3 apos; application of-dimethylallyl genistein to treatment of calcified vascular diseases

Abstract

The invention discloses application of 3 '-dimethyl allyl genistein to treatment of a calcified vascular disease. The 3 '-dimethylallyl genistein is extracted from ficus virens, and the research finds that the natural compound can significantly inhibit the calcification level of vascular smooth muscle cells and mouse blood vessels, can effectively reduce the calcification level of calcified vascular diseases, can be further used as a medicine for treating the calcified vascular diseases, and has broad application prospects. Clinical application of the natural compound to treatment of calcified vascular diseases becomes possible. Meanwhile, the natural compound disclosed by the invention is low in toxicity, so that damage to organisms is avoided.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and more specifically, to the application of 3'-dimethylallyl genistein in the treatment of calcified vascular diseases. Background Art

[0002] Vascular calcification is a common pathological phenomenon in various diseases such as chronic kidney disease (CKD), vascular injury, atherosclerosis, and aging. Vascular calcification is mainly due to the transformation of the osteogenic phenotype of smooth muscle cells in the vascular medium, resulting in calcium and phosphorus deposition, causing the blood vessels to lose their contractile function, leading to hardening of the vascular wall, formation of atherosclerotic plaques, and further aggravating CKD. Therefore, it is of great significance to study drugs and mechanisms for the precise treatment of vascular calcification.

[0003] In the article "Study on the Regulation of Vascular Smooth Muscle Cell Calcification by Genistein", it is disclosed that genistein can significantly inhibit the calcification of mouse vascular smooth muscle cells (VSMCs) in a concentration-dependent manner. The results of fluorescence quantitative PCR and Western Blot show that genistein can induce the expression of OPG and a-SMA, and inhibit the expression of bone formation-related genes ALP and OPN (P < 0.05). The conclusion is that genistein regulates mouse VSMCs through the OPG signaling pathway, providing a new way to inhibit vascular calcification. It can be seen that the prior art discloses that genistein has a therapeutic effect on the calcification of mouse vascular smooth muscle cells, but the application of 3'-dimethylallyl genistein (isowighteone) in the treatment of calcified vascular diseases is not disclosed in the prior art. Summary of the Invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies existing in the prior art, and provide an application of 3'-dimethylallyl genistein in the preparation of a drug for the treatment of calcified diseases.

[0005] The above object of the present invention is achieved by the following technical solutions:

[0006] The research of the present invention shows that 3'-dimethylallyl genistein can significantly inhibit the calcification levels of vascular smooth muscle cells and mouse blood vessels, can effectively reduce the calcification level of calcified vascular diseases, can be further used as a drug for the treatment of calcified vascular diseases, and 3'-dimethylallyl genistein can be extracted from Ficus hispida.

[0007] Therefore, the present invention provides an application of 3'-dimethylallyl genistein in the preparation of a drug for the treatment of calcified diseases, and the 3'-dimethylallyl genistein is shown as the following formula (I):

[0008]

[0009] Furthermore, the calcific disease is a calcific vascular disease.

[0010] Furthermore, the calcific vascular disease is vascular calcification.

[0011] Furthermore, the preparation method of 3'-dimethylallyl genistein (Isowighteone) is to dry and powder the fruits of Ficus hispida, and obtain it through extraction, extraction, elution, and separation. Natural plants have advantages in treating cardiovascular diseases, improving curative effects, and reducing toxicity. With the progress of biochemical and pharmacological research, natural plants are increasingly regarded as a valuable source for alternative and complementary treatments of vascular calcification. Ficus hispida L.f. (Moraceae) is a plant mainly growing in the tropical and subtropical regions of southern China and is well-known for its uses in traditional medicine. Different parts of Ficus hispida are used in medical practice, mainly due to their significant anti-inflammatory and antioxidant properties. However, its active ingredients and the potential mechanisms promoting its therapeutic effects still need to be clearly elucidated. 3'-dimethylallyl genistein extracted from Ficus hispida is an important component and member of 7-hydroxyisoflavone. 7-hydroxyisoflavone has hydroxyl groups substituted at the 5, 7, and 4' positions of isoflavone and a pentyl group substituted at the 3' position.

[0012] Specifically, the preparation method of 3'-dimethylallyl genistein includes the following steps: drying and powdering the fruits of Ficus hispida, extracting by ultrasound, then extracting, performing methanol gradient elution in a liquid chromatograph to obtain 21 components, and then separating the 3'-dimethylallyl genistein by high-performance liquid chromatography.

[0013] Furthermore, the solvent for the ultrasound is ethanol.

[0014] Preferably, the ethanol is 75% ethanol.

[0015] Furthermore, the solvent for the extraction is dichloromethane.

[0016] Furthermore, the elution is performed by successively using methanol solutions of 30% to 100%.

[0017] Furthermore, the separation is performed by using high-performance liquid chromatography.

[0018] Preferably, the conditions for high-performance liquid chromatography separation are a Shimadzu preparative column, 5um, 20×250mm, 70% methanol / water, and a flow rate of 10 mL / min.

[0019] Furthermore, the drug includes but is not limited to granules, instant granules, tablets, and capsules.

[0020] Furthermore, the drug treats calcified diseases by reducing calcium ion levels and inhibiting the expression of calcification genes.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides the application of 3'-dimethylallyl genistein in the treatment of calcified vascular diseases. 3'-dimethylallyl genistein is extracted from Ficus hispida in the present invention. It is found that this natural compound can significantly inhibit the calcification levels of vascular smooth muscle cells and mouse blood vessels, can effectively reduce the calcification level of calcified vascular diseases, and can further be used as a drug for treating calcified vascular diseases, making it possible to clinically use this natural compound to treat calcified vascular diseases. At the same time, the natural compound described in the present invention has low toxicity and avoids damage to the body. Description of the Drawings

[0023] Figure 1 is the extraction process of Isowighteone.

[0024] Figure 2 is the 1 HNMR spectrum of Isowighteone.

[0025] Figure 3 is the change in the calcification level of HASMCs evaluated by alizarin red after treatment with Isowighteone.

[0026] Figure 4 is the calcium ion content of HASMCs quantitatively evaluated by calcium ion after treatment with Isowighteone

[0027] Figure 5 is the change in the expression of calcification genes in HASMCs evaluated by qRT-PCR after treatment with Isowighteone.

[0028] Figure 6 is the change in the expression of calcification proteins in HASMCs evaluated by Western Blot after treatment with Isowighteone.

[0029] Figure 7 is the effect of Isowighteone on the calcification of calcified mouse blood vessels evaluated by alizarin red.

[0030] Figure 8 is the effect of Isowighteone on the calcification of calcified mouse blood vessels quantitatively evaluated by calcium ion.

[0031] Figure 9 is the body weight detection of mice after treatment with Isowighteone. Detailed Implementation Modes

[0032] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments. However, the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0033] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0034] 1. Alizarin Red experimental method:

[0035] 1) Prepare 2% alizarin red: 2 g of alizarin red + 100 mL of ddH2O. After fully dissolving, filter with filter paper and adjust the pH to 4.2.

[0036] 2) Wash the cells 3 times with pre-cooled PBS, and fix the cells with 4% paraformaldehyde (PFA) at room temperature for 15 minutes.

[0037] 3) After fixation, wash once with PBS.

[0038] 4) Stain with 2% alizarin red for 30 minutes.

[0039] 5) After staining, recover the alizarin red (optionally, it can be reused once).

[0040] 6) Wash 3 times with ddH2O (do not use PBS).

[0041] 7) After air drying, take pictures under the microscope.

[0042] 2. Calcium ion quantitative detection method:

[0043] 1) Take out the cells cultured in the six-well plate from the incubator, discard the culture medium, wash 3 times with pre-cooled PBS, add 1 mL of 0.6 M HCl, and place it in the refrigerator at 4°C for 24 hours.

[0044] 2) Collect the HCl. After decalcification, add 200 μL of 1 mM NaOH and 0.1% SDS to each well to scrape the protein, and measure the total protein concentration in each well using a BCA protein quantification kit.

[0045] 3) Measure the free calcium content in the HCl using a calcium ion quantitative colorimetric kit. According to the kit instructions, dilute the calcium ion standard solution to 5 mM (0.2 mg / mL) by mixing 1 μL of the calcium standard solution (500 mM) with 99 μL of deionized water, and mix well.

[0046] 4) Prepare the standard curve: Add 0, 2, 4, 6, 8, and 10 μL of the above diluted calcium ion standard solution to a 96-well plate to produce 0 (measuring the blank), 0.4, 0.8, 1.2, 1.6, and 2.0 μg / well. Add deionized water to make up the volume to 25 μL.

[0047] 5) Sample preparation: Add 5 μL of the sample to each well, add 20 μL of deionized water, and supplement the volume to 25 μL.

[0048] 6) Add 45 μL of the chromogenic reagent to each of the above wells, and additionally add 30 μL of the calcium assay buffer to each well.

[0049] 7) After thorough mixing, incubate in the dark at room temperature for 10 minutes, then measure the absorbance at 575 nm. After calculating the standard curve, calculate the calcium ion concentration of the sample based on the OD value.

[0050] 8) Standardize the calculation with the total protein amount (Ca / mg).

[0051] 3. qRT-PCR method:

[0052] 1) Extract total RNA from HASMCs using Trizol. Specifically, lyse with Trizol for 15 min, then add chloroform for extraction, centrifuge for 15 min, take the upper aqueous phase, add an equal volume of isopropanol and then centrifuge for 15 min, and wash twice with 75% ethanol.

[0053] 2) Quantify and reverse transcribe the RNA using HiScript III RT SuperMix for qPCR (+gDNA wiper).

[0054] 3) Perform qRT-PCR on the QuantStudio 5 real-time system (Life technologies) using ChamQ Universal SYBR qPCR Master Mix.

[0055] Note: Each PCR is repeated three times. All gene expression data are calculated using 2 -ΔΔCT and normalized to β-actin. The control value is represented as 1 to represent the exact fold change value of each gene of interest.

[0056] 4. Western Blot method:

[0057] 1) Sample preparation: Treat HASMCs cells with RIPA lysis buffer for 30 min, then detect the protein concentration using a BCA kit, dilute to a final concentration of 1 mg / mL with PBS, and dilute with loading buffer at 4 gradients: 10 ng, 5 ng, 1 ng, 500 pg.

[0058] 2) Gel preparation: Prepare a 10% separating gel concentration according to the molecular weight of the antigen protein. Use a small vertical electrophoresis tank and 1 mm glass plates. Pour 4.5 mL of the separating gel, add isopropanol to seal the gel. After about 30 minutes, the separating gel solidifies. Pour off the isopropanol and pour in 1 mL (5%) of the stacking gel. Insert the comb. After solidifying for 1 - 2 hours, load the samples (with 4 gradient loading amounts).

[0059] 3) Electrophoresis: The electrophoresis is carried out in a constant voltage mode. The stacking gel is at 80 v. After about 25 minutes, it enters the separating gel, and the voltage is adjusted to 120 v. Stop the electrophoresis when the bromophenol blue runs to the bottom of the separating gel.

[0060] 4) Blotting: Use an NC membrane. After fixing the gel, transfer it in an ice - water bath with a constant current (the maximum voltage, current 300 mA). The current and transfer time are determined according to the molecular weight of the target protein.

[0061] 5) Immune reaction: After successful blotting, clear Marker bands should be observable on the membrane. Place the membrane in 5% skim milk / TBST at room temperature and shake on a shaker for 45 minutes for blocking. Dilute the primary antibody with the blocking solution at a dilution ratio of 1:1000. Pour off the blocking solution and add the primary antibody dilution. Incubate at room temperature on a shaker for 1 hour (or overnight at 4°C). After the primary antibody incubation, wash the membrane 4 times with TBST, 5 minutes each time. Dilute the secondary antibody with TBST at a dilution ratio of 1:5000. Incubate at room temperature on a shaker for 30 minutes. After the secondary antibody incubation, wash the membrane 4 times with PBST, 5 minutes each time.

[0062] 6) Exposure. Add ECL developing solution and expose.

[0063] Example 1 Extraction of Natural Compound (Isowighteone)

[0064] The schematic diagram of the extraction process is as Figure 1 shown, and specifically includes the following steps:

[0065] (1) Collect 10 kg of Ficus hispida fruits, dry them and grind them into powder.

[0066] (2) Add 75% ethanol to the dry powder, use ultrasound 5 times, 48 hours each time, to extract the compounds in the dry powder, and obtain the total ethanol extract after concentration under reduced pressure.

[0067] (3) Extract with dichloromethane, and obtain 80 g of dichloromethane extract after concentration by a rotary evaporator.

[0068] (4) Subject 80 g of the extract to gradient elution (gradient eluent from 30:70 methanol:water to 100:0 methanol:water) using a rapid preparative liquid chromatograph (Santai ODS preparative column, 50 μm, 330 g) to obtain a total of 21 components such as DGO - Ⅰ, DGO - Ⅱ, DGO - Ⅲ... DGO - XXI.

[0069] (5) The DGO-XIV fraction was separated by high performance liquid chromatography (Shimadzu preparative column, 5 μm, 20×250 mm, 70% methanol-water, flow rate of 10 mL / min).

[0070] A total of 30 mg of the target product 3'-dimethylallyl genistein (Isowighteone) was obtained. The 1 1H NMR spectrum of isowighteone is as Figure 2 shown, and the structural formula is as follows:

[0071]

[0072] Example 2 Therapeutic effect of Isowighteone on calcification of smooth muscle cells

[0073] In order to explore the therapeutic effect of Isowighteone on calcification of smooth muscle cells, the present invention also explored the effects of the structural analogs of Isowighteone, 3'-(3-methylbut-2-enyl)biochanin A and Myrsininone A, on the treatment of calcification of smooth muscle cells.

[0074] The structural formula of 3'-(3-methylbut-2-enyl)biochanin A is as follows:

[0075]

[0076] The structural formula of Myrsininone A is as follows:

[0077]

[0078] I. Experimental methods

[0079] 1. Establishment of HASMCs calcification model and in vivo experiment to verify the therapeutic effects of the three compounds

[0080] Primary cultured human aortic smooth muscle cells (HASMCs) were derived from the aortic tissue excised from heart donors in the transplantation surgery center. After removing the intima and adventitia of the aortic tissue in DMEM / F12 medium (SH30023.01; HyClone), the tissue was cut into pieces about 1.5 mm on ice 2Fragments were obtained and transferred to a new culture dish. The tissue blocks were evenly distributed on the bottom of the culture dish and allowed to stand for 30 min, and then an appropriate amount of DMEM / F12 medium supplemented with 10% fetal bovine serum (FBS; 1767839; Thermo Fisher Scientific) and 1% penicillin-streptomycin (15140-122; Thermo Fisher Scientific) was added. After culturing in an incubator for about 7 days, spindle-shaped SMCs grew out from the tissue blocks. Then, when the confluence reached about 80%, HASMCs were used for further experiments.

[0081] HASMCs were induced to calcify with calcification medium (CM), which consisted of medium supplemented with 10% fetal bovine serum, 10 mM β-glycerophosphate, and 250 μM ascorbic acid, and cultured for 7 days.

[0082] HASMCs were divided into five groups. The first group was the control group (Con group), that is, normal HASMCs without treatment; the second group was the blank control group (Cal+PBS group), that is, PBS was added to the calcified HASMCs induced by CM; the third group was the treatment group (Cal+Isowighteone group), that is, Isowighteone was added to the calcified HASMCs induced by CM. The fourth group was the treatment group (Cal+3’-(3-methylbut-2-enyl)biochanin A group), that is, 3’-(3-methylbut-2-enyl)biochanin A was added to the calcified HASMCs induced by CM. The fifth group was the treatment group (Cal+Myrsininone A group), that is, Myrsininone A was added to the calcified HASMCs induced by CM. Alizarin red and calcium ion quantification were used to detect the intracellular calcium ion content. qRT-PCR and Western Blot were used to detect the expression levels of intracellular calcification-related proteins.

[0083] II. Experimental Results

[0084] The results of the alizarin red experiment were as Figure 3 shown. The calcium ion content of the cells in the calcification group (blank control group) was higher than that of the control group, while the calcium ion content of the cells in the third group was lower than that of the calcification group. There was no significant difference in the calcium ion content of the cells in the fourth and fifth groups compared with the calcification group. The experiments were repeated 3 times.

[0085] The results of calcium ion quantification were as Figure 4 shown, which were consistent with the alizarin red results. The calcium ion content of the cells in the calcification group (blank control group) was higher than that of the control group, while the calcium ion content of the cells in the third group was significantly lower than that of the calcification group. There was no significant difference in the calcium ion content of the cells in the fourth and fifth groups compared with the calcification group. The experiments were repeated 3 times.

[0086] The results of qRT-PCR were as follows Figure 5 As shown, the expression levels of RUNX2, BMP2, and MSX2 genes in the cells of the calcification group (blank control group) were higher than those in the control group, while the expression levels of RUNX2, BMP2, and MSX2 genes in the cells of the treatment group (Cal+Isowighteone group) were lower than those in the calcification group. The experiments were repeated 3 times.

[0087] The results of Western Blot were as follows Figure 6 As shown, consistent with the qRT-PCR results, the expression levels of RUNX2, BMP2, and MSX2 proteins in the cells of the calcification group (blank control group) were higher than those in the control group, while the expression levels of RUNX2, BMP2, and MSX2 proteins in the cells of the treatment group (Cal+Isowighteone group) were lower than those in the calcification group.

[0088] In summary, it is shown that Isowighteone can improve the calcification of smooth muscle cells, but its structural analogs 3’-(3-methylbut-2-enyl)biochanin A and Myrsininone A do not have the function of improving the calcification of smooth muscle cells.

[0089] Example 3 Construction of a mouse vascular calcification model and in vitro verification of the therapeutic effect of Isowighteone

[0090] The animals used in this study were the mice raised by Regene Biotech. The experimental protocol was approved by the Medical Research Ethics Committee of Guangdong Provincial People's Hospital and was carried out in accordance with the "Guide for the Care and Use of Laboratory Animals" of the country. The mouse strain used was C57BL / 6J.

[0091] I. Experimental methods

[0092] The method for constructing vascular calcification mice was as follows: After dissolving VitD3 with glucose (Dextrose) and Cremophor, the weight of each mouse was recorded. Each mouse was subcutaneously injected with vitD3 at a dose of 5*10 5 IU / kg for 3 consecutive days. On the seventh day after the injection was completed, the mice were sacrificed by cervical dislocation, and the aorta was taken.

[0093] Three groups of mice were set up: the first group was normal C57BL / 6J mouse model (Con), the second group was vascular calcification mice injected with PBS via the tail vein (Cal+PBS), and the third group was vascular calcification mice orally fed with Isowighteone (Cal+Isowighteone). The same number of male mice, 3 in each group, were included in all experiments.

[0094] The specific experimental process is as follows: Isowighteone was diluted with PBS. Starting from the fourth day of vascular calcification modeling (i.e., the first day after injecting VitD3), the third group of mice was intragastrically treated with a dose of 10 mg / kg per day for 4 consecutive days to treat vascular calcification. The second group of mice was intragastrically administered with the same amount of PBS as the treatment group as the calcification group. The first group of mice was not treated. Alizarin red and calcium ion quantification were used to detect the degree of vascular calcification in mice. The experiments were repeated 3 times.

[0095] To detect the toxicity of Isowighteone to mice, a group of normal C57BL / 6J mice were orally administered PBS for 7 days, and another group of normal C57BL / 6J mice were orally administered Isowighteone for 7 days. The body weight changes were continuously detected.

[0096] II. Experimental Results

[0097] The experimental results of alizarin red are as Figure 7 shown. Isowighteone can significantly improve vascular calcification in mice.

[0098] The results of calcium ion quantification are as Figure 8 shown. Isowighteone can reduce the calcium ion content in the blood vessels of mice.

[0099] The body weight changes of mice are as Figure 9 shown. Isowighteone has no effect on the body weight of mice. Therefore, Isowighteone has no toxic or side effects on mice.

[0100] The above results indicate that the natural compound Isowighteone has a certain therapeutic effect on vascular calcification, and has good safety performance, and is expected to develop small molecule drugs for the treatment of vascular calcification.

Claims

1. Use of 3'-dimethylallyl genistein in the preparation of a medicament for treating calcific diseases, characterized in that, The 3'-dimethylallyl genistein is shown as the following formula (I):

2. The application according to claim 1, characterized in that The calcific disease is a calcific vascular disease.

3. The application according to claim 2, characterized in that, The calcific vascular disease is vascular calcification.

4. The application according to claim 1, wherein The preparation method of the 3'-dimethylallyl genistein is as follows: drying and powdering the fruits of Ficus hispida, and obtaining the product through extraction, extraction, elution, and separation.

5. The application according to claim 4, wherein The solvent for extraction is ethanol.

6. The application according to claim 4, wherein The solvent for extraction is dichloromethane.

7. The application according to claim 4, wherein The elution is carried out successively using a methanol solution of 30% - 100%.

8. The application according to claim 4, characterized in that, The separation is carried out by high performance liquid chromatography.

9. The application according to claim 1, characterized in that The drug is in the form of granules, extracts, tablets, or capsules.

10. The application according to claim 1, wherein The drug treats the calcific disease by reducing the calcium ion level and inhibiting the expression of calcification genes.

Images