阅读说明：本技术 花色苷衍生物用于制备治疗胆固醇代谢异常的药物的应用 (Application of anthocyanin derivative in preparation of medicine for treating cholesterol metabolism disorder ) 是由 白卫滨 焦睿 袁洋冰 李旭升 李娅雯 朱远琴 梁灏甄 于 2021-10-12 设计创作，主要内容包括：本发明公开了羧基吡喃矢车菊素-3-O-葡萄糖苷(Cpycy-3-glu)用于制备治疗胆固醇代谢异常的药物的应用。Cpycy-3-glu可改善由于高胆固醇带来的机体胆固醇代谢异常,可保护因高胆固醇而造成的肝脏和血管损伤。本发明为Cpycy-3-glu作为天然功能因子预防高胆固醇血症提供了理论依据,具有重要的意义。(The invention discloses application of carboxyl cyanidin-3-O-glucoside (Cpycy-3-glu) in preparing a medicament for treating abnormal cholesterol metabolism. Cpycy-3-glu can improve abnormal cholesterol metabolism caused by high cholesterol and protect liver and blood vessel damage caused by high cholesterol. The invention provides a theoretical basis for Cpycy-3-glu serving as a natural functional factor to prevent hypercholesterolemia, and has important significance.)

1. The application of a anthocyanin derivative in preparing a medicament for treating abnormal cholesterol metabolism is characterized in that: the anthocyanin derivative is carboxypyranopyranopyramid-3-O-glucoside (carboxymethyl-Pyranocyanidin-3-O-glucoside, Cpycy-3-glu).

2. Use according to claim 1, characterized in that: the cholesterol metabolism disorder is cholesterol metabolism disorder caused by high cholesterol.

3. Use according to claim 2, characterized in that: the cholesterol metabolism abnormality is cholesterol synthesis abnormality or cholesterol reverse transport abnormality.

4. Use according to claim 1, characterized in that: the medicament is a cholesterol-lowering medicament.

5. Use according to claim 1, characterized in that: the medicament is a medicament for preventing hypercholesterolemia.

6. The use according to claim 4 or 5, wherein the medicament comprises: a therapeutically effective amount of carboxypyranopyramidan-3-O-glucoside, and a pharmaceutically acceptable excipient.

Technical Field

The invention belongs to the field of biological medicines, relates to a new application of a anthocyanin derivative, and particularly relates to an application of the anthocyanin derivative in preparing a medicine for treating cholesterol metabolism disorder.

Background

Hypercholesterolemia is the pathological basis for inducing Cardiovascular diseases (CVD), and the regulation of cholesterol is an effective means for treating hypercholesterolemia and preventing CVD. However, since statins commonly used in clinic bring certain side effects to the body, it is a research hotspot to seek natural functional active substances to improve cholesterol metabolism of the body.

Anthocyanin is a flavonoid compound, widely exists in dark fruits and vegetables, has strong physiological activity, and carboxyl cyanidin-3-O-glucoside is produced as anthocyanin derivative in fermentation and aging process of berries rich in anthocyanin, and has the structure that hydroxyl of original anthocyanin between C4 and C5 is cyclized, added and condensed to form a fourth pyran ring (see figure 1).

Compared with prototype anthocyanin, the carboxyl cyanidin-3-O-glucoside has better stability and good biological activities such as anti-inflammation and antioxidation, and the research report of the metabolism of the carboxyl cyanidin-3-O-glucoside is lacked at present.

Disclosure of Invention

The invention aims to provide application of a anthocyanin derivative in preparing a medicament for treating cholesterol metabolism disorder.

The anthocyanin derivative is Carboxyl Pyranocyanidin-3-O-glucoside (carboxymethyl-pyranocyanidinin-3-O-glucoside, Cpycy-3-glu).

According to a further feature of the present invention, the cholesterol metabolism disorder is a cholesterol metabolism disorder caused by high cholesterol.

According to a further feature of the present invention, the abnormality in cholesterol metabolism is an abnormality in cholesterol synthesis or an abnormality in reverse cholesterol transport.

According to a further feature of the present invention, the drug is a cholesterol-lowering drug.

According to a further feature of the present invention, the medicament is a medicament for preventing hypercholesterolemia.

According to a further feature of the present invention, the medicament includes: a therapeutically effective amount of carboxypyranopyramidan-3-O-glucoside, and a pharmaceutically acceptable excipient.

The inventor shows through experiments that the carboxyl pyranocyanidin-3-O-glucoside (Cpycy-3-glu) can reduce the content of total cholesterol in cells, maintain the balance of cholesterol in organisms and protect the liver and blood vessels from being damaged by high cholesterol by inhibiting the endogenous synthesis of cholesterol, promoting the reverse transport process of the cholesterol and regulating the expression of related proteins of a cholesterol metabolic pathway. Therefore, Cpycy-3-glu can improve abnormal cholesterol metabolism of the body caused by high cholesterol and can protect liver and blood vessel damage caused by the high cholesterol. The invention provides a theoretical basis for Cpycy-3-glu serving as a natural functional factor to prevent hypercholesterolemia, and has important significance.

Drawings

FIG. 1 is a schematic structural diagram of carboxypyranopyramidase-3-O-glucoside (Cpycy-3-glu) according to the invention.

FIG. 2 shows the change of intracellular cholesterol content. Wherein, the effect of C3G and Cpycy-3-glu intervention with A at 200. mu.M on total cholesterol levels in HepG2 cells.

FIG. 3 shows the mRNA expression levels of SREBP2 and HMGCR and the protein expression level of HMGCR in the cholesterol synthesis pathway. Wherein A is the mRNA level of SREBP2, B is the mRNA expression level of HMGCR, and C, D is the protein expression level of HMGCR.

Figure 4 is the expression levels of mRNA and protein of the cholesterol uptake pathway LDLR and PCSK9 during reverse cholesterol transport. Wherein A, B is the mRNA expression level of PCSK9 and LDLR, and C, D, E, F is the protein expression level of PCSK9 and LDLR.

FIG. 5 shows PPAR γ and ABCA1 in the cholesterol efflux pathway during reverse cholesterol transport. Wherein A is the mRNA expression level of PPAR γ and B is the mRNA expression level of ABCA 1.

The English terms in the above drawings have the meanings as follows: "Total Cholesterol" is "Total Cholesterol", "Control" is "Control", "model" is "modeling group", and "Relative mRNA level" is "Relative mRNA level", all terms well known to those skilled in the art.

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Experimental materials:

1. experimental cell lines

Human HepG2 liver cancer cells were purchased from the chinese institute cell dictionary.

2. Experimental intervened object and molding object

The cholesterol and 25-hydroxycholesterol used in the experiment were purchased from Sigma-Aldrich, USA, and C3G and Cpycy-3-glu were obtained from subjects based on the previous experiments.

3. Primer usage information

The primer sequences referred to in the examples are shown in Table 1.

Table 1: PCR primer information

All primers were synthesized by Shanghai Bioengineering, Inc.

Experimental methods

Measurement of Total Cholesterol in cell

1. Determination of total cholesterol content in cells

The method is completed according to the instruction of a Total Cholesterol (TC) kit, and mainly comprises the following steps:

(1) HepG2 cells were harvested in logarithmic growth phase, digested, centrifuged, resuspended, plated in 6-well plates (5X 105/well) in 2 mL/well format, and incubated overnight in a 37 ℃ cell culture chamber.

(2) After plating for 12h, the old culture was aspirated and discarded, washed 2 times with PBS at 1 mL/wash, and cell culture medium containing either mock or Ator or varying concentrations of C3G and Cpycy-3-glu was added to each group and incubated overnight in a 37 ℃ cell incubator.

(3) Adding medicine (C3G or Cpycy-3-glu or Ator + molding substance) for 24h, sucking out old culture solution, discarding, washing with PBS 2 times at a rate of 1mL, adding pancreatin 300 μ L per well, incubating in incubator for 1min, transferring to clean bench, and terminating digestion with culture medium 900 μ L.

(4) The cells were blown and transferred to 1.5mL EP tubes and centrifuged at 1000r/min for 3 min. Then, cell counting was performed.

(5) After the cell counting is finished, the tube after counting is subjected to the centrifugation and supernatant discarding operation, and the corresponding cell lysate is added according to the proportion of 100 mu L lysate/100 ten thousand cells.

(6) And (3) sequentially vortexing the samples added with the cell lysate, repeating the operations for 30s for each sample, and placing the samples on ice for 30min to fully lyse the samples.

(7) At least 75. mu.L of the supernatant after standing was pipetted into a new 1.5mL EP tube (transfer 1), heated in a metal bath at 70 ℃ for 10min, centrifuged at 2000g at room temperature in a low speed centrifuge for 5min, and the previous 1.5mL EP tube was subsequently inserted into an ice box for further use.

(8) The centrifuged supernatant was carefully aspirated, with as little cell debris as possible, and transferred to a new 1.5mlEP tube (transfer 2) for use.

(9) And (3) taking a 5mM cholesterol standard product in the cholesterol kit, diluting the cholesterol standard product with absolute ethyl alcohol, and preparing the cholesterol standard product into gradient concentrations of 0, 39, 78, 156 and 312 mu M for later use.

(10) And preparing the working solution under the condition of keeping out of the sun. Preparing a working solution according to the volume ratio of R1 to R2 to 4 to 1, and uniformly mixing by vortex; adding the working solution into a 96-well plate, adding 183 mu L of the working solution into each well, adding 17 mu L of a sample to be detected, a blank control (namely absolute ethyl alcohol) and cholesterol standards with different concentrations, and marking each group with two multiple wells, wherein each group of the sample group to be detected is provided with three multiple wells.

(11) And then, incubating the 96-well plate in a constant temperature incubator at 37 ℃ for 20min in the dark, shaking the plate by a microplate reader for 15s, determining the OD value at 590nm, and calculating the cholesterol content according to a cholesterol standard curve and a BCA result.

2. Determination of cellular protein concentration

(1) Marking a new 1.5mL EP tube, adding 36 mu L of ultrapure water into each tube, taking 4 mu L of the sample inserted on the ice box before, transferring the sample into the EP tube, finally diluting by 10 times, and uniformly swirling for later use.

(2) The BCA working solution was prepared at a volume ratio of 50:1 of reagent a to reagent B.

(3) The 4mg/mL protein standard is diluted with ultrapure water to prepare gradient standard concentrations of 0, 0.03125, 0.0625, 0.125, 0.25, 0.5 and 1 mg/mL.

(4) The operation process of adding the sample is to add the working solution into the sample. 10 μ L of sample per well, 200 μ L of BCA working solution per well; three duplicate wells were set for each sample set and two duplicate wells for the standard set.

(5) Incubating in a constant temperature incubator at 37 ℃ for 30min, shaking by a microplate reader for 15s before measurement, measuring OD value at 562nm, and calculating the protein concentration of the original sample according to the standard.

Second, RNA extraction and RT-PCR

1. Referring to the operation of the specification of the RNA extraction kit of the Hunan Aikery Biotechnology Limited company, the main reagents are all from the kit, and the main steps are as follows:

(1) HepG2 cells were harvested in logarithmic growth phase, plated in 6-well plates (5X 105/well) after digestion and centrifugation for resuspension, with a total volume of 1mL per well, and incubated overnight in a 37 ℃ cell incubator.

(2) After plating for 12h, the old culture was aspirated and discarded, washed 2 times with PBS 1 mL/time, and the corresponding mold and Cpycy-3-glu cell culture medium were added to each experimental group and incubated overnight in a 37 ℃ cell incubator.

(3) After adding the drug for 24h, the old culture solution was aspirated and discarded, washed 2 times with PBS at a rate of 1 mL/time, and then 1mL of Trizol was added to each well to allow sufficient lysis, and allowed to stand on ice for 20 min.

(4) After lysis was complete, the cells in each well were blown out and transferred separately to a 1.5mL EP tube (time controlled process), followed by addition of 200. mu.L of chloroform, and each tube was vortexed for 15s and then placed on ice for 5 min.

(5) After standing, all samples were centrifuged in a precooled high-speed low-temperature centrifuge at 4 ℃ at 12000g for 10 min. After the centrifugation was completed, the EP tube was carefully removed, 200. mu.L of the upper aqueous phase was taken out, and transferred to a new EP tube (Note: ten million not to be sucked into the cell debris and other impurities below).

(6) Subsequently, an equal volume (200 μ L) of isopropanol was added to a new EP tube, vortexed for 15s, and allowed to stand for 10 min.

(7) Centrifuging the well-standing sample by using a high-speed low-temperature centrifuge under the following centrifugation conditions: 12000g, centrifuging for 15min at 4 ℃ to obtain RNA precipitate.

(8) The now prepared 75% ethanol (prepared with DEPC water and absolute ethanol) was added to the pellet and the pellet was washed by gentle shaking. 12000g, centrifuging for 15min at 4 ℃ and then discarding 75% ethanol. The above operation was repeated once more. The precipitate washing was completed.

(9) The EP tube was inverted on filter paper and air dried for approximately 45min to obtain the final precipitate.

(10) 40-100 μ L of DEPC water was added to dissolve the precipitate according to the amount of the specific precipitate.

(11) mu.L of each RNA stock was taken out of each tube, and the A260/A280 purity and RNA concentration were measured by Nanodrop.

2. Reverse transcription of RNA

The experiment was performed on ice using a reverse transcription kit from echolocation.

(1) DNA removal reaction

According to Table 2, RNase Free dH2O was added according to the calculation results, 5 XgDNA Clean Buffer and gDNA Eraser were added and mixed, and finally different amounts of RNA were added according to the final calculation results. After mixing, performing DNA removal experiment, wherein the reaction conditions are as follows: keeping the temperature at 42 ℃ for 2min and 4 ℃ for standby.

Table 2: DNA removal reaction system

Reagent	Amount of the composition used
		5×gDNA Clean Buffer	2.0μL
gDNA Eraser	1.0μL
		RNase Free dH2O	Make up to 10.0 mu L
Total RNA	Adjusted according to the RNA concentration of the sample to a final concentration of 0.1. mu.g/. mu.L
		Total amount of	10.0μL

(2) Reverse transcription reaction

RNase Free dH was purified according to Table 3₂O, Prime Script RT Enzyme Mix I, 5 XPrimeScript Buffer 2 and RT Primer Mix are added together in advance and mixed, and then transferred into the system of the previous step of reaction. Reverse transcription was performed in a PCR instrument. Reverse transcription conditions: 15min at 37 ℃; 5s at 85 ℃; the cDNA sample is stored at-80 ℃ after being subpackaged at 4 ℃ for 5 min.

Table 3: reverse transcription reaction system

Reagent	Amount of the composition used
		Reaction solution in step (1)	10.0μL
Prime Script RT Enzyme MixⅠ	1.0μL
		RT Primer Mix	1.0μL
5×PrimeScript Buffer 2	4.0μL
		RNase Free dH2O	4.0μL
Total amount of	20.0μL

3. Real-time fluorescent quantitative PCR (Real-time PCR)

In the experiment, the SYBR Premix Ex TaqTM II kit from Escisory was used to perform the Real-time PCR experiment. Samples were loaded according to Table 4 and amplified according to Table 5.

Table 4: real-time PCR reaction system

Table 5: two-step amplification Standard procedure

Reaction step	Reaction conditions
		Stage 1: pre-denaturation	95 ℃ 30s (repeat 1 time)
Stage 2: PCR reaction	15s at 95 ℃; 60 ℃ 30s (repeat 40 times)
		Stage 3：Dissociation

4. Real-time PCR result processing

Analyzing the melting curve and the reaction amplification curve, deriving data, obtaining Ct (target) -Ct (internal reference) as delta Ct according to the obtained Ct value, and then calculating delta Ct and 2-delta Ct.

Third, Western Blot (WB) related reagent

(1) 150g of glycine, 30g of Tris-Base and 10g of SDS are weighed respectively in 10 xSDS-PAGE electrophoresis buffer solution, deionized water is added to the solution to reach the constant volume of 1000mL, and the solution is fully and uniformly mixed and stored at normal temperature for standby.

(2) Taking 200mL of 10 xSDS-PAGE electrophoresis buffer solution from 1 xSDS-PAGE electrophoresis buffer solution, adding 1800mL of deionized water, fully mixing uniformly, and storing at normal temperature for later use.

(3) 144g of glycine and 30g of Tris-Base are respectively weighed by 10 xSDS-PAGE membrane conversion buffer solution, deionized water is added to the solution to be constant volume of 1000mL, and the solution is fully and uniformly mixed and stored at normal temperature for standby.

(4) Taking 160mL of 10 xSDS-PAGE membrane conversion buffer solution as 1 xSDS-PAGE membrane conversion buffer solution, adding 1440mL of deionized water, adding 400mL of methanol, fully mixing uniformly, and placing at 4 ℃ for precooling for later use.

(5) 88g of sodium chloride powder is weighed in 10 times TBS solution, 1000mL of deionized water is added, and the mixture is fully and uniformly mixed and stored at normal temperature for standby.

(6)1 xTBS solution 100mL of 10 xTBS solution was added with 890mL of deionized water and 10mL of Tris-HCl (pH 7.5) to prepare 1000mL of solution, which was then mixed well and stored at room temperature for further use.

(7)1 × TBST solution 100mL of 10 × TBS solution was taken, and 890mL of deionized water and 10mL of Tris-HCl (pH 7.5) were added, followed by addition of 0.5mL of Tween-20, gentle mixing and storage at room temperature.

(8) Weighing 2.5g of skimmed milk powder in 5% of confining liquid, dissolving in 50ml of TBS solution, and mixing uniformly for later use.

(9)1M Tris-HCl (pH8.8) accurately weighing Tris6.06g, adding 40mL of ultrapure water for dissolving, adjusting the pH to 8.8 with 4MHCl, then adding ultrapure water to a constant volume of 50mL, and storing at 4 ℃ for later use.

Fourth, data processing and statistical analysis

The exposed Western Bolot bands were analyzed by ClinxChemi Analysis software, and the data from the experiments were statistically analyzed and graphically represented using Graph Pad Prism 8.0, all data results are expressed as Mean ± standard deviation (Mean ± SD), and One-way Analysis of variance (One-way ANOVA) was used for comparisons between groups. Statistically significant differences were found to be # p < 0.05, # p < 0.01, # p < 0.001, # p < 0.0001.

Results and analysis

1. C3G and Cpycy-3-glu for cholesterol regulation

Selecting 200 mu M of C3G and Cpycy-3-glu, 10 mu M of positive control Ator and 40 mu M of cholesterol +4 mu M of 25-hydroxycholesterol to jointly act on HepG2, incubating for 24h in a cell incubator, and determining TC in each group of cells

The results show that compared with the model group, the positive control Ator group has a significant decrease in intracellular TC content (p < 0.0.1), which is about 44.07%, while the C3G group has a slight decrease but no significant difference, but the intervention of Cpycy-3-glu significantly decreases the intracellular TC content (p < 0.05), with a decrease rate of about 33.33% (FIG. 2A).

0, 50, 100 and 200 mu M of Cpycy-3-glu and 10 mu M of Ator are selected between 0 and 200 mu M concentrations to treat HepG2 cells in a high cholesterol (40 mu M cholestrol +4 mu M25-HC) state for 24 hours, and then the TC content of each group is determined.

The results show that the cholesterol-lowering ability of Cpycy-3-glu at different concentrations is dose-dependent, with substantially no cholesterol-lowering effect at 50. mu.M, but at concentrations up to 100. mu.M, the TC content was reduced by about 10.44%, and at concentrations up to 200. mu.M, the TC content was very significantly reduced (p < 0.0001), with a reduction of 30.46% (FIG. 2B).

2. Regulation of the hepatic cholesterol synthesis pathway by Cpycy-3-glu

In the experiment, different concentrations of Cpycy-3-glu and Ator are respectively used for acting on HepG2 cells in a high cholesterol state for 24 hours.

The results show that in the HepG2 cells with high cholesterol, the mRNA level of SREBP2 is obviously reduced, and HMGCR has a certain reduction trend but has no obvious difference. The intracellular high-cholesterol level environment is shown to feedback-inhibit mRNA expression of SREBP2 and HMGCR, and after positive control Ator and Cpycy-3-glu intervention for 24h, the mRNA expression level of SREBP2 is obviously up-regulated compared with a modeling group, wherein the up-regulation effect of Cpycy-3-glu is in a dose-dependent relationship, and the effect of 200 mu M of Cpycy-3-glu is leveled with the effect of 10 mu M of Ator, which is probably related to negative feedback regulation (FIG. 3A). For HMGCR, both mRNA expression levels were significantly down-regulated, and the down-regulation of Cpycy-3-glu also exhibited a dose-related relationship (FIG. 3B). Meanwhile, the protein expression level of HMGCR is analyzed, and WB bands and mapping results show that the protein expression of HMGCR of the model group has no difference compared with the control group, and after Cpycy-3-glu acts, the protein expression of HMGCR of the model group is remarkably reduced, and the trend is consistent with the result of qPCR (fig. 3C-D). The above results indicate that Cpycy-3-glu can reduce endogenous synthesis of liver cholesterol by inhibiting transcriptional expression of the HMGCR gene.

3. Regulation of hepatic cholesterol uptake pathway by Cpycy-3-glu

LDLR is a membrane surface glycoprotein, can remove LDL-C in plasma through a vesicle endocytosis mechanism, has important significance for maintaining blood cholesterol balance, and PCSK9 is a natural degradation agent thereof. The high cholesterol environment in HepG2 cells caused significant reduction in mRNA levels of LDLR and PCSK9, but the mRNA expression of LDLR was significantly increased after Cpycy-3-glu intervention, reversing the expression down-regulation caused by high cholesterol, while the mRNA level of PCSK9 did not significantly change, indicating that Cpycy-3-glu can up-regulate the gene expression of LDLR, but had no effect on the mRNA level of PCSK9, as analyzed by qPCR and WB detection (fig. 4A-B). With respect to protein results, although protein expression of model PCSK9 was unchanged from control, intervention of Cpycy-3-glu could significantly down-regulate its protein expression. For LDLR, the protein expression of the modeling group is significantly reduced, but after Cpycy-3-glu prediction, the protein expression level of LDLR is obviously up-regulated, which indicates that Cpycy-3-glu can promote the protein expression of LDLR, and the result is consistent with qPCR (FIGS. 4C-F). It was demonstrated above that Cpycy-3-glu plays an important role in promoting the elimination of blood cholesterol intake.

4. Cpycy-3-glu regulation of hepatic cholesterol conversion efflux pathway

ABCA1 is an ATP-dependent membrane-bound transporter that mediates efflux of cholesterol and reverse transport of cholesterol, and is thought to play an important role in anti-atherosclerosis, while PPAR γ, a nuclear receptor, is thought to promote LDLR expression to enhance cholesterol metabolism. The results indicate that the high cholesterol environment slightly reduced PPAR mRNA levels, but PPAR γ mRNA expression was significantly elevated following Cpycy-3-glu intervention (fig. 5A). For ABCA1, the ABCA1 mRNA level of the building block was slightly decreased but not significantly different from that of the control group, but after positive control Ator and Cpycy-3-glu stem prognosis, the mRNA expression was significantly increased, and gradually increased with the increase of Cpycy-3-glu concentration, and the Cpycy-3-glu effect at 200. mu.M was substantially equal to that at 10. mu.M, indicating that Cpycy-3-glu can increase the cholesterol efflux out of the cell by up-regulating the mRNA expression of ABCA1 (FIG. 5B).

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.