| Objective:Ganoderma is a traditional Chinese medicinal material in China,and its fruiting bodies and spores can be used as medicine.Ganoderma lucidum spore powder is not easily absorbed by the human body,and is often used as a wall-breaking spore powder.The products currently on the market have problems such as low industrial production efficiency,high heavy metal residues,and incomplete quality standards.This article screens the best tools for industrial production of Ganoderma lucidum spore powder,enzymes,enzymolysis conditions and process parameters to improve production efficiency and reduce heavy metal residues.Carry out quality control research on traits,identification,inspection,content determination,etc.for Changbai Mountain Provide the basis for the improvement of the quality standard of ganoderma lucidum spore powder;explore the pharmacological effect and preliminary mechanism of Changbai Mountain ganoderma lucidum spore powder on the protection of myocardial injury,and provide data for clinical application of this product.Methods:1.Study on the wall breaking process:(1)According to the structural characteristics of the spore wall,lysozyme,chitinase,cellulase,and β-glucanase were used individually or in combination to break the wall to break the wall.As an indicator,the optimal tool enzyme type was selected through orthogonal design experiments;(2)the wall breakage rate was increased after enzymolysis combined with sonication or high-shear treatment,and single-factor screening was used to determine the process parameters of physical wall breakage after enzymolysis.2.Quality control research:(1)observe the characteristics with an optical microscope and check the wall breaking rate;(2)use the color reaction of potassium iodide solution to identify whether starch-like impurities are doped in the spore powder of broken Ganoderma lucidum;Spore structure to determine the microstructure and identification characteristics;(4)Determination of total polysaccharides by phenolsulfuric acid method to determine the content of main effective components;determination of ganoderma acid A and G content by high performance liquid chromatography to determine representative indicators Ingredient content.3.Study on pharmacological effects:(1)The experimental mice were divided into a blank control group,a model group,and a low-medium high-dose administration group.The wall-breaking sporozoite powder was used as a test drug.One week after the mice were tested with different doses of the drug,isoproterenol hydrochloride was injected subcutaneously on the back of the neck to establish a model of myocardial injury,which was continued for 3 weeks after modeling;(2)The appearance and weight changes were continuously observed after modeling;(3)Determination of superoxide dismutase(SOD),catalase(CAT),trace reduced glutathione(GSH),lactate dehydrogenase(LDH),creatine kinase(CK),aspartate aminotransferase(AST),alanine aminotransferase(ALT),tumor necrosis factor(TNF-α),interleukin-6(IL-6),interleukin-1β(IL-1β)content changes;(4)comparative analysis of test drugs Protective effect on myocardial injury.4.Study on the mechanism of action:extract the cardiac muscle tissue protein of mice,analyze the expression of BCL-2 and BAX protein by Western blot,and explore the mechanism of action of the test drug.Results:1.The best tool enzyme is β-glucanase;the best conditions for hydrolysis are 50℃,pH 5,enzyme concentration 3%,and hydrolysis time 6h;process parameters of ultrasonic treatment after enzymolysis The ultrasonic frequency is 40KHZ,the power is 700W,and the ultrasonic time is 1h.The processing parameters of the high-shear method are a shear speed of 10000rad/min and a shear time of 15min.2.Observed by light microscope,this product has an elliptical structure,and the top is in the shape of a apex.There are broken notches on any side of the cell wall.Check that the wall breakage rate of this product is 95%.Cell wall structure,there is a certain distance between the double walls,and there is a supporting structure between the double walls.After observation,the double walls were damaged.No microscopic characteristics of mycelia and ganoderma mycelium were observed;no starch impurities were identified by physical and chemical identification;UV The average content of total polysaccharides measured by spectrophotometry is 7.727%;the content of ganoderic acid A is 3.67 mg/g and the content of ganoderic acid G is 0.42 mg/g.Degrees,sample stability,and reproducible RSD values were all<2%.The average sample recovery rates of Ganoderma A and G were 98.8%and 100.5%,respectively.3.The protective effect of pre-administration on myocardial injury showed that compared with the model control group,each dose group can significantly increase the content of SOD,CAT,GSH in the serum of mice,and reduce the content of LDH in the serum.Statistical significance(P<0.05),and showed a dose-dependent trend.The content of SOD increased by 12.4%,18.0%,and 21.9%,respectively;the content of CAT increased by 10.7%,25.6%,and 43.5%;the content of GSH increased.33.3%,39.6%,57.3%;LDH content decreased by 13.1%,25.1%,47.3%.Compared with the model group,the levels of CK,AST,and ALT in serum were also significantly reduced,and the levels of TNFα,IL-6,and IL-1β were also reduced to varying degrees.4.Western blot analysis showed that BCL-2 protein expre’ssion was up-regulated and BAX protein expression was decreased in the myocardial tissue of the administration group.Conclusion:Using β-glucanase as a tool enzyme for ganoderma spore powder to break the wall,combined with sonication and high shear treatment,the wall breaking rate can reach 95%,the process is reasonable and feasible;the established quality standards have good repeatability and strong specificity,Can be used for quality control of Changbai Mountain’s broken ganoderma spore powder;the Changbai Mountain’s broken ganoderma spore powder prepared according to the above method has a protective effect on myocardial damage,and its protective effect on myocardial damage may be achieved by intervening the expression of BAX and BCL-2. |
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