Research On The Microanalysis Method Of The Quality Of Traditional Chinese Medicine

With the development of science and technology,the scale of analytical chemistry extends to the microscopic scale.Microanalysis focuses on the microcosmic nature of heterogeneous samples in micro-scale.It extracts spectral information from materials,simultaneously,visualizes the spatial distribution of chemical components.Thus,microanalysis techniques are necessary tools to study the microcosmic properties of materials.The diversity of material triggers the different microanalysis techniques.Microanalysis extends the human visual senses,which highlights the heterogeneity of sample including composition,structure,spatial distribution in the microscopic scale,etc.It is an indispensable means to understand the micro-world.The thesis studied the two apsects of microanalysis.One hand was about the elemental microanalysis based on the "element profiling" by Laser-induced breakdown spectroscopy(LIBS).The other hand focused on the microanalysis of liposomes morphology observations.Moreover,the hepatoprotective activity was performed on the liposomes-based nano-delivery systems.The inorganic elements are the main and basic ingredients of all Chinese medicine,which were related to specific properties of the five-flavor of Traditional Chinese medicine(TCM).Meanwhile,inorganic elements are the core composition of effective drug components.Modern pharmacological and pharmaceutical studies had proved that the mineralogical elements of TCM were not only associated with the pharmacological effects,but also related to the intrinsic quality of TCM.Since the genuine medicinal quality was influenced by the geological background.The elemental fingerprints of herbal medicine were established based on elemental profiling theory using LIBS in the microscopic scale.The following works were done in this thesis.1.Elemental compositions microanalysisLIBS was applied to perform rapid element analysis of four kinds of precious Tibetan Medicine(such as "Renqing Mangjue","Renqing Changjue","Twenty-five taste coral pills"and "Twenty-five flavors pearl pill")and GTso Thal.The specific spectra of four kinds of Tibetan Medicine were established.Based on the National Institute of Standard and Technology(NIST)database,LIBS provides reliable elemental analysis of Tibetan Medicine.Mainly multi-elements including Ca,Na,K,Mg,C-N molecular band,etc.were detected in four kinds of Tibetan Medicine.Fe was detected in "Renqing Changjue" and "Twenty-five flavors pearl pill".Moreover,heavy mental elements such as Hg and Cu were shown in the LIBS spectra of "Renqing Mangjue" and "Renqing Changjue".Ag was found in the LIBS spectra of "Renqing Changjue".The similar elements were summarized in "Renqing Mangjue","Renqing Changjue" and GTso Thal.The results demonstrated that LIBS is a viable technique for analysis of Tibetan Medicine.LIBS has unique advantages,such as no or little sample preparation,real-time,in situ and rapid multi-element analysis.It has prospects in the study of Tibetan Medicine.(i)Rapid element microanalysis and provenance study of Blumea balsamifera DC were performed by LIBS.Principal component analysis(PCA)and partial least squares discriminant analysis(PLS-DA)were implemented to exploit the multivariate nature of the LIBS data.The PLS-DA model using complete spectra as input variables had similar discrimination performance to using selected spectral lines as input variables.The down-selection of spectral lines was specifically focused on the major elements of B.balsamifera samples.Results indicated that PLS-DA algorithm showed good classification performance by the miacronalysis of LIBS.(?)To explore the linear and non-linear nature of LIBS data marix,Least squares support vector machines(LS-SVM)were employed to exploit the multivariate characters of the LIBS data and multi-class discriminate among mint samples from five different provinces.The experiments revealed that in addition specific mineralogical elements,active organic ingredients were present in mint samples as well.Specifically,the performance of linear and non-linear LS-SVM algorithms was explored in sensitivity and robustness tests.In the sensitivity test,the average total correct classification rates of Lin_kernel LS-SVM and RBF_kernel LS-SVM were 96.10%and 94.38%,respectively.In the robustness test,the total unclassification rates were 12.73%in the RBF_kernel LS-SVM and 9.57%in the Lin_kernel LS-SVM.And the total correct classification rates were 87.71%and 86.58%,respectively.Generally,Lin_kernel LS-SVM equally outperformed RBF_kernel LS-SVM.Both LS-SVM algorithms exhibited excellent performance in sensitive test and good performance(a little inferior)in robustness test duo to the intrinsic difference of heterogeneous samples.The results indicated the potential application in herbal medicine,especially for in situ monitoring application.2.The elemental quantitative microanalysisThe special attention was paid to the negative effects of LIBS microanalysis,such as the matrix effect and self-absortion effect.The matrix-assisted corrected method was presented and analyzed to reduce the influence of matrix effect.The calibration curve analysis was introduced for the quantitative microanalysis.In detail,starch and Copper oxide(CuO)were used as matrix-assisted reagents at ambient.Furthermore,Lorentz and Gauss fitting curve of analytical spectrum were performed to correct the line intensity.The result showed that CuO was effective to inprove the calibrating measurement based on the Gauss fitting curve.The quantitative microanalysis results of Arsenic and mercury were at a high confidence level with correlation coefficient 0.9659 and 0.9962,respectively.The limits of detection of Arsenic and mercury were 2.073 mg·g-1g and 2.224 mg·g-1,respectively.Based on certified experimental results,the relative error of Arsenic and mercury were less than 10%in sample 1#and sample 3#.Howerver,the relative error of both elements was more than 10%in sample 2#.The heterogeneous of sample 2#maybe caused the high relative error of two elements.3.Elemental time series microanalysisBlending is a significant pharmaceutical unit,which also is a time serial process.LIBS was used to assess the cinnabar and realgar blending of An-Gong-Niu-Huang Wan(AGNH)in a pilot-scale experiment,including the blending end-point.The blending variability of two mineral medicines,cinnabar and realgar,was measured by signal relative intensity changing rate(RICR)and moving window standard deviation(MWSD)based on LIBS.Meanwhile,relative concentration changing rate(RCCR)was obtained based on the reference method involving inductively coupled plasma optical emission spectrometry(ICP-OES).The LIBS result was consistent with ICP-OES at blending end-point determinations of both mineral medicines.Unlike the ICP-OES method,LIBS does not need an elaborate digestion procedure,which is a promising and rapid technique to understand the blending process of Chinese Materia Medica(CMM)containing cinnabar and realgar.Meanwhile,the pearl powder,a main component of AGNH,showed similar blending behavior to the cinnabar and realgar.The proposal blending end point was 38 min.4.Microanalysis of elemental spatial distributionThe unique multi-elemental characteristics of Juncus effusus L.were deciphered in micro-scale by LIBS.The microanalysis and multimode sampling methodologies were designed in this study.The relative standard deviation(RSD)approach was performed to optimize the multi-shot measurements.Taking advantage of the capability with no or minimal sample pre-treatment of LIBS,thermodynamic chart(or hot map)of four elements(Mg,Ca,Ba,and Na)was created from twelve collection regions.The diagram of elemental distribution in micro-scale was generated to explore the nature of Juncus effusus L.by LIBS.The results demonstrated that LIBS is a promising technique for rapid elemental microanalysis of heterogeneous samples.5.Y08 loaded liposomes microanalysisThis study focused on the microanalysis of Y08 loaded liposome(Y08-liposome)based on the "liver-based therapy theory".The transmission electron microscopy(TEM),scanning electron microscope(SEM)and laser light scattering(LLS)were used to study the morphology observations of liposomes in microscopic scale.Meanwhile,Fluorescence live imaging was implemented to visualize the spatio-temporal biodistribution of Y08-liposome.Furthermore,the hepatoprotective potency of Y08-liposome in vitro was investigated.Furthermore,the hepatoprotective activity and inhibition accumulation of cytoplasmic lipid droplet were studied.The TEM image showed that most liposomes were spherical particles with similar size and uniform dispersion.Indeed,the drug displayed sustained release trend in liposomal formulation indicating that Y08 was completely entrapped in the liposome.The in vivo and in vitro fluorescence imaging study exhibited hepatic targeting of liposomes through enhanced permeation and retention(EPR).Except control group,L02 cells were pretreated by different concentrations of Y08-liposome and Y08 compound for 48h and exposed to 200?M H2O2 for another 6 h to induce oxidative damage.The cells vitality of Y08-liposome at 1.1 ?M and 3.3?M was 92.87%and 96.20%,respectively,which was 6.31%and 9.64%higher than the control group.Meanwhile,the robust phenotype-based HCS imaging microassay was developed to study the suppression lipid droplet accumulation of drug loaded liposome in 3T3-L1.The IC50 of inhibiting lipid droplet accumulation effect of Y08-Liposome and Y08 compound was 8.68 and 31.08 ?M,respectively.The inhibiton rate of 10 ?M Y08-liposome was ahove 90%,which was even superior to the effect of 30 ?M Y08.Moreover,the hepatoprotective effect exhibited a dose dependent in vitro.Neither Y08-liposome nor Y08 compound had not apparent cytotoxicity in the whole range of examined concentrations.The nano-delivery systems exhibited definite hepatoprotective activity agaist the H2O2 induced damage and the suppressed accumulation of cytoplasmic lipid droplet than the original compound.In summary,microanalysis of TCM was foused on elemental compositions,pattern recognition,elemental spatial distribution,time series microanalysis and quantitative microanalysis.Morever,the morphology microanalysis was performend in active component of Chinese herb loaded liposome.Microanalysis is a challenging field of analytical chemistry.