Quality Control Method Of Freeze-dried Garlic Powder And Its Improvement Effect On Acute Myocardial Ischemia Of Rats

Garlic is the bulb of Allium sativum L.,as an edible medicinal plant,it has a long history.It has various pharmacological activities such as antibacterial,antitumour,anti-atherosclerosis,prevention of cardiovascular and cerebrovascular diseases,hepatoprotective,etc.It contains active ingredients such as alliin,garlic polysaccharides,garlic saponins,as well as nutritional components such as amino acids,inorganic elements and protein.Freeze-dried garlic powder is a slightly garlicky white solid product,which is freezed,crushed and freeze-dried at low temperature.The freeze-dried garlic powder used in this study is provided by Xinjiang Tefeng Pharmaceutical Co.,Ltd.This manufacturer uses freeze-dried garlic powder as an intermediate to produce garlic enteric-coated tablets(National Medicine Permission Number H65020356),which is used in clinic as anti-infective drugs for the treatment of acute and chronic bacillary dysentery,enteritis,whooping cough,fungal infections of the lung and digestive tract,etc.The main quality control standard of the freeze-dried garlic powder in manufacturer is to determine of content of alliin,but because of the complex biotransformation problems,the method needs to be improved.In addition,garlic enteric-coated tablets are only used as anti-infective drugs in clinic,their activities have not been fully exploited.In order to improve the quality control standards and methods and to explore other pharmacological effects of freeze-dried garlic powder,this study looked into the determination of main active components and fingerprint,the contents of amino acids,inorganic elements,heavy metal elements and the pharmacological effects and mechanisms on improving acute myocardial ischemia of rats of the freeze-dried garlic powder.A new method to determine alliin was established by high performance liquid chromatography(HPLC)to solve the biotransformation problems in the existing method.Nine enzyme inactivation conditions and 9 doses of allinase inhibitor(carboxymethoxylamine hemihydrochloride)were screened to determine the best way and parameter to inactivate alliinase.A quantitative calibration model for the determination of alliin by near infrared spectroscopy(NIRS)was also established,which can be used for the rapid determination of alliin content at production site.The pretreatment methods of garlic polysaccharides and garlic saponins were optimized,the contents of garlic polysaccharides and garlic saponins were determined by ultraviolet-visible spectrophotometry(UV-vis),which supplemented the quality control content.The fingerprint of freeze-dried garlic powder was established,so that the overall quality control of freeze-dried garlic powder could be guaranteed.The contents of free and hydrolyzed forms of amino acids such as aspartic acid and threonine and 10 elements such as Ca,Mg and P in freeze-dried garlic powder were determined,which could provide a basis for the interpretation of its nutritional compositions and the risk management of heavy metal contamination.The myocardial ischemia model was established by ligating the left anterior descending coronary artery of SD rats.By examining the incidence of arrhythmia,myocardial infarction rate,myocardial enzyme indexes,oxidative stress factors and inflammatory factors,the pharmacological effect of freeze-dried garlic powder on improving acute myocardial ischemia of rats was clarified,and it could improve acute myocardial ischemia by regulating PI3K/Akt/Nrf2 signaling pathway,which laid a foundation for exploring new clinical indications of freeze-dried garlic powder.The main contents and results are as follows:1 Study on the determination of main active components and fingerprint of freeze-dried garlic powder1.1 Determination of alliin in freeze-dried garlic powder by HPLCA method for the determination of alliin in freeze-dried garlic powder was established by HPLC.By examining the column,flow rate,detection wavelength and mobile phase,the chromatographic conditions were determined as follows:Hypersil BDS C18 column(4.6 mmx250 mm,5 μm),and the mobile phase was methanol-0.28%potassium dihydrogen phosphate+0.2%sodium heptanes sulfonate(pH=2.1)=10:90;the flow rate was 0.5 mL/min and the detection wavelength was 210 nm;the injection volume was 5 μL and the column temperature was 35℃.The activity of alliinase must be inactived before determining the content of alliin.Nine enzyme inactivation conditions and 9 doses of allinase inhibitor(carboxymethoxylamine hemihydrochloride)were screened to determine the pretreatment method of the sample.Paired sample Wilcoxon signed-rank test was used to compare the determination results of the method established in this study and the derivatization method of the United States Pharmacopeia.The results showed that there was no significant difference between the method established in this study and the derivatization method of the United States Pharmacopoeia(P>0.05).The content of alliin in 15 batches of freeze-dried garlic powder ranged from 3.31%to 3.88%.The method established in this study could avoid the biotransformation problems in the method established by the manufacturer.It could provide a simple,easy and reliable method for manufacturer to control the quality of the freeze-dried garlic powder effectively.1.2 Determination of alliin in freeze-dried garlic powder by NIRSA rapid method for the determination of alliin in freeze-dried garlic powder was established by NIRS.The NIRS of 50 batches of freeze-dried garlic powder were scanned and the contents of alliin were determined by HPLC.A quantitative calibration model between contents and NIRS was established using partial least squares(PLS),and the model was optimized and evaluated.The content of alliin in freeze-dried garlic powder was predicted by the model in order to test the accuracy of the model.The results showed that the r of the calibration model was 0.9987,the RMSEC was 0.0245 and the RMSECV was 0.104.The average recovery rate of the model was 99.72%.Compared to HPLC,the method was rapid without destroying samples.The analysis process was not limited by the site,and this method did not consume chemical reagents or produce any pollution.It was suitable for rapid determination of alliin in multi-batches of freeze-dried garlic powder,especially the quality control in the production process.1.3 Determination of garlic polysaccharides in freeze-dried garlic powder by UV-visThe determination method of garlic polysaccharides in freeze-dried garlic powder was established by UV-vis.By screening the extraction method,extraction time and the volume of extraction solvent,the preparation method of the solution was determined as follows:25 mg freeze-dried garlic powder was purified with ethanol,and then refluxed with 50 mL water for 30 min.The coloration and determination of the solution were carried out by phenol-sulfuric acid method.The refined garlic polysaccharides were prepared.Via calculation,the reduction coefficient between glucose and refined garlic polysaccharides was 1.35.The contents of garlic polysaccharides in 15 batches of freeze-dried garlic powder ranged from 66.62%to 73.90%.The establishment of the determination method of garlic polysaccharides could provide basis for supplementing the quality control standard of freeze-dried garlic powder.1.4 Determination of garlic saponins in freeze-dried garlic powder by UV-visThe determination method of garlic saponins in freeze-dried garlic powder was established by UV-vis.By screening the extraction method,extraction solvent,the volume of extraction solvent,extraction time and extraction times,the preparation method of the solution was determined as follows:200 mg freeze-dried garlic powder was refluxed with 40 mL methanol for 1 h,and extracted with water-saturated n-butanolc for 4 times.The coloration and determination of the solution were carried out by sulfuric acid-methanol method.The contents of garlic saponins in 15 batches of freeze-dried garlic powder ranged from 1.66%to 2.24%.The establishment of the determination method of garlic saponins could provide basis for supplementing the quality control standard of freeze-dried garlic powder.1.5 Establishment of fingerprint of freeze-dried garlic powder by HPLCFingerprint of freeze-dried garlic powder was established by HPLC.By examining the pH,mass fraction and gradient elution conditions of the mobile phase,the chromatographic conditions of the fingerprint were determined.Fingerprints of 15 batches of the freeze-dried garlic powder were established.The results showed that the chemical components of 15 batches of freeze-dried garlic powder were basically consistent,and the similarities between 15 batches of the freeze-dried garlic powder and their control fingerprint were all≥0.996.Eleven common peaks were determined.Compared with the reference substance,aspartic acid and alliin were identified.The establishment of the fingerprint was conducive for manufacturer to control the quality of the freeze-dried garlic powder comprehensively.2 Analysis of amino acids,inorganic elements and heavy metal elements in freeze-dried garlic powder2.1 Determination of amino acids in freeze-dried garlic powderThe contents of hydrolysed tryptophan in 15 batches of freeze-dried garlic powder were determined by HPLC.The contents of free and hydrolyzed forms of 17 amino acids such as aspartic acid and threonine in 15 batches of garlic freeze-dried powder were determined by amino acid auto-analyser.The contents of hydrolysed tryptophan were measured to be 1.4 mg/g,the average contents of 17 hydrolysed amino acids were measured to be 112.0 mg/g,and the total contents of 18 hydrolysed amino acids were 113.4 mg/g;the total contents of 15 free amino acids were 36.1 mg/g,methionine and phenylalanine were not detected.The status,types and contents of some of the amino acids contained in freeze-dried garlic powder were clarified,which provided a basis for the clarification of the nutritional compositions of freeze-dried garlic powder.2.2 Determination of the contents of inorganic elements and heavy metal elements in freeze-dried garlic powderThe contents of Ca,Mg and P in 15 batches of freeze-dried garlic powder were determined by inductively coupled plasma emission spectrometry(ICP-OES),and the contents of Mn,Fe,Cu,Zn,Cd,Pb and Se in 15 batches of freeze-dried garlic powder were determined by inductively coupled plasma mass spectrometry(ICP-MS).The results showed that the average contents of Ca,Mg,P,Mn,Fe,Zn and Se were 9.13×102,8.10×102,6.18×103,8.38,35.65,23.14 and 0.05 mg/kg,respectively,and the contents of heavy metal elements Cu,Cd and Pb were all in compliance with the limits in the 2020 edition of the Chinese Pharmacopoeia.The contents of some inorganic elements and heavy metals in freeze-dried garlic powder were clarified,which provided a basis for the study of the types and contents of elements and the risk management of heavy metal contamination of freeze-dried garlic powder.3 The improvement effects and mechanisms on acute myocardial ischemia of rats of the freeze-dried garlic powderThe myocardial ischemia model was established by ligating the left anterior descending coronary artery of SD rats.The experimental results showed that freeze-dried garlic powder could reduce the incidence of ventricular premature,ventricular tachycardia,ventricular fibrillation and reduce the rate of myocardial infarction.Freeze-dried garlic powder could reduce creatine kinase(CK),creatine kinase-MB(CK-MB),lactate dehydrogenase(LDH),cardiac troponin T(cTnT),malonaldehyde(MDA),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)levels,elevate total antioxidant capacity(T-AOC)and superoxide dismutase(SOD)levels in serum.Freeze-dried garlic powder could also increase the expressions of phosphorylation of phosphatidylinositol-3kinase(p-PI3K),phosphorylation of protein kinase B(p-Akt),nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase-1(HO-1)and decrease the expression of kelch-like ECH-associated protein 1(Keapl)in myocardial tissue.Freeze-dried garlic powder could improve acute myocardial ischemia by reducing the release of serum myocardial enzymes and exerting antioxidant and anti-inflammatory effects,and it could improve acute myocardial ischemia by regulating PI3K/Akt/Nrf2 signaling pathway.The results laid the foundation for expanding the clinical indications of freeze-dried garlic powder.ConclusionThe determination method of alliin in freeze-dried garlic powder was established by HPLC,which avoided the biotransformation problems in the method established by the manufacturer.The content of alliin in 15 batches of freeze-dried garlic powder ranged from 3.31%to 3.88%.The quantitative calibration model of alliin was established by NIRS,which was convenient for the rapid determination of alliin of multiple batches of samples.The determination method of garlic polysaccharides and garlic saponins in freeze-dried garlic powder was established by UV-vis,which provided basis for supplementing the quality control standard of freeze-dried garlic powder.The contents of garlic polysaccharides in 15 batches of freeze-dried garlic powder ranged from 66.62%to 73.90%,and the contents of garlic saponins in 15 batches of freeze-dried garlic powder ranged from 1.66%to 2.24%.Fingerprint of freeze-dried garlic powder was established,the similarities between 15 batches of the freeze-dried garlic powder and their control fingerprint were all≥0.996.The quality standard of freeze-dried garlic powder had been improved.The total contents of 18 hydrolysed amino acids were 113.4 mg/g,and the total contents of 15 free amino acids were 36.1 mg/g,methionine and phenylalanine were not detected.The contents of heavy metal elements Cu,Cd and Pb were all in compliance with the limits in the 2020 edition of the Chinese Pharmacopoeia.The study clarified some of the nutrients contained in freeze-dried garlic powder and provided a basis for the risk management of heavy metal contamination.Freeze-dried garlic powder could reduce the incidence of ventricular premature,ventricular tachycardia,ventricular fibrillation and the myocardial infarction rate of acute myocardial ischemia rats.Freeze-dried garlic powder could improve acute myocardial ischemia by reducing the release of serum myocardial enzymes and exerting antioxidant and anti-inflammatory effects,and it could improve acute myocardial ischemia by regulating PI3K/Akt/Nrf2 signaling pathway.It clarified that freeze-dried garlic powder could improve acute myocardial ischemia.