Preparation method of cyanidin and its application in preventing ischemic stroke and Alzheimer's disease

Abstract

The invention discloses a preparation method and use of blue flower pseudoindigo, and the preparation method comprises the following steps: 1) extracting the ghost arrow Caragana medicinal material crushed into small particles with 95% ethanol hot reflux twice, filtering, combining the filtrate, and then concentrating under reduced pressure to dry, to obtain a 95% ethanol extract ECJ; 2) subjecting the extract ECJ to silica gel column chromatography, gradient eluting with dichloromethane-methanol of different volume ratios, detecting by thin layer chromatography, comparing with standard products, combining the blue flower pseudoindigo fractions, and obtaining component ECJ1; 3) taking ECJ1 and then subjecting it to Sephadex LH20 column chromatography, eluting with pure methanol, detecting by thin layer chromatography, comparing with standard products, combining the blue flower pseudoindigo fractions, and obtaining component ECJ1-1 containing crystals; 4) taking ECJ1-1, adding methanol, filtering out the crystals, and obtaining blue flower pseudoindigo. The compound of the present invention has the effect of significantly inhibiting acetylcholinesterase activity and resisting ischemic stroke.

Description

Technical Field

[0001] The present invention relates to the field of pharmacy, and in particular to a preparation method and new use of cyanobacterium indigoferum, especially to the use of cyanobacterium indigoferum in preventing ischemic stroke and Alzheimer's disease. Background Art

[0002] Stroke is a major chronic non-communicable disease that seriously endangers the health of Chinese people. It is the leading cause of death and disability among adults in my country. It has five major characteristics: high morbidity, high disability rate, high mortality rate, high recurrence rate, and high economic burden. Ischemic stroke (IS) accounts for about 80% of the total number of strokes. Currently, the main treatments for ischemic brain injury are thrombolytic therapy and neuroprotective agent intervention. Thrombolysis is one of the most effective treatments for ischemic stroke, but its therapeutic window is narrow and may be accompanied by serious adverse reactions such as bleeding risks and vasogenic edema. Neuroprotective agents also have defects that cannot be ignored, such as low solubility, short half-life, and poor permeability of the blood-brain barrier in the body, and their clinical efficacy is poor. Therefore, it is urgent to find new anti-ischemic stroke drugs.

[0003] Alzheimer's disease (AD) is a neurodegenerative disease. Studies have shown that the concentration of acetylcholine in the brain of people with dementia is greatly reduced, leading to a series of typical AD symptoms. In the human body, cholinesterase is responsible for the hydrolysis and metabolism of acetylcholine to maintain its stable concentration level. The most important and important one is acetylcholinesterase. Acetylcholinesterase is the main enzyme for hydrolyzing acetylcholine. Inhibiting acetylcholinesterase can alleviate the clinical symptoms of AD.

[0004] Natural products have long been an important source for the discovery of new drugs or lead compounds for the treatment of nervous system diseases due to their novel chemical structure, drug-like properties and unique pharmacological effects. Texasin, also known as texasin in English, is an isoflavone compound isolated from the xylem heartwood of the medicinal plant Caragana fasciata. Its structural formula is:

[0005]

[0006] Research on the biological activity of cyanobacterium is very limited. Currently, it is only reported that it has the activity of inhibiting tyrosinase and leukocyte 5-esteroxygenase. Whether cyanobacterium has the effect of preventing ischemic stroke and inhibiting acetylcholinesterase has not been reported. Summary of the invention

[0007] Purpose of the invention: In order to solve the above technical problems, the first purpose of the present invention is to provide a three-step method for quickly and efficiently obtaining a high-yield preparation method of cyanobacterium indigo.

[0008] The second object of the present invention is to provide the use of cyanobacterium indigofera in the preparation of drugs for treating and / or preventing ischemic stroke and / or drugs for Alzheimer's disease.

[0009] Technical solution: The present invention provides a method for preparing blue flower pseudoindigo, comprising the following steps:

[0010] 1) The powdered Caragana fasciata medicinal material is extracted twice with 95% ethanol under hot reflux, filtered, the filtrate is combined, and then concentrated and dried under reduced pressure to obtain a 95% ethanol extract ECJ; in this step, extracting twice can ensure that the extraction is relatively complete, and on the other hand, it saves time and reagents;

[0011] 2) separating the extract ECJ by silica gel column chromatography, using dichloromethane-methanol in different volume ratios for gradient elution, detecting by thin layer chromatography, comparing with a standard substance of cyanidin, combining the fractions containing cyanidin, and obtaining component ECJ1;

[0012] 3) ECJ1 was subjected to Sephadex LH20 column chromatography, eluted with pure methanol, detected by thin layer chromatography, compared with a standard of cyanobacterium indigoferum, and the fractions containing cyanobacterium indigoferum were combined to obtain a component ECJ1-1 containing crystals;

[0013] 4) Take ECJ1-1, add methanol, filter out the crystals, and obtain blue flower pseudoindigo.

[0014] Wherein, step 2) gradient elution with dichloromethane-methanol in different volume ratios includes: first eluting with dichloromethane-methanol in a volume ratio of 100:1, and then eluting with dichloromethane-methanol in a volume ratio of 50:1. In this step, selecting dichloromethane-methanol in a volume ratio of 100:1 can elute impurities with low polarity, and then using dichloromethane-methanol in a volume ratio of 50:1 can ensure that blue flower pseudoindigo can be eluted without introducing too many impurities, thereby reducing the subsequent purification steps of blue flower pseudoindigo.

[0015] The present invention also includes the use of cyanobacterium indigofera in the preparation of drugs for treating and / or preventing ischemic stroke.

[0016] The present invention also includes the use of cyanobacterium indigofera in the preparation of drugs for treating and / or preventing Alzheimer's disease.

[0017] Wherein, the medicine is a single component or a compound preparation.

[0018] The dosage form of the drug is one of tablets, capsules, oral liquid, syrup, pills, injection liquid or freeze-dried powder injection.

[0019] In general, compounds are separated by extracting the medicinal materials, sequentially extracting with organic reagents of different polarities (such as petroleum ether, ethyl acetate and n-butanol) to obtain different extraction parts, and then systematically separating the extraction parts. This approach easily leads to the dispersion of compounds, that is, the compounds may be present in different extraction parts, but the amount of presence is different. The present invention directly uses a silica gel chromatographic column for separation after extraction, without the extraction step, thus avoiding the dispersion of the compounds. In addition, Sephadex LH20 is used in the second step of treatment. Taking into account the molecular sieve principle of Sephadex LH20, the compounds can be eluted in descending order according to the molecular weight. At the same time, the gel also has a good pigment effect. Figure 6 It can be seen that after separation by Sephadex LH20 column chromatography, cyanobacterium pseudoindigoferin was further enriched.

[0020] Beneficial effects: The present invention can quickly prepare high-yield cyanobacterium from Caragana fasciata medicinal materials in just three steps. The present invention has experimentally verified that cyanobacterium has significant effects of inhibiting acetylcholinesterase activity and resisting ischemic stroke, and can be used to treat ischemic stroke, Alzheimer's disease and other diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 , UPLC analysis of methanol extract of Caragana fasciata;

[0022] Figure 2 , UPLC analysis of 95% ethanol extract of Caragana fasciata;

[0023] Figure 3 , UPLC analysis of Caragana acetone extract;

[0024] Figure 4 , UPLC analysis of Caragana fasciata anhydrous ethanol extract;

[0025] Figure 5 , thin layer chromatogram of ECJ after separation by silica gel column chromatography (the fractions framed by the first frame are combined to form ECJ1, and the second frame is the standard product of cyanobacterium indigoferum);

[0026] Figure 6 , thin layer chromatogram of ECJ1 after separation by Sephadex LH20 gel column (the fractions in the first frame are combined to form ECJ1-1, and the second frame is the standard product of cyanobacterium indigoferum);

[0027] Figure 7 , the hydrogen spectrum of cyanidin;

[0028] Figure 8, carbon spectrum of blue flower pseudoindigo;

[0029] Fig. 9 , high-resolution mass spectrometry of cyanidin;

[0030] Fig.10 , UPLC analysis chart of blue flower pseudoindigo standard.

[0031] Fig.11 , the protective effect of cyanobacterium on OGD / R damaged PC12 cells. DETAILED DESCRIPTION

[0032] Sephadex LH20 (18-111 μm) used in the present invention was purchased from GE Heakhcare, USA; the medicinal materials of Caragana fasciata were collected from Tibet in January 2021; Preparation of the blue flower pseudoindigo standard: Blue flower pseudoindigo was homemade in the laboratory, and the purity was 98.01% according to HPLC analysis, and it was dissolved in methanol when used.

[0033] Example 1 Comparison of the total amount of blue pseudoindigo obtained by extracting Caragana fasciata with different solvents

[0034] Four 5g portions of Caragana fasciata medicinal materials crushed into small particles were extracted with methanol, 95% ethanol, acetone and anhydrous ethanol at 85°C for hot reflux, respectively, and the extraction was performed twice, and the filtrates were combined. After filtering the filtrate, 1μl was taken for UPLC analysis at 254nm. The chromatographic peak of cyanobacterium indigoferum in the extract was determined by comparing the retention time with that of the cyanobacterium indigoferum standard, and the cyanobacterium indigoferum content in the extract was evaluated based on the peak area. At the same time, the solvent was recovered from the extract to obtain an extract, and the yield was calculated. UPLC analysis of methanol extract of Caragana fasciata, UPLC analysis of 95% ethanol extract of Caragana fasciata; UPLC analysis of acetone extract of Caragana fasciata; UPLC analysis of anhydrous ethanol extract of Caragana fasciata, see Figure 1 to Figure 4 , methanol extraction yield 25.58%, peak area 2947808; acetone extraction yield 27.37%, peak area 3224894; 95% ethanol extraction yield 30.74%, peak area 3059710; anhydrous ethanol extraction yield 24.84%, peak area 3079465. From the yield point of view, 95% ethanol> acetone> methanol> anhydrous ethanol; from the peak area point of view, acetone> anhydrous ethanol> 95% ethanol> methanol. The yield of 95% ethanol is 1.12 times that of acetone; the peak area of ​​acetone is 1.05 times that of 95% ethanol; therefore, the total amount of blue flower pseudoindigo obtained by 95% ethanol extraction is the largest. At the same time, 95% ethanol is a green and environmentally friendly solvent and does not pollute the environment, so 95% ethanol is selected as the extraction solution.

[0035] Example 2 Preparation of Pseudoindigoferin

[0036] 1. 5 g of Caragana fasciata powder was crushed into small particles and extracted twice with 95% ethanol under hot reflux at 85°C, filtered, the filtrates were combined, and then concentrated and dried under reduced pressure to obtain 95% ethanol extract ECJ;

[0037] 2. Separate ECJ by silica gel column chromatography, elute with dichloromethane-methanol in a volume ratio of 100:1, and combine to obtain 5 fractions; then isocratically elute with dichloromethane-methanol in a volume ratio of 50:1, detect by thin layer chromatography, compare with a standard substance of texasin, combine the fractions containing texasin to obtain ECJ1; see Figure 5 ;

[0038] 3. Take ECJ1 and perform Sephadex LH20 column chromatography separation, elute with pure methanol, detect by thin layer chromatography, compare with the standard of cyanobacterium indigo, combine the cyanobacterium indigo fractions, and obtain ECJ1-1, which contains crystals; see Figure 6 ;

[0039] 4. Take ECJ1-1, add methanol, filter out the crystals, and obtain blue flower pseudoindigo (86.77 mg) with a yield of 1.74%. The hydrogen spectrum, carbon spectrum and high-resolution mass spectrum of the prepared blue flower pseudoindigo are shown in Figure 7 , Figure 8 and Fig. 9 , HPLC analysis see Fig.10 , with a purity of 98.01%.

[0040] Comparative Example 1 Preparation of Pseudoindigoferin

[0041] 2.7 kg of Caragana davidiana was crushed and refluxed with 95% ethanol at 85°C for 5 times. The filtrates were combined and concentrated under reduced pressure to obtain 1 kg of total extract. The extract was separated by D101 macroporous resin column chromatography and eluted with different concentrations of ethanol to obtain 50% ethanol fraction, 75% ethanol fraction and 100% ethanol fraction of macroporous resin. The 50% ethanol fraction (673 g) was separated by silica gel column chromatography and eluted with dichloromethane-methanol gradient with a product ratio of (100:1 to 0:1). After thin layer chromatography detection, it was combined into 10 fractions (Fr.1-Fr.10). Fr.3 was separated by silica gel column chromatography and eluted with petroleum ether-ethyl acetate gradient with a product ratio of (10:1 to 0:1) and combined into 7 fractions (Fr.3.1-Fr.3.7). Fr.3.6 contained crystals, which were washed out and identified as cyanobacterium indigoferum (303 mg) by NMR and mass spectrometry. This method was used to prepare cyanobacterium indigoferum with a yield of 0.01%.

[0042] Example 3 Evaluation of the protective effect of cyanobacterium on OGD / R damaged PC12 cells

[0043] Cytotoxicity evaluation: PC12 cells in the logarithmic growth phase were taken and 2×10 5 The cells were planted at a density of 100 μg / mL in a 96-well plate, with 200 μL per well, and cultured in a cell culture incubator. After 24 hours of cell culture, the culture medium was discarded and the cells were divided into the following groups: blank control group, high (100 μg / ml), medium (50 μg / ml), and low (25 μg / ml) dose groups of blue flower pseudoindigo. The cell viability of each group was detected after 24 hours of culture. Blue flower pseudoindigo had no effect on the viability of normally cultured PC12 cells at 25-100 μM ( Fig.11 A).

[0044] Protective effect on OGD / R-injured PC12 cells: The drugs were divided into the following groups: control group, oxygen glucose deprivation / reperfusion (OGD / R) group, OGD / R+blue flower pseudoindigo high-dose group (25μg / ml), OGD / R+blue flower pseudoindigo medium-dose group (12.5μg / ml), OGD / R+blue flower pseudoindigo low-dose group (6.25μg / ml) and OGD / R+edaravone group (6.25μg / ml). 100μL complete medium was added to the normal group and OGD / R group, and 100μL of drug-containing complete medium was added to each drug-treated group. After 24 hours of pre-administration, the culture medium was aspirated and washed twice with 100 μL of PBS. The control group was added with 100 μL of complete culture medium and placed in a cell culture incubator for culture; the remaining groups were added with 100 μL of sugar-free culture medium and placed in a three-gas incubator at 37°C, 1% O2, 5% CO2, and 94% N2 for 6 hours. After the modeling was completed, the sugar-free culture medium was aspirated, and complete culture was added to continue culturing in a 37°C carbon dioxide incubator for 24 hours. The CCK8 method was used to determine the cell survival rate of each group. 12.5 and 25 μM cyanidin can increase the viability of PC12 cells damaged by OGD / R, and the effect is equivalent to that of 50 μM positive drug Edaravone ( Fig.11 B) This suggests that cyanobacterium has a protective effect on neuronal cells injured by oxygen-glucose deprivation / reperfusion.

[0045] Example 4

[0046] Acetylcholinesterase activity assay: 100 μl PBS (PH = 8.0) was added to each well of a 96-well plate, and 20 μl of iodothioacetylcholine was added at a concentration of 1.2 mM. The reaction was carried out at 37°C for 30 min, and then 20 μl of 4% sodium dodecyl sulfate (SDS) was added to terminate the reaction, and 20 μl of 0.6 mM display solvent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) was added, and then the absorbance OD1 value was immediately measured at 405 nm. 20 μl PBS buffer was used to replace the enzyme working solution, and the absorbance OD2 was measured; 20 μL PBS was used to replace the sample solution, and the absorbance OD3 was measured; 20 μL PBS was used to replace the sample solution, and 20 μL PBS buffer was used to replace the enzyme working solution, and the absorbance OD4 was measured. Since the sample has color, OD4 was set to eliminate the influence of color on the absorbance value, so that the result is more accurate. The inhibition rate of acetylcholinesterase activity was calculated as follows: SR% = [1-(OD1-OD2) / (OD3-OD4)] × 100%.

[0047] IC of acetylcholinesterase inhibited by cyanobacterium 50 It was 5.16±0.22μg / mL, and the positive drug galantamine was 1.84±0.01μg / mL.

[0048] Table 1. Inhibitory activity of acetylcholinesterase (IC 50 , μg / mL)

[0049]

Claims

1. A method for preparing blue flower pseudoindigo, characterized in that: The following steps are involved: 1) The powdered Caragana fasciata medicinal material was extracted twice with 95% ethanol under hot reflux, filtered, the filtrates were combined, and then concentrated and dried under reduced pressure to obtain 95% ethanol extract ECJ; 2) The extract ECJ was separated by silica gel column chromatography, and gradient elution was performed with dichloromethane-methanol in different volume ratios. After detection by thin layer chromatography, the standard substance of cyanobacterium indigo was used for comparison, and the fractions containing cyanobacterium indigo were combined to obtain component ECJ1; 3) Taking component ECJ1 and subjecting it to Sephadex LH20 column chromatography, eluting it with pure methanol, detecting it by thin layer chromatography, comparing it with a standard substance of cyanobacterium indigoferum, combining the cyanobacterium indigoferum-containing fractions, and obtaining component ECJ1-1 containing crystals; 4) Take component ECJ1-1, add methanol, filter out the crystals, and obtain cyanidin.

2. The method for preparing cyanobacterium pseudoindigo according to claim 1, characterized in that: Step 2) gradient elution with dichloromethane-methanol in different volume ratios includes: first eluting with dichloromethane-methanol in a volume ratio of 100:1, and then eluting with dichloromethane-methanol in a volume ratio of 50:

1.

3. Use of cyanobacterium indigofera in the preparation of drugs for treating and / or preventing Alzheimer's disease.

4. The use according to claim 3, characterized in that: The medicine is a single component or a compound preparation.

5. The use according to claim 3, characterized in that: The dosage form of the drug is one of tablets, capsules, oral liquid, syrup, dripping pills, injection liquid dosage form or freeze-dried powder injection dosage form.

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