Lipid-lowering application of triterpenic acid monomeric compound in sea-buckthorn

Abstract

The invention relates to application of a series of compounds in preparation of lipid-lowering drugs. The compounds can reduce lipid accumulation and lipid metabolism related enzyme activity, and can be used for preventing or treating various high-fat related diseases.

Description

Technical Field

[0001] The present invention relates to the field of natural medicines. Background Art

[0002] Hyperlipidemia, also known as high blood lipids or dyslipidemia, usually refers to elevated triglycerides and (or) total cholesterol in plasma, elevated low-density lipoprotein cholesterol and decreased high-density lipoprotein cholesterol. The disease can occur in people of different ages and genders, especially in people aged 50 to 69, with a clear genetic tendency. The causes of the disease include gene mutations, various environmental factors, such as poor eating habits, lack of physical activity, obesity, etc., and other diseases such as diabetes, nephrotic syndrome, liver disease, etc.

[0003] Obesity refers to a pathological state in which excessive body fat accumulates or is abnormally distributed, leading to excessive weight gain. Those without obvious endocrine or metabolic causes are considered simple obesity. The World Health Organization lists obesity as one of the five major diseases that seriously endanger human health. With the development of obesity, clinical symptoms such as fatigue, palpitations, shortness of breath when moving, and decreased physical strength may appear. Over time, it is easy to induce hypertension, hyperlipidemia, cardiovascular and cerebrovascular diseases, and infectious diseases, which not only seriously affect the quality of human life, but also accelerate the aging of the body and increase the incidence of sudden death. Summary of the invention

[0004] The invention provides a series of compounds for use in lipid-lowering and the like.

[0005] Specifically, the present invention provides the use of one of the following compounds or a pharmaceutically acceptable salt thereof in the preparation of a lipid-lowering drug:

[0006]

[0007]

[0008] Furthermore, the compound is selected from:

[0009]

[0010] Furthermore, the drug is a drug that reduces lipid accumulation.

[0011] TC: total cholesterol; TG: triglycerides; LDLC: low-density lipoprotein cholesterol. When one or more of total cholesterol (TC) or low-density lipoprotein cholesterol (LDLC) or triglycerides (TG) exceeds the upper limit of normal values, it can be indicated as a hyperlipidemia patient. The present invention has found that the drug can reduce TG, TC and / or LDLC, indicating that the compound of the present invention can effectively reduce lipids.

[0012] FAS: fatty acid synthase; ACC: acetyl-CoA carboxylase; SCD: stearoyl-CoA desaturase.

[0013] ACC / FAS / SCD plays a key role in the de novo synthesis of fatty acids and is an important metabolic control point. Inhibiting its expression is beneficial for the treatment of lipid metabolism-related diseases. ACC is a key enzyme in the lipogenesis process, which catalyzes acetyl CoA to produce malonyl CoA (Li S, Zhang Q, Wang J, et al. Recent progress in metabolic engineering of Saccharomyces cerevisiae for the production of malonyl-CoA derivatives [J]. Journal of Biotechnology, 2021, 325: 83-90.). FAS uses acetyl CoA and malonyl CoA as substrates and further processes them into palmitic acid (Rudolph MC, Maluf NK, Wellberg EA, et al. Mammalian fatty acid synthase activity from crude tissue lysates tracing 13C-labeled substrates using gas chromatography–mass spectrometry [J]. Analytical biochemistry, 2012, 428 (2): 158-166.). SCD can catalyze the biosynthesis of saturated fatty acids such as palmitic acid, which are synthesized de novo or extracted from the diet, into monounsaturated fatty acids (Paton CM, Ntambi JM. Biochemical and physiological function of stearoyl-CoA desaturase [J]. American Journal of Physiology-Endocrinology and Metabolism, 2009, 297 (1): E28-E37.).

[0014] The present invention has found that the compound can reduce the expression of ACC, FAS and SCD, indicating that the compound can effectively inhibit the generation of fatty acids in the body and reduce lipids in the body.

[0015] Furthermore, the drug is a drug for preventing or treating hyperlipidemia or obesity.

[0016] Hyperlipidemia, also known as high blood lipids or dyslipidemia, usually refers to elevated triglycerides and / or total cholesterol, elevated low-density lipoprotein cholesterol and decreased high-density lipoprotein cholesterol in plasma.

[0017] Obesity refers to excessive fat storage in the body. It is a chronic metabolic disease caused by the interaction of multiple factors such as genetic factors and environmental factors. Its occurrence mechanism is that energy intake exceeds energy consumption, resulting in excessive accumulation of body fat and abnormal weight. Obesity is often accompanied by metabolic disorders while active lipid metabolism, such as hypertriglyceridemia, hypercholesterolemia, high low-density lipoprotein cholesterol, low high-density lipoprotein cholesterol, etc.

[0018] The term "salt" refers to a compound containing a cation and an anion, which can be generated by protonation of a proton-acceptable site and / or deprotonation of a proton-donating site. It is worth noting that protonation of a proton-acceptable site results in the formation of a cationic species, the charge of which is balanced by the presence of a physiological anion, while deprotonation of a proton-donating site results in the formation of an anionic species, the charge of which is balanced by the presence of a physiological cation.

[0019] The term "pharmaceutically acceptable salt" means that the salt is pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to:

[0020] Acid addition salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or with organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, gluconic acid, salicylic acid, benzenesulfonic acid, benzenesulfonic acid, etc.; or with acidic amino acids such as aspartic acid and glutamic acid.

[0021] Base addition salts are formed with the conjugate base of any of the above-mentioned inorganic acids, wherein the conjugate base comprises a cationic component selected from Na+, K+, Mg2+, Ca2+. It should be understood that all references to pharmaceutically acceptable salts include solvent addition forms (solvates) or crystal forms (polymorphs) defined herein of the same acid addition salt.

[0022] The administration method of the compound or pharmaceutical composition of the present invention is not particularly limited, and representative administration methods include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0023] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0024] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0025] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, etc.

[0026] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0027] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances.

[0028] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0029] Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0030] The compounds of the present invention can also be used in injection preparations, wherein the injection is selected from liquid injection (water injection), sterile powder for injection (powder injection) or injection tablets (referring to molded tablets or machine-pressed tablets made by aseptic operation of drugs, dissolved in injection water before use, for subcutaneous or intramuscular injection).

[0031] The powder for injection contains at least an excipient in addition to the above-mentioned compound. The excipient in the present invention is a component intentionally added to the drug, which should not have pharmacological properties in the amount used, but the excipient can help the processing, dissolution or dissolution of the drug, delivery of the drug through a targeted administration route or stability.

[0032] The term "treatment" as used herein includes, but is not limited to, curing a patient's disease, or alleviating the severity of the disease, or delaying the progression of the disease.

[0033] The term "prevention" used in this application refers to intervening and preventing healthy people or asymptomatic patients before the disease occurs to avoid the occurrence of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 , Flow chart of extraction and separation of 4 monomer compounds

[0035] Figure 2 , 4 liquid phase chromatograms of monomer compounds, among which the top picture is the liquid phase diagram from the F4 component, (1) is the chromatogram of the F4-4-1 monomer compound, (2) is the chromatogram of the F4-3-1 monomer compound, (3) is the chromatogram of the F4-2-1 monomer compound, and (4) is the chromatogram of the F4-5-1 monomer compound.

[0036] Figure 3, high-resolution mass spectra of four monomer compounds, Figure 3 (1) is the mass spectrum of the F4-4-1 monomer compound, Figure 3 (2) is the mass spectrum of the F4-3-1 monomer compound, Figure 3 (3) is the mass spectrum of the F4-2-1 monomer compound, and Figure 3 (4) is the mass spectrum of the F4-5-1 monomer compound.

[0037] Figure 4, 1H NMR spectra of four monomer compounds, Figure 4 (1) is the hydrogen spectrum of the monomer compound F4-4-1, Figure 4 (2) is the hydrogen spectrum of the monomer compound F4-3-1, Figure 4 (3) is the hydrogen spectrum of the monomer compound F4-2-1, and Figure 4 (4) is the hydrogen spectrum of the monomer compound F4-5-1

[0038] Figure 5, 13C NMR spectra of four monomer compounds, Figure 5 (1) is the carbon spectrum of the monomer compound F4-4-1, Figure 5 (2) is the carbon spectrum of the monomer compound F4-3-1, Figure 5 (3) is the carbon spectrum of the monomer compound F4-2-1, and Figure 5 (4) is the carbon spectrum of the monomer compound F4-5-1

[0039] Figure 6 Effects of triterpenoid monomers on cell viability

[0040] Figure 7 Effects of triterpenoid monomer compounds on TC, TG and LDLC in HepG2 cells

[0041] Figure 8 , the activity of triterpenoid monomer compounds in alleviating liver damage and oxidative stress

[0042] Fig. 9 , the effects of triterpenoid monomer compounds on the expression of key enzymes in fat metabolism, where Con is the normal group, Mod is the model group, Orlistat is orlistat, BA is betulinic acid, MA is maslinic acid, and JA is jacaranda triterpenoid acid. SREBP1: sterol regulatory element binding protein 1; FAS: fatty acid synthase; ACC: acetyl-CoA carboxylase; SCD: stearoyl-CoA desaturase. DETAILED DESCRIPTION

[0043] Sources of the compounds of the present invention: 4 monomer components 3-O-cis-p-coumaryl maslinic acid, 3-O-cis-p-coumaryl corosolic acid, 3-O-trans-p-coumaryl maslinic acid, and jacaranda triterpenoid acid were made in the laboratory. Ursolic acid and oleanolic acid were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0044] The cell sources used in the present invention: HepG2 cell line was obtained from the Cell Resource Center of Shanghai Institute for Biological Sciences, Chinese Academy of Sciences (Shanghai, China).

[0045] The preparation methods of some compounds can be as follows:

[0046] The seabuckthorn fruit is extracted with 95% ethanol and concentrated to obtain the seabuckthorn extract, distilled water is added, heated in a water bath at 60°C until the extract is dissolved, allowed to stand and precipitated, filtered and separated, and the precipitate is dried to obtain the crude extract of seabuckthorn triterpenic acid. The crude extract of triterpenic acid is added to a dry ODS column, eluted with methanol aqueous solution of different concentrations, and the fraction is collected. After elution with 100% methanol solution, it is concentrated and dried to obtain the F4 fraction. The F4 fraction of the seabuckthorn extract is taken and eluted with 90% MeOH + 0.2% phosphoric acid to obtain F4-1, F4-2, F4-3, F4-4, and F4-5 fractions in turn. NaOH is added to each fraction for neutralization before concentration. Each fraction is subjected to secondary preparation, and the preparation conditions are 84% MeOH + 0.2% phosphoric acid, 180mL / min. Four monomer compounds, F4-2-1, F4-3-1, F4-4-1, and F4-5-1, were obtained in sequence, with purities of 98.002%, 96.683%, 95.626%, and 98.023%, respectively. After structural analysis by high-resolution mass spectrometry, 1H, and 13CNMR, the four monomer components were determined to be 3-O-cis-p-coumaryl hawthorn acid, 3-O-cis-p-coumaryl corosolic acid, 3-O-trans-p-coumaryl hawthorn acid, and jacaranda triterpene acid.

[0047]

[0048] Example 1

[0049] (1) MTT assay to determine cell viability

[0050] HepG2 cells at a concentration of 1×105 / mL were inoculated into a 96-well plate. After culturing for 24 hours until the cells were completely attached to the wall, the old culture medium was discarded, and 6 replicate wells were set for each concentration. 100 μL of DEME complete culture medium containing different concentrations of seabuckthorn triterpenic acid monomer compounds was added to each well. The concentrations of the monomer compounds were 0, 0.5, 1, 10, 20, 50, 100, and 200 μM, respectively. After continuing to culture for 24 hours, the old culture medium was discarded, and new DEME complete culture medium was added. 10 μL of MTT solution was added to each well, and the culture was continued for 4 hours. The absorbance was then measured at 490 nm to calculate the cell viability.

[0051] (2) Determination of biochemical indicators

[0052] HepG2 cells at a concentration of 1×105 / mL were inoculated in a 6-well plate. After culturing for 24 hours, each well was set as a normal group, a model group (0.5mM sodium oleate), a drug-treated group (0.5mM sodium oleate + 20μM monomer compound), and a positive group (0.5mM sodium oleate + 20μM orlistat). The cells were further cultured for 24 hours, and after trypsin digestion, the cells were extracted by centrifugation at 1000r / min for 10min. After adding 50μL of PBS for cell homogenization, the contents of various biochemical indicators in the cells were determined using TC, TG, LDLC, ALT, AST, SOD, MDA, and GSH kits.

[0053] (3) Western Blot

[0054] HepG2 cells at a concentration of 1×105 / mL were inoculated in a 6-well plate. After culturing for 24 hours, each well was set to the normal group, model group (0.5mM sodium oleate), drug group (0.5mM sodium oleate + 20μM seabuckthorn triterpene acid monomer compound), and positive group (0.5mM sodium oleate + 20μM orlistat) and continued to be cultured for 24 hours. The culture medium was aspirated with a suction pump, washed twice with PBS buffer, and the PBS was aspirated. Cell lysis solution was added, the 6-well plate was shaken to mix, placed on ice for lysis for 10 minutes, and the cells were scraped off with a cell scraper and collected in a centrifuge tube. The centrifuge tube was placed on ice and continued to lyse for 30 minutes. After the lysis was completed, the supernatant containing protein was collected in a new EP tube at 12000r / min and 4°C using a low-temperature centrifuge. The protein content was quantitatively determined using a BCA protein detection kit. Take the diluted protein sample, add protein loading buffer and mix well, denature in a metal bath at 100℃ for 15min. Separate the protein by 10% SDS-PAGE and then transfer to PVDF membrane. The blocking solution is 5% skim milk prepared with 1×TBST. After the transfer, take out the PVDF membrane and put it into the incubation box, add skim milk until it covers the PVDF membrane, place it on a shaker at room temperature and shake slowly for 1h, pour out the skim milk, and wash it with 1×TBST for 10min, and wash it 3 times. Dilute the primary antibody at a ratio of 1:1000 with antibody diluent, put the PVDF membrane in the diluted primary antibody solution, incubate the primary antibody at a low speed on a shaker at 4℃ overnight, recover the primary antibody solution, wash it with 1×TBST for 10min, and wash it 3 times. Put the PVDF membrane in a 1:5000 diluted horseradish peroxidase-labeled secondary antibody and incubate it at room temperature for 1h. After incubation, recover the secondary antibody solution and wash the membrane 3 times at room temperature with 1×TBST, each time for 10min. The ECL chemical development method was used for development. The developer A and B were mixed at a ratio of 1:1, and 100 μL was added to the PVDF membrane. The developer was developed and photographed. Western blot data were collected and processed using Image J software, and the grayscale value of protein bands was quantitatively analyzed. The grayscale analysis of protein bands was performed using β-actin as an internal reference. Graphpad prism 8.0 statistical software was used for statistical analysis, and the data were expressed as mean ± standard deviation (X ± s). One-way ANOVA was used for the mean values ​​of each group, and P < 0.05 was considered statistically significant.

[0055] 3. Experimental results

[0056] like Figure 6 As shown, the four seabuckthorn triterpene acid extracts of the present invention have no toxicity to cells at concentrations below 20 μM, but reduce cell survival rates at concentrations above 20 μM. Therefore, the unified concentration for administration is 20 μM.

[0057] like Figure 7As shown in the results, compared with the normal group, the contents of TC, TG and LDLC in HepG2 cells after 24 hours of sodium oleate treatment were significantly different (P<0.01). In terms of TG, the triterpenic acid of high-grade jacaranda was significantly different from that of the model group at a concentration of 20 μM (P<0.01), while 3-O-cis-p-coumaryl maslinic acid was significantly different from that of the model group (P<0.05). In terms of TC, at a concentration of 20 μM, all six monomers were significantly different from those of the model group (P<0.01). In terms of LDL-C, the triterpenic acid of high-grade jacaranda was significantly different from that of the model group (P<0.05), while 3-O-cis-p-coumaryl corosolic acid and 3-O-trans-p-coumaryl maslinic acid were significantly different from those of the model group at a concentration of 20 μM (P<0.01). Therefore, 3-O-cis-p-coumarylmaslinic acid, 3-O-cis-p-coumarylcorosolic acid, 3-O-trans-p-coumarylmaslinic acid, and jacaranda triterpenoid acids have the effect of improving oleic acid-induced lipid accumulation in HepG2 cells.

[0058] like Figure 8 As shown in the results, compared with the normal group, the ALT, AST, and MDA contents of the 0.5 mM oleic acid modeling group increased significantly (P<0.05), while the SOD and GSH contents decreased significantly (P<0.05). In terms of ALT, 3-O-trans-p-coumaryl maslinic acid significantly alleviated the rising trend of ALT compared with the model group. In terms of AST, except for 3-O-trans-p-coumaryl maslinic acid, the other compounds could significantly reduce the AST content compared with the model group (P<0.05). In terms of the three indicators of relieving oxidative stress, SOD, MDA, and GSH, compared with the model group, the triterpenic acid of high-grade blue jacaranda had extremely significant differences in the three indicators (P<0.01), and could significantly alleviate oxidative stress damage. Therefore, the triterpenic acid of high-grade blue jacaranda can be used as an effective triterpenic acid monomer compound to reduce lipid accumulation, alleviate liver damage, and relieve oxidative stress.

[0059] like Fig. 9 As shown, in order to clarify the mechanism of the effect of the potent compounds on lipid accumulation, the protein level of the AMPK-SREBP1 pathway signal was verified by Western blotting. The protein expression of SREBP1, ACC, FAS, and SCD was detected. As shown in the figure, the protein expression of SREBP1, ACC, FAS, and SCD in cells was significantly increased after OA stimulation (P<0.05). Compared with the model group, the trend of the high-added jacaranda triterpenic acid, betulinic acid, and hawthorn acid administration groups was reduced, especially the high-added jacaranda triterpenic acid was able to significantly alleviate the increase in SREBP1, ACC, FAS, and SCD protein expression (P<0.01). These results indicate that the AMPK-SREBP1 signaling pathway may play an important role in the lipid-lowering effect of triterpenic acid.

Claims

1. Use of one of the following compounds or a pharmaceutically acceptable salt thereof in the preparation of a lipid-lowering drug:

2. The use according to claim 1, characterized in that The compound is selected from:

3. The use according to claim 1, characterized in that The drug is a drug that reduces lipid accumulation.

4. The use according to any one of claims 1 to 3, characterized in that: The medicine is a medicine for reducing one or more of triglycerides, cholesterol and low-density lipoprotein cholesterol.

5. The use according to any one of claims 1 to 3, characterized in that: The drug is a drug for reducing the expression of one or more of fatty acid synthase, acetyl-CoA carboxylase and stearoyl-CoA desaturase.

6. The use according to any one of claims 1 to 5, characterized in that: The medicine is a medicine for preventing or treating hyperlipidemia.

7. The use according to any one of claims 1 to 5, characterized in that: The medicine is a medicine for preventing or treating obesity.

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