| In this paper,the method of detecting the 4 kinds of alkaloids in the stems and leaves of Macleaya cordata from different habitats was investigated by using the method of Macleaya cordata leaves and stems as raw materials.The contents of four alkaloids in different raw materials were detected,and the differences were analyzed.Established the Stem-and-Leaf fingerprint.Secondly,compared with the acid water percolation and acid alcohol reflux method,the efficiency of the sanguinarine in the stems and leaves was extracted.The optimal extraction conditions of the two extraction methods were determined by orthogonal test and response surface optimization experiments.Then,the stability of the two extraction methods was verified by using different raw materials,and then the stems and leavs extract were ob-tained by the alkali sedimentation test.Finally,the stability of hematoxylin was investigated.The structure and morphology of hematobase in different pH environments were explored by HPLC and HPLC-Q/TOF-MS.The possible structural forms were inferred.We get the follow-ing results:1.The HPLC method for determination of 4 kinds of alkaloids in the stems and leaves of Macleaya cordata was determined and the methodological investigation was carried out.The results show that the precision,stability,sample preparation method and reproducibility of the analytical method are all good.Secondly,the contents of four alkaloids in the stems and leaves from 10 different habitats were detected.It was found that different geographical environments had significant effects on the content of 4 kinds of alkaloid in the stems and leaves of Macleaya cordata.Then,using the characteristic fingerprint similarity evaluation system of traditional Chinese medicine,the HPLC chromatograms of the leaves and leaves of 10 different habitats were compared and analyzed in detail,and the characteristic fingerprints of the stems and leaves of Macleaya cordata were established.Finally,the similarity evaluation of the HPLC chromatograms of the stems and leaves of Macleaya cordata was compared with the control chromatogram.The results showed that the similarity of the raw materials of most of the pro-ducing areas and the control characteristic fingerprints were above 90%,which can be used for the identifying the quality of the stems and leaves of Macleaya cordata.2.Compare the acid water percolation method and the acid alcohol reflux method to extract the stems and leaves of Macleaya cordata.First,a single factor test was performed on the two extraction methods,and the range of the interval was selected.Then,the orthogonal test design and response surface optimization test design were carried out for acid water seep-age extraction and acid alcohol reflux extraction.The experimental results show that the opti-mal extraction process for acid water percolation extraction is as follows:1%sulfuric acid is used as the percolation solution,the soaking time is 30 min,the solvent dosage is 30 times,and the percolation rate is 0.5 mL·min-1.In the verification test under this condition,the trans-fer rate of sanguinarine was 83.63±2.09%?n=5,RSD=2.49%?;the optimal extraction process for acid alcohol reflux extraction was:ethanol volume fraction 90%,liquid-solid ratio 30:1mL·g-1,sulfuric acid concentration 0.12%,extraction temperature 80°C,extraction time 2 h,under this condition,the average transfer rate of sanguinarine can reach 89.36±0.49%?n=5,RSD=0.54%?.Then,the two extraction methods were verified by using different raw materials.The results showed that the extraction methods of the raw materials from different places were stable.The alkali precipitation test was carried out on the two extraction methods of raw ma-terial from Henan Luoyang with the highest content of sanguinarine.The acid water percola-tion solution was subjected to alkali sedimentation,filtration and drying,and finally the con-tent of sanguinarine in Macleaya cordata extract in the final extract was 2.25±0.11%?n=5,RSD=4.88%?,the average yield was 21.64±0.78%?n=5,RSD=3.61%?,and the average trans-fer rate of sanguinarine was 75.06±1.87%?n=5,RSD=2.49%?.The acid alcohol extract is sub-jected to a series of processes such as recovery solvent,reconstitution,filtration,alkali precip-itation,filtration,drying,etc.,and the content of sanguinarine in the extract obtained from the final is 1.65±0.06%?n=5,RSD=3.36%?,the yield was 24.27±0.92%?n=5,RSD=3.77%?,and the sanguinarine transfer rate was 61.82±1.58%?n=5,RSD=2.56%?.It can be seen from the results that if the sanguinarine in the Stems and leaves of Macleaya cordata is extracted by the acid water percolation method,the operation steps are less,the sanguinarine loss is lower,and the obtained sanguinarine content in the Macleaya cordata extract is higher,and the pro-duction cost is higher.Lower,more suitable for application requirements in industrial produc-tion.3.By investigating the stability of sanguinarine in different temperature,light and pH environment,it was found that Macleaya cordata in hydrochloric acid aqueous solution,hy-drochloric acid methanol solution,Macleaya cordata stem and leaf osmosis liquid and acidic ethanol Macleaya cordata stem and leaf extract,Among them,thermal stability and light sta-bility performed well.The sanguinarine remains stable under acidic conditions,and when the pH is adjusted to neutral or alkaline,the color of the solution changes,or a white flocculent precipitate appears.The performance of sanguinarine is very unstable in different pH environ-ments.Next,the sanguinarine in different pH environments was tested with full-wavelength of the mobile phase corresponding to pH,and the structure of hematoxylin was inferred based on the maximum absorption wavelength.The HPLC-Q/TOF-MS technique was used in dif-ferent pH environments.The structure of sanguinarine is detected and judged.The inference results are:in liquid chromatography,with the change of mobile phase system,hematoxylin may exist in three structural forms:?max=274nm of sanguinarine,?max=278nm of 6-meth-oxysanguinarine and?max=286nm of 6-hydroxysanguinarine.However,under the detection of mass spectrometry conditions,only hematoxylin and 6-methoxysanguinarine were searched.Moreover,the content of 6-methoxysanguinarine is very low,and the 6-hydroxysanguinarine and 6-methoxysanguinarine bond may be very unstable,and it has been basically converted back to sanguinarine when it is detected by primary mass spectrometry.It is difficult to inves-tigate the structural morphology of hematoxine in different environments under the conditions of ordinary mass spectrometry.In the future,nuclear magnetic resonance or other techniques can be considered for further research. |
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