CN115969893B

CN115969893B - Extraction method and application of total glucoside of common head cabbage

Info

Publication number: CN115969893B
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: 2024-01-26
Anticipated expiration: 2042-12-28
Also published as: CN115969893A

Abstract

The invention belongs to the technical field of natural product extraction, and relates to an extraction method and application of total glucoside of common head cabbage. Mixing common head cabbage with ethanol, extracting to obtain crude extract, adding lead acetate solution into the crude extract for sedimentation, concentrating the filtrate under reduced pressure after sedimentation, lyophilizing, extracting with ethanol, separating, purifying, and eluting to obtain solid, namely common head cabbage total thioglycoside. The method provided by the invention can realize the efficient extraction of the sulfatides in the common head cabbage, the content of the obtained total sulfatides is 1.43+/-0.08 mmol/g, and the obtained total sulfatides account for 17.00+/-1.41% of all sulfatides in the extract before separation. Meanwhile, the application of the total glucoside of the common head cabbage in preparing the heart failure resistant medicament is evaluated for the first time, the ideal effect is obtained, and the result shows that the total glucoside of the common head cabbage shows remarkable heart failure resistant effect on the in-vivo and in-vitro level and has wide application prospect.

Description

Extraction method and application of total glucoside of common head cabbage

Technical Field

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

Background

The common head cabbage (Brassica oleracea L.var.capitata L.) is used as a high-yield economic crop and 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 in a wide distribution. Therefore, the common head cabbage is cultivated in all places of China and is one of main vegetables in northern China. Research reports that common head cabbage is rich in ascorbic acid, B vitamins, folic acid, polyphenol and other substances, especially Glucosinolates (GLs) and hydrolysate Isothiocyanate (ITCs) is the most bioactive substance with anticancer effect found in vegetables so far. In addition, polyphenols have free radical scavenging and antioxidant effects, and can inhibit bacteria, diminish inflammation, resist viruses, and reduce cardiovascular diseases and cancer incidence. Therefore, the common head cabbage is a vegetable worthy of promoting a large amount of eating, and can reduce the occurrence risk of cardiovascular diseases and cancers to a certain extent.

GLs is classified into three major classes, aliphatic, aromatic and indolyl, depending on the branching, which are rapidly hydrolyzed by myrosinase into ITCs, thiocyanates and nitrile salts. Research shows that ITCs can effectively prevent DNA damage caused by substances such as polycyclic aromatic hydrocarbon, heterocyclic amine, nitrosamine and the like in diet, and have obvious blocking effect on liver cancer, lung cancer, breast cancer and esophageal cancer. ITCs also have bactericidal, anti-insect, antioxidant, platelet aggregation inhibiting, etc. effects. In recent years, studies have shown that methanol extracts of common head cabbage can effectively inhibit the damage of H9C2 cells by oxidative stress at in vitro level, and the protection is closely related to the protein expression levels of ERK1/2, JNK and p 38. GLs, which is the most abundant high active ingredient in common head cabbage, may be of potential value in myocardial preservation.

At present, the extraction and application of GLs mainly aims at brassica plants (broccoli, radish seeds, broccoli and the like), one of which is: the GLs contained in different plants are different in variety and content, so obvious differences exist in the extraction process, the existing process for other plants cannot be directly adopted in the extraction of GLs in the common head cabbage, for example, the content of GLs in the obtained product in the broccoli extraction process is only 414.98 mu mol/g, and the content of GLs in the radish seed process product is only about 53%; and two,: GLs of different sources and compositions has a large difference in application, such as GLs-enriched broccoli extract mainly applied to insulin resistance related diseases; and thirdly,: although the mu yield of the fresh common head cabbage is about 2500-4000 kg/mu, about 40% of leaves are discarded or used as animal feed in the processing and transportation processes, so that the fresh common head cabbage can provide sufficient raw material sources for large-scale production on the premise of not affecting the normal common head cabbage supply market; fourth, it is: the common head cabbage has strong adaptability and is cultivated in various places in China, so that the restriction on the places for selecting planting and deep processing is small, and the common head cabbage has good popularization prospect; fifth, it is: although the research on the protection effect of the part GLs on the cardiovascular system is available, the research report on the heart failure of GLs is lacking, particularly the report on the heart failure of GLs in the common head cabbage is lacking, the development is carried out with the aim of total GLs in the common head cabbage, the market value of the common head cabbage can be better excavated, and the economic benefit is generated.

Therefore, the content of GLs in the common head cabbage is researched, the optimal extraction process is determined, and the heart failure resistance in vivo and in vitro is measured, so that the method not only can provide data support for popularization and application of the common head cabbage GLs, but also is a theoretical basis for deep mining of the biological utilization value and economic development potential of the common head cabbage.

Disclosure of Invention

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

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

an extraction method of GLs in common head cabbage comprises the following steps:

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

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

(3) Extracting the crude total glucoside of the common head cabbage obtained in the step (2) by using absolute ethyl alcohol, and separating and purifying the crude total glucoside of the common head cabbage by using column chromatography.

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%.

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

Preferably, the mass-volume ratio of the common head cabbage leaf powder to the ethanol solution in the step (1) is 1 g/13 mL.

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

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

Further, in the step (2), the concentration of the lead acetate aqueous solution is 0.01-0.05 mmol/L, and the mass-volume ratio of the total extract of the common head cabbage to the lead acetate aqueous solution is 1 g/20 mL.

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 12 hours.

Further, the mass volume ratio of the crude total glucoside of the common head cabbage to the absolute ethyl alcohol in the step (3) is 1g (5-13) mL; the material selected during the separation and purification of the column is silica gel, wherein the eluent during the separation and purification of the silica gel is a mixed solution of ethyl acetate and methanol in a volume ratio of 5:1 and 3.5:1.

Preferably, the mass-volume ratio of the crude total glucoside of the common head cabbage to the absolute ethyl alcohol in the step (3) is 1g to 12mL.

Further, GLs in the common head cabbage extracted by the above method.

Further, the common head cabbage GLs is applied to preparation of the heart failure resistant medicine.

Further, the common head cabbage GLs is applied to the preparation of the medicines for improving heart failure by regulating abnormal activation of glycolysis of myocardial cells.

The invention has the following beneficial effects:

1. the invention firstly carries out process research with GLs in the common head cabbage as a target and provides basis for developing the potential value of the common head cabbage. The research results show that: the cost of separating by silica gel column chromatography is lower than that of other column chromatography, and the separation cost is low. The content of GLs after separation was 1.43.+ -. 0.08mmol/g, and the total GLs obtained after separation accounted for 17.00.+ -. 1.41% of all the sulfanyls in the pre-separation extract.

2. The invention definitely evaluates the application of the sulfan component in the aspect of heart failure resistance for the purpose of non-disease diagnosis or treatment for the first time, obtains ideal effect, and the research result shows that: the common head cabbage GLs has remarkable inhibition effect on the expression level of blood factors such as BNP, concentration of NO, degree of cell hypertrophy, concentration of lactic acid and concentration of pyruvic acid in a heart failure model, and has remarkable cytoprotective effect. The invention provides a research foundation for the application of GLs in the aspect of heart failure resistance.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of BNP assay in an in vitro medium according to an embodiment of the present invention.

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

FIG. 3 shows the results of the test for the hypertrophy of H9C2 cells according to the application example of the present invention.

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

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

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

FIG. 7 shows the results of BNP assay in serum of rats according to the application example of the present invention.

FIG. 8 shows the results of measuring NO content in serum of rat in the application example of the present invention.

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

FIG. 10 shows the results of detecting the content of pyruvic acid in the serum of rats according to the application example of the invention.

FIG. 11 shows the result of immunohistochemical staining of myocardial tissue in rat according to the application example of the present invention.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without any inventive effort, are intended to be within the scope of the invention.

Example 1

The extraction method of the common head cabbage GLs of the embodiment comprises the following steps:

taking dry common head cabbage leaves, breaking the wall, crushing, sieving with a 100-mesh sieve, and drying common head cabbage leaf powder at 65 ℃ for 24 hours. 5g of the dry powder was precisely weighed, and 100mL of each concentration of ethanol solution was added in a weight-to-volume ratio of 1g to 20mL. Extracting under reflux for 3 hr. Filtering to remove residue, and concentrating the filtrate at 60deg.C under reduced pressure to obtain viscous total extract. The total extract was dried at-80℃for 48h with a freeze dryer. The total extracts were weighed precisely and the content of GLs in the extracts was measured by thymol assay. The test results are shown in Table 1: in the concentration range of 0-50% ethanol, the yield of the total extract is increased from 46.6+/-3.2% to 55.4+/-2.2%, and as the concentration of ethanol is increased, the yield of the total extract is continuously reduced, and the yield is reduced to 41.9+/-3.1% in 100% extraction. Meanwhile, the detection results of GLs content of different extracts show that the GLs content in the extracts is above 0.15mmol/g under the extraction condition of 20% -80% ethanol.

TABLE 1 influence of ethanol of different concentrations on extraction yield of common head cabbage and total content of glycosides in the extract

Example 2

precisely weighing 5g of dried powder of common head cabbage leaf, and extracting with 50% ethanol under reflux for 3 hr. The weight-volume ratio is 1g:5 mL-1 g:20mL. Filtering to remove residue, and concentrating the filtrate at 60deg.C under reduced pressure to obtain viscous total extract. The total extract was dried at-80℃for 48h with a freeze dryer. The weight of each total extract was precisely weighed. The test results are shown in Table 2: in the material ratio range of 1:5-1:13, the yield of the total extract is continuously increased along with the increase of the volume of the extraction solution. However, even if the volume of the extraction solution is continuously increased after exceeding the ratio in the detection range, the yield of the extract remains unchanged in a stable range.

TABLE 2 influence of different Material ratios on total extract yield of Cryptographic head cabbage

Example 3

precisely weighing 5g of dried common head cabbage leaf powder, and carrying out hot reflux extraction with 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 60deg.C under reduced pressure to obtain viscous total extract. The total extract was dried at-80℃for 48h with a freeze dryer. The weight of each total extract was precisely weighed. The test results are shown in Table 3: the extraction time is within 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. After that, even if the extraction time is increased, the yield does not significantly change in the detection range.

TABLE 3 influence of different extraction times on total extract yield of common head cabbage

Example 4

precisely weighing 5g of dried powder of common head cabbage leaves, and carrying out thermal reflux for 2.5h according to the weight-volume ratio of 50% ethanol to 1 g/13 mL. Filtering to remove residue, and concentrating the filtrate at 60deg.C under reduced pressure to obtain viscous total extract. The total extract was dried at-80℃for 48h with a freeze dryer. 1g of the dry total extract is precisely weighed, and the dry extract is dissolved in lead acetate solutions with different concentrations according to the weight-volume ratio of 1g to 20mL. Standing at room temperature for 12 hr, and settling to remove protein and part of polyphenols in the extract. Vacuum filtering, concentrating the filtrate under reduced pressure, lyophilizing at-80deg.C for 48 hr, and drying the residue at 60deg.C for 24 hr. And obtaining the total extract residual rate after lead acetate sedimentation through calculation. The test results are shown in Table 4: by precipitating non-thio-glycoside impurities in the total extract by using lead acetate solutions of different concentrations, about 17.2% of impurities in the total extract were removed when the concentration of lead acetate was 0.04mmol/L, and the residual rate of the total extract was 82.8.+ -. 2.7%, in which case the precipitation was considered complete and the amount of precipitated impurities was not increased even if the concentration of lead acetate was increased.

TABLE 4 effect of different concentrations of lead acetate solution on impurity removal in common head cabbage extract

Example 5

a crude extract containing 77.1% of the sample and 24.6% of the lead acetate inorganic salt was obtained by the above procedure after 0.04mol/L of lead acetate was sedimented and dried. 1g of sample is precisely weighed, solid-liquid extraction is carried out by absolute ethyl alcohol, and inorganic salt is removed. The extract was concentrated under reduced pressure at 60℃and dried at-80℃for 48h to give crude GLs. The crude GLs yield and GLs content are shown in table 5: when the ratio of the absolute ethyl alcohol of the extract liquid is continuously increased, the yield of the crude GLs is continuously increased, the corresponding GLs content is continuously changed, and when the material ratio is 1:12, the yield of the absolute ethyl alcohol extraction is 10.4+/-0.3%, and the content of GLs in the extract reaches the highest value of 0.56+/-0.02 mmol/g. While the yield of absolute ethanol extract is still increasing as the volume of absolute ethanol increases, the content of GLs contained therein begins to decrease.

TABLE 5 influence of different solid to liquid ratios on crude Total sulfan yield and content in common head cabbage

Example 6

The embodiment is an extraction method of the common head cabbage GLs, which comprises the following steps:

the crude head cabbage prepared in example 5 (GLs g) was separated with different column chromatography packing (DEAE Sephadex A-25, sephadex LH-20, dowex 50, activated carbon and silica gel) and eluted with the corresponding eluent, respectively. Wherein DEAE Sephadex A-25 is eluted with a water solution gradient of pH 9-2, sephadex LH-20 is eluted with a 50% methanol water solution isocratic, dowex 50 is eluted with a water solution gradient of pH 2-7; gradient elution of active carbon with 0-95% ethanol water solution, and respectively using ethyl acetate to obtain silica gel: methanol=5:1 to 3.5:1 gradient elution. The results show that: (1) The DEAE Sephadex A-25 has no eluting effect in the range of pH=9 to pH=3 when eluting with the aqueous solution of pH 9-2, and crude GLs is completely eluted at pH=2, so that the DEAE Sephadex A-25 has no obvious separation and purification effect on GLs in the common head cabbage; (2) When the Sephadex LH-20 is eluted with 50% methanol-water solution at equal degree, the crude GLs has no obvious separation effect, so that the Sephadex LH-20 has no obvious separation and purification effects on GLs in the common head cabbage; (3) When the Dowex 50 is eluted by the aqueous solution with the pH of 2-7, the pH=2 elutes all crude GLs, so the Dowex 50 has no obvious separation and purification effects on the common head cabbage GLs; (4) When the active carbon is eluted by 0-95% ethanol water solution, the eluted part of the 0% ethanol water solution obtains 311+/-38 mg of recovered product, the GLs content is only 0.04+/-0.03 mmol/g, the eluted part of the 10-95% ethanol water solution has no eluting effect, and GLs in the crude GLs is lost due to adsorption by the active carbon; (5) In silica gel column chromatography, collecting eluent of ethyl acetate and methanol=3.5:1 part, drying, and cleaning with methanol to obtain transparent crystal, namely purified total glucoside of common head cabbage (Brassica oleracea glucosinolates, BOG). The BOG weight collected was 172.+ -. 14mg and GLs content was 1.43.+ -. 0.08mmol/g, and GLs after isolation accounted for 17.00.+ -. 1.41% of all GLs in the pre-isolation extract.

In conclusion, the invention researches the extraction method of the total glucoside of the common head cabbage, and the research results show that:

(1) And (3) optimizing extraction conditions: the ethanol with different concentrations is used for extraction, the yield of the extract is highest when the ethanol with 50% is used for extraction, the extract reaches 55.5+/-2.2%, and meanwhile, the extraction of the total thioglycoside under the condition is more complete, and the total thioglycoside is 0.16+/-0.02 mmol/g.

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

(3) The ethanol thermal reflux extraction time is optimal for 2.5 hours, and the total extract yield is 55.6+/-1.3%.

(4) Settling with 0.04mmol/L lead acetate solution according to the proportion of 1g to 20mL, and obtaining the crude extract of the total glucosides of the common head cabbage with the yield of 82.8+/-2.7%.

(5) The filtrate after lead acetate sedimentation is concentrated under reduced pressure, and when the weight of the powder after freeze drying is extracted by absolute ethyl alcohol, the volume (g/mL) of absolute ethyl alcohol is=1:12, the yield of crude GLs is 10.4+/-0.3%, wherein the content of total sulfatides is 0.56+/-0.02 mmol/g.

(6) The total glucoside of the common head cabbage is further separated and purified by using DEAE Sephadex A-25, sephadex LH-20, dowex 50, activated carbon and silica gel respectively. The research finds that: when the silica gel is used for purification, after partial impurities are removed by ethyl acetate and methanol=5:1, the silica gel is eluted by ethyl acetate and methanol=3.5:1, and the eluent is collected, dried and then washed by methanol, so that transparent crystals, namely the purified BOG, can be obtained. Through inspection, the content of GLs in the BOG after purification is 1.43+/-0.08 mmol/g, and 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 thioglycoside in the aspect of heart failure resistance comprises the following steps:

1. in vitro experiments

1.1 method: cultured rat cardiomyocytes H9C2 (2-1) were inoculated into 96-well plates at 10000/well after digestion and counted, and cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at 37℃in an incubator. After cells grow to 70-80% density, they are replaced with 2% FBS-containing medium, and BOG is diluted to the desired concentration with medium and added to wells for pretreatment. After pretreatment for 3 hours, an inducer isoprenaline (ISO; 50. Mu.M) (pure water as solvent) was added for co-culture for 48 hours to construct a three-dimensional external heart failure model. A Control group (Control) was set up, and each group of experiments was performed 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, respectively)

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 can intuitively reflect whether the establishment of a heart failure model is successful or not, so that the content of BNP in a detection culture medium can be used for representing the damage degree of myocardial cells, after co-culture for 48 hours, the culture medium of each group of cells is taken to detect according to the operation steps of a BNP ELISA detection kit, an enzyme-labeled instrument detects the absorption value at 450nm, data processing is carried out by Granpad Prism 5 software, and the significance difference (Unpaired t-test) is calculated.

As shown in fig. 1, BNP expression levels in the model group were significantly increased compared to the control group, while BNP expression levels in the BOG-treated group were significantly decreased compared to the model group and exhibited concentration dependence. The BNP reduction level was the greatest in the BOG (100. Mu.g/mL) treated group, i.e., the most significant protection of the cells. Errors identify the standard error of the average (SEM), #: significant differences relative to control group,: the significance differences were relative to the model set.

1.2.2NO concentration detection

NO is a critical blood factor that maintains normal physiological function of the cardiovascular system. When myocardial cells are damaged, the level of NO expression increases, resulting in an increase in the concentration of NO in the medium, and thus detecting the amount of NO in the medium can be used to characterize the extent of damage to myocardial cells. After 48h of co-culture, the culture medium of each group of cells was taken and tested according to the procedure of BNP ELISA test kit, the absorbance was measured at 550nm by an ELISA reader, data processing was performed with GraphPad Prism 5 software, and the significance difference (Unpairedt-test) was 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 significant in BOG (100 μg/mL) treated group, i.e. the most significant protection to cells. Error marks are mean Standard Error (SEM), #: significant differences relative to control group, ×: the significance differences were relative to the model set.

1.2.3 cell hypertrophy experiments

Cell hypertrophy is a common hallmark problem of heart failure, which can reflect the extent to which heart failure occurs. Cells were treated with actin-tracker green stain and the staining results were indicative of the extent of cell enlargement. Cells were stained following the kit procedure after molding, observed and photographed under a microscope after staining was completed, stained sections were quantified with Image J software, data processed with GraphPad Prism 5 software, and a significant difference (uinpairedt-test) was 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 reduced compared to the model group, and showed concentration dependence, and the area of the BOG (100 μg/mL) treated group was most reduced, i.e., the cell protection effect was most significant. Error marks are mean Standard Error (SEM), #: significant differences relative to control group, ×: the significance differences were relative to the model set.

1.2.4 lactic acid concentration detection

Lactic acid is an important metabolite of glycolysis, at concentrations that are at levels that are responsive to glycolysis levels of cardiomyocytes. When heart failure occurs, the glycolysis of the cardiomyocytes increases, and the lactic acid expression level increases, resulting in an increase in the lactic acid concentration in the medium, so detecting the amount of lactic acid in the medium can be used to characterize the degree of abnormal increase in glycolysis in the cardiomyocytes. After 48h of co-culture, the culture medium of each group of cells is taken to be detected according to the operation steps of the lactic acid detection kit, the absorption value is detected at 440nm by an enzyme-labeled instrument, the data processing is carried out by using GraphPad Prism 5 software, and the significance difference (Unpairedt-test) is 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 reduced level of lactate, i.e. the most pronounced protective effect on cells, was found in the BOG (100 μg/mL) treated group. Error marks are mean Standard Error (SEM), #: significant differences relative to control group, ×: the significance differences were relative to the model set.

1.2.5 detection of pyruvate concentration

Pyruvic acid is an important metabolite of glycolysis, and its concentration is high or low to reflect glycolysis level of cardiomyocytes. When heart failure occurs, the glycolysis of the cardiomyocytes increases, and the expression level of pyruvic acid increases, resulting in an increase in the concentration of pyruvic acid in the medium, so detecting the amount of pyruvic acid in the medium can be used to characterize the degree of abnormal increase in glycolysis in the cardiomyocytes. After 48h of co-culture, the culture medium of each group of cells is taken to be detected according to the operation steps of the pyruvic acid detection kit, the absorption value is detected at 505nm by an enzyme-labeled instrument, the data processing is carried out by using GraphPad Prism 5 software, and the significance difference (Unpaired 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, whereas the pyruvate expression level of the BOG-treated group was significantly decreased compared to the model group, and exhibited concentration dependence. The most reduced levels of pyruvate in the BOG (100 μg/mL) treated group, i.e. the most pronounced protective effect on cells. Error marks are mean Standard Error (SEM), #: significant differences relative to control group, ×: the significance differences were relative to the model set.

2. In vivo experiments

2.1 model construction method

SPF-class Male Wistar rats 40 (200-250 g) purchased from Jinanmingyue laboratory animal breeding Co., ltd. After one week of acclimation, the animals were randomly divided into four groups of ten animals. The blank group was irrigated with physiological saline for 14 days and was intraperitoneally injected with physiological saline on days 13 and 14; the model group was perfused with physiological saline for 14 days and injected intraperitoneally with ISO physiological saline (85 mg/kg. Day) on days 13 and 14; ME group (60 mg/kg. Day) and BOG group (75 mg/kg. Day and 150 mg/kg. Day) were continuously lavaged for 14 days and ISO saline solution (85 mg/kg. Day) was intraperitoneally injected on days 13 and 14. After 12 hours of feeding interruption, blood is taken from the orbit on the 15 th day, upper serum is taken after centrifugation at 12000g and 4 ℃ and stored at-80 ℃ for blood factor detection. The rat was sacrificed by cervical removal, the heart was dissected and removed, residual blood in the heart was removed on ice in 4 ℃ pre-chilled PBS solution, transection was performed in the middle of the heart, and the area near the apex of the heart was fixed with 4% paraformaldehyde solution for slice detection.

2.2 results

2.2.1HE dyeing

Fig. 6 is a representative image of HE staining of heart sections to assess the morphology of tissue. The obvious damage of the myocardial cells in the model group can be seen, and partial positive cell infiltration is accompanied, and the BOG administration group can lighten the damage degree of the myocardial cells, has regular and compact cell arrangement and plays a role in protecting myocardial tissues.

2.2.2BNP, NO, lactate and pyruvate detection

FIGS. 7-10 show the results of BNP, NO, lactate, and pyruvate detection in rat serum. 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 dependence. The most reduced level of BOG (150 mg/kg day) treatment group, i.e. the most significant protective effect on cells. Error identifies mean Standard Error (SEM), #: significant difference relative to control group, x: the significance differences were relative to the model set.

2.2.3 immunohistochemistry

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

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims

1. The application of the common head cabbage total thioglycoside in preparing the heart failure resistant medicament is characterized in that: the extraction steps of the total glucoside of the common head cabbage are as follows:

(1) Adding the wall-broken crushed and dried common head cabbage leaf powder into an ethanol solution, carrying out hot reflux extraction and suction filtration on the ethanol, and concentrating the filtrate under reduced pressure to obtain a common head cabbage total extract; wherein the volume fraction of the ethanol solution is 20-80%; the mass volume ratio of the common head cabbage leaf powder to the ethanol solution is 1 (g) (5-20) mL; the hot reflux extraction time of the ethanol is 0.5-3 h, and the hot reflux extraction temperature of the ethanol is 85 ℃;

(2) Dissolving the total extract of the common head cabbage obtained in the step (1) by using a lead acetate aqueous solution, standing at room temperature, performing suction filtration, concentrating filtrate under reduced pressure, and freeze-drying to obtain crude total glucoside of the common head cabbage; wherein the concentration of the lead acetate aqueous solution is 0.02-0.05 mmol/L, and the mass volume ratio of the total extract of the common head cabbage to the lead acetate aqueous solution is 1g:20 mL;

(3) Extracting crude total glucoside of the common head cabbage obtained in the step (2) by absolute ethyl alcohol, and separating and purifying by column chromatography to obtain purified total glucoside of the common head cabbage; the mass volume ratio of the crude total glucoside of the common head cabbage to the absolute ethyl alcohol is 1 (g) (5-13) mL; the material selected during the separation and purification of the column is silica gel, wherein ethyl acetate is used during the separation and purification of the silica gel: and (3) gradient eluting with methanol=5:1-3.5:1, and collecting eluent of the ethyl acetate-methanol=3.5:1 part.

2. The use according to claim 1, characterized in that: the standing time at room temperature in the step (2) is 12h.

3. Use according to claim 1 or 2, characterized in that: the common head cabbage total thioglycoside is used for improving heart failure by regulating abnormal activation of glycolysis of myocardial cells.