were restored. The results of the administration showed that the TC and TG levels of the model group mice were approximately 5 times and 8 times higher than those of the normal group, respectively. After administration of the extract at 200, 100, and 50 mg / kg, TC and TG levels were significantly restored, with a stronger effect than the positive control fenofibrate.

[0143] Furthermore, elevated ALT is a key marker of acute liver cell damage. AST levels are normally low in the blood. Once tissues and organs such as the liver are damaged or diseased, the body releases more AST into the blood. As shown in Table 3-9, compared with the normal group, serum ALT and AST levels in the model group mice were significantly elevated, indicating severe liver cell damage. Treatment with the drug significantly reduced serum ALT and AST levels in the mice, indicating recovery from liver damage.

[0144] Table 3 Effects of AMG on serum TG, TC, LDL-C, HDL-C, ALT and AST in mice

[0145]

[0146]

[0147] Note: Data are mean ± SEM (n = 6). Compared with the model group, there were significant differences: ***P < 0.001, **P < 0.05, *P < 0.01.

[0148] (2) Effect of AMG on serum SOD activity in mice

[0149] Superoxide dismutase (SOD) is an important enzyme molecule that has the effect of scavenging free radicals in the human body. Its activity level can reflect the body's ability to eliminate free radicals. Low SOD content in the body means that the body's ability to eliminate harmful superoxide anion free radicals is reduced, which will cause damage to the liver cell membrane and lead to liver cell degeneration and necrosis. Since SOD can dismutate O2 into H2O2, once O 2- Produced in large quantities in the body, SOD rapidly and specifically captures O 2- , forming the first line of defense against free radical damage to the body. Therefore, SOD can effectively reduce the free radical content in the blood, reduce the burden on the liver, and improve liver function. Figure 8 As shown in the results, after tyloxapol administration, the SOD activity level in the model group decreased significantly, while the SOD activity in the AMG 200 mg / kg administration group was significantly improved, indicating that it has a significant effect on improving blood lipids in hyperlipidemic mice, has significant lipid-lowering activity and antioxidant capacity, and can reduce the burden on the liver to a certain extent.

[0150] (3) Effect of AMG on liver steatosis in mice

[0151] like Figure 9 As shown in the figure, the liver morphology and appearance of the normal group mice were obviously darker in color. The liver color and state of the model group after tyloxapol stimulation were yellower and the liver was obviously enlarged compared with the normal group. After administration of AMG, the liver returned to normal color, and the liver index also gradually returned to normal with the administration concentration ( Figure 10 ), indicating that AMG has the effect of alleviating the liver color, morphology and liver index of fatty liver; in addition, liver HE staining ( Figure 11 ), the liver lobule cells of the normal group were arranged regularly, while the liver cells of the model group were arranged irregularly, and the inflammatory cell infiltration and fatty degeneration were significantly aggravated, with a large number of white fat vacuoles; by staining the liver lobule sections with oil red O ( Figure 12) found that the liver lobule structure in the normal group was normal, while the liver of the model group had a large amount of red lipid accumulation. After administration, the pathological state of the liver lobule structure was significantly improved. Even at a concentration of 200 mg / mL, the effect on the liver was stronger than that of the positive control group, tyloxapol.

[0152] (4) Effects on biochemical indicators in the liver

[0153] As shown in Table 4, compared with the normal group, the model group mice showed slightly elevated levels of TG, TC, and LDL-C in the liver, while HDL-C levels decreased. Administration of high, medium, and low doses of AMG (200, 100, and 50 mg / kg) decreased TC, TG, and LDL-C levels in the liver, while increasing HDL-C levels. Furthermore, compared with the normal group, the model group mice showed significantly elevated levels of ALT and AST in the liver, which were significantly reduced after compound administration.

[0154] Table 4 Effects of AMG on TG, TC, LDL-C, HDL-C, ALT and AST in mouse liver

[0155]

[0156]

[0157] Note: Data are mean ± SEM (n = 6). Compared with the model group, there were significant differences: ***P < 0.001, **P < 0.05, *P < 0.01.

[0158] (5) Effects on MPO activity in the liver

[0159] Myeloperoxidase (MPO) in the liver is an enzyme related to the development of liver disease. Inflammation or liver disease in the body will increase the activity of MPO. Experiments have confirmed that the activity of MPO in the liver of patients with fatty liver increases. Figure 12 It can be seen that the extract has a significant reducing effect on MPO levels after administration. The reduction in MPO levels means that AMG can play a positive role in the regulation of liver inflammation.

[0160] (6) Effects on inflammatory factor IL-6 in the liver

[0161] A series of pathological conditions induced by NAFLD will lead to changes in the expression of many related inflammatory factors in the body, including interleukin 6 (IL-6), an important inflammatory factor that has both anti-inflammatory and pro-inflammatory properties. IL-6 can act as an important metabolic regulator in lipid metabolism. It can mediate inflammatory reactions, participate in the inflammatory damage process, and further damage liver cells. Increased levels of inflammatory factors are a common phenomenon in liver damage, and their expression gradually increases as the degree of fatty liver gradually worsens. IL-6 has the effect of accelerating protein synthesis in liver cells in emergency situations, and its increased level is related to the liver's decreased ability to clear inflammatory factors. For example Figure 13 As shown in the results, after administration of 200, 100 and 50 mg / kg AMG, IL-6 levels decreased by 23.68%, 22.44 and 18.03%, respectively, indicating that it can slow down the increase of inflammatory factors caused by liver damage, indicating that it has the effect of treating non-alcoholic fatty liver inflammation and liver damage.

[0162] (7) Effects of AMG on epididymal fat in mice

[0163] like Figure 14 As shown in the results, compared with the normal group, the epididymal fat in the model group increased significantly. After administration of AMG, the size of epididymal white adipose tissue was reduced. Analysis of the epididymal white adipose tissue index (ratio to body weight) of mice showed that high dose (200 mg / kg) significantly improved the increase of epididymal white fat in mice ( Figure 15 ).

[0164] These results demonstrate that AMG inhibits pancreatic lipase activity in vitro and reduces intracellular lipid accumulation. In vivo, it significantly reduces elevated serum and liver biochemical markers such as triglycerides (TG), TC, LDL-C, ALT, and AST, alleviates the decrease in HDL-C, and improves hyperlipidemia to a certain extent. It also increases serum SOD activity in mice and improves the elevated activity of liver inflammatory factors IL-6 and MPO caused by fat accumulation. It also restores liver morphology, steatosis, inflammation, and oxidative damage in mice. Furthermore, it improves the enlargement of epididymal adipose tissue. Therefore, these in vitro and in vivo studies indicate that AMG has a positive impact on the treatment of hyperlipidemia and non-alcoholic fatty liver disease, posing a valuable opportunity for further development and application.

[0165] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for preparing flavonoid glycosides from Chrysanthemum morifolium, characterized by: The synthetic route of the preparation method of the flavonoid glycosides is: The flavonoid glycoside is apigenin 7-O-(6"-O-malonyl)-β-D-glucoside, and its structural formula is:

2. The preparation method according to claim 1, wherein: The steps include: (1) Synthesis of Compound 1: Apigenin and silver carbonate were dissolved in quinoline, acetyl bromide-α, D-glucose was added at room temperature, and the mixture was reacted at 50°C for 0.5 h. The reaction was completed when the starting material disappeared after TLC monitoring. The reaction solution was poured into 6M hydrochloric acid solution and extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Compound 1 was obtained after purification by silica gel column chromatography as a yellow solid with a yield of 40.3%. The ratio of apigenin: silver carbonate: quinoline: acetyl bromide-α,D-glucose: hydrochloric acid solution is 2.7:5.5:100:4.5:200 in g:g:mL:g:mL; (2) Synthesis of Compound 2: Compound 1 and potassium carbonate were dissolved in N,N-dimethylformamide, benzyl bromide was added, and the mixture was reacted at 50°C for 3 h. The reaction was monitored by TLC. The reaction solution was poured into a 2M dilute hydrochloric acid solution and extracted with ethyl acetate. The organic phases were combined and washed three times with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Compound 2 was purified by silica gel column chromatography to obtain a yellow solid with a yield of 72.5%. The ratio of compound 1: potassium carbonate: N, N-dimethylformamide: benzyl bromide: dilute hydrochloric acid solution (g:g:mL:g:mL) is 1.4:1.3:20:0.8:200; (3) Synthesis of Compound 3: Compound 2 was dissolved in methanol, sodium methoxide was added at room temperature, and the reaction was allowed to proceed at room temperature for 1 hour. The reaction was monitored by TLC. The reaction solution was poured into a 2M dilute hydrochloric acid solution and extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Compound 3 was obtained after column chromatography separation and purification as a light yellow solid with a yield of 85.4%. Among them, the ratio of compound 2: methanol: sodium methoxide: dilute hydrochloric acid solution is 1:20:277:20 in g:mL:mg:mL; (4) Synthesis of Compound 4: Compound 3 was dissolved in acetonitrile and N,N-dimethylformamide, malonic acid and tert-butyl isocyanide were added to the solution, and the mixture was reacted at 80°C for 8 h. The reaction was monitored by TLC. The reaction solution was poured into water and extracted with ethyl acetate. The organic phases were combined and washed three times with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Compound 4 was obtained after column chromatography purification as a light yellow solid with a yield of 40.6%. Among them, the ratio of compound 3: acetonitrile: N,N-dimethylformamide: malonic acid: tert-butyl isocyanide: water in mg:mL:mL:mg:mg:mL is 670:5:5:208:249:20; (5) Synthesis of apigenin 7-O-(6"-O-malonyl)-β-D-glucoside: Compound 4 was dissolved in ethanol, and 5% to 15% palladium carbon was added. The mixture was reacted at room temperature under a pressure of 15 psi in a hydrogen atmosphere for 16 h. The reaction solution was filtered through diatomaceous earth, the filter cake was rinsed three times, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was then separated and purified by a high-performance liquid phase preparative column to obtain the compound apigenin 7-O-(6"-O-malonyl)-β-D-glucoside as a yellow solid with a yield of 27.0%. The ratio of compound 4: ethanol: palladium carbon in mg: mL: mg is 310:10:

20.

3. Use of a flavonoid glycoside from Goldenrod in the preparation of a drug for treating hyperlipidemia and / or fatty liver; the flavonoid glycoside is apigenin 7-O-(6"-O-malonyl)-β-D-glucoside derived from Goldenrod, and has the structural formula:

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