CN111166735B - Use of bis- (2-ethylheptyl) phthalate for inhibiting fat accumulation - Google Patents

Abstract

The invention discloses an application of monomer compound phthalic acid-bis- (2-ethylheptyl) ester from Potentilla mucilaginosa in inhibiting fat accumulation. The structural formula of the compound is shown as formula I. The compound is derived from natural plants, has the characteristic of low toxicity, has strong inhibition effect on the differentiation of 3T3-L1 cells and the accumulation of fat, and can be used for preventing/treating fat accumulation.

Description

Use of bis- (2-ethylheptyl) phthalate for inhibiting fat accumulation

Technical Field

The invention belongs to the field of medicines, and relates to a preparation method and application of a monomer compound phthalic acid-bis- (2-ethylheptyl) ester derived from Potentilla mucilaginosa for preventing/treating lipopexia.

Background

Excessive fat accumulation causes various health problems in humans, such as obesity, fatty liver disease, hyperlipidemia, hypertension, diabetes, etc., and brings more and more serious trouble to modern people. Prevention and treatment of fat accumulation is critical to solving these health problems.

In the case of obesity, it is less than 1 million people worldwide who are obese for half a century ago. The threat of human health is also becoming more serious with the faster and faster steps of economic development, wherein obesity and its complications are acting as an "amorous killer" and seriously jeopardizing human health. Currently, the number of obese patients is increasing worldwide.

In western countries such as europe and america, obesity has become a global 4 th medical society problem in recent years, and drug absorption, alcoholism and aids are the first three of them. In the united states, in particular, there are 7800 thousands of obese people counted and accounting for 13% of the total number of obese people worldwide, and studies have shown that direct consumption of up to 520 billion dollars per year is used for obesity, and that indirect use costs can reach 470 billion dollars, and that these consumption dates account for 4% of the total national production economy in the united states, and that the obese people in china are ranked the second worldwide, and that the number of people is increasing.

Obesity has become a serious risk factor for life safety. With the development of society, the dietary structure of people is changed, and the people are mainly high-fat, high-energy and low-fiber foods, so that the number of obese people is gradually increased, and by the year 2016, the latest investigation shows that more than ten percent of people in about 70 hundred million people worldwide are suffering from various distress and puzzles caused by obesity. Meanwhile, 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 a key factor in obesity is the accumulation of adipose tissue.

Currently, there are various ways of treating obesity, for example, reducing food or energy intake, stimulating energy production by lipid metabolism, inhibiting pancreatic lipase and adipocyte differentiation, wherein inhibiting adipocyte differentiation is the most common way. The massive accumulation of adipose tissue is mainly caused by the increase of the number of fat cells and the increase of the volume of fat cells, the quantity of the fat cells is realized by the differentiation of the fat cells to a great extent, and the increase of the differentiation of the fat cells can lead to the hypersecretion of fat factors, which play a vital role in the development process of obesity and related diseases, so that the regulation of the differentiation of the fat cells can provide help for effectively controlling the obesity.

Many traditional drugs are used to treat fat accumulation. Wherein the medicinal plant herba Potentillae chinensis (Potentilla longifolia Willd. Ex Schlecht.) is perennial herb of Potentilla of Rosaceae, and is called hepatitis grass in Korean medicine. The medicinal books are recorded that the hepatitis grass can be used as a whole herb, collected in summer and autumn, and used for treating hepatitis in fresh or dried mode.

Disclosure of Invention

The invention aims to provide an active ingredient derived from natural plants and having a fat accumulation inhibiting effect, and a preparation method and application thereof. The invention successfully separates the monomer compound phthalic acid-bis- (2-ethylheptyl) ester from Potentilla mucilaginosa, and the compound has good anti-fat accumulation effect and can be applied to the prevention and treatment of fat accumulation.

In one aspect, the invention provides an application of a compound shown as a formula I in preparing a preparation for preventing/treating fat accumulation:

in the above technical scheme, the compound shown as the formula I can be directly or prepared into pharmaceutically acceptable salts to be contained in the preparation for preventing/treating the fat accumulation. The pharmaceutically acceptable salts include inorganic salts or organic salts. Such as, but not limited to, hydrochloride, sulfate, phosphate, diphosphate, and the like. Such as, but not limited to, salts, succinates, citrates, acetates, lactates, and the like. The content of the compound represented by the formula I and its pharmaceutically acceptable salt in the agent for preventing/treating lipopexia is not particularly limited, and may be appropriately selected depending on the specific condition of application and the specific condition to be administered to a subject, and preferably, the amount of the compound of the present invention is at least 0.001% by mass of the agent.

In the above-described embodiments, the fat accumulation refers to an accumulation disorder caused by fat, such as obesity, hyperlipidemia, hypertension, diabetes, and the like.

In a second aspect of the invention, a pharmaceutical composition for treating lipopexia is provided, which consists of the compound shown in the formula I and pharmaceutically acceptable auxiliary materials. The pharmaceutically acceptable auxiliary materials are selected from at least one of diluents, binders, disintegrants, surfactants, coating materials, capsule materials and film forming materials. The pharmaceutical composition can be granule, pill, capsule, tablet, oral liquid preparation or freeze-dried powder preparation. The content of the compound represented by formula I in the pharmaceutical composition is not particularly limited, and may be appropriately selected according to the specific condition of application and the specific condition to be administered to a subject, and preferably the amount of the compound of the present invention is at least 0.001% by mass of the pharmaceutical composition.

In a third aspect of the invention, there is provided the use of a compound of formula I as described above in a food or beverage for inhibiting fat accumulation. The compound represented by the formula I or a pharmaceutically acceptable salt thereof of the present invention can be contained in a food or beverage as an active ingredient for inhibiting fat accumulation. The shape and properties of the food and drink are not particularly limited as long as the effect of the compound as an active ingredient is not impaired, and the food and drink may be produced by a conventional method by including a raw material used in usual foods and drinks in addition to the compound of the present invention. The content of the compound represented by formula I in the food or beverage is not particularly limited, and may be appropriately selected. The food and drink can be used for preventing or improving symptoms caused by fat accumulation, such as obesity, hyperlipidemia, hypertension, diabetes, etc.

In a fourth aspect of the present invention, there is provided a method for preparing the above compound of formula i, 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.

In the above technical scheme, the Potentilla mucida is an above-ground part or root of the Potentilla mucida collected dry or fresh.

The compound shown in the formula I is derived from natural plants, has the characteristic of low toxicity, has strong inhibition effect on differentiation of 3T3-L1 cells and accumulation of fat, and can be used for preventing/treating fat accumulation.

Drawings

FIG. 1 shows the effect of compound 9 on preadipocyte survival in 3T3-L1 mice.

FIG. 2 is a photograph showing the result of oil red O staining of preadipocytes of 3T3-L1 mice induced to differentiate after treatment with Compound 9.

FIG. 3 shows the results of measuring absorbance at 540nm after staining preadipocytes of 3T3-L1 mice induced to differentiate after treatment with Compound 9 with oil red O and decolorizing with isopropyl alcohol.

FIG. 4 shows the results of measuring the triglyceride content in 3T3-L1 preadipocytes induced to differentiate after treatment with Compound 9.

FIG. 5 shows the docking of Compound 9 with AMPK and SCD 1.

Detailed Description

The invention is described in detail below with reference to the attached drawings and the specific embodiments, but does not limit the scope of the invention. Unless otherwise specified, the experimental methods adopted by the invention are all conventional methods, and all experimental equipment, materials, reagents and the like used can be purchased from chemical companies.

The materials used in the following examples:

the aerial parts and roots of Potentilla mucida (Potentilla longifolia Willd. Ex Schlecht.) collected in the Yanbian region of Jilin province were protected from the sun and air dried for use.

EXAMPLE 1 isolation and purification of Compound 9 from Potentilla mucida root

(1) Weighing 10kg of dry roots of Potentilla mucida, crushing the dry roots into four parts, soaking each part in distilled water for 2 hours, decocting the parts in a vacuum concentration decoction machine for 2 hours, repeatedly extracting for 3 times, filtering absorbent cotton while the absorbent cotton is hot, merging filtrate, concentrating the filtrate under reduced pressure (45 r/min) to obtain 1633g of crude extract, taking out 55g for pharmacological experiments, and using the rest 1578g for separation and purification;

(2) Dissolving extract (1578 g) in 3L distilled water under heating and ultrasonic, dividing into three parts, separating stationary phase with D101 macroporous adsorbent resin, sequentially adding H ₂ Eluting with O, 25%, 50%, 75%, and 95% ethanol, eluting with each gradient until the eluate is colorless, concentrating under reduced pressure, and drying to obtain H ₂ 434g of O elution part, 526g of 25% ethanol elution part, 139g of 50% ethanol elution part, 8g of 75% ethanol elution part and 2.8g of 95% ethanol elution part;

(3) Separating and purifying 5.2g of a 75% ethanol elution part at the root of the potentilla anserina by normal phase silica gel column chromatography, loading the column by a wet method, and sequentially using a dichloromethane-methanol system with volume ratios of 20:1, 10:1, 5:1 and 3:1 and a dichloromethane-methanol-water system with volume ratios of 5:1:0.1, 3:1:0.1 and 1:1:0.1 as mobile phases to perform gradient elution, combining the same fractions according to a thin layer chromatography result to obtain 15 fractions, namely fractions 1-15;

(4) Separating and purifying fraction 4 (84 mg) by normal phase silica gel column chromatography, sequentially performing gradient elution by using a dichloromethane-methanol system with volume ratio of 35:1 and 100% methanol as mobile phase, and combining the same fractions according to thin layer chromatography result to obtain compound 9 (10.4 mg), wherein the structure is shown as formula I:

example 2 chemical nature and chemical Structure identification of Compound 9

Structural resolution of compound 9: molecular formula C ₂₆ H ₄₂ O ₄ Molecular weight 418, colorless solid. ¹ H-NMR(300MHz，CDCl ₃ ) Delta 7.71 (dd, j=5.6, 3.4hz,2 h), 7.53 (dd, j=5.8, 3.3hz,2 h) suggests a substitution of 2 on the benzene ring, a substitution of 4 enal hydrogen signals on the benzene ring in the ortho position, and it can be further speculated that the ortho positions of the benzene ring are each linked to 2 identical substituent groups. δ4.23 (dd, j=5.8, 3.7hz,4 h) suggests two methylene groups. ¹³ C-NMR(125MHz,CDCl ₃ ) Delta 167.71 is carbonyl carbon and delta 68.18 suggests methylene, and the above data are consistent with those reported in the reference (Ma Yu et al, herbal, 2006,37 (9): 1315-1317), and therefore identified as bis (2-ethylheptyl) phthalate, under the English name bis (2-ethylhepyl) phytate.

EXAMPLE 3 Effect of Compound 9 on 3T3-L1 mouse preadipocyte viability

Cytotoxicity of compound 9 was detected by MTT assay:

(1) 3T3-L1 cell culture

3T3-L1 mouse preadipocytes were cultured in DMEM medium containing 10% calf serum (FCS), 1% penicillin-streptomycin diabody cocktail in a 37℃incubator containing a humidity and 5% carbon dioxide.

(2) Cytotoxicity experiment (MTT)

In the experiment, a cytotoxicity experiment is carried out on the monomer compound 9, 100 mu L of culture solution containing 3T3-L1 cells is added into a 96-well plate, and after the cells are attached, 100 mu L of compound 9 with corresponding concentration is added into each well, and the concentration of the compound 9 for treating the cells is respectively 0, 10, 20, 40 and 80 mu M. 3 parallel wells were set for each concentration gradient, and the treated 3T3-L1 cells were placed and cultured at 37℃under 5% carbon dioxide. After 96h, the culture broth was discarded, 10. Mu.L of MTT solution was added to each well, left at 37℃for 4h under dark conditions, 100. Mu.L of dimethyl sulfoxide was added thereto, and after shaking for 10min with a shaker, the absorbance A was measured at a wavelength of 540nm to calculate the Cell viability (Cell viability) of 3T3-L1 cells, and the results were shown in FIG. 1.

As can be seen in fig. 1, compound 9 was not toxic in the range of 0 to 80 μm. Compound 9 was selected at a concentration of 40 μm in the subsequent study.

Example 4 Effect of Compounds on 3T3-L1 mouse preadipocyte differentiation

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

Subculturing 3T3-L1 preadipocytes in a 6-well plate, culturing with culture solution containing 10% calf serum (FCS), inducing differentiation when 3T3-L1 cells are full (defined as day 0), and respectively setting normal groups (CON group, 10% FCS culture solution); the differentiation-inducing agent I (5% FBS+DMEM+1. Mu.M dexamethasone+500. Mu.M 3-isobutyl-1-methylxanthine+10. Mu.g/ml insulin)) was added to the control group (DM group (differentiation medium)) alone; pioglitazone control group: (PIO group (pioglitazone), added with induced differentiation agent i+10 μm pioglitazone); compound 9 dosing group (induced differentiation agent i + compound 9:40 μm); after 2 days of culture, the same differentiation inducing agent was replaced as described above. After 4 days of culture, the culture medium was changed, the CON group was changed to a fresh 10% FCS culture medium, and the DM group, PIO group, and each of the administration groups were changed to an induced differentiation agent II: 10 μg/ml insulin+5% FBS+DMEM. After 2 days, the CON group was replaced with fresh 10% fcs broth and the remaining groups were replaced with induced differentiation agent iii: 5% FBS+DMEM. The liquid is changed every 2 days, and the culture is continued until the 8 th day, thus completing the induction process.

(2) Oil red O dyeing

After completion of the cell induced differentiation, the medium in the 6-well plate was discarded, and washed 3 times with PBS. After fixing with 10% paraformaldehyde for 1h at room temperature, PBS was used for 2 times, 1ml of an oil red O dye reagent prepared in advance was added to each well, and after dyeing at room temperature for 2h, the dye in the plate was washed with distilled water, and the differentiation of 3T3-L1 cells was observed in an inverted microscope, and photographs were taken, and the results were shown in FIG. 2. After the stained 6-well plate was dried overnight at room temperature, it was decolorized with isopropyl alcohol (IPA) and the absorbance was measured at 540nm, and the results are shown in FIG. 3.

The oil red O is a very strong fat solvent and a fat dyeing agent, is combined with triglyceride to form a small fat drop shape, fat drops in cells or tissues are dyed to form orange, in order to screen monomers capable of inhibiting 3T3-L1 preadipocyte differentiation, 3T3-L1 cells are differentiated while being treated with medicines with corresponding concentrations, after differentiation is finished, the oil red O is used for dyeing, the differentiation condition of the 3T3-L1 cells is judged according to the dyeing condition, the dyed cells are decolorized by isopropanol, the absorbance is measured, and the differentiation condition of the 3T3-L1 cells can be also indicated by the absorbance value. From the results of oil red O staining (FIG. 2), it can be seen that 3T3-L1 cells in the DM group differentiated from long spindle-shaped cells into mature adipocytes, and that lipid droplets were accumulated in large amounts, forming a "ring-like structure", as compared with the CON group; the DM group showed no significant difference in cell differentiation compared to the PIO group, thus indicating complete differentiation of 3T3-L1 cells. The accumulation of oil droplets in the compound 9-dosed group was significantly reduced and the differentiation of 3T3-L1 cells was significantly inhibited compared to the DM group. The measurement of absorbance after decolorization of oil red O-stained cells with isopropanol shows that, compared with DM group, compound 9 administration group has an absorbance value of 76%, and it can be preliminarily determined that compound 9 has a strong inhibitory effect on fat accumulation.

In fig. 3, # # represents p <0.001 compared to the normal control group; * Represents p <0.001 compared to the model (DM) group.

(3) TG content determination

Triglyceride (TG) is a constituent of lipid in the human body, is the lipid with the largest content in the human body, and is an index for clinical examination of lipid-related diseases. The effect of compound 9 on TG content in 3T3-L1 was examined using the following method:

adding 120 μl of cell lysate [ (25 mM sucrose, 20mM triaminomethane, 1mM ethylenediamine tetraacetic acid, 1mM ethylene glycol bis (2-aminoethyl ether) tetraacetic acid ] into each well of the 6-well plate sample subjected to induced differentiation according to the step (1), scraping the cells, placing the cells into a 1.5ml test tube, placing the test tube into a refrigerated centrifuge at 4 ℃ and 13000rpm, centrifuging for 20min, taking the supernatant after centrifugation, quantifying the protein, drawing a standard curve (performing three parallel experiments), and measuring the concentration of the sample, taking 80 μg of the protein, quantifying to 30 μl, and measuring the absorbance values of the calibrated well and each well sample at 510nm after the operation according to the specification of the kit, and calculating the TG content of the sample according to the absorbance values, wherein the result is shown in FIG. 4.

The results in fig. 4 show that the compound 9 administration group showed a significant difference from the DM group, reduced to 60%, indicating that compound 9 had a stronger inhibitory effect on triglyceride accumulation.

In fig. 4, # # represents p <0.001 compared to the normal control group; * Represents p <0.001 compared to the model (DM) group.

From the above experimental results, it was revealed that the monomer compound 9 has a strong inhibitory effect on accumulation of triglycerides and the like, and that the compound 9 has a remarkable inhibitory effect on both differentiation of 3T3-L1 cells and accumulation of fats. Example 5 investigation of the mechanism of action of Compound 9 by molecular docking

AMP-activated protein kinase (AMPK) is closely related to the metabolism of fat and carbohydrates. After activation, AMPK inhibits the expression of sterol regulatory element binding protein (sREBP 1C), peroxisome proliferator activated receptor gamma (pparγ), CCAAT/enhancer binding protein (C/ebpα) and its downstream proteins such as stearoyl-coa dehydrogenase-1 (SCD 1) and Fatty Acid Synthase (FAS). These proteins are involved in the biosynthesis of Triglycerides (TGs) and fatty acids and in adipocyte maturation, thereby inhibiting adipogenesis. Meanwhile, molecular docking studies are generally used to examine the position of compounds at protein binding sites, and are a computer simulation method, which can be regarded as an optimization option, and the conformation of protein-ligand complexes can be automatically determined.

Thus, this example further illustrates the presence or absence of interactions between compound 9 and AMPK (or SCD 1) using molecular docking methods, specifically:

the crystal structures of the SCD1 (PDB ID:4 YMK) and AMPK (PDBID: 5T 5T) proteins were downloaded from the RCSB protein database, and the 3D structure of Compound 9 was converted from chem3D 14.0.0.117. Compounds and proteins were then prepared with Discovery studio4.0 and molecular docking studies were performed with Libdock therein.

The results are shown in FIG. 5, wherein FIG. 5A shows the interaction of compound 9 with AMPK, FIG. 5B shows a two-dimensional view of the interaction of compound 9 with AMPK, FIG. 5C shows the interaction of compound 9 with SCD1, and FIG. 5D shows a two-dimensional view of the interaction between compound 9 and SCD 1. As shown in FIGS. 5A and 5B, when the compound 9 is butted with AMPK, a carbon-hydrogen bond is formed with the residue Amp-402, an alkyl group is formed with the residues Leu-276, val-296, ala-294, leu-314, leu-172, val-292 and Leu-291, and pi cation or pi anion is formed with the residues Lys-169 and So-4404. Meanwhile, as shown in FIGS. 5C and 5D, when the compound 9 and SCD1 are butted, a conventional hydrogen bond is formed with residues Asn-261 and Trp-149, pi alkyl or alkyl is formed with residues Ala-288, his-156, his-294, his-153, val-260, phe-142, leu-254, trp-258 and Leu-181, pi-pi accumulation or pi-pi t form is formed with residues Trp-180. Due to these ligand-protein interactions, compound 9 may bind effectively to AMPK and SCD1, activating the activity of AMPK and its downstream kinase SCD1, thereby significantly inhibiting fat accumulation.

The data statistics and analysis method according to the above embodiment is as follows: all data are expressed as "MEAN ± standard deviation" (MEAN ± SE), sigma plot 12.5 statistical software was used to process the data, and one-way anova-student's t-test was used for the comparison analysis between the groups. When P <0.05, there was a difference between groups, when P <0.01, the difference between groups was remarkable, and when P <0.001, the difference between groups was extremely remarkable.

The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the embodiments described herein, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the 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.