CN115969893A

CN115969893A - Extraction method and application of total glucosinolates of common head cabbages

Info

Publication number: CN115969893A
Application number: CN202211699997.5A
Authority: CN
Inventors: 张宝宝; 栾会玲; 侯保华; 陈思思
Original assignee: Henan University of Technology
Current assignee: Henan University of Technology
Priority date: 2022-12-28
Filing date: 2022-12-28
Publication date: 2023-04-18
Anticipated expiration: 2042-12-28
Also published as: CN115969893B

Abstract

The invention belongs to the technical field of natural product extraction, and relates to an extraction method and application of common head cabbage total glucosinolates. Mixing the common head cabbage with ethanol, extracting to obtain a crude extract, then adding a lead acetate solution into the crude extract for sedimentation, concentrating the filtrate under reduced pressure after the sedimentation is finished, freeze-drying, extracting with ethanol, and finally separating, purifying and eluting to obtain a solid, namely the common head cabbage total glucosinolate. The method provided by the invention can realize the high-efficiency extraction of the glucosinolates in the common head cabbages, the content of the obtained total glucosinolates is 1.43 plus or minus 0.08mmol/g, and the obtained total glucosinolates account for 17.00 plus or minus 1.41 percent of all the glucosinolates in the extract before separation. Meanwhile, the application of the total glucosinolate components of the common head cabbage in the aspect of preparing the heart failure resistant medicine is clearly evaluated for the first time, so that the ideal effect is achieved, and the result shows that the total glucosinolate of the common head cabbage shows the obvious heart failure resistant effect in vitro and in vivo, so that the method has a wide application prospect.

Description

Extraction method and application of total glucosinolates of common head cabbages

Technical Field

The invention belongs to the technical field of natural product extraction, and relates to an extraction method and application of common head cabbage total glucosinolates.

Background

Common head cabbage (Brassicaoleracea L. Var. Capitatal.) as a high-yield commercial crop has the characteristics of cold resistance, disease resistance, storage resistance, transportation resistance, high yield and the like. Compared with the special requirements of many crops on the environment, the strong adaptability of the common head cabbage enables the common head cabbage to grow widely. Therefore, common head cabbage is cultivated in all parts of China, and is one of the main vegetables in northern China. The research reports that common head cabbage is rich in substances such as ascorbic acid, B vitamins, folic acid and polyphenol, particularly Glucosinolates (GLs), and the hydrolysate Isothiocyanate (ITCs) of the common head cabbage is the bioactive substance with the most anticancer effect found in vegetables so far. In addition, the polyphenol substances have the capabilities of removing free radicals and resisting oxidation, and can inhibit bacteria, diminish inflammation, resist viruses and reduce the morbidity of cardiovascular diseases and cancers. Therefore, the common head cabbage is a vegetable which is worthy of advocating large-scale eating and can reduce the occurrence risk of cardiovascular diseases and cancers to a certain extent.

GLs can be classified into three major groups, aliphatic, aromatic and indole according to the difference of branched chains, and they can be rapidly hydrolyzed by myrosinase into ITCs, thiocyanate and nitrile salts. Research shows that the ITCs can effectively prevent DNA damage caused by polycyclic aromatic hydrocarbons, heterocyclic amine, nitrosamine and other substances in food and have obvious blocking effect on the formation of liver cancer, lung cancer, breast cancer and esophagus cancer. The ITCs also have the effects of sterilization, insect resistance, oxidation resistance, platelet aggregation inhibition and the like. In recent years, studies have shown that methanol extracts of common head cabbage can effectively inhibit the damage of oxidative stress to H9C2 cells at an in vitro level, and the protective effect is closely related to the protein expression levels of ERK1/2, JNK and p 38. GLs may have potential value in terms of myocardial protection as the most abundant highly active ingredient in common head cabbage.

At present, the extraction and application of GLs are mainly directed to brassica plants (broccoli, radish seed, broccoli and the like), and one of the methods is as follows: the types and the contents of GLs contained in different plants are different, so the extraction process has obvious difference, the existing process aiming at other plants cannot be directly adopted in the extraction of the GLs in the common head cabbage, for example, the content of the GLs in the obtained product in the broccoli extraction process is only 414.98 mu mol/g, and the content of the GLs in the radish seed process product is only about 53 percent; and the second step is as follows: GLs of different sources and compositions have more differences in their application, for example, the extract of broccoli with abundant GLs is mainly used for insulin resistance related diseases; and thirdly: although the yield per mu of the fresh common head cabbage is about 2500-4000 kg/mu, about 40 percent of leaves are discarded or used as animal feed in the processing and transportation process, so that sufficient raw material sources can be provided for large-scale production on the premise of not influencing the supply market of the normal common head cabbage; and the fourth step: the common head cabbage has strong adaptability, and is cultivated all over the country, so that the restriction on the places for selective planting and deep processing is small, and the popularization prospect is good; and fifthly: although partial research on the protective effect of GLs on the cardiovascular system exists, research reports on heart failure of GLs are still lacking, particularly the effect of GLs in the common head cabbage on heart failure is lacking, the development is carried out by taking total GLs in the common head cabbage as a target, the market value of the common head cabbage can be better excavated, and economic benefits are generated.

Therefore, the research on the content of GLs in the common head cabbage, the determination of the optimal extraction process and the measurement of the in vivo and in vitro heart failure resistance of the common head cabbage not only can provide data support for the popularization and the application of the GLs of the common head cabbage, but also can be a theoretical basis for deeply excavating the biological utilization value and the economic development potential of the common head cabbage.

Disclosure of Invention

The invention provides an extraction method and application of total glucosinolates of common head cabbages, aiming at the technical problems of low extraction rate, expensive separation materials, low product content and the like in the prior art for extracting GLs. The content of the common head cabbage GLs obtained by the thymol method detection is up to 1.43mmol/g, and the common head cabbage GLs have excellent performance in the aspect of heart failure resistance and have wide application prospect.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

a method for extracting GLs in common head cabbage comprises the following steps:

(1) Adding the wall-broken and dried powder of the common head cabbage leaves into an ethanol solution, performing hot reflux extraction and suction filtration on the ethanol solution, and concentrating the filtrate under reduced pressure to obtain a total common head cabbage extract;

(2) Dissolving the total common head cabbage extract obtained in the step (1) by using a lead acetate aqueous solution, standing at room temperature, performing suction filtration, and performing reduced pressure concentration and freeze drying on the filtrate to obtain crude total glucosinolates of the common head cabbage;

(3) And (3) performing solid-liquid extraction on the crude total glucosinolates of the common head cabbages obtained in the step (2) by using absolute ethyl alcohol, and separating and purifying by using column chromatography to obtain the total glucosinolates of the common head cabbages.

Further, the volume fraction of the ethanol solution in the step (1) is 0-100%.

Preferably, the volume fraction of the ethanol solution in the step (1) is 50%.

Furthermore, the mass volume ratio of the cabbage leaf powder to the ethanol solution in the step (1) is 1g (5-20) mL.

Preferably, the mass-to-volume ratio of the cabbage leaf powder to the ethanol solution in the step (1) is 1g.

Further, the ethanol hot reflux extraction time in the step (1) is 0.5-3 h, and the ethanol hot reflux extraction temperature is 85 ℃.

Preferably, the ethanol hot reflux extraction time in the step (1) is 2.5h.

Further, the concentration of the lead acetate aqueous solution in the step (2) is 0.01-0.05 mmol/L, and the mass volume ratio of the common head cabbage total extract to the lead acetate aqueous solution is 1g.

Preferably, the concentration of the lead acetate aqueous solution in the step (2) is 0.04mmol/L.

Further, the standing time at room temperature in the step (2) is 12h.

Further, the mass volume ratio of the crude total glucosinolates of the kohlrabi in the step (3) to the absolute ethyl alcohol is 1g (5-13) mL; the substance selected for the column separation and purification is silica gel, wherein the eluent for the silica gel separation and purification is a mixed solution of ethyl acetate and methanol in a volume ratio of 5.

Preferably, the mass-to-volume ratio of the crude total glucosinolates of the kohlrabi to the absolute ethyl alcohol in the step (3) is 1g.

Furthermore, the GLs in the common head cabbage are extracted by the method.

Furthermore, the common head cabbage GLs are applied to the preparation of anti-heart failure medicines.

Furthermore, the application of the common head cabbage GLs in preparing the medicine for improving the heart failure by regulating the abnormal activation of the glycolysis of the myocardial cells.

The invention has the following beneficial effects:

1. the invention carries out process research by taking GLs in the common head cabbage as a target for the first time, and provides a basis for developing the potential value of the common head cabbage. The research result shows that: the price of the silica gel column chromatography is lower and the separation cost is lower compared with other column chromatographs. The content of separated GLs is 1.43 plus or minus 0.08mmol/g, and the total GLs obtained after separation accounts for 17.00 plus or minus 1.41 percent of all glucosinolates in the pre-separation extract.

2. The invention firstly and definitely evaluates the application of the nucleoside component in the aspect of heart failure resistance aiming at non-disease diagnosis or treatment, obtains more ideal effect, and research results show that: the common head cabbage GLs have obvious inhibition effects on the expression level of various blood factors such as BNP, the concentration of NO, the degree of cell hypertrophy, the concentration of lactic acid and the concentration of pyruvic acid in a heart failure model, and have obvious cell protection effects. The invention provides a research basis for the application of GLs in the aspect of resisting heart failure.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows the results of the measurement of BNP content in the in vitro culture medium according to the application example of the present invention.

FIG. 2 shows the result of measuring the NO content in the culture medium in vitro according to the application example of the present invention.

FIG. 3 shows the results of the H9C2 cell hypertrophy test in the application example of the present invention.

FIG. 4 shows the results of measuring the content of lactic acid in the culture medium in vitro according to the application example of the present invention.

FIG. 5 shows the result of measuring the content of pyruvic acid in the in vitro culture medium according to the application example of the present invention.

FIG. 6 shows the results of HE detection of rat myocardial tissue in accordance with the present invention.

FIG. 7 shows the measurement results of BNP content in rat serum in the application example of the present invention.

FIG. 8 shows the result of detecting NO content in rat serum.

FIG. 9 shows the results of measuring the lactic acid content in rat serum, which is an application example of the present invention.

FIG. 10 shows the result of measuring the pyruvic acid content in the serum of rat in accordance with the present invention.

FIG. 11 shows the results of immunohistochemical staining of rat myocardial tissue in an example of application of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art based on the embodiments of the present invention without inventive step, are within the scope of the present invention.

Example 1

The method for extracting the common head cabbage GLs comprises the following steps:

taking dried cabbage leaf, breaking the wall, grinding, sieving with 100 mesh sieve, and drying the cabbage leaf powder at 65 ℃ for 24h. 5g of the dried powder was precisely weighed, and 100mL of ethanol solutions of respective concentrations were added in a weight-to-volume ratio of 1g. Extracting under hot reflux for 3h. Filtering to remove residue, and concentrating the filtrate at 60 deg.C under reduced pressure to obtain viscous total extract. Drying the total extract with freeze drier at-80 deg.C for 48 hr. The total extracts were weighed precisely and the extract was tested for GLs content by thymol assay. The results are shown in table 1: in the range of 0-50% ethanol concentration, the yield of the total extract is increased from 46.6 +/-3.2% to 55.4 +/-2.2%, the yield of the total extract is continuously reduced along with the increase of the ethanol concentration, and the yield is reduced to 41.9 +/-3.1% when 100% is extracted. Meanwhile, the detection results of the GLs content of different extracts show that the GLs content in the extracts is more than 0.15mmol/g under the extraction condition of 20-80% ethanol.

TABLE 1 influence of ethanol concentrations on the extraction yield of Brassica oleracea and the total glucosinolate content of the extract

Example 2

precisely weighing 5g of dry powder of nodulized cabbage leaves, and extracting for 3h by hot reflux with 50% ethanol. The weight volume ratio is 1g:5 mL-1g. Filtering to remove residue, and concentrating the filtrate at 60 deg.C under reduced pressure to obtain viscous total extract. Drying the total extract with freeze drier at-80 deg.C for 48 hr. Each total extract was weighed precisely. The results are shown in table 2: within the range of material ratio of 1. However, the yield of the extract remains constant within a stable range even if the volume of the extraction solution is continuously increased after exceeding the ratio within the detection range.

TABLE 2 influence of different material ratios on the yield of total extract of common head cabbage

Example 3

precisely weighing 5g of dry powder of the nodulized cabbage leaves, and performing hot reflux extraction by 50% ethanol according to the weight-volume ratio of 1g to 13mL, wherein the extraction time is 0.5-3 h respectively. Filtering to remove residue, and concentrating the filtrate at 60 deg.C under reduced pressure to obtain viscous total extract. Drying the total extract with freeze drier at-80 deg.C for 48 hr. Each total extract was weighed precisely. The results are shown in table 3: the extraction time is in the range of 0.5-2.5 h, and the yield of the extract is increased from 48.9 +/-2.9% to 55.7 +/-1.3% along with the increase of the extraction time. Even if the extraction time is increased thereafter, the yield does not change significantly within the detection range.

TABLE 3 Effect of different extraction times on the Total extract yield of Brassica oleracea

Example 4

5g of dried powder of the noduliflower cabbage leaves is precisely weighed and thermally refluxed for 2.5h by 50% ethanol according to the weight-volume ratio of 1g. Filtering to remove residue, and concentrating the filtrate at 60 deg.C under reduced pressure to obtain viscous total extract. Drying the total extract with freeze drier at-80 deg.C for 48 hr. Precisely weighing 1g of dry total extract, and dissolving the dry extract by lead acetate solutions with different concentrations according to the weight-volume ratio of 1g. Standing at room temperature for 12 hr, and settling to remove protein and part of polyphenols in the extract. Filtering, concentrating the filtrate under reduced pressure, drying with a freeze dryer at-80 deg.C for 48 hr, and drying the residue at 60 deg.C for 24 hr. The residual rate of the total extract after the lead acetate is settled is obtained by calculation. The results are shown in table 4: the non-glucosinolate impurities in the total extract were settled by lead acetate solutions of different concentrations, and when the concentration of lead acetate was 0.04mmol/L, about 17.2% of the impurities in the total extract were removed, and the remaining rate of the total extract was 82.8. + -. 2.7%, in which case the settlement was considered complete, and even if the concentration of lead acetate was increased, the amount of the settled impurities was not increased.

Table 4 influence of lead acetate solutions of different concentrations on the clearance of impurities from common head cabbage extract

Example 5

the crude extract obtained after sedimentation and drying of 0.04mol/L lead acetate is obtained according to the above operation method, and the crude extract contains 77.1 percent of sample and 24.6 percent of lead acetate inorganic salt. 1g of a sample was precisely weighed, and subjected to solid-liquid extraction with anhydrous ethanol to remove inorganic salts. Concentrating the extractive solution at 60 deg.C under reduced pressure, and drying with freeze drier at-80 deg.C for 48 hr to obtain crude GLs. The yield of crude GLs and the content of GLs are shown in table 5: when the proportion of the absolute ethyl alcohol in the extraction liquid is continuously increased, the yield of the crude GLs is continuously increased, the corresponding content of the GLs in the crude GLs is continuously changed, when the material ratio is 1. Although the yield of the absolute ethanol extract is still continuously increased with the increase of the volume of the absolute ethanol, the content of GLs contained therein starts to decrease.

TABLE 5 influence of different solid-liquid ratios on the yield and content of crude total glucosides in common head cabbage

Example 6

The present embodiment is a method for extracting common head cabbage GLs, comprising the following steps:

crude GLs 1g of common head cabbage prepared in example 5 was separated with different column chromatography packing materials (DEAE Sephadex a-25, sephadex LH-20, dowex 50, activated carbon and silica gel) and eluted with the respective eluents. Wherein DEAE Sephadex A-25 is eluted by water solution gradient with pH 9-2, sephadex LH-20 is eluted by 50% methanol water solution isocratic, dowex 50 is eluted by water solution gradient with pH 2-7; the activated carbon is eluted in a gradient manner by 0 to 95 percent of ethanol water solution, and the silica gel is respectively eluted by ethyl acetate: methanol =5, gradient elution from 1 to 3.5. The results show that: (1) When DEAE SephadexA-25 is eluted by a pH 9-2 aqueous solution, no elution effect exists in the range of pH =9 to pH =3, and crude GLs are completely eluted when pH =2, so that DEAE SephadexA-25 has no obvious separation and purification effect on GLs in the kohlrabi; (2) When the Sephadex LH-20 is eluted isocratically by 50% methanol-water solution, the crude GLs have no obvious separation effect, so the Sephadex LH-20 has no obvious separation and purification effect on the GLs in the common head cabbage; (3) When Dowex 50 is eluted by an aqueous solution with pH 2-7, all crude GLs are eluted at pH =2, so that Dowex 50 has no obvious separation and purification effect on the cabbage GLs; (4) When the activated carbon is eluted by 0-95% ethanol water solution, the elution part of the 0% ethanol water solution obtains 311 +/-38mg of a recovered product, the content of GLs is only 0.04 +/-0.03 mmol/g, the elution part of the 10-95% ethanol water solution has no elution effect, and GLs in crude GLs are lost due to the adsorption of the activated carbon; (5) Collecting eluent of a part 1 of ethyl acetate and methanol =3.5 in silica gel column chromatography, drying, and washing with methanol to obtain transparent crystals, namely purified common head cabbage total glucosinolates (BOG). The collected BOG had a weight of 172. + -. 14mg, a GLs content of 1.43. + -. 0.08mmol/g, and the GLs obtained after isolation accounted for 17.00. + -. 1.41% of all GLs in the pre-isolation extract.

In conclusion, the method for extracting the total glucosinolates of the common head cabbages is researched, and the research result shows that:

(1) Optimizing extraction conditions: the ethanol with different concentrations is used for extraction, the extract yield is the highest when the ethanol with 50 percent is used for extraction, the extract reaches 55.5 +/-2.2 percent, and the total glucosinolate is completely extracted under the condition and is 0.16 +/-0.02 mmol/g.

(2) Material ratio: the best extraction effect is achieved by dissolving 1g of powder in 13mL of alcohol, and the yield is 55.6 +/-1.2%.

(3) The ethanol hot reflux extraction time is 2.5h, and the yield of the total extract is 55.6 +/-1.3%.

(4) The crude extract of the total glucosides of the common head cabbage is settled according to the proportion of 1g to 20mL by using 0.04mmol/L lead acetate solution, and the yield of the crude extract of the total glucosides of the common head cabbage is 82.8 +/-2.7 percent.

(5) And (3) after lead acetate is settled, concentrating the filtrate under reduced pressure, and after freeze drying, extracting the powder by absolute ethyl alcohol, wherein when the volume (g/mL) = 1.

(6) And further separating and purifying the total glucosides of the common head cabbage by using DEAE Sephadex A-25, sephadex LH-20, dowex 50, activated carbon and silica gel respectively. The research finds that: when purification is performed with silica gel, after removing part of impurities with ethyl acetate: methanol = 5. The content of GLs in the purified BOG is 1.43 +/-0.08 mmol/g through inspection, and the content of GLs in the BOG obtained after purification accounts for 17.00 +/-1.41% of all GLs in the extract before separation.

Application example

The application of the common head cabbage total glucosinolate in the aspect of resisting heart failure comprises the following steps:

1. in vitro experiments

1.1 method: cultured rat cardiomyocytes H9C2 (2-1) were digested and counted, and then inoculated into a 96-well plate in the number of 10000/well, and cultured in a DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37 ℃ in an incubator. After the cells had grown to a density of 70-80%, they were replaced with 2% FBS-containing medium, and BOG was diluted with the medium to the desired concentration and added to the wells for pretreatment. After 3h of pretreatment, isoproterenol (ISO; 50. Mu.M) as an inducer is added for co-culture for 48h to establish an in vitro heart failure model. A blank Control (Control) was set up and each experiment was run in triplicate. The drug groups were metoprolol (ME; 100. Mu.M) and BOG (25, 50 and 100. Mu.g/mL) (the solvents were pure water)

1.2 results

1.2.1BNP concentration detection

BNP is B-type natriuretic peptide, is a marker substance of heart failure, and the expression level of the BNP can intuitively reflect whether the establishment of a heart failure model is successful, so that the content of the BNP in a detection culture medium can be used for representing the damage degree of myocardial cells, after the culture medium is cultured for 48 hours, the culture medium of each group of cells is taken to carry out detection according to the operation steps of a BNP ELISA detection kit, an enzyme-labeling instrument detects the absorption value at 450nm, graphPad Prism 5 software is used for carrying out data processing, and the significance difference (Unaccessing t-test) is calculated.

As shown in fig. 1, BNP expression level in the model group was significantly increased compared to the control group, while BNP expression level in the BOG-treated group was significantly decreased compared to the model group and exhibited concentration dependence. The BNP reduction level was the most in the BOG (100 μ g/mL) treated group, i.e. the most significant protective effect on cells. Errors are identified as mean Standard Error (SEM), # #: significant differences were compared to control groups,: significant differences were relative to the model group.

1.2.2NO concentration detection

NO is a key blood factor in maintaining normal physiological functions of the cardiovascular system. When the myocardial cells are injured, the expression level of NO is increased, so that the concentration of NO in the culture medium is increased, and therefore, the content of NO in the culture medium can be detected to be used for representing the injury degree of the myocardial cells. After the co-culture is carried out for 48h, the culture medium of each group of cells is taken to carry out detection according to the operation steps of the BNP ELISA detection kit, an enzyme-linked immunosorbent assay (ELISA) instrument is used for detecting the absorption value at 550nm, graphPad Prism 5 software is used for carrying out data processing, and the significance difference (Unpairedt-test) is calculated.

As shown in fig. 2, the NO expression level of the model group was significantly increased compared to the control group, while the NO expression level of the BOG-treated group was significantly decreased compared to the model group and exhibited concentration dependence. The NO reduction level was the most in the BOG (100 μ g/mL) treated group, i.e. the protective effect on cells was the most significant. Errors are identified as mean Standard Error (SEM), # #, significant differences versus control group, #: significant differences were relative to the model group.

1.2.3 cell hypertrophy test

Cellular hypertrophy is a common hallmark problem of heart failure, which may reflect the extent to which heart failure occurs. Cells were treated with actin-tracker green stain and the staining results were indicative of the degree of cell enlargement. After molding, cells are stained according to the kit operation method, after staining, the cells are observed and photographed under a microscope, the stained parts are quantified by Image J software, data processing is carried out by GraphPad Prism 5 software, and the significance difference (Unpairedt-test) is calculated.

As shown in fig. 3, the cell staining area of the model group was significantly increased compared to the control group, while the cell staining area of the BOG-treated group was decreased compared to the model group and was concentration-dependent, and the area of the BOG (100 μ g/mL) treated group was decreased the most, i.e., the protection effect on the cells was the most significant. Errors are identified as mean Standard Error (SEM), # #, significant differences versus control group, #: significant differences were relative to the model group.

1.2.4 detection of lactic acid concentration

Lactic acid is an important metabolite of glycolysis, and its concentration is high or low in response to the glycolytic level of myocardial cells. When heart failure occurs, myocardial cell glycolysis increases and lactate expression levels increase, resulting in an increase in lactate concentration in the culture medium, and thus measuring the lactate content in the culture medium can be used to characterize the degree of abnormal increase in glycolysis in the myocardial cell. After 48h of co-culture, the culture medium of each group of cells was taken and detected according to the operation procedure of the lactic acid detection kit, the absorbance was detected at 440nm with a microplate reader, data was processed with GraphPad Prism 5 software, and the significance difference (Unpairedt-test) was calculated.

As shown in fig. 4, the lactate expression level of the model group was significantly increased compared to the control group, while the lactate expression level of the BOG-treated group was significantly decreased compared to the model group and exhibited concentration dependence. The most lactic acid reduction, i.e. the most significant protective effect on the cells, was observed in the BOG (100 μ g/mL) treated group. Errors are identified as mean Standard Error (SEM), ## # significant differences versus control group, #:: significant differences were relative to the model group.

1.2.5 detection of pyruvate concentration

Pyruvate is an important metabolite of sugar, and its concentration can reflect glycolytic levels of cardiomyocytes. When heart failure occurs, glycolysis of the myocardial cells is increased, the expression level of pyruvic acid is increased, and the concentration of pyruvic acid in the culture medium is increased, so that the detection of the content of pyruvic acid in the culture medium can be used for representing the abnormal increasing degree of glycolysis in the myocardial cells. After the co-culture is carried out for 48h, the culture medium of each group of cells is taken to carry out detection according to the operation steps of a pyruvic acid detection kit, an enzyme-labeling instrument is used for detecting an absorption value under 505nm, graphPad Prism 5 software is used for carrying out data processing, and the significance difference (Unaccessing t-test) is calculated.

As shown in fig. 5, the pyruvate expression level of the model group was significantly increased compared to the control group, while the pyruvate expression level of the BOG-treated group was significantly decreased compared to the model group and exhibited concentration dependence. The most decreased pyruvate in the BOG (100 μ g/mL) treated group, i.e. the most significant protective effect on cells. Errors are identified as mean Standard Error (SEM), # #, significant differences versus control group, #: significant differences were relative to the model group.

2. In vivo experiments

2.1 model construction method

Male Wistar rats of SPF grade 40 (200-250 g) were purchased from jinamingye laboratory animals breeding limited. After acclimatizing feeding for one week, the animals were randomly divided into four groups of ten animals. The blank group was gavaged with normal saline for 14 days, and was injected with normal saline intraperitoneally on days 13 and 14; the model group was gavaged with saline for 14 days, and was intraperitoneally injected with ISO saline solution (85 mg/kg day) on days 13 and 14; the ME group (60 mg/kg. Day) and the BOG group (75 mg/kg. Day and 150 mg/kg. Day) were continuously gavaged for 14 days, and were intraperitoneally injected with ISO normal saline solution (85 mg/kg. Day) on days 13 and 14. After eating for 12 hours, blood is taken from the orbit on the 15 th day, the upper layer serum is taken out after 12000g and 4 ℃ centrifugation and stored at-80 ℃ and used for detecting blood factors. The rats were sacrificed by decapitation, the hearts were dissected away, the residual blood in the hearts was removed by ice-freezing in 4 ℃ pre-cooled PBS solution, a transverse cut was made in the middle of the hearts, and the area near the apex of the heart was fixed with 4% paraformaldehyde solution for slice examination.

2.2 results

2.2.1HE staining

Fig. 6 is a picture of representative HE staining of heart sections to assess tissue morphology. It can be seen that the myocardial cells of the model group are obviously damaged and are accompanied by partial positive cell infiltration, while the BOG administration group can reduce the degree of myocardial cell damage, the cells are regularly and compactly arranged, and the protective effect is achieved on myocardial tissues.

2.2.2BNP, NO, lactate and pyruvate assays

FIGS. 7-10 show the results of BNP, NO, lactic acid and pyruvic acid assays in rat sera. The expression level in serum of the model group was significantly increased compared to the control group, while the expression level of the BOG-treated group was significantly decreased compared to the model group and exhibited concentration dependency. The most decreased levels, i.e., the most significant protective effect on cells, were found in the BOG (150 mg/kg day) treated group. Errors are identified as mean Standard Error (SEM), #:significantdifferences versus control group, #: significant differences were relative to the model group.

2.2.3 immunohistochemistry

FIG. 11 shows the results of expression of proteins involved in glycolysis in rat myocardial tissue. The expression levels of glycolysis-related proteins HIF-1 a, GLUT1 and PFKFB3 in myocardial tissues of the model group were significantly increased, while the expression levels of the BOG-treated group were significantly decreased compared to the model group and exhibited concentration dependence. The most decreased levels, i.e., the most significant protective effect on cells, were found in the BOG (150 mg/kg day) treated group.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A method for extracting total glucosinolates of common head cabbages is characterized by comprising the following steps:

(2) Dissolving the total common head cabbage extract obtained in the step (1) by using a lead acetate aqueous solution, standing at room temperature, carrying out suction filtration, and carrying out reduced pressure concentration and freeze drying on the filtrate to obtain crude total glucosinolates of the common head cabbage;

(3) And (3) performing solid-liquid extraction on the crude total glucosinolates of the common head cabbages obtained in the step (2) by using absolute ethyl alcohol, and separating and purifying by using column chromatography to obtain purified total glucosinolates of the common head cabbages.

2. The method for extracting total glucosinolates of common head cabbage according to claim 1, wherein the method comprises the steps of: the volume fraction of the ethanol solution in the step (1) is 0-100%.

3. The method for extracting total glucosinolates of common head cabbage according to claim 1 or 2, wherein the method comprises the following steps: the mass volume ratio of the cabbage leaf powder to the ethanol solution in the step (1) is 1g (5-20) mL.

4. The method for extracting total glucosinolates of common head cabbage according to claim 3, wherein the method comprises the following steps: in the step (1), the hot reflux extraction time of the ethanol is 0.5-3 h, and the hot reflux extraction temperature of the ethanol is 85 ℃.

5. The method for extracting total glucosinolates of common head cabbage according to any one of claims 1, 2, or 4, wherein the total glucosinolates of common head cabbage are selected from the group consisting of: in the step (2), the concentration of the aqueous solution of lead acetate is 0.01-0.05 mmol/L, and the mass volume ratio of the total common head cabbage extract to the aqueous solution of lead acetate is 1g.

6. The method for extracting total glucosinolates of common head cabbage according to claim 5, wherein the method comprises the following steps: and (3) standing at room temperature in the step (2) for 12h.

7. The method for extracting total glucosinolates of common head cabbage according to claim 6, wherein the method comprises the steps of: the mass volume ratio of the crude total glucosinolates of the kohlrabi in the step (3) to the absolute ethyl alcohol is 1g (5-13) mL; the substance selected for the column separation and purification is silica gel, wherein the eluent for the silica gel separation and purification is a mixed solution of ethyl acetate and methanol in a volume ratio of 5.

8. Total glucosinolates of common head cabbage extracted by the method of any one of claims 1, 2, 4, 6 or 7.

9. The use of the total glucosides of cabbage as claimed in claim 8 in the manufacture of a medicament for the treatment of heart failure.

10. Use of the total glucosides of common head cabbage according to claim 8 for the preparation of a medicament for ameliorating heart failure by modulating the abnormal activation of myocardial cell glycolysis.