Application of polyketone natural product enterocin in preparation of lipid-lowering medicine

Abstract

The invention discloses application of a polyketone natural product enterocin in preparation of a lipid-lowering medicine, and relates to the technical field of medicine preparation. Research finds that the compound enterocin can regulate a cell ASGR1 target spot and promote cholesterol to flow out of cells, and is different from the action mechanism of statins; the polyketone enterocin can reduce the levels of lipid, TC, TG, LDL-C and the like in an animal body, improve the HDL-C content and promote excretion of TC and TG, part of indexes are equivalent to that of a clinical medicine atorvastatin calcium, part of indexes have remarkable advantages, and the polyketone enterocin has a good application prospect in preparation of the lipid-lowering medicine, and the application proposes that the polyketone natural product enterocin is applied to the lipid-lowering medicine for the first time, so that the application prospect of the polyketone enterocin is wide. A new development direction is provided for the source of the lipid-lowering medicine.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparation, and particularly relates to the application of the polyketide natural product enterocin in the preparation of lipid-lowering drugs. Background Art

[0002] In China, under the circumstances of accelerating population aging and urbanization, the incidence and mortality of cardiovascular diseases are on the rise year by year, higher than those of malignant tumors, which is a major public health problem. Research shows that abnormal elevation of lipid contents such as low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG) in serum is the main factor leading to cardiovascular diseases. In particular, atherosclerosis induced by hypercholesterolemia, and statin drugs that inhibit hepatic cholesterol synthesis are currently first-line lipid-lowering drugs.

[0003] Enterocin is isolated from Streptomyces in the bacterial kingdom and belongs to polyketide natural products, but it is significantly different from statin drugs in structure. The structure of Enterocin is very special, having a unique and highly oxidized tricyclic cage-like core skeleton, with an α-pyranone unit and a benzyl group. The biosynthetic pathway of this compound starts with the assembly of benzoic acid units by ligases, and then through decarboxylative Claisen condensation reactions with malonyl coenzyme A, it is synthesized through seven iterative extension steps mediated by type II polyketide synthase. Particularly noteworthy is the oxidation reaction of the C4 methylene group catalyzed by flavoprotein, which further triggers a Favosky-like rearrangement and two intramolecular aldol condensation reactions, thus constructing the tricyclic cage-like skeleton.

[0004] Currently, chemists and biochemists have spent a great deal of effort in studying the structure and biosynthetic characteristics of enterocin, but the biological functions of its clinical therapeutic potential are still unclear. It is reported in the literature that enterocin has a series of weak to moderate biological activities, including antibacterial efficacy, cytotoxicity against HeLa and HepG2 cancer cells, herbicidal activity, and inhibitory activity against β-amyloid protein fibrillation, etc. However, there has been no report on the lipid-lowering related biological activity of enterocin.

[0005] In summary, currently, the international community has insufficiently developed the biological functions of polyketide natural products such as enterocin with unique structures, and there is no research report on lipid-lowering functions. Therefore, in-depth research to discover the lipid-lowering activity of enterocin and compare its efficacy with clinical drug molecules (such as atorvastatin calcium) will serve the research of innovative drugs and has important research value and significance. Summary of the Invention

[0006] In view of the above deficiencies in the prior art, the present invention provides the use of the polyketide natural product enterocin in the preparation of lipid-lowering drugs to solve the problems raised in the above background art.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The use of the polyketide natural product enterocin in the preparation of lipid-lowering drugs, wherein the chemical formula of the polyketide natural product enterocin is C 22 H 20 O 10 , and its structural formula is shown in Formula I:

[0009]

[0010] Preferably, the lipid-lowering drug is a drug that promotes cholesterol metabolism.

[0011] Preferably, the lipid-lowering drug improves cholesterol metabolism by regulating the ASGR1 (asialoglycoprotein receptor 1) pathway.

[0012] Preferably, the lipid-lowering drug is a drug that reduces subcutaneous and visceral fat levels.

[0013] Preferably, the lipid-lowering drug is a drug that reduces blood lipid levels.

[0014] Preferably, the lipid-lowering drug is a drug that reduces liver fat accumulation and liver lipid levels.

[0015] Preferably, the drug contains the polyketide natural product enterocin.

[0016] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0017] Preferably, the pharmaceutically acceptable excipients include one or more of excipients, binders, disintegrants, lubricants, coating agents, solvents, cosolvents, suspending agents, thickeners, and surfactants.

[0018] Preferably, the dosage form of the lipid-lowering drug is a capsule, tablet, pill, granule, oral liquid preparation, or injection. Compared with the prior art, the beneficial effects of the present invention are:

[0019] (1) The present invention for the first time discloses that the polyketide natural product enterocin can be applied to the preparation of lipid-lowering drugs, providing a new development direction for the source of existing lipid-lowering drugs.

[0020] (2) It was found in the present invention that the polyketide natural product enterocin may improve cholesterol metabolism by regulating the ASGR1 pathway, and this mechanism of action is different from the mode of action of statin drugs in inhibiting cholesterol synthesis clinically.

[0021] (2) Through research, it was found that when the dosage of the polyketide natural product enterocin was 10 mg / kg, the effect of reducing subcutaneous fat was comparable to that of atorvastatin calcium, and the effect of reducing visceral fat was significantly better than that of atorvastatin calcium; the levels of TC, TG, and LDL-C in the blood of high-fat diet mice decreased significantly, approaching those of the positive control group of atorvastatin calcium, and the level of "good" cholesterol HDL-C was higher than that of atorvastatin calcium; enterocin could significantly reduce the fat accumulation in the liver of high-fat diet mice, with an effect comparable to that of atorvastatin calcium; the levels of TC, TG, and LDL-C in the liver of high-fat diet mice decreased, approaching those of the positive control group of atorvastatin calcium, and the level of "good" cholesterol HDL-C was higher than that of atorvastatin calcium; enterocin could significantly promote the lipid excretion of high-fat diet mice, with an effect better than that of atorvastatin calcium. Description of the Drawings

[0022] Figure 1 Results of promoting cholesterol efflux in ASGR1 overexpressing HEK-293T cells by the natural product enterocin. Among them, ASGR1: asialoglycoprotein receptor 1; OE: overexpression of ASGR1;

[0023] Figure 2 Micro-CT modeling images of mice; among them, yellow represents subcutaneous fat, green represents visceral fat, ND: normal diet mouse control; HFD: high-fat diet mouse; Atorvastatin: atorvastatin calcium;

[0024] Figure 3 Statistics of subcutaneous fat and visceral fat levels; among them, A is the statistics of subcutaneous fat level, B is the statistics of visceral fat level, HFD: high-fat diet mouse; ATOR: atorvastatin calcium;

[0025] Figure 4 The natural product enterocin reduces the blood lipid levels of high-fat diet mice; among them, (A) TC content in serum, (B) TG content in serum, (C) LDL-C content in serum, (D) HDL-C content in serum, HFD: high-fat diet mouse; ATOR: atorvastatin calcium;

[0026] Figure 5 The natural product enterocin reduces the liver fat accumulation of high-fat diet mice; among them, ND: normal diet mouse control; HFD: high-fat diet mouse; Atorvastatin: atorvastatin calcium;

[0027] Figure 6 For the natural product enterocin to reduce the liver lipid level in high-fat diet mice; among them, (A) TC content in the liver, (B) TG content in the liver, (C) LDL-C content in the liver, (D) HDL-C content in the liver, HFD: high-fat diet mice; ATOR: atorvastatin calcium;

[0028] Figure 7 For the natural product enterocin to promote cholesterol excretion in high-fat diet mice; among them, (A) TC content in the feces, (B) TG content in the feces, HFD: high-fat diet mice; ATOR: atorvastatin calcium. Detailed implementation manners

[0029] The technical solutions of the present invention will be further described in detail below in conjunction with the specific implementation manners.

[0030] The CAS number of the polyketide natural product enterocin of the present invention is 59678-46-5, which is commercially available. It can be purchased from many commercial companies, such as Merck KGaA in Darmstadt, Germany (Merck & Co., Inc.), and the product number of enterocin in this company is SML3845.

[0031] Example: Lipid-lowering test of the polyketide natural product enterocin

[0032] 1. Test method

[0033] 1.1 Construction of cholesterol efflux model

[0034] Take the experimental HEK-293T cells in the logarithmic growth phase, count them using a Vi-Cell cell counter, and inoculate them in a 96-well plate at 0.2×10 6 / mL, 100 μL of serum-free medium per well, and culture for 24 h. Pipette 30 μL of Lipofectamine TM 2000 transfection reagent and 2470 μL of serum-free medium and mix gently. After gentle oscillation, let it stand at room temperature for 5 min. This is working solution A. Take 20 μg of ASGR1 plasmid and add serum-free medium to 2500 μL, mix gently and let it stand at room temperature for 5 min. This is working solution B. Add working solution B to an equal volume of working solution A, carefully pipette and mix well, pay attention not to generate bubbles during the process, and after standing for 20 min, obtain the transfection working solution. Discard the old medium from the cells and add 50 μL of serum-free medium, then add 50 μL of the mixed transfection working solution, and culture for 24 h to construct ASGR1-overexpressing HEK-293T cells.

[0035] Before the experiment, the marker Bodipy-Cholesterol (0.0625 μM) and enterocin were diluted to the required concentration with 0.1% BSA-DMEM medium. The carrier solution was prepared from serum-free medium and dimethyl sulfoxide. The control group, model group, and experimental group were set up. The control group was added with HEK-293T cells without overexpression of ASGR1 in the carrier solution, the model group was added with HEK-293T cells with overexpression of ASGR1 in the carrier solution, and the experimental group was added with HEK-293T cells with overexpression of ASGR1 in the carrier solution and then enterocin. The specific experimental steps of the experimental group were as follows: 50 μL of the marker solution and the drug solution with the corresponding concentration were added to each well containing HEK-293T cells with overexpression of ASGR1. After mixing, they were placed in a cell culture incubator for 24 h. In each of the above groups, 1% mouse serum was used to induce cholesterol efflux and incubated for 6 h. After incubation, 80 μL of the extracellular fluid was collected into a black 96-well plate, and the remaining culture medium was discarded. 100 μL of 10% SDS cell lysate was added to each well, and the 96-well plate oscillator was used to shake for 6 min to fully lyse the cells. 80 μL of the intracellular fluid was collected into the black 96-well plate to make the liquid evenly distributed, and the fluorescence value was read using Flexstation3 (Ex / Em = 482 / 515 nm).

[0036] 1.2 Construction of high-fat diet mouse model

[0037] First, the high-fat diet contains 17.4% protein, 54.4% carbohydrates, 3.5% fat, 8.2% other components, and added components of 10% lard, 3% cholesterol, and 0.5% bile salts in the basic components. The preparation method of the high-fat diet is as follows: Place the lard (10%) in a heating device at 100 - 150 °C until it completely melts. Slowly add cholesterol (3%) to the melted lard while continuously stirring to ensure complete dissolution of cholesterol. Subsequently, add bile salts (0.5%) and still maintain the stirring state to evenly disperse the bile salts in the oil. After the bile salts are evenly dispersed, add the basic mouse diet and stir-fry it, ensuring that the oil fully penetrates the diet and the nutritional components are evenly distributed. The prepared high-fat diet should be divided into portions according to the daily requirement and refrigerated (4 °C or -20 °C) for subsequent use. Secondly, a high-fat diet mouse model was constructed by inducing C57BL / 6J mice with a high-fat diet. The specific method is to gradually increase the proportion of the high-fat diet on the basis of the normal diet. The high-fat diet and the normal diet are mixed in the ratios of 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, and 4:1 in sequence, and the ratio is adjusted daily until finally completely transitioning to the high-fat diet. The experiments were divided into a normal diet group (ND, Normal Diet), a high-fat diet group (HFD), an atorvastatin calcium administration group (Atorvastatin, ATOR, 1 mg / kg), and an enterocin administration group (1 mg / kg, 10 mg / kg). All groups were given the corresponding drugs or control solvents (equal volume of 0.3% CMC-Na solution) by gavage for 18 weeks. The body weight and food intake were recorded 1 - 2 times a week, and the time was uniformly arranged between 3 - 4 pm. At this time, the mice have not entered the active feeding period, and the body weight fluctuation is small, which can effectively reduce the measurement error. In addition, the feed replacement and gavage operations were both completed synchronously during this period to utilize the characteristics of the mice's active metabolism at night to promote drug absorption and efficacy.

[0038] 1.3 Preparation and Detection of Serum, Liver, and Fecal Samples

[0039] Serum sample preparation: Before the experiment, the mice need to fast for 12 hours. Take a glass capillary with an inner diameter of 1.0 - 1.5 mm and break it into segments 2 - 2.5 cm long before use. When collecting blood, first anesthetize the mice with ether. Fix its head with the left hand and gently press both sides of the neck to hinder venous return, causing congestion in the orbital venous plexus. Hold the capillary with the right hand and insert it into the conjunctiva from the inner canthus, slowly push it towards the bottom of the eye, and gently rotate it to cut the venous plexus, allowing the blood to flow into the EP tube. After blood collection, gently press the eye with a gauze to stop bleeding. When collecting whole blood, the orbital enucleation method can be used. After the mice are anesthetized, fix its body with one hand and cut off the whiskers to avoid blood contamination. Gently press the skin of the eye on the blood collection side to make the eyeball congest and protrude, quickly remove the eyeball with a curved forceps, and at the same time gently press the heart area with the middle finger of the left hand to accelerate blood flow. After the blood has drained, euthanize the mice by dislocation. The collected blood is allowed to stand at room temperature for 30 minutes, then centrifuged at 4°C and 13428 g for 15 minutes, and the supernatant is aliquoted and stored in an -80°C refrigerator. Liver sample preparation: Take out the liver tissue from the -80°C refrigerator, thaw it on ice, accurately weigh the tissue weight, and place it in an EP tube. Clamp in two grinding beads, and according to the ratio of tissue weight (g): volume (mL) = 1:9, add 9 times the volume of homogenization medium (absolute ethanol). Homogenize mechanically at 60 Hz for 30 s × 2 times under an ice-water bath condition, and then centrifuge at 685 g for 10 min. Take the supernatant and put it into an -20°C refrigerator for aliquot storage. Fecal sample preparation: Collect fresh feces from the mice and remove the moisture using a freeze dryer. Accurately weigh 100 mg of the freeze-dried feces, and according to the ratio of weight (g): volume (mL) = 1:9, add 9 times the volume of homogenization medium (absolute ethanol). Homogenize mechanically at 60 Hz for 30 s × 2 times under an ice-water bath condition, and then centrifuge at 685 g for 10 min. Take the supernatant and store it in an -20°C refrigerator after aliquoting. Detection of indicators such as TC, TG, HDL-C, and LDL-C levels in the samples is processed using internationally recognized reagent kits or working solutions, then the absorbance is measured using an enzyme-linked immunosorbent assay reader, and finally data processing is carried out.

[0040] 1.4 In vivo small animal CT imaging

[0041] Three mice were randomly selected from each group and anesthetized with inhaled isoflurane gas using a WD R500 small animal anesthesia machine. Connect the power supply of the air pump and turn on the air pump switch. Rotate and adjust the gas source valve at the front end of the oxygen flow meter so that the output gas reaches the required flow rate. Generally, the oxygen flow rate for mice is 0.3 - 0.5 L / min. The specific required gas flow rate is mainly determined by the body weight and the state of the animal. Wait until the anesthetic fills the induction chamber. After about 1 minute, place the mice in the induction chamber, then close the induction chamber and wait for the animals to be fully anesthetized (this process takes about 2 - 3 minutes). The induction chamber can be gently shaken to check if the mice are fully anesthetized. If the animal's body topples to a side position and does not try to resume its lying position, it indicates that the animal is fully anesthetized. After anesthesia, use a live small animal CT instrument to perform a whole-body scan and take images of subcutaneous and visceral fat in the mice.

[0042] 2. Experimental Results

[0043] 2.1 Natural product enterocin promotes cholesterol efflux in ASGR1 overexpressing HEK-293T cells

[0044] HEK-293T cells were transfected to overexpress ASGR1, and the model was verified by western blot experiment. Compared with the control group, cholesterol efflux in HEK-293T cells overexpressing ASGR1 in the model group decreased.

[0045] The results showed (see details in Figure 1 ), that the natural product enterocin at 10 μM could significantly promote cholesterol efflux in ASGR1 overexpressing HEK-293T cells, indicating that enterocin might improve cholesterol metabolism by regulating the ASGR1 pathway. This mechanism of action is different from the mode of action of clinically used statin drugs that inhibit cholesterol synthesis. All data are expressed as mean ± standard deviation, ***p < 0.001.

[0046] 2.2 Natural product enterocin reduces subcutaneous and visceral fat levels in high-fat diet mice

[0047] The effects of the natural product enterocin on subcutaneous and visceral fat levels in high-fat diet (HFD) mice were measured. The experiment was divided into a normal diet group (ND), a high-fat diet group (HFD), an atorvastatin calcium administration group (Atorvastatin, ATOR, 1 mg / kg), and an enterocin administration group (1 mg / kg, 10 mg / kg).

[0048] Figure 2 Micro-CT modeling images of the mice. The results showed (see details inFigure 3 ), enterocin at 10 mg / kg can significantly reduce the subcutaneous and visceral fat levels in high-fat diet mice. The effect of reducing subcutaneous fat is comparable to that of atorvastatin calcium, and the effect of reducing visceral fat is significantly better than that of atorvastatin calcium. All data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s. indicates no statistical significance (one-way ANOVA).

[0049] 2.3 Natural product enterocin reduces blood lipid levels in high-fat diet mice

[0050] The effect of enterocin on reducing the serum TC, TG, LDL-C, and HDL-C levels in high-fat diet mice was detected. The mice were fed a high-fat diet for 18 weeks. Before detection, the mice were fasted for 12 h. After tail bleeding, the serum lipid content was detected using an in vitro assay kit (immunoenzymatic method).

[0051] The results showed (see details in Figure 4 ), compared with the high-fat control group, after administration of 10 mg / kg of enterocin, the TC, TG, and LDL-C levels in high-fat diet mice decreased, and the decreasing effect was close to that of the positive control group atorvastatin calcium. Notably, after administration of enterocin, the level of HDL-C, a "good" cholesterol, increased significantly, and the effect was significantly better than that of atorvastatin calcium. All data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s. indicates no statistical significance (one-way ANOVA).

[0052] 2.4 Natural product enterocin reduces liver fat accumulation in high-fat diet mice

[0053] Multidimensional analysis of the liver was performed. Morphological findings showed that the livers of mice in the ND group were reddish-brown, while those of mice in the HFD group turned yellowish-brown compared with them, indicating that a high-fat diet can cause lipid accumulation and pathological changes in the livers of mice. After administration (atorvastatin calcium and enterocin), the results showed (see details in Figure 5 ), enterocin can significantly reduce liver fat accumulation in high-fat diet mice, and the effect is comparable to that of atorvastatin calcium when the dosage of enterocin is 10 mg / kg.

[0054] 2.5 Natural product enterocin reduces liver lipid levels in high-fat diet mice

[0055] The effects of enterocin on the levels of liver lipids TC, TG, LDL-C, and HDL-C in high-fat diet mice were detected. The results showed (see details in Figure 6 ), compared with the high-fat control group, after administration of 10 mg / kg enterocin, the levels of liver lipids TC, TG, and LDL-C in high-fat diet mice decreased, approaching those of the positive control group of atorvastatin calcium. Notably, after enterocin administration, the level of HDL-C, the "good" cholesterol, increased significantly, with an effect significantly better than that of atorvastatin calcium. All data are expressed as mean ± standard deviation, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. indicates no statistical significance (one-way ANOVA).

[0056] 2.6 Natural product enterocin promotes cholesterol excretion in high-fat diet mice

[0057] The effects of enterocin on cholesterol excretion in high-fat diet mice were determined. The experiment evaluated the ability of the compound to promote lipid excretion by detecting the contents of total cholesterol TC and triglyceride TG in feces. After the administration, biochemical analysis of fresh feces from mice was performed using an in vitro assay kit (immunoenzymatic method).

[0058] The results showed (see details in Figure 7 ), when enterocin was at 10 mg / kg, the excretion amount of TC was significantly higher than that of the positive control group of atorvastatin calcium, and the excretion amount of TG was also slightly higher than that of the positive control group of atorvastatin calcium, indicating that enterocin can significantly promote lipid excretion in high-fat diet mice, with an effect better than that of atorvastatin calcium. All data are expressed as mean ± standard deviation, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. indicates no statistical significance (one-way ANOVA).

[0059] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of enterocin, a polyketide natural product, in the preparation of lipid-lowering drugs, characterized in that: The structural formula of the polyketide natural product enterocin is shown in Formula I:

2. The use according to claim 1, characterized in that The lipid-lowering drug is a drug that promotes cholesterol metabolism.

3. The use according to claim 2, characterized in that The lipid-lowering drug improves cholesterol metabolism by regulating the ASGR1 pathway.

4. The use according to claim 1, characterized in that The lipid-lowering drug is a drug that reduces subcutaneous and visceral fat levels.

5. The use according to claim 1, characterized in that The lipid-lowering drug is a drug that lowers blood lipid levels.

6. The use according to claim 1, characterized in that The lipid-lowering drug is a drug that reduces liver fat accumulation and liver lipid levels.

7. A lipid-lowering drug, characterized in that: The drug contains enterocin, a polyketide natural product.

8. The lipid-lowering drug according to claim 7, characterized in that The drug also includes pharmaceutically acceptable excipients.

9. The lipid-lowering drug according to claim 8, characterized in that The pharmaceutically acceptable excipients include one or more of excipients, binders, disintegrants, lubricants, coating agents, solvents, cosolvents, suspending agents, thickeners and surfactants.

10. The lipid-lowering drug according to any one of claims 7 to 9, characterized in that: The dosage form of the lipid-lowering drug is capsule, tablet, pill, granule, oral liquid preparation or injection.

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