Research On Quality Control Methods Of Zhuang Medicine Cardiospermum Halicacabum L.

Objective:Research on the quality control methods of the Zhuang Medicine Cardiospermum halicacabum L.to provide a basis for further research on the quality standards.Methods:1.According to the method prescribed in the Chinese Pharmacopoeia of2020 edition,the relevant inspection items of C.halicacabum L of different origins are determined.2.The TLC was used to identify the chemical constituents of C.halicacabum L and different habitats,different temperature and humidity was used to investigate the adaptability of the method.3.The different habitats fingerprints of C.halicacabum L was established by HPLC,and corresponding mathematical models was established to classify and analyze for the fingerprints.4.Combining the Box-Behnken response surface methodology,The HPLC was utilized to determine the content of 6 known components in the C.halicacabum L,and conduct methodological investigations,on this basis,analyze the components of the different medicinal materials of C.halicacabum L,the distribution law was studied,and QAMS was established for the six components of C.halicacabum L.Results:1.The moisture content,total ash content,acid-insoluble ash,extract of C.halicacabum L were measured.The results showed that the moisture content of 10 batches was in the range of 2.62% ～ 5.24%,the total ash content was in the range of 3.88% ～ 7.62%,acid-insoluble ash was in the range of0.32% ～ 1.46%,the extract content was in the range of 20.62% ～ 28.78%.2.The TLC method of C.halicacabum L was established,The chloroform: ethyl acetate: formic acid = 6:4:2 as the development system,and spotting 2 μL of the reference solution and the test solution on the silica gel G plate,upwards unfold,evaporate dry after unfolding,then spray with 5%sulfuric acid ethanol solution,heat at 105 °C for 2 min,and inspect under UV lamp 366 nm.The test product can show fluorescent spots with green and blue colors consistent with the same quantity and shape of the reference product.The four components of luteolin-7-O-glucuronide,apigenin-7-Oglucuronide,quercitrin and chrysoeriol were identified by the comparison method of the reference substance.The method adaptability test shows that it should be carried out under normal temperature and humidity.3.Measure the medicinal materials from different origins by HPLC,10 origins fingerprints was established by the fingerprint software,11 common peaks was calibrated by the similarity software,and the similarity is greater than 0.9;combined with HPLC-DAD refers to 6 known components,include of isoquercitrin,luteolin-7-O-glucuronide,quercitrin,apigenin-7-Oglucuronide,apigenin,chrysoeriol.The cluster analysis of the C.halicacabum L from 10 producing areas is performed by SPSS software,the 15-square euclidean distance is used as the basis to subdivide into three categories: the first category is S2,S4,the second category is S8,S9,and the third category is S1,S3,S5,S6,S7,S10;Principal component analysis has three principal components,which are consistent with the results of cluster analysis.The results of principal component rotation matrix show that quercitrin and apigenin contribute significantly to principal component analysis.4.Based on the results of the experimental investigation,combined with the Box-Behnken response surface methodology,the material-to-liquid ratio（X1）,ethanol concentration（X2）,and extraction time（X3）are used as the inspection indicators,and the mass fraction of flavonoids（Y,The sum of the mass fractions of 6 flavonoids）is the response value for the Central Composite design,and finally the optimal extraction conditions for the test product are determined as: 1 g of the medicinal material is accurately weighed,placed in a stoppered conical flask,and 65% is precisely added 30 m L of ethanol,weigh it,heat it to reflux for 60 minutes,let it cool,and then weigh it.Use 65% ethanol to make up the lost weight,shake well,filter,and take the additional filtrate through a 0.22 microporous membrane to obtain the test solution.Determine the content of 6 components of 10 origins.The methodological investigation results show that the precision of the instrument is good.The test solution is basically stable within 24 hours.The linearity investigation shows that the 6 components have a good linear relationship within a certain range.The sample recovery test shows that the method is accurate and feasible.The content distribution of the six components in the stems,leaves and fruits of the medicinal materials of C.halicacabum L was researched,and the distribution of the six components was significantly different.QAMS for the 6 components of C.halicacabum L was established.The apigenin is used as the internal reference because of cheap,easy-to-obtain and stable,and the relative correction factors of the 5 components are obtained by the multi-point calibration method,which are f _{isoquercitrin/apigenin}=0.9442,f_{luteolin-7-O-glucuronide/apigenin}=0.9505,f_{quercetrin/apigenin}=0.9497,f_{apigenin-7-O-glucuronide/ apigenin}=0.5934,fchrysoeriol/ apigenin=1.1678.Different concentrations of reference materials,different manufacturers of chromatographic columns and different manufacturers of HPLC was used to investigate the obtained relative correction factors,the results show that its reproducibility and accuracy all conform the relevant requirements.Conclusions:1.The test item experiment shows that most items are not much different from the existing Guangxi Zhuang medicine standard,and the selected method in the extract item is better than the existing standard,suggesting that the current standard has possibility for optimization.2.The TLC method is simple and feasible,and the results are accurate and credible.The comparison of 4 known components increases the accuracy of the method and can be used for qualitative identification.3.The fingerprint spectrum established based on the HPLC method has good reproducibility,accurate results and high sensitivity,and can be used for the qualitative identification of the medicinal materials of C.halicacabum L.4.The content determination method established based on the HPLC method has good reproducibility and high accuracy.The Box-Behnken response surface methodology is adopted to improve the extraction efficiency of the test product;the content determination experiment of different medicinal materials provides more for research reference;the establishment of the QAMS simplifies the measurement method and reduces the cost.The experiment can systematically and normatively study the quality control methods of the C.halicacabum L through a number of indicators,and provide a certain scientific basis for further research on the quality standards in the future,and beneficial for development and utilization of the C.halicacabum L resources.