| Objective:To establish an ultra high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry based metabolomics approach for qualitative analysis and chemical markers screening of flavonoids in Lysimachiae Herba（LH）and Desmodii Styracifolii Herba（DSH）,and to preliminarily explore the mechanism of Lysimachiae Herba in the intervention of renal calcium oxalate stone formation in rats.Methods:12 batches of LH and DSH were ultrasonically extracted and centrifuged,and the supernatant was gradient eluted with an Agilent SB-C18（100 mm×2.1 mm,2.7μm）column and 0.1%aqueous acetic acid-acetonitrile mobile phase.The mass spectrometry used an electrospray ion source for detection in negative ion mode.The obtained data were compared with 28kinds of reference materials and self-built library secondary fragments by Peakview,and qualitative analysis was carried out based on the cleavage rules of various types of flavonoids.After the raw data was imported into the MarkerView software for etention time correction,peak normalization and elimination of isotope peaks,it was imported into SIMCA-P for multivariate statistical analysis,and the compounds with VIP value greater than 5 were selected as marker compounds of two medicinal materials.The rats were fed1%ethylene alcohol and 2%ammonium chloride to cause renal calculus model.All the rats were randomly divided into 4 groups（n=8 each）:the blank control group,the model group,the low-dose group and the high-dose group.After 28 days of feeding,urine,serum and kidney tissues of rats in each group were collected and the contents of oxalic acid and citric acid in urine during24 hours were measured.Various chemical elements in urine and serum were determined by ICP-MS method and PCA analysis was performed on the results to observe the distribution trend of each group.Calcium oxalate crystals in urine smears and kidney tissue sections,as well as tubular dilation in kidney tissue sections,were observed with a microscope.Results:A total of 113 flavonoids（80 kinds of LH,61 in DSH）were identified.In addition,by screening VIP values of the raw data,21 potential marker compounds were finally obtained,among which 15 kinds of flavonoids（6 in LH,9 in DSH）were identified as marker compounds of the two herbs,and it was found that the 9 flavonoid-C-glycosides selected were all found in DSH,and the 6 makers of LH were all flavonols or its O-glycosides.The intervention of Lysimachiae Herba in renal calcium oxalate stone showed that compared with the model group（P<0.05）,the 24-hours urine volume and urine citric acid concentration were significantly increased,and the urine oxalic acid concentration were significantly decreased in rats with the high-dose medication.Compared with the control group（P<0.05）,the urine Ca,Ni,As,Se,Pd and Hg in the high-dose group were significantly increased,and the serum Mg,Cr,Fe,As,Cd were significantly decreased.Compared with the model group（P<0.05）,the levels of urine Al,Mn,Fe,Ni,Se,Mo,Pd and Hg in the high-dose group were significantly decreased,and the levels of serum Fe,Cu were significantly increased.The results of microscopic observation showed that LH could reduce the crystallization of calcium oxalate in urine and kidney tissue of rats with kidney stone,and relieve the dilation of renal tubules.Conclusion:In this study,the UHPLC-Q-TOF-MS/MS based metabolomics approach was firstly established for qualitative analysis and chemical markers screening of flavonoids in LH and DSH,which provided a chemical basis for the quality evaluation and the difference of pharmacological action mechanism.LH has a significant inhibitory effect on the formation of calcium oxalate stone.The possible mechanism was to reduce the urinary calcium oxalate saturation by excretion of diuretic and supplementation of citrate,and to make various elements of calcium oxalate model rats tend to normal level by affecting the homeostasis of calcium,magnesium and vanadium in vivo.In addition,polyphenols in LH could reduce oxidative stress and protect renal epithelial cells,thus inhibiting the formation of calcium oxalate stones. |
PDF Full Text Request