CN111166735B - Application of Phthalate-Bis-(2-Ethylheptyl) Ester in Inhibiting Fat Accumulation - Google Patents

Abstract

The invention discloses the application of monomeric compound phthalate-bis-(2-ethylheptyl) ester derived from Potentilla villosa in inhibiting fat accumulation. The structural formula of the compound is shown in formula I. The compound of the present invention is derived from natural plants, has the characteristics of low toxicity, has a strong inhibitory effect on the differentiation of 3T3-L1 cells and fat accumulation, and can be used for the prevention/treatment of fat accumulation disease.

Description

Application of Phthalate-Bis-(2-Ethylheptyl) Ester in Inhibiting Fat Accumulation

technical field

The invention belongs to the field of medicine, and relates to a preparation method and application thereof of a monomer compound phthalate-bis-(2-ethylheptyl) ester derived from Potentilla villosa for preventing/treating fat accumulation disease.

Background technique

Excessive fat accumulation has caused many human health problems, such as obesity, fatty liver disease, hyperlipidemia, high blood pressure and diabetes, etc., which has brought more and more serious troubles to modern people. Preventing and treating fat accumulation is key to addressing these health concerns.

Take obesity as an example. Half a century ago, there were less than 100 million obese people in the world. Along with the faster and faster pace of economic development, the threats to human health are becoming more and more serious. Among them, obesity and its complications are acting as a "ruthless killer", seriously endangering human health. Currently, the number of obese patients is increasing worldwide.

In Western countries such as Europe and the United States, obesity has become the fourth largest medical and social problem in the world in recent years, and the top three are drug abuse, alcoholism and AIDS. Especially in the United States, there are 78 million obese people according to statistics, accounting for 13% of the total number of obese people in the world. Studies have shown that as much as 52 billion U.S. dollars are spent on obesity each year, and the indirect costs will reach 47 billion U.S. dollars. These consumptions will account for about 4% of the gross national product of the United States. China's obese population ranks second in the world, and the number continues to increase.

Obesity has become a risk factor that seriously threatens people's life safety. With the development of society, people's dietary structure has changed, and they are gradually focusing on high-fat, high-energy, and low-fiber foods, which has gradually increased the number of obese people. As of 2016, the latest survey shows that the global More than 10% of the 7 billion people are suffering from various distresses and problems caused by obesity. At the same time, obesity is also an important cause of various chronic metabolic diseases, such as diabetes, fatty liver, cardiovascular and cerebrovascular diseases, atherosclerosis, musculoskeletal diseases and certain cancers. It is well known that the key factor causing obesity is the accumulation of adipose tissue.

At present, there are many ways to treat obesity, such as reducing food or energy intake, stimulating energy production through lipid metabolism, inhibiting pancreatic lipase and adipocyte differentiation, and inhibiting adipocyte differentiation is the most commonly used method. The massive accumulation of adipose tissue is mainly caused by the increase in the number of adipocytes and the increase in the volume of adipocytes, and the number of adipocytes is largely achieved through the differentiation of adipocytes, and the increase of adipocyte differentiation will lead to adipokines These factors play a vital role in the development of obesity and related diseases, so regulating the differentiation of adipocytes can help effectively control obesity.

Many traditional medicines are used to treat fat accumulation. Among them, the medicinal plant Potentilla longifolia Willd.ex Schlecht. is a perennial herb of the family Rosaceae, Potentilla longifolia Willd. ex Schlecht. It is recorded in medical classics that the whole herb can be used as medicine, collected in summer and autumn, and used fresh or dried for the treatment of hepatitis.

Contents of the invention

The object of the present invention is to provide an active ingredient derived from natural plants and capable of inhibiting fat accumulation, as well as its preparation method and application. The present invention successfully separates and obtains the monomer compound phthalate-bis-(2-ethylheptyl) ester from sticky spinach. The compound has a good anti-fat accumulation effect and can be applied to the prevention/prevention of fat accumulation disease and treatment.

One aspect of the present invention provides a compound as shown in formula I in the preparation of preparations for the prevention and/or treatment of fat storage disease:

In the above technical scheme, the compound represented by formula I can be included in the preparation for preventing/and treating fat accumulation disease directly or after being made into a pharmaceutically acceptable salt. The pharmaceutically acceptable salts include inorganic salts or organic salts. The inorganic salts are, for example, hydrochloride, sulfate, phosphate, diphosphate, etc., but are not limited thereto. The organic salts include salt, succinate, citrate, acetate, lactate, etc., but are not limited thereto. The content of the compound shown in formula I and its pharmaceutically acceptable salts in the preparation for preventing/and treating liposis is not particularly limited, and can be appropriately selected according to the specific application symptoms and the specific conditions of the subject. , preferably, the amount of the compound of the present invention accounts for at least 0.001% of the mass of the preparation.

In the above technical solution, the fat storage disease refers to a disease caused by the accumulation of fat, such as obesity, hyperlipidemia, hypertension and diabetes.

The second aspect of the present invention provides a pharmaceutical composition for treating fat accumulation disease, which consists of the compound represented by formula I and pharmaceutically acceptable excipients. The pharmaceutically acceptable auxiliary material is at least one selected from diluents, binders, disintegrants, surfactants, coating materials, capsule materials and film-forming materials. The pharmaceutical composition can be in the form of granules, pills, capsules, tablets, oral liquid preparations or freeze-dried powder preparations. The content of the compound shown in formula I in the pharmaceutical composition is not particularly limited, and can be appropriately selected according to the specific application symptoms and the specific conditions of the subject. Preferably, the amount of the compound of the present invention accounts for at least 0.001% by mass of the composition.

The third aspect of the present invention provides an application of the above-mentioned compound represented by formula I in food and drink for inhibiting fat accumulation. The compound represented by formula I of the present invention or a pharmaceutically acceptable salt thereof can be contained in food and drink as an active ingredient for inhibiting fat accumulation. As long as the effect of the compound as an active ingredient is not impaired, the shape or properties of the food and drink are not particularly limited. In addition to the compound of the present invention, the food and drink can contain ingredients commonly used in food and drink, and can be prepared according to a conventional method. The content of the compound represented by formula I in the food and drink is not particularly limited and can be appropriately selected. The food and drink can be used to prevent or improve symptoms caused by fat accumulation, such as obesity, hyperlipidemia, hypertension, diabetes and the like.

In the fourth aspect of the present invention, there is provided a method for preparing the above-mentioned compound represented by formula I, comprising the following steps:

(1) The water extract of Potentilla villosa is separated by using D101 type macroporous adsorption resin, and in the separation process, water, 25% ethanol, 50% ethanol, 75% ethanol, and 95% ethanol are used as eluents successively , carry out gradient elution, each gradient is eluted until the eluent is colorless, the eluent of each gradient is concentrated under reduced pressure and dried to obtain water eluted extract, 25% ethanol eluted extract, 50% ethanol extract Ethanol eluted extract, 75% ethanol eluted extract and 95% ethanol eluted extract;

(2) The 75% ethanol eluted extract is separated by normal phase silica gel column chromatography, and in the separation process, dichloromethane with a volume ratio of 20:1, 10:1, 5:1, 3:1 is used successively - Methanol system and dichloromethane-methanol-water system with a volume ratio of 5:1:0.1, 3:1:0.1, 1:1:0.1 as mobile phase, gradient elution, according to the results of thin layer chromatography, combined For the same fraction, 15 fractions are obtained, that is, fractions 1 to 15;

(3) Fraction 4 is separated by normal phase silica gel column chromatography, and in the separation process, the dichloromethane-methanol system with a volume ratio of 35:1 and 100% methanol is used as the mobile phase successively to carry out gradient elution, according to According to the results of thin layer chromatography, the same fractions were combined to obtain the compound shown in formula I.

In the above-mentioned technical scheme, the sticky Potentilla villosa is the aerial part or root of the dried or freshly collected sticky spinach spinach.

The compound represented by formula I of the present invention is derived from natural plants, has the characteristics of low toxicity, and has a strong inhibitory effect on the differentiation of 3T3-L1 cells and fat accumulation, and can be used for the prevention/treatment of fat accumulation disease.

Description of drawings

Figure 1 shows the effect of Compound 9 on the survival rate of preadipocytes in 3T3-L1 mice.

Figure 2 shows the photos of Oil Red O staining results of induced differentiation of 3T3-L1 mouse preadipocytes treated with Compound 9.

Figure 3 shows the result of measuring the absorbance at 540nm after being treated with Compound 9 to induce differentiation of 3T3-L1 mouse preadipocytes, stained with Oil Red O, and then decolorized with isopropanol.

Fig. 4 shows the measurement results of triglyceride content in 3T3-L1 preadipocytes induced to differentiate after treatment with compound 9.

Figure 5 shows the docking effect of compound 9 with AMPK and SCD1.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention will not be limited. Unless otherwise specified, the experimental methods used in the present invention are conventional methods, and the experimental equipment, materials, reagents, etc. used can be purchased from chemical companies.

Materials used in the following examples:

The aerial parts and roots of Potentilla longifolia Willd. ex Schlecht. (commonly known as Hepatitis Grass) collected in Yanbian area, Jilin Province were protected from the sun, air-dried, and set aside.

Example 1 Separation and Purification of Compound 9 in Sticky Potentilla Root

(1) Weigh 10kg of dried root of Potentilla villosa, divide it into four equal portions after crushing it, soak each equal portion with distilled water for 2 hours, then decoct for 2 hours with a vacuum concentrating decoction machine, and repeat the extraction for 3 Once, the filtrate was combined after the absorbent cotton was filtered while it was hot, and the filtrate was concentrated under reduced pressure (45r/min) to the extractum shape, and the crude extract extractum 1633g was obtained, and 55g was taken out for pharmacological experiments, and the remaining 1578g was used for separation and purification;

(2) Heat and ultrasonically dissolve the extract (1578g) in 3L distilled water, and then divide it into three parts. The stationary phase is _D101 macroporous adsorption resin. % ethanol for elution, each gradient was eluted until the eluent was colorless, then the eluent of each gradient was concentrated under reduced pressure and dried to obtain 434 g of H ₂ O eluted fraction and 526 g of 25% ethanol eluted fraction , 50% ethanol eluted fraction 139g, 75% ethanol eluted fraction 8g, 95% ethanol eluted fraction 2.8g;

(3) Separating and purifying 5.2 g of the 75% ethanol-eluted part of the root of the root part of the lingering root, using normal phase silica gel column chromatography, the particle size of the silica gel used is 200 to 300 mesh, and the column is packed in a wet method. After loading the sample in a dry method, sequentially Use dichloromethane-methanol system at volume ratios of 20:1, 10:1, 5:1, 3:1 and dichloromethane at volume ratios of 5:1:0.1, 3:1:0.1, 1:1:0.1 The methane-methanol-water system was used as the mobile phase for gradient elution. According to the results of thin layer chromatography, the same fractions were combined to obtain 15 fractions, namely fractions 1-15;

(4) Fraction 4 (84 mg) was separated and purified by normal phase silica gel column chromatography, and the dichloromethane-methanol system with a volume ratio of 35:1 and 100% methanol was used as the mobile phase in sequence to carry out gradient elution, according to thin As a result of layer chromatography, the same fractions were combined to obtain compound 9 (10.4 mg), whose structure is shown in formula I:

Chemical properties and chemical structure identification of embodiment 2 compound 9

Structural analysis of compound 9: the molecular formula is C ₂₆ H ₄₂ O ₄ , the molecular weight is 418, and it is a colorless solid. ¹ H-NMR (300MHz, CDCl ₃ ) δ7.71 (dd, J = 5.6, 3.4Hz, 2H), 7.53 (dd, J = 5.8, 3.3Hz, 2H) suggests that it is 2 substitutions on the benzene ring, which is the ortho position The 4 alkene hydrogen signals on the benzene ring are replaced, and it can be further speculated that the ortho positions of the benzene ring are respectively connected with 2 identical substituent groups. δ4.23 (dd, J=5.8, 3.7Hz, 4H) suggests that there are two methylene groups. ¹³ C-NMR (125MHz, CDCl ₃ ) shows that δ167.71 is carbonyl carbon, and δ68.18 suggests methylene, the above data and references (Ma Yu et al., Chinese herbal medicine, 2006,37(9):1315-1317) The report is consistent, so it is identified as bis-(2-ethylheptyl) phthalate, and its English name is bis(2-ethylheptyl)phythalate.

Example 3 Effect of compound 9 on the survival rate of preadipocytes in 3T3-L1 mice

The cytotoxicity of compound 9 was detected by MTT assay:

(1) 3T3-L1 cell culture

3T3-L1 mouse preadipocytes were cultured in a 37°C incubator with a certain humidity and 5% carbon dioxide in a DMEM medium containing 10% calf serum (FCS) and 1% penicillin-streptomycin double antibody mixture .

(2) Cytotoxicity test (MTT)

In this experiment, the cytotoxicity test of monomer compound 9 was carried out. 100 μL of culture medium containing 3T3-L1 cells was added to a 96-well plate. After the cells adhered to the wall, 100 μL of the corresponding concentration of compound 9 was added to each well. The concentrations were 0, 10, 20, 40, 80 μM, respectively. Three parallel wells were set up for each concentration gradient, and the treated 3T3-L1 cells were kept at 37° C. and 5% carbon dioxide for further culture. After 96 hours, discard the culture medium, add 10 μL of MTT solution to each well, place it in the dark at 37°C for 4 hours, then add 100 μL of dimethyl sulfoxide, shake it with a shaker for 10 minutes, and measure the absorbance A at a wavelength of 540 nm. Value, calculate the cell viability of 3T3-L1 cells (Cell viability), the results are shown in Figure 1.

As can be seen from Figure 1, compound 9 has no toxicity in the range of 0-80 μM. In the next study, compound 9 was selected at a concentration of 40 μM.

Effect of Example 4 compound on the differentiation of preadipocytes in 3T3-L1 mice

(1) 3T3-L1 cell differentiation and drug treatment

3T3-L1 preadipocytes were subcultured in a 6-well plate, cultured with a medium containing 10% calf serum (FCS), and induced to differentiate when 3T3-L1 cells were confluent (defined as day 0). A normal group (CON group, added with 10% FCS culture medium) was set up; a control group (DM group (differentiation medium) given induction differentiation medium alone, was added with differentiation induction agent Ⅰ (5% FBS+DMEM+1 μM dexamethasone+500 μM3 -isobutyl-1-methylxanthine+10 μg/ml insulin)); pioglitazone control group: (PIO group (pioglitazone), adding differentiation-inducing agent Ⅰ+10 μM pioglitazone); compound 9 administration group (differentiation-inducing agent Ⅰ + compound 9: 40 μM); after 2 days of culture, the same differentiation-inducing agent was replaced as above. After 4 days of culture, the culture medium was changed, the CON group was replaced with fresh 10% FCS culture medium, and the DM group, PIO group, and each administration group were replaced with differentiation-inducing agent II: 10 μg/ml insulin + 5% FBS + DMEM. After 2 days, the CON group was replaced with fresh 10% FCS culture medium, and the other groups were replaced with differentiation-inducing agent Ⅲ: 5% FBS+DMEM. The medium was changed every 2 days, and the culture was continued until the 8th day to complete the induction process.

(2) Oil red O staining

After the induction and differentiation of cells was completed, the medium in the 6-well plate was discarded and washed 3 times with PBS. After fixing with 10% paraformaldehyde at room temperature for 1 h, wash with PBS twice, add 1 ml of Oil Red O dye reagent prepared in advance to each well, and stain at room temperature for 2 h, then wash the dye in the plate with distilled water, and The differentiation of 3T3-L1 cells was observed under an inverted microscope, and photographs were taken. The results are shown in Figure 2. After the stained 6-well plate was dried overnight at room temperature, it was decolorized with isopropanol (IPA), and the absorbance was measured at 540 nm. The results are shown in Figure 3.

Oil Red O is a strong fat solvent and fat staining agent. It combines with triglyceride to form small lipid droplets. The lipid droplets in cells or tissues are stained orange red after being stained with it. In order to screen for the ability to inhibit 3T3-L1 preadipocytes Differentiated monomers, 3T3-L1 cells were differentiated while being treated with corresponding concentrations of drugs. After the differentiation was completed, they were stained with Oil Red O, and the differentiation of 3T3-L1 cells was judged according to the staining status. The stained cells were stained with isopropanol Decolorize, measure its absorbance, and the value of absorbance can also explain the differentiation of 3T3-L1 cells. From the results of Oil Red O staining (Fig. 2), it can be seen that compared with the CON group, the 3T3-L1 cells in the DM group differentiated from long-spindle cells into mature adipocytes, and a large number of lipid droplets gathered to form a "ring". "-like structure; compared with the PIO group, there was no significant difference in cell differentiation between the DM group and the PIO group, which indicated that the 3T3-L1 cells were completely differentiated. Compared with the DM group, the accumulation of oil droplets in the compound 9 administration group was significantly reduced, and the differentiation of 3T3-L1 cells was significantly inhibited. The cells stained with Oil Red O were decolorized with isopropanol to measure the absorbance, and the results of the absorbance value showed that (Fig. 3), compared with the DM group, the absorbance value of the compound 9 administration group reached 76%. Fat accumulation has a strong inhibitory effect.

In Fig. 3, ### indicates p<0.001 compared with the normal control group; *** indicates p<0.001 compared with the model (DM) group.

(3) Determination of TG content

Triglyceride (TG) is a component of lipids in the human body. It is the most abundant lipid in the human body, and it is also an inspection index for lipid-related diseases in clinical practice. The following method was used to detect the effect of compound 9 on the TG content in 3T3-L1:

Add 120 μl of cell lysate [(25 mM sucrose, 20 mM trisaminomethane, 1 mM ethylenediaminetetraacetic acid, 1 mM ethylene glycol bis(2-aminoethane) to each well of the 6-well plate samples that have been induced and differentiated according to step (1). base ether) tetraacetic acid], the cells were scraped off, put into a 1.5ml test tube, placed in a refrigerated centrifuge, 4°C, 13000rpm, and centrifuged for 20min. After the centrifugation was completed, the supernatant was taken to quantify the protein, and a standard curve was drawn (do three parallel experiments), measure the sample concentration. Get 80 μ g of protein quantitatively to 30 μ l; After the operation according to the kit instructions, measure the calibration hole and each hole sample absorbance value under the 510nm condition. Calculate the TG content of the sample according to the absorbance value, the result is as follows Figure 4.

The results in Figure 4 show that, compared with the DM group, there was a significant difference between the compound 9 administration group and the DM group, which was reduced to 60%, indicating that compound 9 has a strong inhibitory effect on the accumulation of triglycerides.

In Fig. 4, ### indicates p<0.001 compared with the normal control group; *** indicates p<0.001 compared with the model (DM) group.

Based on the above experimental results, the monomeric compound 9 has a strong inhibitory effect on the accumulation of triglyceride, etc., and the compound 9 has a significant inhibitory effect on the differentiation of 3T3-L1 cells and the accumulation of fat. Example 5 Study on the Mechanism of Compound 9 by Molecular Docking

AMP-activated protein kinase (AMPK) is closely related to the metabolism of fat and carbohydrates. Upon activation, AMPK inhibits sterol regulatory element-binding protein (sREBP1c), peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein (C/EBPa) and their downstream proteins such as stearoyl Expression of coenzyme A dehydrogenase-1 (SCD1) and fatty acid synthase (FAS). These proteins are involved in the biosynthesis of triglycerides (TGs) and fatty acids and adipocyte maturation, thereby inhibiting adipogenesis. Meanwhile, molecular docking studies, commonly used to examine the position of compounds at protein binding sites, are a computer simulation method that can be viewed as an optimization option that automatically determines the conformation of protein-ligand complexes.

Therefore, this example uses the method of molecular docking to further clarify whether there is an interaction between compound 9 and AMPK (or SCD1), specifically:

The crystal structures of SCD1 (PDB ID: 4YMK) and AMPK (PDB ID: 5T5T) proteins were downloaded from the RCSB protein database, and the 3D structure of compound 9 was transformed by Chem3D14.0.0.117. Then, prepare compounds and proteins with Discovery Studio4.0, and carry out molecular docking research with Libdock.

The results are shown in Figure 5, where Figure 5A shows the interaction between Compound 9 and AMPK, Figure 5B shows the two-dimensional view of the interaction between Compound 9 and AMPK, Figure 5C shows the interaction between Compound 9 and SCD1, and Figure 5D shows the interaction of Compound 9 A two-dimensional view of the interaction between and SCD1. As shown in Figures 5A and 5B, when compound 9 docks with AMPK, it forms a carbon-hydrogen bond with residue Amp-402, and with residues Leu-276, Val-296, Ala-294, Leu-314, Leu-172, Val- 292, Leu-291 form an alkyl group, and dock with residues Lys-169 and So-4404 to form π cations or π anions. At the same time, as shown in Figure 5C and 5D, when compound 9 docked with SCD1, it formed conventional hydrogen bonds with residues Asn-261 and Trp-149, and with residues Ala-288, His-156, His-294, and His-153 , Val-260, Phe-142, Leu-254, Trp-258, Leu-181 form π alkyl or alkyl, and form π-π stacking or π-πt shape with residue Trp-180. Due to these ligand-protein interactions, compound 9 may effectively bind to AMPK and SCD1, activate the activity of AMPK and its downstream kinase SCD1, and thus significantly inhibit fat accumulation.

The data statistics and analysis methods involved in the above-mentioned embodiments are as follows: all data are represented by "average value ± standard deviation" (MEAN ± SE), and Sigma plot 12.5 statistical software is selected to process the data, and comparative analysis between multiple groups adopts single factor Analysis of variance (one-way anova-student's t-test). When P<0.05, there is a difference between the groups; when P<0.01, the difference between the groups is significant; when P<0.001, the difference between the groups is extremely significant.

The above description of the embodiments is for those of ordinary skill in the art to understand and apply the present invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described here to other embodiments without creative efforts. Therefore, the present invention is not limited to the embodiments herein. Improvements and modifications made by those skilled in the art according to the disclosure of the present invention without departing from the scope of the present invention should fall within the protection scope of the present invention.

Claims (3)

1. Use of a compound of formula I:

2. a process for the preparation of a compound of formula I as claimed in claim 1, comprising the steps of:

(1) Separating water extract of Potentilla mucida with D101 macroporous adsorbent resin, sequentially eluting with water, 25% ethanol, 50% ethanol, 75% ethanol, and 95% ethanol as eluent in the separation process, eluting until the eluent is colorless, concentrating under reduced pressure, and drying to obtain water-eluted extract, 25% ethanol-eluted extract, 50% ethanol-eluted extract, 75% ethanol-eluted extract, and 95% ethanol-eluted extract;

(2) Separating the 75% ethanol eluted extract by normal phase silica gel column chromatography, in the separation process, sequentially using a dichloromethane-methanol system with the volume ratio of 20:1, 10:1, 5:1 and 3:1 and a dichloromethane-methanol-water system with the volume ratio of 5:1:0.1, 3:1:0.1 and 1:1:0.1 as mobile phases, carrying out gradient elution, and combining the same fractions according to the thin layer chromatography result to obtain 15 fractions, namely fractions 1-15;

(3) Separating fraction 4 by normal phase silica gel column chromatography, sequentially eluting with dichloromethane-methanol system with volume ratio of 35:1 and 100% methanol as mobile phase, and mixing the same fractions according to thin layer chromatography result to obtain compound shown in formula I.

3. The method of claim 2, wherein the Potentilla mucida is a dry or freshly harvested portion or root of Potentilla mucida.