CN106008483A - Preparation and application of isovitexin - Google Patents

Abstract

The invention belongs to the field of medicine and natural product chemistry, and in particular relates to a preparation method of flavonoid carbon glycosides and a new application thereof. In the present invention, the normal phase (silica gel) column chromatography is used for separation, and then the reverse phase (open ODS) column chromatography is used for separation, and the isovitexin crystals are naturally precipitated. When the silica gel column chromatography is used for separation, the sample is loaded by dry method; when the open ODS column chromatography is used for separation, the sample is loaded by wet method. The invention also provides the cardioprotective use of isovitexin.

Description

Preparation and use of isovitexin

technical field

The invention belongs to the field of medicine and natural product chemistry, and specifically relates to a preparation method of flavonoid carbon glycosides and a new application thereof.

Background technique

Isovitexin is a typical flavonoid carbon glycoside, which widely exists in vegetables, fruits and Chinese herbal medicines, such as the leaves of Vitex littoralis A. Cunn. of Verbenaceae, Gleditsia sinensis Lam .) and the leaves of Lespedezacuneata G.Don, and the seeds of mung bean (Vigna radiate L.). A large number of studies have shown that isovitexin has anti-oxidation, antibacterial, anti-pathogen and hypoglycemic activities, but the cardioprotective effect of isovitexin has not been reported yet.

In recent years, fillers such as silica gel and ODS have been widely used in the extraction, separation and purification of natural pharmaceutical chemical components, preparation process reform, preparation quality analysis, etc., and have clearly shown their unique functions. Utilizing the opposite adsorption function of silica gel and ODS and the principle of similarity and compatibility of substances, the active ingredients or effective parts can be separated and refined from the extract of traditional Chinese medicine, and the roughness and essence can be removed to the greatest extent, so as to promote the development of modernization research of Chinese medicine.

Administration of isoprenaline (ISO) can induce acute myocardial ischemic injury in rats, and its changes in electrocardiogram, myocardial enzymes and histopathology are similar to the lesion characteristics of human acute myocardial ischemia. An important animal model in the field of vascular disease research.

Contents of the invention

One object of the present invention is to provide a preparation method of isovitexin. Specific steps are as follows:

(1) Take an appropriate amount of mung beans, extract with ethanol or methanol, filter, and combine the filtrates to obtain an alcohol extract; reduce the alcohol extract to recover alcohol until it has no alcohol smell, and obtain a crude extract;

(2) The obtained crude extract is sequentially extracted with different polar organic solvents, the extract is discarded, then extracted with n-butanol, the n-butanol extract is combined, the n-butanol is recovered under reduced pressure, and the mung bean extract is obtained;

(3) The obtained mung bean extract is dispersed with an appropriate amount of methanol, added with an appropriate amount of silica gel, stirred evenly, loaded into a pre-treated silica gel column, and mixed with two organic solvents (100:0～0:100) Carry out gradient elution, collect the mixed organic solvent (50:50～30:70) eluate, concentrate until thick, add appropriate amount of ethanol or methanol to disperse; open the ODS column on the top, and use alcohol-water (0:100～100 :0), preferably alcohol-water (10:90～40:60), carry out gradient elution, collect the eluate of alcohol-water (35:65～40:60), leave it overnight to precipitate yellow crystals, which is iso Vitexin, the purity is above 95%.

In the present invention, the concentration of ethanol or methanol in step (1) is 30%-100%.

In the present invention, the extraction described in step (1) is to use 5 to 20 times the amount of 30% to 100% ethanol or methanol to flash extract 1 to 3 times, and combine the extracts.

In the present invention, the different polar organic solvents described in step (2) include, but are not limited to: petroleum ether, cyclohexane, methylene chloride, chloroform, ethyl acetate.

In the present invention, the amount of different polar organic solvents and n-butanol described in the step (2) is 0.5 to 2 times the volume, and the number of extractions is 1 to 5 times.

In the present invention, the mixed organic solvent described in step (3) is dichloromethane-methanol, or chloroform-methanol, or dichloromethane-ethanol, or chloroform-ethanol.

In the present invention, the alcohol-water described in step (3) is methanol-water or ethanol-water.

In the present invention, the normal phase (silica gel) column chromatography is used first for separation, and then the reverse phase (open ODS) column chromatography is used for separation, and isovitexin crystals are naturally precipitated.

In the present invention, when adopting silica gel column chromatographic separation, adopt dry method to load the sample; when adopting open ODS column chromatographic separation, adopt wet method to load the sample.

Another object of the present invention is to provide the cardioprotective application of isovitexin.

Description of drawings

Fig. 1 is the hydrogen spectrogram of isovitexin.

Figure 2 is the carbon spectrum of isovitexin.

Fig. 3 is a flowchart of the extraction and separation of isovitexin.

Figure 4 is the effect of isovitexin on the pathomorphology of myocardial tissue in ISO-induced myocardial ischemia rats (HE staining).

detailed description

Embodiment 1, the separation and preparation of isovitexin

1. Plant material: seeds of leguminous mung bean (Vigna radiate L.).

2. Separation and preparation method: 50 kg of mung bean seeds, reflux extraction with 10, 10, and 10 times the amount of 95% ethanol for 3 times, filter, combine the filtrates, recover the ethanol under reduced pressure until there is no alcohol smell, and obtain about 5 L of concentrated solution. Extract with 5 L of petroleum ether, 5 L of dichloromethane, and 5 L of n-butanol respectively three times, combine the n-butanol extracts, recover n-butanol under reduced pressure, and obtain mung bean extract. Dissolve the mung bean extract with an appropriate amount of methanol, and take an appropriate amount of silica gel to mix the sample. Put the mixed sample into a silica gel column, and use dichloromethane-methanol (100:0-0:100) gradient elution to obtain 5 fractions, collect fraction 4 (45:55-35:65), Load the sample on an open ODS column, elute with methanol-water (10:90-40:60) gradient, collect the methanol-water (40:60) eluate, let it stand, and precipitate a yellow solid, which is isovitexin. Example 2. Myocardial protective activity test of isovitexin

1. Experimental animals: SD rats, weighing 200-220 g, provided by the Animal Center of Shenyang Pharmaceutical University; certificate number: SCXK (Liao) 2015-0001.

2. Reagents: ISO injection (Shanghai Hefeng Pharmaceutical Co., Ltd., batch number: 41150402), urethane (China Pharmaceutical (Group) Shanghai Chemical Reagent Company), creatine kinase (CK), lactate dehydrogenase (LDH), aspartate Transaminase (AST), malondialdehyde (MDA), superoxide dismutase (SOD) kits (Nanjing Jiancheng Bioengineering Institute).

The isovitexin used in the experiment is self-made by the inventor, and its chemical structure is confirmed by hydrogen spectrum and carbon spectrum, and its HPLC purity is above 98%.

3. Experimental method: 18 SD rats were randomly divided into three groups: blank group, model group and isovitexin group, 6 rats in each group. The blank group and the model group were given continuous gastric administration of normal saline for 7 days, the isovitexin group was given continuous gastric administration of isovitexin solution (30mg/kg) for 7 days, and the model group and the isovitexin group were administered on the 6th and 7th days. Five minutes after the administration, ISO 2 mg/kg was administered intraperitoneally, and 2 hours after the administration of ISO on the seventh day, blood was collected from the orbital venous plexus, and the serum was separated to determine serum creatine kinase (CK), lactate dehydrogenase (LDH), aspartate Transaminase (AST) activity. Rats were sacrificed after blood collection, and the heart was quickly removed. Part of the heart tissue homogenate was taken, centrifuged (3500r/min, 15min), and the myocardial homogenate supernatant was taken out to measure the contents of SOD and MDA in the myocardial homogenate. The experimental results were statistically processed by Excel, and the significance was judged by t test. Another part of heart tissue was fixed in 10% formalin solution, routinely dehydrated, embedded in paraffin, sectioned, stained with hematoxylin-eosin (HE), and observed histopathological changes under a light microscope.

4. Test results: As shown in Table 2, compared with the blank group, CK, LDH and AST in the serum of the rats in the model group were all significantly increased, indicating that the leakage of the three from the cells increased, and the myocardial tissue of the rats in the model group MDA significantly increased, and SOD decreased significantly. The above indicators all indicated that the model of ISO-induced myocardial ischemia in rats was established. Compared with the model group in the isovitexin group, the CK, LDH and AST in the serum of the isovitexin group were all significantly decreased, which inhibited the leakage of the three cells, and the MDA in the myocardial tissue of the rats in the isovitexin group was significantly decreased. SOD increased significantly, suggesting that isovitexin can protect ischemic myocardium by enhancing antioxidant capacity.

Table 2. Effect of isovitexin on myocardial enzyme activity in ISO-induced myocardial ischemia rats

Observed under an optical microscope, the myocardium of the rats in the blank group had no pathological changes, and the rats in the model group could see myocardial rupture, myocardial interstitial edema, and inflammatory cell infiltration. The pathological damage of myocardial tissue in the isovitexin group was reduced compared with that in the model group, no myocardial interstitial edema was observed, and inflammatory cell infiltration was rarely seen. The results are shown in Figure 4, suggesting that isovitexin can significantly reduce the degree of myocardial ischemia injury induced by ISO in rats.

The test results of the invention show that the isovitexin has cardioprotective activity, so the compound has good application and development prospects.

Claims (10)

1. the preparation method of isovitexin, comprises the following steps: (1) Preparation of the mung bean crude extract: take an appropriate amount of mung beans, extract with ethanol or methanol under reflux, filter, combine the filtrates, and obtain the alcohol extract, and recover the alcohol under reduced pressure from the alcohol extract until it has no alcohol smell, to obtain the crude extract; (2) Preparation of mung bean extract: The mung bean crude extract is extracted with different polar organic solvents successively, the extract is discarded, and then extracted with n-butanol, the n-butanol extract is combined, and n-butanol is recovered under reduced pressure to obtain Mung bean plant extract; (3) Refining of isovitexin: the mung bean extract of gained is dispersed with appropriate amount of methanol, adds appropriate amount of silica gel, stirs, packs in pre-treated silica gel column, mixes with two kinds of organic solvents mixed organic solvent (100 :0～0:100) for gradient elution, collect the mixed organic solvent (50:50～30:70) eluate, concentrate until thick, add appropriate amount of ethanol or methanol to disperse; open ODS column on top, use alcohol- Water (0:100～100:0), preferably alcohol-water (10:90～40:60), carry out gradient elution, collect the eluate of alcohol-water (35:65～40:60) and leave it overnight Precipitation of yellow crystals is isovitexin. 2. the preparation method as claimed in claim 1 is characterized in that, the concentration of ethanol or methanol described in step (1) is 30%～100%, adopts 30%～20 times of amount in step (1). Extract with 100% ethanol or methanol for 1 to 3 times, and combine the extracts. 3. The preparation method according to claim 1, characterized in that, in step (2), first extract 1 to 5 times with 0.5 to 2 times the volume of petroleum ether or cyclohexane, and then use 0.5 to 2 times the volume of dichloro Extract with methane or chloroform or ethyl acetate for 1 to 5 times, and finally extract with 0.5 to 2 times the volume of n-butanol for 1 to 5 times. 4. the preparation method as claimed in claim 1 is characterized in that, the mixed organic solvent gradient elution described in step (3), its mixed organic solvent is dichloromethane-methanol, or chloroform-methanol, or dichloromethane Methane-ethanol, or chloroform-ethanol. 5. The preparation method according to claim 1, characterized in that, the alcohol-water described in step (3) is methanol-water or ethanol-water. 6. A pharmaceutical composition, comprising isovitexin prepared by the preparation method described in any one of claims 1-5 and a pharmaceutically acceptable carrier. 7. The use of isovitexin in the preparation of cardioprotective medicine. 8. The use according to claim 7, characterized in that the myocardial protection is the protective effect on isoproterenol-induced myocardial ischemia. 9. purposes as claimed in claim 7, is characterized in that, described cardioprotective medicine is heart disease, especially the medicine of the angina pectoris prevention and symptom relief that myocardial ischemia causes. 10. The use according to claim 9, characterized in that the medicine is tablet, capsule, granule, oral solution, sterile powder for injection or injection.