TW202112381A - Use of compounds for enhancing expression of genes involved in cholesterol metabolism - Google Patents

Abstract

The present invention provides use of lariciresinol 4'-O-β-D-glucopyranoside or adenoside in manufacture of a pharmaceutical composition for enhancing expression of genes involved in cholesterol metabolism. The cholesterol metabolism related genes include SCARB1, APOA1, and LDLR. By promoting the expression of these genes, 4'-O-β-D-glucopyranoside and adenoside can reduce the risk of cardiovascular diseases in individuals with poor cholesterol metabolism.

Description

Use of compounds that promote the expression of cholesterol metabolism-related genes

The present invention relates to the use of a compound for the preparation of a blood cholesterol content regulator, in particular to the use of a glycoside or nucleoside compound for the preparation of a pharmaceutical composition for promoting the expression of genes related to cholesterol metabolism.

Cholesterol metabolism in the human body involves multiple organs, of which the most important organ is the liver. According to previous studies, the cholesterol content of cells is regulated by a complex balance, which involves the endogenous synthesis of cholesterol, the uptake of cholesterol by cells, and the excretion of intracellular cholesterol from the cells into the blood.

When cholesterol is transported in the blood, it is packaged in vesicles together with other lipids and proteins in the form of cholesterol lipids. The intestine assembles cholesterol ingested through diet into chylomicrons, which are transported by the blood and finally absorbed by the liver. The liver is also the main place for cholesterol synthesis in the body. The liver packs dietary and newly synthesized cholesterol into low-density lipoproteins (LDL), which are secreted into the blood for transport to other tissues. Cells of these tissues absorb LDL through endocytosis mediated by LDL receptors, thereby obtaining and using cholesterol from LDL. On the other hand, high-density lipoprotein (HDL) promotes blood vessel health by obtaining excess cholesterol from surrounding tissues and transporting it to the liver. In the liver, cholesterol is converted into bile acids and excreted from the body.

Cholesterol homeostasis is essential to human health. When cholesterol metabolism is defective, it can cause too much cholesterol to circulate in the blood. For example, defects in LDL receptors will reduce the uptake of LDL by cells, which will allow LDL to accumulate in the blood, leading to cholesterol deposits in the arterial wall, and ultimately causing atherosclerosis and coronary artery disease. Due to the strong negative correlation between blood cholesterol level and coronary artery disease, blood cholesterol level is an important index for clinical evaluation of cardiovascular disease risk.

In view of this, in order to reduce the risk of individuals suffering from cardiovascular diseases due to poor cholesterol metabolism, it is really necessary to develop a composition that can promote cholesterol metabolism.

For this reason, one object of the present invention is to provide a composition for the preparation of a pharmaceutical product for promoting the expression of cholesterol metabolism-related genes, wherein the composition comprises a pharmaceutically acceptable carrier, and one selected from the group consisting of larisinol 4'-O-β-D-glucopyranoside, adenosine, and a combination of compounds consisting of a group.

In an embodiment of the present invention, the cholesterol metabolism-related gene regulated by the composition encodes a low-density lipoprotein receptor (LDLR), and the concentration of the compound is at least 50 μg/mL.

Another object of the present invention is to provide a kind of lariciresinol 4'-O-β-D-glucopyranoside (lariciresinol 4'-O-β-D-glucopyranoside) for preparing the expression of genes related to promoting cholesterol metabolism Use of pharmaceutical composition.

In an embodiment of the present invention, the cholesterol metabolism-related gene regulated by larisinol 4'-O-β-D-glucopyranoside encodes a scavenger receptor class B type 1 (scavenger receptor class B). member 1, SRB1) or a low-density lipoprotein (LDL) receptor (LDLR).

In one embodiment of the present invention, the pharmaceutical composition contains laricinol 4'-O-β-D-glucopyranoside at a concentration of at least 50 μg/mL.

Another object of the present invention is to provide a use of adenosine (adenoside) for preparing a pharmaceutical composition for promoting the expression of genes related to cholesterol metabolism.

In one embodiment of the present invention, the cholesterol metabolism-related gene regulated by adenosine encodes a low-density lipoprotein receptor (LDLR).

In an embodiment of the present invention, the pharmaceutical composition contains adenosine at a concentration of at least 50 μg/mL.

This article discloses that administering an effective amount of laricinol 4'-O-β-D-glucopyranoside or adenosine can significantly promote the expression of genes related to cholesterol metabolism in cells. Therefore, the two compounds can be used to prepare a composition that promotes the expression of cholesterol metabolism-related genes, which can reduce the risk of individuals suffering from cardiovascular diseases due to poor cholesterol metabolism, thereby improving the cardiovascular system of those with abnormal cholesterol metabolism or those on a high-cholesterol diet. health. The composition can be powder, granule, solution, colloid or paste, and can be made into medicines, foods, beverages, or nutritional supplements, and administered to a body by oral or other methods.

The following will further illustrate the implementation of the present invention in conjunction with the drawings. The following examples are used to illustrate the features and applications of the present invention, rather than to limit the scope of the present invention. Anyone familiar with the art will not depart from Within the spirit and scope of the present invention, some changes and modifications can be made. Therefore, the scope of protection of the present invention shall be subject to those defined by the attached patent scope.

Figure 1 shows the relative expression levels of SCARB1, APOA1, and LDLR genes in HepG2 cells treated with the tartary buckwheat seed coat extract or its secondary extract of an embodiment of the present invention for 24 hours relative to the control group cells (not treated) ; FT stands for tartary buckwheat seed coat extract, FTE stands for ethyl acetate layer extract, FTB stands for n-butanol layer extract, FTW stands for water layer extract.

Figure 2 shows the mass spectrum of compound FT-01.

Figure 3 shows the hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectrum of compound FT-01.

Figure 4 shows the mass spectrum of compound FT-02.

Figure 5A shows the ¹ H-NMR spectrum of compound FT-02.

Figure 5B shows the carbon nuclear magnetic resonance ( ¹³ C-NMR) spectrum of compound FT-02.

Figure 5C shows the hydrogen-hydrogen correlation (COSY) spectrum of compound FT-02.

Figure 5D shows the carbon-hydrogen heteronuclear single quantum correlation (HSQC) spectrum of compound FT-02.

Figure 5E shows the carbon-hydrogen heteronuclear multiple bond correlation (HMBC) spectrum of compound FT-02.

Figure 6 shows the mass spectrum of compound FT-03.

Figure 7 shows the ¹ H-NMR spectrum of compound FT-03.

Figure 8 shows the relative expression levels of SCARB1, APOA1, and LDLR genes in HepG2 cells treated with compounds FT-01, FT-02, or FT-03 for 24 hours relative to control group cells (not treated).

The present invention provides a use of laricinol 4'-O-β-D-glucopyranoside or adenosine for preparing a pharmaceutical composition for promoting the expression of cholesterol metabolism-related genes. The present invention also provides a use of a composition comprising a pharmaceutically acceptable carrier, and any of the foregoing compounds or combinations thereof for the preparation of pharmaceuticals for promoting the expression of genes related to cholesterol metabolism. The following examples show that administering any of the aforementioned compounds or compositions containing these compounds to hepatocytes can promote the expression of genes including SCARB1, APOA1, and LDLR.

definition

Unless otherwise specified, "a", "the" and similar terms used in this article should be understood to include both singular and plural forms.

The numerical values used herein are approximate values, and all experimental data are expressed in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

The bitter buckwheat described herein is an edible plant belonging to the genus Fagopyrum of Polygonaceae (Polygonaceae), and its scientific name is Fagopyrum tataricum , also known as Tartary buckwheat or green buckwheat. Compared with common buckwheat ( Fagopyrum esculentum ) of the same genus, tartary buckwheat has a bitter taste and contains more rutin, but lacks salicylaldehyde and naphthalene. Tartary buckwheat and ordinary buckwheat are generally regarded as grains, but they are different from common grains belonging to the Poaceae family, so they have nothing to do with wheat.

The pharmaceutical composition or medicine described herein can be prepared into a dosage form suitable for parenterally or orally administration by using techniques well known to those skilled in the art, which includes But not limited to: injection (for example, sterile aqueous solution or dispersion), sterile powder, tablet, troche, lozenge (lozenge), pill (pill), capsule (capsule), dispersible powder (dispersible powder), fine particle (granule), solution, suspension (suspension), emulsion (emulsion), syrup (syrup), elixir (elixir) ), slurry and the like.

The pharmaceutical compositions described herein can be administered by parenteral routes, including but not limited to: intraperitoneal injection, subcutaneous injection, intramuscular injection, and Intravenous injection.

The pharmaceutical composition described herein may include a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing technology. The pharmaceutically acceptable carrier can be Contains one or more reagents selected from the group consisting of solvent (solvent), emulsifier (emulsifier), suspending agent (suspending agent), decomposer (decomposer), binding agent (binding agent), excipient Excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant, absorption delay agent ( absorption delaying agent), liposomes and the like. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

The aforementioned pharmaceutically acceptable carrier includes a solvent selected from the group consisting of water, normal saline (normal saline), phosphate buffered saline (PBS), sugar-containing solution, and alcohol-containing solution. Aqueous solution containing alcohol, and combinations thereof.

Materials and Methods material

DMEM medium (Gibco Dulbecco's modified Eagle's medium), fetal bovine serum (FBS), penicillin/streptomycin (Gibco penicillin/streptomycin), and phosphate buffered saline (Gibco PBS) were purchased from Thermo Fisher Scientific.

Solvents were purchased from Merck, Taiwan, including ethyl acetate, methanol, ethanol, acetonitrile, acetone, dichloromethane, and chloroform- d ₁ (degree of deuteration 99.5%), methanol - d ₆ (deuterated degree 99.5%), heavy water (deuterium oxide, deuterated degree> 99.8%), and dimethyl sulfoxide _{- d 6 (dimethyl sulfoxide- d 6} , Degree of deuteration>99.9%).

Chemical analysis equipment

The compound separation system utilizes column chromatography (column chromatography) and thin layer chromatography (thin layer chromatography, TLC). The medium pressure liquid chromatography (MPLC) system is Combi Flash ^® Rf+ (Teledyne ISCO); the column packing material is selected from Sephadex LH-20 (Amersham Biosciences), Diaion HP-20 (Mitsubishi Chemical) , Merck Kieselgel 60 (40-63μm, Art.9385), and Merck LiChroprep® RP-18 (40-63μm, Art.0250). The high performance liquid chromatography (HPLC) system is equipped with Hitachi L-2310 series pumps, Hitachi L-2420 ultraviolet-visible light detector (detection wavelength is 200nm to 380nm), and D-2000 Elite data Processing software; the column system is selected from the analytical grade Discovery® HS C ₁₈ (250×4.6mm, 5μm; SUPELCO) and Mightysil RP-18 GP 250 (250×4.6mm, 5μm; Kanto Chemical), and the semi-preparative Discovery® HS C ₁₈ (250×10.0mm, 5μm; SUPELCO) and preparative Discovery® HS C ₁₈ (250×21.0mm, 5μm; SUPELCO). Thin-layer chromatography film is coated with silicone 60 F ₂₅₄ (0.25mm; Merck) or RP-18 F _254S (0.25mm; Merck) aluminum film, which is used with UV lamp UVP UVGL-25 (wavelength: 254nm and 365nm) analysis.

The chemical structure of the compound is determined by mass spectrometry (MS) and nuclear magnetic resonance spectrometry (NMR). Specifically, electrospray ionization tandem mass spectrometry (ESI-MS/MS) and two-dimensional ion trap tandem Fourier transform mass spectrometry (Bruker amaZon SL system and Thermo Scientific Orbitrap Elite system) were used; and 400MHz Varian 400 FT-NMR was used to obtain one Two-dimensional and two-dimensional NMR spectroscopy, with tetramethylsilane (TMS) as the internal standard (δ=0). NMR spectrum uses δ to represent chemical shift (chemical shift), the unit is ppm; coupling constant ( J ) is in Hz; s represents singlet (singlet), d represents doublet (doublet), d represents triplet (triplet) , Q means quartet, p means quintet, m means multiplet, and brs means broad peak.

Cell culture

The cell line used in the following examples was purchased from the human liver cancer cell line HepG2 (ATCC HB-8065) of the American Type Culture Collection (ATCC). HepG2 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin under the conditions of 37°C and 5% carbon dioxide, hereinafter referred to as cell culture medium.

Gene expression analysis

Based on quantitative polymerase chain reaction (qPCR) to determine the expression level of cholesterol metabolism-related genes in HepG2 cells, the steps are briefly described as follows. According to the manufacturer’s instructions, use RNA Extraction Kit (Geneaid) to isolate ribonucleic acid (RNA) from cells, and reverse transcribe 2000ng RNA into cDNA at 37°C with the reverse transcriptase SuperScript® III Reverse Transcriptase (Invitrogen) . Afterwards, use the qPCR kit (KAPA CYBR FAST qPCR Kit (2X); KAPA Biosystems) and the primer pair of the target gene and the Glyceraldehyde 3-phosphate dehydrogenase gene GAPDH as an internal control (Table 1) Perform qPCR on the aforementioned cDNA in a PCR reaction machine (Step One Plus Real-Time PCR system; Applied Biosystems), and analyze the melting curve of the PCR product.

Finally, the 2- ^△△CT method was used to determine the relative expression level of the target gene. In this method, the cycle threshold (C _T ) of the GAPDH gene is used as the cycle threshold of the reference gene of the internal control, and the relative fold change is calculated according to the following formula:

△ C _T = experimental group or control group gene of C _T - C _T internal control

△△ C _T = test group △ C _T - △ C _T of the control group

Multiple change=2 ^{-△△Ct average value}

The data are expressed as mean±standard deviation (SD). The statistical analysis department uses the STDEV function in Excel software to calculate the standard deviation of the relative expression of each gene, and uses the one-tailed student t test (TTEST) to determine the statistical significance of the differences between the data.

Example 1 Preparation of compounds with cholesterol-lowering activity

In order to obtain compounds with cholesterol-lowering activity, first prepare a tartary buckwheat seed coat extract. In short, first wash and dry the whole tartary buckwheat husk, which is the seed coat of tartary buckwheat, and crush it with a grinder. Secondly, the crushed tartary buckwheat seed coat is extracted with water as a solvent. The mixing weight ratio of the solvent and the pulverized tartary buckwheat seed coat ranges from 15:1 to 10:1. The extraction temperature is between 70°C and 90°C. The extraction time in this embodiment is 1 to 2 hours.

In order to remove residual solids, the tartary buckwheat seed coat extract obtained by the above extraction step is cooled to room temperature, and then filtered with a 400 mesh filter to obtain a supernatant. The supernatant can be further concentrated under reduced pressure at 40°C to 60°C to obtain a concentrated product (FT).

Thereafter, a secondary extract rich in cholesterol-lowering active ingredients is further separated from the tartary buckwheat seed coat extract, and the steps are briefly described as follows. First, extract 10L of tartary buckwheat seed coat extract three times by means of liquid phase distribution in equal proportions of ethyl acetate and water (volume ratio 1:1). The obtained ethyl acetate layer extracts were combined, and then concentrated and dried under reduced pressure to obtain about 1.9 g of an ethyl acetate layer extract (FTE). The remaining aqueous layer extracts are extracted three times with a liquid phase distribution method in the same ratio of n-butanol and water (volume ratio 1:1). The obtained n-butanol layer extract and the aqueous layer extract were combined separately, and then concentrated and dried under reduced pressure to obtain about 11.5 g of an n-butanol layer extract (FTB) and about 75.0 g of an aqueous layer extract (FTW).

In order to evaluate the cholesterol-lowering effect of human liver cancer cell line HepG2, 2 mL of cell culture medium containing 20μg/mL of the aforementioned tartary buckwheat seed coat extract, ethyl acetate layer extract, n-butanol layer extract, or water layer extract is applied, or Treat the cells with cell culture medium only (control group). After culturing cells in each group at 37 ℃ 24 hours are spent amount of gene expression analysis, a gene includes a coding scavenger receptor class B type 1 (SRB1) SCARB1 the gene encoding the apolipoprotein A1 (apolipoprotein A1, ApoA1) gene of APOA1 , And the LDLR gene encoding low-density lipoprotein receptor (LDLR).

The reason for using the above three genes as index genes for cholesterol metabolism is that SRB1 is a receptor required for liver cells to take up high-density lipoprotein (HDL), and its presence helps cholesterol from surrounding tissues to enter liver cells to be metabolized through HDL; ApoA1 It is one of the main constituent proteins of HDL. Its production facilitates the formation of HDL and therefore promotes the transport of cholesterol by HDL to liver cells for further metabolism; LDLR is a receptor for low-density lipoprotein (LDL), and its presence helps liver cells and Other cells take up LDL in the blood to reduce the accumulation of cholesterol in the blood.

Figure 1 shows the relative expression levels of each target gene in the HepG2 cells of the aforementioned groups; ^{* **} and ^{*** in the} figure indicate p<0.05, p<0.01, and p<0.001 compared to the control group, respectively. According to Figure 1, compared with the control group, the administration of tartary buckwheat seed coat extract (FT) can only improve the expression of the LDLR gene in HepG2 cells; the administration of ethyl acetate layer extract significantly reduced the expression of SCARB1, APOA1, and LDLR gene; However, the treatment of n-butanol layer extract (FTB) increased the expression of SCARB1, APOA1, and LDLR genes by about 18%, 23%, and 60%, and the treatment of water layer extract (FTW) increased SCARB1, APOA1, and The expression of the LDLR gene was approximately 119%, 51%, and 56%, respectively.

Therefore, according to the bioassay guided fractionation method, the cholesterol-lowering active ingredient is further separated from the n-butanol layer extract and the aqueous layer extract. Using a column packed with Sephadex LH-20 and methanol extraction liquid, the n-butanol layer extract (about 11.5 g) was subjected to column chromatography, and 6 divided layers (labeled BF1 to BF6 respectively) were obtained. Afterwards, using a mixture of dichloromethane and methanol in a volume ratio of 7:1 as the eluent, the BF2 layer was subjected to silica gel column chromatography, and the resulting effluent was separated by thin-layer chromatography to obtain 10 Divide the layers (labeled BF2-1 to BF2-10 respectively). BF2-5 is divided into layers with a mixture of water and methanol in a medium-pressure liquid chromatography system for declining polarity, and the resulting effluent is separated by thin-layer chromatography to obtain 6 divided layers (respectively labeled as BF2- 5-1 to BF2-5-6). BF2-5-6 is divided into layers and further uses a mixture of methanol and water in a volume ratio of 3:7 as the mobile phase for high performance liquid chromatography (using a C18 column), and about 8.0 mg of compound FT-02 can be separated.

In addition, using a column filled with Diaion HP-20 and an eluent mixed with a decreasing polarity gradient of water and methanol, the aqueous layer extract (approximately 75.0g) was subjected to column chromatography to separate 6 divided layers (respectively labeled as WF1 to WF6). After that, WF2 was divided into layers to obtain about 200 mg of compound FT-01 by the methanol recrystallization method. Using a mixture of dichloromethane and methanol in a volume ratio of 7:1 as the eluent, silica gel column chromatography was performed on the mother liquor after WF2 was divided into layers and recrystallized, and about 5.0 mg of compound FT-03 was isolated.

The chemical structures of compounds FT-01, FT-02, and FT-03 were determined by mass spectrometry and nuclear magnetic resonance spectroscopy (NMR). Figures 2 and 3 show the mass spectrum and NMR spectrum of FT-01, respectively; Figure 4 and Figures 5A-5E show the mass spectrum and NMR spectrum of FT-02, respectively; Figure 6 and Figure 7 show the mass spectrum of FT-03, respectively Figure and NMR spectrum. According to Figure 2-3, the compound FT-01 is determined to be rutin; according to Figures 4 and 5A-5E, the compound FT-02 is determined to be lariciresinol 4'-O-β-D-glucopyranoside (lariciresinol). 4'-O-β-D-glucopyranoside); According to Figure 6-7, the compound FT-03 was determined to be adenosine (adenoside). Table 2 below shows the structure of each of the compounds.

Example 2 Validation of the compound's cholesterol-lowering activity

In order to verify the effects of the compounds FT-01, FT-02, and FT-03 in regulating the expression of cholesterol metabolism-related genes, the human hepatocarcinoma cell line HepG2 was administered with 2 mL of cell culture medium containing 50 μg/mL of any of the three compounds, or only Cell culture medium treated cells (control group). Cells in each group were cultured at 37°C for 24 hours and then used for gene expression analysis. The target genes included SCARB1, APOA1, and LDLR genes.

Figure 8 shows the relative expression levels of each target gene in the HepG2 cells of the aforementioned groups; ^{* **} and ^{*** in the} figure indicate p<0.05, p<0.01, and p<0.001 compared to the control group, respectively. According to Figure 8, compared with the control group, administration of FT-01 (i.e. rutin) could not improve the performance of SCARB1, APOA1, and LDLR genes. In contrast, the administration of FT-02 (i.e. larisinol 4'-O-β-D-glucopyranoside) significantly increased the performance of SCARB1 and LDLR genes by approximately 34% and 127%, respectively. In addition, the treatment of FT-03 (namely adenosine) also significantly increased the expression of the LDLR gene by approximately 135%. This result verified the effect of FT-02 and FT-03 on promoting the expression of cholesterol metabolism-related genes.

In summary, the present invention discloses that administering an effective amount of laricinol 4'-O-β-D-glucopyranoside and adenosine can significantly promote the expression of genes related to cholesterol metabolism in cells. Therefore, the two compounds can be used to prepare a composition for promoting the expression of cholesterol metabolism-related genes, which can reduce the risk of individuals suffering from cardiovascular diseases due to poor cholesterol metabolism, thereby improving the cardiovascular health of people with abnormal cholesterol metabolism or those with high cholesterol diets. The composition can be powder, granule, solution, colloid or paste, and can be made into medicines, foods, beverages, or nutritional supplements, and administered to a body by oral or other methods.

<110> Dajiang Biomedical Co., Ltd.

<120> Use of compounds that promote the expression of cholesterol metabolism-related genes

<130> 108B0461-I1

<160> 8

<170> PatentIn version 3.5

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Claims (10)

The use of a composition for the preparation of pharmaceuticals for promoting the expression of cholesterol metabolism-related genes, wherein the composition comprises a pharmaceutically acceptable carrier, and one selected from the group consisting of larisinol 4'-O-β-D-pyridine Glucopyranoside, adenosine, and a combination of compounds formed by the group. The use described in item 1 of the scope of patent application, wherein the cholesterol metabolism-related gene encodes a low-density lipoprotein receptor (LDLR). The use as described in item 1 of the scope of the patent application, wherein the concentration of the compound is at least 50 μg/mL. A use of larisinol 4'-O-β-D-glucopyranoside for preparing a pharmaceutical composition for promoting the expression of cholesterol metabolism-related genes. The use described in item 4 of the scope of patent application, wherein the cholesterol metabolism-related gene encodes a scavenger receptor type B type 1 (SRB1). The use described in item 4 of the scope of patent application, wherein the cholesterol metabolism-related gene encodes a low-density lipoprotein receptor (LDLR). The use described in item 4 of the scope of the patent application, wherein the concentration of the larisinol 4'-O-β-D-glucopyranoside is at least 50 μg/mL. An adenosine is used to prepare a pharmaceutical composition for promoting the expression of genes related to cholesterol metabolism. The use described in item 8 of the scope of patent application, wherein the cholesterol metabolism-related gene encodes a low-density lipoprotein receptor (LDLR). The use as described in item 8 of the scope of patent application, wherein the concentration of the adenosine is at least 50 μg/mL.

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