Application of dimeric dihydrochalcone compound in preparation of lipid-lowering product

Abstract

The invention discloses application of a dimeric dihydrochalcone compound in preparation of a lipid-lowering product, which is characterized in that HepG2 cells are induced by oleic acid to obtain a high-lipid model, and the high-lipid model is treated by the dimeric dihydrochalcone compound to obtain the lipid-lowering product. Experimental results show that the dimeric dihydrochalcone compound can effectively down-regulate accumulation of TG in HepG2 cells treated by oleic acid at low concentration and reduce the number of lipid droplets, the effect of the dimeric dihydrochalcone compound is superior to that of a positive drug fenofibrate, and the result shows that the dimeric dihydrochalcone compound has a lipid-lowering effect and can be used for preparing a novel anti-tumor drug. The invention provides a new way for developing high-efficiency and low-toxicity lipid-lowering products.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a new use of a dimerized dihydrochalcone compound in the preparation of lipid-lowering products. Background Art

[0002] Hyperlipidemia is a disease characterized by abnormally elevated plasma lipid levels caused by abnormal lipid metabolism or transport. Hyperlipidemia can lead to a variety of complications, including arteriosclerosis, coronary heart disease, cerebral infarction, cardiovascular and cerebrovascular disease, and visual impairment. Because statins can cause liver damage when used to treat hyperlipidemia, natural products have become a research hotspot in the lipid-lowering therapeutic field due to their potential therapeutic effects and minimal side effects.

[0003] O's tea is a kind of tea with the leaves of Begonia variegata ( Malus toringoides (Rehd.) Hughes) is a healthy tea made from the tender leaves of the Chinese yew tree. Malus variegata is a plant used for both medicinal and edible purposes, primarily found in Tibet, Sichuan, Gansu, and Qinghai provinces in China. Ose tea resembles a pine needle and has a delicate, sweet aroma. Its buds and leaves are delicate, curved, and lustrous, while its tea is tender green, with a lustrous, evenly green leaf base. Ose tea has a long history of consumption. Its sweet, slightly bitter flavor and cooling properties are associated with heat-clearing and detoxifying effects, lowering blood pressure and lipids, and providing antioxidant benefits. It has traditionally been used to treat hypertension and hyperlipidemia, and these medicinal properties are documented in the book "Compendium of Tibetan Medicine." Modern phytochemical research reveals that it primarily contains flavonoids and phenolic acids, while modern pharmacological studies demonstrate its antioxidant, blood sugar-lowering, anti-inflammatory, immune-enhancing, and lipid-lowering properties. Summary of the Invention

[0004] The present invention provides a dimeric dihydrochalcone compound (3-hydroxy-phloridzin-( I -5′, II -6)-phloretin-4- O -glucoside), namely its use in preparing lipid-lowering products, the chemical structural formula of the dimer dihydrochalcone compound is as follows: .

[0005] The lipid-lowering product of the present invention comprises a dimerized dihydrochalcone compound (or active ingredient), and one or more pharmaceutically or functional food-acceptable excipients may be added, or the compound may be combined with other active ingredients to exert therapeutic effects. In addition to being formulated into pharmaceutical or functional food tablets, the compound may also be formulated into pills, powders, capsules, granules, oral liquids, and the like.

[0006] The invention relates to a dimerized dihydrochalcone compound isolated from O's tea, and a high-fat model is obtained by inducing HepG2 cells with oleic acid. The high-fat model is then treated with the dimerized dihydrochalcone compound. Experimental results show that the dimerized dihydrochalcone compound can effectively downregulate the accumulation of TG in human liver cancer cells (HepG2) treated with oleic acid at a low concentration, and reduce the number of lipid droplets. The effect is better than that of the positive drug fenofibrate, indicating that the dimerized dihydrochalcone compound has a lipid-lowering effect. The invention provides a new approach for developing high-efficiency and low-toxic lipid-lowering products. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 The cytotoxicity of dimeric dihydrochalcone compounds to HepG2 cells; Figure 2 The results show the effect of dimeric dihydrochalcone compounds on TG accumulation in HepG2 cells induced by oleic acid; Figure 3 The results show the effect of dimeric dihydrochalcone compounds on oil droplets in HepG2 cells induced by oleic acid. DETAILED DESCRIPTION

[0008] The present invention will be further described below with reference to the examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are conventional instruments, reagents, and materials already available in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are conventional experimental methods and detection methods already available in the prior art.

[0009] Example 1: Obtaining a Dimeric Dihydrochalcone Compound 1. After pulverizing 10 kg of Ose tea with a powder grinder, ultrasonically extract with 80% methanol aqueous solution for 3 times, each time for 30 min. The combined extracts were collected and concentrated by rotary evaporation at 55 ° C to obtain 1.2 kg of Ose tea extract; 2. Dissolve the Russian tea extract in water and extract with ethyl acetate five times. Collect and combine the ethyl acetate phases and remove the solvent by rotary evaporation at 50°C to obtain 260 g of crude extract. 3. The crude extract was chromatographed on a silica gel column using dichloromethane-methanol (100:1-1:1) as the eluent. Thin layer chromatography (TLC) was used for detection. The eluates containing the same fractions were combined and the solvent was removed by rotary evaporation at 50°C to obtain seven components, Fr.1-Fr.7. (4) Component Fr.7 (7.1 g) was separated and purified by medium pressure preparative chromatography using a gradient elution of 10%, 30%, 50%, 70% and 90% methanol in water. The eluate was collected and rotary evaporated at 50°C to obtain five components (Fr. AE). Among them, Fr. B (1.2 g) was separated by silica gel column and eluted with chloroform and acetone (15:1, 10:1 and 5:1) to obtain three components (Fr. ac). Component Fr. c was then further separated and purified by preparative liquid chromatography to obtain a dimerized dihydrochalcone compound (5 mg).

[0010] The structure of the dimerized dihydrochalcone compound is as follows: 3-hydroxy-phloridzin-( I -5′, II -6)-phloretin-4- O -glucosid, light yellow powder. HR-ESI-MS m / z : 435.4172 [MH] - ;Molecular formula: C 21 H 24 O 10 . 1 H NMR (400 MHz, DMSO) d H :6.64 (1H, s, H- I -2′), 6.32 (1H, s, H- I -6′), 2.48 (2H, m, H- I -2), 3.28 (2H,m, H- I -3), 6.10 (1H, d, J = 1.2 Hz, H- I -6), 5.90 (1H, d, J = 1.2 Hz, H- I -6), 7.05 (2H, d, J = 8.4 Hz, H- II -2′, II -6′), 6.64 (2H, d, J = 8.4 Hz, H- II -3′, II -5′), 2.79 (2H, m, H- II -2), 3.35 (2H, m, H- II-3), 6.28 (1H, s, H- II -8); 13 C NMR (125MHz, DMSO) d C : 26.2 (t, C- I -2), 43.9 (t, C- I -3), 205.0(s, C- I -4), 160.9 (s, C- I -5), 94.3 (d, C- I -6), 164.2 (s, C- I -7), 96.8 (d,C- I -8), 165.1 (s, C- I -9), 105.2 (s, C- I -10), 131.6 (s, C- I -1′), 115.0 (d,C- I -2′), 144.2 (d, C- I -3′), 142.5 (s, C- I -4′), 123.3 (s, C- I -5′), 118.5(s, C- I -6′), 100.9 (d, C- I -1′′), 73.2 (d, C- I -2′′), 77.3 (d, C- I -3′′),69.3 (d, C- I -4′′), 76.8 (d, C- I -5′′), 60.6 (t, C- I -6′′), 29.1 (t, C- II -2), 45.1 (t, C- II -3), 204.9 (s, C- II -4), 163.6 (s, C- II -5), 110.0 (s, C- II -6), 159.5 (s, C- II -7), 94.3 (d, C- II -8), 162.1 (s, C- II -9), 104.4 (s,C- II-10), 131.6 (s, C- II -1′), 129.2 (d, C- II -2′, II -6′), 115.0 (d, C- II -3′, II -5′), 155.2 (s, C- II -4′), 100.9 (d, C- II -1′′), 73.0 (d, C- II -2′′),77.3 (d, C- II -3′′), 69.0 (d, C- II -4′′), 76.8 (d, C- II -5′′), 60.6 (t, C- II -6′′).

[0011] Example 2: Effect of dimeric dihydrochalcone compounds on triglyceride (TG) accumulation and oil droplet formation in oleic acid-treated human hepatoma cells (HepG2) A high-lipid model was established using HepG2 cells induced by oleic acid. Oleic acid stimulation causes lipid deposition in HepG2 cells, mimicking the pathogenesis of fatty liver disease caused by lipid metabolism disorders and resulting in the accumulation of large amounts of triglycerides (TG). A triglyceride (TG) assay kit was used to measure TG levels in the control and experimental groups to assess the inhibitory effect of the compounds on TG accumulation.

[0012] 1. Cell culture Human hepatic cell lines (HepG2, purchased from the Kunming Institute of Zoology, Chinese Academy of Sciences) were used for the experiments. The cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (Gibco, Grand Island, NY, USA) in an incubator at 37°C and 5% CO ; 2. Cytotoxicity evaluation 200 μL HepG2 cells were seeded into 96-well cell culture plates (2×10 4cells), incubated at 37°C for 24 hours, then 200 μL of different concentrations of dihydrochalcone compounds (10-100 μM) were added to a 96-well plate. After incubation for 24 hours, the culture medium was removed, and 200 μL of MTT (0.5 mg / mL) solution dissolved in PBS was added to the 96-well cell culture plate. The plate was placed in a 37°C, 5% CO2 incubator for further culturing for 4 hours, and then 200 μL of DMSO was added for 10 minutes. The absorbance was measured at 490 nm using a microplate reader, and the cell viability was calculated. The toxicity of dihydrochalcone compounds on HepG2 cells was evaluated by MTT assay. Cell viability (%) = (A 样品 / A 空白 )×100%, where A 样品 is the absorbance value of the experimental group with the addition of dimerized dihydrochalcone compound, A 空白 is the absorbance value of the blank control group without adding the dimerized dihydrochalcone compound; See the results Figure 1 ,The results in the figure show that the dimeric dihydrochalcone compounds showed no toxicity to HepG2 cells within the concentration range of 10-100 μM; 3. Evaluation of triglyceride (TG) accumulation HepG2 cells in the logarithmic growth phase were seeded in 6-well cell culture plates (3×10 5 After incubation at 37°C for 12 h, the cells in the culture plate were washed with PBS and complete medium containing 0.4 mM oleic acid was added. The cells were cultured at 37°C for 24 h. Different concentrations of dimeric dihydrochalcone compounds (10, 20, 40, 80, 100 μM) and fenofibrate (FNB, 40 μmol / L) were then added to the culture medium. The cells were cultured at 37°C and 5% CO2 for 24 h. The accumulation of triglycerides (TG) in the cells was determined using a triglyceride (TG) content detection kit. See the results Figure 2 The dimerized dihydrochalcone compound had a significant inhibitory effect on the accumulation of TG at concentrations of 80μM and 100μM, and the effect was better than that of the positive drug fenofibrate, especially at 100μM, it showed good inhibitory activity.

[0013] 4. Oil Red O staining HepG2 cells (2 mL, concentration 3 × 10 5HepG2 cells were seeded into 6-well plates (100 μg / mL) and cultured at 37°C for 24 hours. Next, 2 mL of complete medium containing oleic acid (400 μM) was added to the 6-well plates and cultured at 37°C for another 24 hours. Afterwards, the cells were treated with 2 mL of complete medium containing a dihydrochalcone compound (40 μM) and fenofibrate (40 μM) and cultured for an additional 24 hours. HepG2 cells were stained with Oil Red O staining solution according to the kit instructions. Finally, cells were observed and photographed at 200x magnification using an Olympus microscope. See the results Figure 3 After treatment with 400 μM oleic acid, HepG2 cells showed dense accumulation of lipid droplets, indicating successful establishment of a lipid deposition model. In contrast, treatment with dimeric dihydrochalcone (40 μM) reduced the number of lipid droplets to varying degrees, demonstrating an inhibitory effect on lipid deposition, even comparable to that of the positive control drug fenofibrate. These experimental results demonstrate that the compounds of this invention possess potent lipid-lowering activity, providing a new avenue for the development and utilization of osecha and therapeutic lipid-lowering products.

Claims

1. Use of a dimer dihydrochalcone compound having the structural formula shown below in the preparation of a lipid-lowering product; 。

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