A natural flavonoid glycoside and its application in preparing medicine for treating glucose and lipid metabolism disorder

Abstract

The invention discloses a natural flavonoid glycoside. The Chinese name of the natural flavonoid glycoside is luteolin 7-O-(6"-O-malonyl)-β-D-glucoside, the English name is Luteolin-7-O-6"-malonylglucoside, and the structural formula is: The invention synthesizes the compound for the first time through a 5-step synthesis process with a total yield of 2.5%, and finds for the first time that the compound has the activity of reducing hyperlipidemia and hyperglycemia in vitro and in vivo. Therefore, the compound can be used in the preparation of products for treating hyperlipidemia, hyperglycemia and other related diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of natural medicines, in particular to a natural flavonoid glycoside and its application in preparing medicines for treating glucose and lipid metabolism disorders. Background Art

[0002] Luteolin 7-O-(6”-O-malonylglucoside)-β-D-glucoside, English name: Luteolin-7-O-6”-malonylglucoside (hereinafter referred to as LMG), CAS: 98767-38-5. It is reported that LMG has been isolated from plants such as Cynara cardunculus L., Celery, Chrysanthemum, cardoon leaves, Tunisian globe artichoke, lupin, Osmanthus fragranslour, and Bryum capillare. This compound has antioxidant, anti-inflammatory, anti-herpetic, anti-tumor cell proliferation, and antimicrobial activities, and can be used to treat arthritis and autoimmune diseases, and can also be used as a stimulant for black swallowtail butterfly egg-laying. So far, there are no reports on the chemical synthesis of this compound, as well as reports on its hypoglycemic and / or hypolipidemic activities.

[0003] The structural formula of luteolin 7-O-(6”-O-malonyl)-β-D-glucoside is as follows:

[0004]

[0005] Through searching, the following public documents related to the patent application of the present invention are found:

[0006] 1. Journal article: Identification of major flavonoids in petals 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 was found to have antioxidant activity and activity to alleviate carbon tetrachloride-induced liver injury. Carbon tetrachloride-induced liver injury is a stress disease that is essentially different from hyperlipidemia, such as liver phenotype, fatty degeneration, liver inflammation, and overall effects on blood fat content in organisms.

[0007] 2. Journal article: Phenolic acids and flavonoids in leaf and floral stem of cultivated and wild Cynara cardunculus L. genotypes. Food Chemistry (2011), 126 (2), 417-422. This article reports the natural extraction and separation method, and that the compound has antioxidant activity. This article is only an in vitro experiment of antioxidant activity, and there is no report on the treatment of related diseases.

[0008] In the prior art, luteolin 7-O-(6”-O-malonyl)-β-D-glucoside has antioxidant, anti-inflammatory, anti-herpes, anti-tumor cell proliferation, anti-microbial and other activities, and can be used to treat arthritis and autoimmune diseases, and can also be used as a stimulant for black swallowtail butterfly egg-laying, but there are no reports on its activity in lowering blood lipids and blood sugar. In addition, apigenin 7-O-(6”-O-malonyl)-β-D-glucoside has been isolated from a variety of plants, but there are no reports on its chemical synthesis. Summary of the invention

[0009] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a natural flavonoid glycoside and its application in the preparation of drugs for treating glucose and lipid metabolism disorders.

[0010] The technical solution adopted by the present invention to solve its technical problem is:

[0011] A natural flavonoid glycoside, the Chinese name of the natural flavonoid glycoside is: Luteolin 7-O-(6"-O-malonyl)-β-D-glucoside, the English name is: Luteolin-7-O-6"-malonylglucoside, and the structural formula is:

[0012]

[0013] Furthermore, the luteolin 7-O-(6"-O-malonyl)-β-D-glucoside can inhibit pancreatic lipase in vitro, reduce cellular lipid accumulation, and has lipid-lowering activity; the compound can promote cellular glucose consumption, alleviate cellular insulin resistance, and also has blood sugar-lowering effect; the compound can also reduce hyperglycemia and hyperlipidemia symptoms in mice.

[0014] Furthermore, the natural flavonoid glycoside is obtained by using luteolin as a starting material through a 5-step reaction.

[0015] Furthermore, the natural flavonoid glycoside is chemically synthesized using luteolin as a raw material, with a total yield of 2.5%.

[0016] Furthermore, the synthetic route of the chemical synthesis is:

[0017]

[0018] Furthermore, the chemical synthesis method comprises the following steps:

[0019] (1) Synthesis of Compound 1: Luteolin and dichlorodiphenylmethane were dissolved in diphenyl ether, and the reaction solution was placed in an oil bath at 180°C for 2 hours; after the reaction was completed, the reaction solution was poured into water, first extracted with petroleum ether, and the aqueous phase after extraction was extracted three times with ethyl acetate. The organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Compound 1 was obtained after column chromatography purification as a yellow solid with a yield of 50.8%;

[0020] Among them, the ratio of luteolin: dichlorodiphenylmethane: diphenyl ether g: g: mL is 1: 1.2: 20;

[0021] (2) Synthesis of Compound 2: Compound 1, potassium carbonate and tetrabutylammonium bromide were dissolved in N,N-dimethylformamide (DMF), acetyl bromide-α,D-glucose was added, and the mixture was reacted in a microwave reactor at 50°C for 1 h. The reaction was monitored by thin layer chromatography (TLC) until completion; the reaction solution was poured into a 2M dilute hydrochloric acid solution, extracted with ethyl acetate, the organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and purified by column chromatography to obtain Compound 2 as a light yellow solid with a yield of 62.2%;

[0022] Wherein, the ratio of compound 1: potassium carbonate: tetrabutylammonium bromide: N,N-dimethylformamide: acetyl bromide-α,D-glucose: dilute hydrochloric acid solution g: mg: mg: mL: mg: mL is 0.8:483:10:10:779:40;

[0023] (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 for 1 h at room temperature. The reaction was monitored by TLC until completion. The reaction solution was poured into a 2M dilute hydrochloric acid solution, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Compound 3 was purified by column chromatography as a light yellow solid with a yield of 88.2%.

[0024] Among them, the ratio of compound 2: methanol: sodium methoxide: dilute hydrochloric acid solution is 420:10:114:10 in mg:mL:mg:mL;

[0025] (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 in sequence, and the mixture was reacted at 80°C for 8 hours. The reaction was monitored by TLC to be complete. 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 then evaporated under reduced pressure. Compound 4 was obtained after column chromatography purification as a white solid with a yield of 35.7%.

[0026] Among them, the ratio of compound 3: acetonitrile: N,N-dimethylformamide: malonic acid: tert-butyl isocyanide: water in mg: mL: mg: mg: mL is 270: 5: 5: 76: 175: 10;

[0027] (5) Synthesis of compound luteolin 7-O-(6"-O-malonyl)-β-D-glucoside: Compound 4 was dissolved in ethanol, 15% palladium carbon was added, and the reaction was carried out at room temperature for 16 hours under a hydrogen environment at a pressure of 15 psi; the reaction solution was filtered, the filter cake was washed three times, and the filtrate was concentrated under reduced pressure to obtain a crude product; the crude product was separated by a high-performance liquid phase preparative column to obtain compound luteolin 7-O-(6"-O-malonyl)-β-D-glucoside as a yellow solid with a yield of 25.0%, i.e., a natural flavonoid glycoside was obtained;

[0028] Among them, the ratio of compound 4: ethanol: palladium carbon in mg: mL: mg is 110:10:10.

[0029] The use of the natural flavonoid glycosides as described above in the preparation of drugs for treating glucose and lipid metabolism disorders.

[0030] The use of the natural flavonoid glycosides as described above in the preparation of medicines and / or functional foods for treating hyperlipidemia and / or hyperlipidemia.

[0031] The advantages and positive effects achieved by the present invention are:

[0032] 1. The present invention synthesizes the compound for the first time through a 5-step synthesis process with a total yield of 2.5%, and discovers for the first time that the compound has the activity of reducing hyperlipidemia and hyperglycemia in vitro and in vivo. Therefore, the compound can be used in the preparation of products for treating hyperlipidemia, hyperglycemia and other related diseases.

[0033] 2. The present invention synthesized the natural product LMG for the first time through a 5-step reaction, and evaluated the hypoglycemic and hypolipidemic activities of the compound in vitro and in vivo. The results showed that the compound could inhibit pancreatic lipase and reduce cellular lipid accumulation in vitro, indicating that the compound had hypolipidemic activity; the compound could promote cellular glucose consumption and alleviate cellular insulin resistance, indicating that the compound also had hypoglycemic efficacy. In addition, further in vivo experiments in mice confirmed that the compound could reduce the symptoms of hyperglycemia and hyperlipidemia. Therefore, the natural product can be chemically synthesized and has hypoglycemic and hypolipidemic activities in vivo and in vitro, indicating that the compound has good application prospects.

[0034] 3. The present invention realizes the first chemical synthesis and provides a new preparation method, which can provide compounds of sufficient quality for the in-depth study of subsequent drugs and / or functional foods. Therefore, luteolin 7-O-(6"-O-malonyl)-β-D-glucoside can be used as a functional food and / or drug for treating hyperlipidemia and / or hyperglycemia. The preparation method is simple and feasible, and it has the characteristics of low food-borne toxicity and side effects, and has huge market potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the synthetic intermediate 1 in the present invention;

[0036] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of the synthetic intermediate 2 in the present invention;

[0037] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of the synthetic intermediate 3 in the present invention;

[0038] Figure 4 is the hydrogen nuclear magnetic resonance spectrum of the synthetic intermediate 4 in the present invention;

[0039] Figure 5 is the LMG hydrogen nuclear magnetic resonance spectrum of the present invention;

[0040] Figure 6 It is the LMG carbon nuclear magnetic resonance spectrum of the present invention;

[0041] Figure 7This is a graph showing the effect of LMG on the triglyceride (TG) content in HepG2 cells in the present invention; compared with the model group, ***P<0.001;

[0042] Figure 8 The figure is the effect of LMG on insulin resistance of HepG2 cells in the present invention; compared with the model group,

[0043] ***P < 0.01, **P < 0.05;

[0044] Fig. 9 This is a graph showing the effect of LMG on postprandial blood glucose in mice; compared with the model group, **P<0.05, *P<0.01;

[0045] Fig.10 The figure is the area under the curve of LMG on postprandial blood glucose in mice in the present invention; compared with the model group, **P<0.05, *P<0.01. DETAILED DESCRIPTION

[0046] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0047] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0048] A natural flavonoid glycoside, the Chinese name of the natural flavonoid glycoside is: Luteolin 7-O-(6"-O-malonyl)-β-D-glucoside, the English name is: Luteolin-7-O-6"-malonylglucoside, and the structural formula is:

[0049]

[0050] Preferably, the luteolin 7-O-(6"-O-malonyl)-β-D-glucoside can inhibit pancreatic lipase in vitro, reduce cellular lipid accumulation, and has lipid-lowering activity; the compound can promote cellular glucose consumption, alleviate cellular insulin resistance, and also has blood sugar-lowering effect; the compound can also improve hyperglycemia and hyperlipidemia symptoms in mice.

[0051] Preferably, the natural flavonoid glycoside is obtained by a 5-step reaction using luteolin as a starting material.

[0052] Preferably, the natural flavonoid glycoside is chemically synthesized using luteolin as a raw material, with a total yield of 2.5%.

[0053] Preferably, the synthetic route of the chemical synthesis is:

[0054]

[0055] Preferably, the chemical synthesis method comprises the following steps:

[0056] (1) Synthesis of Compound 1: Luteolin and dichlorodiphenylmethane were dissolved in diphenyl ether, and the reaction solution was placed in an oil bath at 180°C for 2 hours; after the reaction was completed, the reaction solution was poured into water, first extracted with petroleum ether, and the aqueous phase after extraction was extracted three times with ethyl acetate. The organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Compound 1 was obtained after column chromatography purification as a yellow solid with a yield of 50.8%;

[0057] Among them, the ratio of luteolin: dichlorodiphenylmethane: diphenyl ether g: g: mL is 1: 1.2: 20;

[0058] (2) Synthesis of Compound 2: Compound 1, potassium carbonate and tetrabutylammonium bromide were dissolved in N,N-dimethylformamide (DMF), acetyl bromide-α,D-glucose was added, and the mixture was reacted in a microwave reactor at 50°C for 1 h. The reaction was monitored by thin layer chromatography (TLC) until completion; the reaction solution was poured into a 2M dilute hydrochloric acid solution, extracted with ethyl acetate, the organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and purified by column chromatography to obtain Compound 2 as a light yellow solid with a yield of 62.2%;

[0059] Wherein, the ratio of compound 1: potassium carbonate: tetrabutylammonium bromide: N,N-dimethylformamide: acetyl bromide-α,D-glucose: dilute hydrochloric acid solution g: mg: mg: mL: mg: mL is 0.8:483:10:10:779:40;

[0060] (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 for 1 h at room temperature. The reaction was monitored by TLC until completion. The reaction solution was poured into a 2M dilute hydrochloric acid solution, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Compound 3 was purified by column chromatography as a light yellow solid with a yield of 88.2%.

[0061] Among them, the ratio of compound 2: methanol: sodium methoxide: dilute hydrochloric acid solution is 420:10:114:10 in mg:mL:mg:mL;

[0062] (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 in sequence, and the mixture was reacted at 80°C for 8 hours. The reaction was monitored by TLC to be complete. 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 then evaporated under reduced pressure. Compound 4 was obtained after column chromatography purification as a white solid with a yield of 35.7%.

[0063] Among them, the ratio of compound 3: acetonitrile: N,N-dimethylformamide: malonic acid: tert-butyl isocyanide: water in mg: mL: mg: mg: mL is 270: 5: 5: 76: 175: 10;

[0064] (5) Synthesis of compound luteolin 7-O-(6"-O-malonyl)-β-D-glucoside: Compound 4 was dissolved in ethanol, 15% palladium carbon was added, and the reaction was carried out at room temperature for 16 hours under a hydrogen environment at a pressure of 15 psi; the reaction solution was filtered, the filter cake was washed three times, and the filtrate was concentrated under reduced pressure to obtain a crude product; the crude product was separated by a high-performance liquid phase preparative column to obtain compound luteolin 7-O-(6"-O-malonyl)-β-D-glucoside as a yellow solid with a yield of 25.0%, i.e., a natural flavonoid glycoside was obtained;

[0065] Among them, the ratio of compound 4: ethanol: palladium carbon in mg: mL: mg is 110:10:10.

[0066] The use of the natural flavonoid glycosides as described above in the preparation of drugs for treating glucose and lipid metabolism disorders.

[0067] The use of the natural flavonoid glycosides as described above in the preparation of medicines and / or functional foods for treating hyperlipidemia and / or hyperlipidemia.

[0068] Specifically, the relevant preparation and detection are as follows:

[0069] Example 1 Study on the Synthesis Process of Compound LMG

[0070] The present invention fully synthesized LMG for the first time, using luteolin as the starting material, through 5 steps of reaction, with a total yield of 2.5%. The synthesis was obtained through the following route:

[0071]

[0072] The specific steps include:

[0073] (1) Synthesis of Compound 1: Luteolin (1 g, 3.5 mmol, 1.0 equivalent) and dichlorodiphenylmethane (1.2 g, 5.2 mmol, 1.5 equivalent) were dissolved in 20 mL of diphenyl ether, and the reaction solution was placed in an oil bath at 180°C for 2 h. After the reaction was completed, the reaction solution was poured into water and first extracted with 200 mL of petroleum ether. The aqueous phase after extraction was extracted three times with 40 mL of ethyl acetate. The organic phases were combined and washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. After purification by column chromatography, 0.8 g of compound 1 was obtained as a yellow solid with a yield of 50.8%.

[0074] The spectrum data of compound 1 are as follows: 1 H NMR (400 MHz, DMSO-d 6 )δ12.86(s,1H),10.92(br s,1H),7.79(d,J=1.8Hz,1H),7.71(dd,J=1.8,8.3Hz,1H),7.59-7.53(m,4H),7.51-7.44(m,6H),7.23(d,J=8.4Hz,1H),6.89(s,1H),6.52(d,J=2.1Hz,1H),6.20(d,J=2.1Hz,1H). H NMR spectrum is shown in Figure 1 .

[0075] (2) Synthesis of Compound 2: Compound 1 (0.8 g, 1.7 mmol, 1.0 equivalent), potassium carbonate (483 mg, 3.5 mmol, 2.0 equivalent) and tetrabutylammonium bromide (10 mg) were dissolved in 10 mL of N,N-dimethylformamide (DMF), and acetyl bromide-α,D-glucose (779 mg, 1.9 mmol, 1.1 equivalent) was added. The reaction was carried out in a microwave reactor at 50°C for 1 h. The reaction was monitored by thin layer chromatography (TLC). The reaction solution was poured into a 2M dilute hydrochloric acid solution (40 mL), extracted with ethyl acetate (20 mL×2), the organic phases were combined and washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. 420 mg of Compound 2 was obtained by column chromatography as a light yellow solid with a yield of 62.2%.

[0076] The spectrum data of compound 2 are as follows: 1 H NMR (400 MHz, CDCl 3)δ12.79(s,1H),7.61-7.57(m,3H),7.46(dd,J=1.8,8.3Hz,1H),7.43-7.37( m,6H),7.00(d,J=8.3Hz,1H),6.59-6.53(m,2H),6.45(d,J=2.1Hz,1H),5.41 -5.27(m,3H),5.23-5.09(m,2H),4.33-4.26(m,1H),4.24-4.18(m,1H),3.95 (ddd,J=2.4,5.9,10.0Hz,1H),2.13-2.10(m,3H),2.09-2.05(m,9H),2.03(br d, J = 4.5 Hz, 1H). H NMR spectrum is shown in Figure 2 .

[0077] (3) Synthesis of compound 3: Compound 2 (420 mg, 0.5 mmol, 1.0 equivalent) was dissolved in 10 mL of methanol, and sodium methoxide (114 mg, 2.1 mmol, 4.0 equivalent) was added in batches at room temperature. The reaction was allowed to react for 1 h at room temperature, and the reaction was completed after monitoring by TLC. The reaction solution was poured into a 2M dilute hydrochloric acid solution (10 mL), extracted with ethyl acetate (5 mL × 2), the organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Column chromatography was used to purify to obtain 270 mg of compound 3 as a light yellow solid with a yield of 88.2%.

[0078] The spectrum data of compound 3 are as follows: 1 H NMR (400MHz, MeOD) δ=7.65-7.55 (m, 6H), 7.42 (brd, J=5.3Hz, 6H), 7.06 (d, J=8.1Hz, 1H), 6.82 (s, 1H), 6.68 (s, 1H), 6.49 (s, 1H), 5.12-5.02 (m, 1H), 3.94 (br d, J=10.6Hz, 1H), 3.72 (dd, J=5.8, 12.1Hz, 1H), 3.61-3.47 (m, 3H), 3.45-3.37 (m, 1H). H NMR spectrum is shown in Figure 3 .

[0079] (4) Synthesis of compound 4: Compound 3 (270 mg, 0.4 mmol, 1.0 equivalent) was dissolved in 5 mL of acetonitrile and 5 mL of N,N-dimethylformamide, and malonic acid (76 mg, 0.8 mmol, 2.0 equivalent) and tert-butyl isocyanide (175 mg, 2.4 mmol, 3.0 equivalent) were added to the solution in sequence, and the mixture was reacted at 80°C for 8 h. The reaction was monitored by TLC. The reaction solution was poured into 10 mL of water and extracted with ethyl acetate (10 mL × 2). The organic phases were combined and washed three times with saturated brine (10 mL), dried over anhydrous sodium sulfate, and then evaporated under reduced pressure. After purification by column chromatography, 110 mg of compound 4 was obtained as a white solid with a yield of 35.7%.

[0080] The spectrum data of compound 4 are as follows: 1 H NMR (400 MHz, DMSO-d 6 )δ=7.80(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 4 .

[0081] (5) Synthesis of compound LMG: Compound 4 (110 mg, 0.2 mmol, 1.0 equivalent) was dissolved in 10 mL of ethanol, and 10 mg of 15% palladium carbon was added. The mixture was reacted at room temperature for 16 h in a hydrogen atmosphere under a pressure of 15 psi. The reaction solution was filtered, the filter cake was washed three times, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated by a high-performance liquid phase preparative column to obtain 21 mg of compound LMG as a yellow solid with a yield of 25.0%.

[0082] The LMG nuclear magnetic resonance hydrogen spectrum, carbon spectrum and mass spectrum data are as follows: 1H NMR (400MHz, MeOD) δ7.46-7.37(m,2H),7.12-7.04(m,1H),6.92(d,J=8.9Hz,1H),6. 77(d,J=2.1Hz,1H),6.61(s,1H),6.51(d,J=2.1Hz,1H),5.09-5.04(m,1H),4.54(br dd,J=1.6,11.9Hz,1H),4.31(dd,J=6.8,11.8Hz,1H),3.84-3.74(m,1H),3.54-3.48(m,2H),3.44-3.38(m,2H). 13 C NMR(400MHz,MeOD)δ184.1(C-4),168.9(COOH),167.0(COOR),164.5(C-2),162. 8(C-7),158.9(C-5),151.1(C-4'),147.0(C-9),123.5(C-2',6'),120.6(C-1') ,116.9(C-3'),114.4(C-5'),107.1(C-l0),104.2(C-3),101.4 / 101.1(C-6,Gl) ,96.3(C-8),77.7(G-3),75.5(G-5),74.7(G-2),71.4(G-4),65.4(G-6),48.4(CH 2 )。 MS (ESI): m / z [M+H + ]=535.1. H NMR spectrum Figure 5 , NMR carbon spectrum see Figure 6 .

[0083] Example 2 Evaluation of LMG pancreatic lipase inhibitory activity

[0084] Lipase is an enzyme necessary for the gastrointestinal tract to break down fat. It can bind to the serine residues of gastric and pancreatic lipase, inactivating lipase and preventing it from breaking down fat in food into free fatty acids, thereby inhibiting the utilization and absorption of fat. Therefore, lipase inhibitors can promote the utilization and absorption of fat.

[0085] The present invention adopts a microplate method to detect the inhibitory activity of LMG of different concentrations on pancreatic lipase, with orlistat as a positive control and 4-methylumbelliferyl oleate as a substrate, and the method is as follows:

[0086] (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.

[0087] (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.

[0088] (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 for use.

[0089] (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.

[0090] (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 value of 4.2 and a concentration of 0.1 mM.

[0091] (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.

[0092] Tris-HCl buffer, substrate solution and different concentrations of the test solution were added in sequence, placed in a 96-well ELISA plate, mixed thoroughly, and then the enzyme solution was 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, and then the absorbance (OD) was immediately measured with an ELISA reader at 340 nm and 460 nm emission wavelengths. The pH value of Tris-HCl buffer was 8.0, and the experimental results were obtained from 3 independent experiments, each with 2 parallels.

[0093] The inhibition rate of pancreatic lipase activity was calculated as follows: Inhibition rate / % = 1-(OD A -OD B ) / (OD C -OD D )]×100%.

[0094] Table 1 Effect of LMG on the inhibition of pancreatic lipase activity

[0095]

[0096] Note: a The data are the average of three independent experiments.b Orlistat was used as a positive control and the tested concentration was 0.08 μM.

[0097] As can be seen from Table 1, LMG has a certain pancreatic lipase inhibitory activity, and the inhibition rate can reach 78.3% at 250 μM.

[0098] Example 3 Effect of LMG on lipid content in HepG2 cells

[0099] First, the cytotoxicity of 250μM and 50μM LMG on HepG2 cells was evaluated. There was no obvious cytotoxicity at this test concentration. Then, the activity of different concentrations of LMG in reducing fat content at the cellular level was evaluated by Oil Red O staining. Lovastatin was used as a positive control. The specific method is as follows:

[0100] HepG2 cells in the logarithmic growth phase were taken and cultured in DMEM low-glucose medium at a cell density of 1×10 5 The cells were inoculated with 2 mL / mL in a 6-well plate, 2 mL in each 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 h, 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 h. 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 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 continued to be cultured for 24 hours, then washed three times with 1×PBS, fixed with 2 mL of 4% paraformaldehyde per well for 30 minutes, washed three times with 1×PBS, and acted on the cells with 2 mL of 60% isopropanol per well for 5 minutes to increase the permeability of the cells. They were stained with 2 mL of Oil Red O per well for 1 hour in the dark at room temperature, washed with distilled water for 4 times, and then 1 mL of isopropanol was added to each well for binding for 10 minutes, washed out with shaking, and the absorbance was measured at 492 nm with an enzyme reader.

[0101] Table 2 Effect of LMG on lipid content of HepG2 cells

[0102]

[0103] Note: a The data are the average of three independent experiments. b Lovastatin was used as positive control. Compared with the model group, there was a significant difference***P<0.001.

[0104] As shown in Table 2, LMG can significantly reduce the ability of cellular fat content accumulation induced by sodium oleate and sodium palmitate, and compared with the positive control lovastatin, the effect of reducing fat content at the tested concentration is more obvious.

[0105] Example 4 Effect of LMG on triglyceride (TG) content in HepG2 cells

[0106] The cell culture and induction methods are the same as those in Example 3, but the total lipid content is determined by the Oil Red O method in Example 3, while the triglyceride content is determined by the kit method in Example 4. After 24 hours of cell induction, the drug is added for 24 hours, and then the HepG2 cells are digested and centrifuged to keep the precipitate below. The cells are evenly mixed with the lysate and lysed at room temperature for 35 minutes, and the intracellular TG content is directly determined using a triglyceride detection kit. Figure 7 It can be seen that compared with the normal group, after the model group was treated with the inducer mixed with sodium oleate and sodium palmitate for 24 hours, the intracellular TG level increased significantly. After 24 hours of administration, the TG content was significantly reduced at drug concentrations of 50, 5, 1, and 0.2 μM, and the activity at 0.2 μM was still equivalent to that of 10 μM lovastatin.

[0107] Example 5 Effect of LMG on HepG2 insulin resistance

[0108] Insulin is the only hormone in the body that lowers blood sugar, and it is also the only hormone that promotes the synthesis of glycogen, fat, and protein. Insulin resistance can cause disorders in the body's glucose and lipid metabolism. For the HepG2 insulin resistance experiment, metformin was used as a positive control. The specific method is as follows:

[0109] HepG2 cells were evenly inoculated in a 96-well cell culture plate, and 100 μL of cell fluid was given to each well. A normal group, a model group, and a drug-treated group were set up and placed in a constant temperature incubator for 24 hours. After multiple washes with an appropriate amount of PBS solution, the cells were starved with a serum-free culture medium. After overnight culture, 100 μL of a mixture of 0.5 μM insulin and culture medium was added to the model group and the drug-treated group to induce hyperinsulinemia. Then, different concentration gradients of the extract to be tested were added and three replicate wells were set up. The positive control was metformin. After 24 hours of drug administration, the glucose consumption of the cells was measured using a glucose determination kit (oxidase method). Glucose consumption can indirectly reflect insulin resistance. Figure 8As shown in the figure, a high concentration of insulin and culture medium mixture was added to the model group and the drug group, and hyperinsulinemia was induced after 24 hours, which reduced the sensitivity to insulin, indicating that the model group had a resistance to insulin. After treatment with different concentrations of drug administration, it was found that the consumption of cellular glucose increased significantly, indicating that it can alleviate the phenomenon of insulin resistance. It is worth noting that LMG is also significant at a concentration of 0.2μM, and 5μM LMG has the same effect as the positive control metformin at a concentration of 1mM. Therefore, this compound can alleviate the phenomenon of insulin resistance in vitro and has the potential to reduce blood sugar and blood lipids.

[0110] Example 6 Effect of LMG on Glucose Consumption in HepG2 Cells

[0111] Glucose consumption experiments were used to evaluate the glucose consumption promoted by different concentrations of LMG, thereby reflecting the hypoglycemic activity at the cellular level. Metformin was used as a positive control, and the specific method was as follows:

[0112] HepG2 cells in the logarithmic growth phase were routinely digested and cultured in DMEM low-glucose medium. The inoculation cell density was 5×10 4 100 μL / mL was added to a 96-well culture plate, and each well was placed in an incubator for overnight culture. After the cells adhered to the wall, the supernatant was discarded, washed once with 1×PBS, starved for 24 hours with serum-free DMEM high-glucose medium, washed once with 1×PBS, and cultured with DMEM high-glucose medium. A drug control group (culture medium + test substance + cells), a negative control group (culture medium + DMSO + cells), a blank control group (simple culture medium + test substance), and a positive control group (culture medium + metformin + cells) were set up. Three replicate wells were set up for each concentration, and 0.5 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 continued to be cultured. After 24 hours, the relative glucose consumption was detected using a glucose determination kit.

[0113] Table 3 Effect of LMG on relative glucose consumption of HepG2 cells

[0114]

[0115] Note: a The data are the average of three independent experiments. b As positive control.

[0116] Before testing the effect on glucose consumption of HepG2 cells, the toxicity of LMG to HepG2 cells was determined, and the results showed that the cell survival rate was still greater than 75% at a maximum concentration of 500 μM. As shown in Table 3, LMG can inhibit the glucose consumption activity of cells at a concentration of 500 μM, indicating that the compound has in vitro hypoglycemic activity.

[0117] Example 7 Effects of hypoglycemic and hypolipidemic activities in mice

[0118] The evaluation method of the hyperglycemic and hyperlipidemia mouse model is as follows:

[0119] (1) Establishment of mouse model

[0120] Male C57BL / 6J mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. Six mice were fed with low-fat diet as the normal group, and the remaining 40 mice were fed with 60% high-fat diet (TP23300) as the model group and the drug-treated group for 27 consecutive days. The model was considered successful when the blood glucose level was higher than 11 mM.

[0121] (2) Drug dissolution and administration method

[0122] LMG powder and positive control were dissolved or dispersed in physiological saline and administered to mice by oral gavage for 15 consecutive days.

[0123] (3) Animal grouping

[0124] In addition to the normal group, the mice with successful modeling were randomly divided into 5 groups, 6 mice in each group, a total of 6 groups. Group 1: normal mouse group (low-fat diet); Group 2: model mouse group (high-fat diet); Group 3: high-dose administration group (high-fat diet + LMG 400 mg / kg); Group 4: low-dose administration group (high-fat diet + LMG 200 mg / kg); Group 5: acarbose hyperglycemia positive control group (high-fat diet + acarbose 100 mg / kg); Group 6: fenofibrate hyperlipidemia positive control group (high-fat diet + fenofibrate 100 mg / kg).

[0125] (4) Mouse Experiment Process

[0126] Fifteen days after intragastric administration, blood was collected from the mice's eye sockets and their blood sugar was tested using a blood glucose meter. Blood was then removed from the eyeballs and the serum was collected by centrifugation to measure blood lipid indicators.

[0127] like Fig. 9 As shown in Figure 2, after 4 weeks of high-fat diet feeding, the blood glucose level in the model group increased significantly, and after 15 days of LMG feeding, the blood glucose level decreased significantly, with significant differences between high and low doses (see Figure 2). Fig.10 ), which indicated that the compound has the ability to lower blood sugar in hyperglycemic mice.

[0128] As shown in Table 4, after 4 weeks of high-fat feeding, the blood lipids of the model group were significantly increased. After LMG was given, triglycerides (TG) and cholesterol (TC) were significantly reduced, indicating that LMG can not only relieve hyperglycemia in mice but also reduce the blood lipid levels of mice.

[0129] Table 4 Effects of LMG on serum triglyceride (TG) and cholesterol (TC) in mice (n=6)

[0130] Group TC(mmol / L) TG (mmol / L) Normal group <![CDATA[2.44±0.18 *** ]]> <![CDATA[5.69±0.61 *** ]]> Model Group 13.37±0.42 42.20±9.41 LMG high dose group <![CDATA[3.34±0.40 *** ]]> <![CDATA[14.20±9.08 *** ]]> LMG low dose group <![CDATA[6.55±1.52 * ]]> <![CDATA[19.82±12.13 *** ]]> Fenofibrate <![CDATA[4.90±2.52 *** ]]> <![CDATA[13.00±5.31 *** ]]>

[0131] Note: Data are mean ± SEM. Compared with the model group, there is a significant difference ***P<0.001, *P<0.01.

[0132] In summary, the present invention synthesized the natural product LMG for the first time through a 5-step reaction, and evaluated the hypoglycemic and hypolipidemic activities of the compound in vitro and in vivo. The results showed that the compound could inhibit pancreatic lipase and reduce cellular lipid accumulation in vitro, indicating that the compound had hypolipidemic activity; the compound could promote cellular glucose consumption and alleviate cellular insulin resistance, indicating that the compound also had hypoglycemic efficacy. In addition, further in vivo experiments in mice confirmed that the compound could reduce the symptoms of hyperglycemia and hyperlipidemia. Therefore, the natural product can be chemically synthesized and has hypoglycemic and hypolipidemic activities in vivo and in vitro, indicating that the compound has good application prospects.

[0133] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate 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 synthesizing natural flavonoid glycosides, characterized in that: The natural flavonoid glycoside is obtained by using luteolin as the starting material through a 5-step reaction; The natural flavonoid glycosides are chemically synthesized using luteolin as a raw material, with a total yield of 2.5%; The chemical synthesis route of the synthetic method is: The chemical synthesis method comprises the following steps: (1) Synthesis of Compound 1: Luteolin and dichlorodiphenylmethane were dissolved in diphenyl ether, and the reaction solution was placed in an oil bath at 180°C for 2 hours; after the reaction was completed, the reaction solution was poured into water, first extracted with petroleum ether, and the aqueous phase after extraction was extracted three times with ethyl acetate. The organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Compound 1 was obtained after column chromatography purification as a yellow solid with a yield of 50.8%; Among them, the ratio of luteolin: dichlorodiphenylmethane: diphenyl ether g: g: mL is 1: 1.2: 20; (2) Synthesis of Compound 2: Compound 1, potassium carbonate and tetrabutylammonium bromide were dissolved in N,N-dimethylformamide, acetyl bromide-α,D-glucose was added, and the mixture was reacted at 50°C for 1 h in a microwave reactor. The reaction was monitored by thin layer chromatography. The reaction solution was poured into a 2M dilute hydrochloric acid solution, extracted with ethyl acetate, the organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Compound 2 was purified by column chromatography as a light yellow solid with a yield of 62.2%. Wherein, the ratio of compound 1: potassium carbonate: tetrabutylammonium bromide: N,N-dimethylformamide: acetyl bromide-α,D-glucose: dilute hydrochloric acid solution g: mg: mg: mL: mg: mL is 0.8:483:10:10:779:40; (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 for 1 h at room temperature. The reaction was monitored by TLC until completion. The reaction solution was poured into a 2M dilute hydrochloric acid solution, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Compound 3 was purified by column chromatography as a light yellow solid with a yield of 88.2%. Among them, the ratio of compound 2: methanol: sodium methoxide: dilute hydrochloric acid solution is 420:10:114:10 in mg: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 in sequence, and the mixture was reacted at 80°C for 8 hours. The reaction was monitored by TLC to be complete. 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 then evaporated under reduced pressure. Compound 4 was obtained after column chromatography purification as a white solid with a yield of 35.7%. Among them, the ratio of compound 3: acetonitrile: N,N-dimethylformamide: malonic acid: tert-butyl isocyanide: water in mg: mL: mg: mg: mL is 270: 5: 5: 76: 175: 10; (5) Synthesis of compound luteolin 7-O-(6"-O-malonyl)-β-D-glucoside: Compound 4 was dissolved in ethanol, 15% palladium carbon was added, and the mixture was reacted at room temperature for 16 hours under a hydrogen environment at a pressure of 15 psi; the reaction solution was filtered, the filter cake was washed three times, and the filtrate was concentrated under reduced pressure to obtain a crude product; the crude product was separated by a high performance liquid phase preparative column to obtain compound luteolin 7-O-(6"-O-malonyl)-β-D-glucoside as a yellow solid with a yield of 25.0%, i.e., a natural flavonoid glycoside was obtained; Wherein, the ratio of compound 4: ethanol: palladium carbon in mg: mL: mg is 110: 10: 10; The Chinese name of the natural flavonoid glycoside is: Luteolin 7-O-(6"-O-malonyl)-β-D-glucoside, the English name is: Luteolin-7-O-6"-malonylglucoside, and the structural formula is:

2. A natural flavonoid glycoside for use in the preparation of a drug for treating glucose and lipid metabolism disorders, wherein the natural flavonoid glycoside is named in Chinese as Luteolin 7-O-(6"-O-malonyl)-β-D-glucoside, in English as Luteolin-7-O-6"-malonylglucoside, and has the structural formula:

3. Use of a natural flavonoid glycoside in the preparation of a drug for treating hyperlipidemia, wherein the natural flavonoid glycoside has the Chinese name: Luteolin 7-O-(6"-O-malonyl)-β-D-glucoside, the English name: Luteolin-7-O-6"-malonylglucoside, and the structural formula is:

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