| Urinary stone,a common and frequently-occurring disease in the world and China has become one of the three major areas of urolithiasis in the world.Kidney stone is the disease with the highest incidence of urinary stones in our country,of which more than 80%kidneys stones are calcium oxalate stones.At present,because of its obvious advantages such as non-invasiveness,remarkable curative effect and small recurrence rate,traditional Chinese medicine for kidney stones has been accepted by more and more patients in clinical practice.The usage and dosage of traditional Chinese medicine is concluded based on ancient works and the long-term clinical practice experience,which needs a systematic and scientific analysis.Therefore,it is necessary to study Chinese medicine that can prevent kidney stones.Herba Desmodii Styracifolii?D.styracifolium?,originates from the dried Desmodium styracifolium?Osb.?Merr.?Leguminosae?plants,one of the popular Traditional Chinese medicines?TCMs?,has been utilized to treat anti-inflammatory,antipyretic and diuretic effects and is well known because of the treatment of urinary calculi.There are flavonoids,saponins,phenols and alkaloids in D.styracifolium,and flavonoids are considered as the major bioactive compounds.It is reported that D.styracifolium has antioxidative effects,obvious diuresis and renal protective effects on rats and can inhibit the formation of kidney stones.However,its inhibition mechanism is not yet clear,so further studies are still needed.On this study,UHPLC-Q-TOF-MS/MS technology was used to qualitatively analyze the flavonoids in D.styracifolium,which laid a foundation for further study of the pharmacodynamic substances.To provide a basis for evaluating efficacy and mechanism of the anti-kidney stones of D.styracifolium,a HPLC method for simultaneous determination of oxalate and citrate level in urine and an ICP-MS method for simultaneous determination of elements in urine and serum of rats were developed and validated,respectively.Then,D.styracifolium extract was administered to rats by oral gavage to observe its effect on related indicators in urine and serum of rat kidney calcium oxalate stone model and the formation of renal calcium oxalate crystals.The effect and mechanism of D.styracifolium on kidney stones was preliminarily explored.Part one Simultaneous qualitative analysis of 51 flavonoids in Herba Desmodii Styracifolii by UHPLC-Q-TOF-MS/MSObjective:To develop an ultra-high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry?UHPLC-Q-TOF-MS/MS?method for the simultaneous determination of flavonoids in D.styracifolium.Methods:The study was performed on an Agilent SB-C18 reversed phase UHPLC column?100×2.1 mm,2.7?m?using gradient elution with mobile phase of acetonitrile?A?and 0.1%acetic acid?B?at the flow rate of 0.4mL/min.The injection volume was 2?L,electrospray ion source,negative ion mode.Firstly,the standard solution of 23 known flavonoids of D.styracifolium were pumped into the mass spectrometer and studied by full scan,product ion scan and precursor ion scan.The fragmentation rules of flavonoids were summarized by analysising fragments of the known compounds.The extracts of D.styracifolium were analyzed by mass spectrometry after liquid separation and the results were analyzed with“IDA Explore”and“MasterView”of PeakView 1.2 softwares.According to the fragmentation rules of known compounds and the fragment information of unknown compounds,the structure of flavonoids in D.styracifolium was identified.Results:Finally,51 compounds were simultaneously identified or tentatively characterized,including 4 flavonoids,26 flavone-C-glycosides,5 flavonoid-O-glycoside,3 flavonol,3 flavonol-O-glycoside,2 flavonone,1flavonone-O-glycoside,1 isoflavone,2 isoflavone-O-glycosid and 4isoflavanone.23 of them were identified by comparing with the standards,and the other 28 compounds were identified by analyzing the information of the fragments,the error tolerance is±5 ppm.Conclusion:An effective and reliable UHPLC-QTOF-MS method was first established for the simultaneous identification of flavonoids in D.styracifolium,which laid a foundation for further study of the pharmacodynamic substances.Part two Simultaneous determination of oxalate and citrate level in rat urine by HPLCObjective:To establish an HPLC method for simultaneous determination the content of oxalic acid and citric acid in rat urine.Methods:The separation was performed on a Cosmosil PBr column?250×4.6 mm,5?m?,eluted with 0.02 mol/L of potassium dihydrogen phosphate?0.01 mol/L IPC-TBA-HA,2.69×105 mol/L EDTA?at 1 mL/min,and the detection wavelength was set at 210 nm.The urine samples of rats were prepared by sulfonyl salicylic acid to precipitate protein.Results:The peak area and concentration of oxalate and citrate in urine have a good linear relationship within a certain range,r>0.99.The range of intra-and inter-batch RSD%were 3.7%7.8%and 3.2%5.1%,RE%was within±15%.The average extraction recoveries were 85.9%91.6%and RSD was less than 8.3%.Conclusion:The HPLC method was sensitive,specific and simple for quantifying oxalate and citrate in rat urine,which lays a foundation for the study of the mechanism of the anti-kidney stone of D.styracifolium.Part three Simultaneous determination of elements in rats by ICP-MSObjective:To establish an inductively coupled plasma mass spectrometry?ICP-MS?method for the simultaneous determination of various elements in urine and serum of rats.Methods:The means of sample pretreatment was direct dilution method.Urine and serum samples were diluted 10-fold and 20-fold respectively with2%nitric acid,mixed before use.The working parameters of ICP-MS were as follows:RF power:1500 W,reflected power:2 W,carrier gas flow rate:1.1L/min,plasma flow rate:15 L/min,pump speed:0.3 r/min,spray chamber temperature:2°C,calibration method:internal standard method.Results:The results showed that the linearity of 18 chemical elements were good in the range of the measured concentration,and the correlation coefficients were all more than 0.99.The limits of detection,limits of quantitation,instrument precision,repeatability,and stability all meet the requirements.The recovery ranges of the urine samples and serum samples were 99.1%104.1%and 93.1%114.3%,respectively and RSD was less than15%.Conclusion:The method is sensitive,reliable and can rapidly detect a variety of elements in urine and serum.By comparing the changes of the contents of various elements in rats under different conditions,it can lay a foundation for the efficacy evaluation and mechanism study of D.styracifolium anti kidney stones.Part four A study on the effect of Herba Desmodii Styracifolii on inhibiting the formation of kidney calcium oxalate stone in ratsObjective:To study the effect of D.styracifolium on the formation of kidney oxalate calcium stone in rats and to explore its possible mechanism.Methods:The rats were fed 1%ethylene alcohol and 2%ammonium chloride to cause renal calculus model.All the rats were randomly divided into4 groups?n=8 each?:the blank control group?free drinking water?,the stone formation group?1%ethylene alcohol and 2%ammonium chloride?,the low dose group and high dose group?1%ethylene alcohol and 2%ammonium chloride+different doses of D.styracifolium extract?.After 28 days of feeding,the urine and serum of rats were collected,the concentration of urinary oxalate and citrate during 24 h and various elements in urine and serum were detected.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:Compared with the stone formation group,in the low dose and high dose groups,the concentration of oxalate reduced significantly?P<0.05?but the concentration of citrate increased significantly?P<0.05?in 24 h urine;the levels of Mg,Fe and Mo increased but the levels of Ca,Cu reduced in urine.Compared with the stone formation group,the concentration of As increased?P<0.05?but Pb reduced?P<0.05?in serum in the high dose group.There was no significant difference in other elements in serum of the four groups of rats?P>0.05?.The results of microscopic observation showed that D.styracifolium 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:D.styracifolium can inhibit the formation of calcium oxalate stone in rats via reducing the concentration of urinary oxalate,adjusting the leves of Ca,Mg and other trace elements.In addition,the diuretic effect of D.styracifolium may promote the expulsion of calcium oxalate stone. |
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