| Based on the literature,this paper summarizes the resource distribution of Leonurus japonicus and studies its status of chemical resources.On the basis,systematic research and analysis were carried out on the resources of L.japonicus by the research ideas of Chinese medicine resources chemistry.Then,a new tactic of the development and utilization on the resources of L.japonicus.This thesis is divided into four chapters.In the first chapter,we reviewed the herbalogical study,progress of L.japonicus resources and the resources chemistry research of L.japonicus.Leonurus japonicus Houtt.is recorded in Chinese herbal medicine in past dynasties and has many kinds of components.It is rich of alkaloids,diterpenes,flavonoids,phenolic acids,phenylpropanoids,nucleosides,amino acids and inorganic elements.Alkaloids are the main active ingredients which of high resource value.It was called as magic medicine of gynae and it has other pharmacological effects,such as cardiovascular protective effect,antibacterial,anlgesics and anti-inflammation,and antitumor.L.japonicus distributes all over the country,however,its utilization percent is low that we need to do many work to reduce the waste of resources of L.japonicus.This chapter aimed to do more researches on L.japonicus and provide theoretical basis and scientific basis for the further development and usage of L.japonicus based on the literature.The second chapter systematically analyzed and evaluated the various chemical components in L.japonicus from different origins.Response surface methodology was used to optimize the extraction of L.japonicus.And,UPLC-TQ/MS2 and ICP-MS were applied for the determination of chemical components of different origin of L.japonicus,including alkaloids,flavonoids,phenolic acids,phenylpropanoids,iridoid glycosides,amino acids,nucleosides and inorganic elements content.Define the differences chemical compounds in L.japonicus from different areas in order to provide new direction for the resource utilization of L.japonicus.(1)The analysis of the chemical constituents of the alkaloids,flavonoids,phenolic acids,phenylpropanoids and iridoid glycosides:a rapid method for the simultaneous determination of 24 components in L.japonicus was developed.The method was applied for the determination of three alkaloids,four phenolic acids,five phenylpropanoids,eleven flavonoids and one iridoid glycoside in L.japonicus that from 38 different regions.The result showed that the L.japonicus was rich of the 24 chemical compounds,and the content of stachydrine hydrochloride was highest for 20.41～433.41 μg·g-1,the highest content of total alkaloids of L.japonicus of Hubei was up to 564.89μg·g-1,the highest content of total phenolic acids of L.japonicus of Henan was up to 177.61μg·g-1,the highest content of total phenylpropanoids and iridoid glycoside of L.japonicus of Jilin were 20.91μg·g-1 and 2.89μg·g-1 respectively,and the highest content of total flavonoids of L.japonicus of Zhejiang was 211.33 μg·g-1.The differences of the contents between the different samples may be related to the region according to the result of the hierarchical clustering analysis and principal components analysis.The differences of environment may result in the differences of chemical components.The result provided theoretical basis for the pertinent development of L.japonicus.(2)The analysis of the chemical composition of nucleosides and amino acids:a new method was established to determine the content of 22 nucleosides and 21 amino acids in 38 samples of L.japonicus from 11 different regions by UPLC-TQ/MS2.The results showed that L.japonicus was rich of nucleosides and amino acids resources.The differences between the 11 regions were the contents of nucleosides and amino acids.Among the 22 nucleosides,the content of Ura was the highest,with an average content of 36.96μg·g-1,the Adeo(0.06 μg·g-1)and Camp were the lowest.The total nucleosides content of L.japonicus of Gansu were the highest,they were 495.33 μg·g-1,493.96μg·g-1 and 483.83μg·g-1 respectively,and the total nucleosides content of L.japonicus of Gansui was the lowest,only 16.98 μg·g-1.Among the 21 amino acids,the content of Asn was the highest,with an average content of 95.43 μg·g-1,and the Hpro(0.93μg·g-1)was the lowest.Meanwhile,the total amino acids contents of Hubei were the highest,reaching 3013.22 μg·g-1,2136.42 μg·g-1 and 2124.07 μg·g-1 respectively.The total content of Anhui was also high,reaching 1158.12 μg·g-1,other regions were in the range of 100～600μg·g-1,and the total amino acids contents of Gansu was the lowest,which was only 80.86μg·g-1.Based on the result of principal components analysis,it showed that the content of Hubei,Henan and Anhui were similar and higher than other areas.Therefore,the differences of the contents of amino acids and nucleosides may be related to the regions.The Hubei,Henan and Anhui were the biggest region of L.japonicus nowadays,which may be the reason for their higher contents of amino acids and nucleosides.This section can provide a new way for the further utilization and scientific basis for the clinical application of L.japonicus.(3)The results of the analysis of inorganic elements:a simple and rapid method was developed for the determination of 27 inorganic elements in L.japonicus by ICP-MS.With Ge,In and Bi as internal standard,the method was applied for the determination of 38 samples of L.japonicus from 11 different areas.And obtained the fingerprint of 27 inorganic elements and carried out the correlation analysis,hierarchical clustering analysis and principal components analysis.There were abundant inorganic elements in L.japonicus and remarkable differences between different samples.Among the 27 inorganic elements,the content of K was the highest,which was up to 68682.57 μg·g-1 in average,the Mg(5291.45μg·g-1),P(5281.62 μg·g-1),Al(1249.85 ug·g-1),Fe(1198.43 μg·g-1),Na(229.57 μg·g-1),Ba(115.74 μg·g-1)and Mn(97.34μg·g-1)were followed,the others were all under the content of 80.00μg·g-1 and the Be(0.11μg·g-1)was the lowest.The content of K,P,Mg were high in all of the samples,while the average content of Mn in Guangxi was the highest,the average content of Na,A1 and Ti in Gansu was the highest,the average content of Pb,Zn and Sr in Jilin was the highest.Among the heavy metals and harmful elements,Hg was over the limit without exception.For the other elements,Guangxi,Gansu and Jilin were over the limit and the rest were match with the standard.142 pairs of elements showed positive correlation significantly,which indicated that their establishment are of mutual promotion.According to the hierarchical clustering analysis and principal components analysis,the differences of the content of inorganic elements in L.japonicus between the different regions may be the environment.In this experiment,we detected inorganic elements in L.japonicus including heavy metals and harmful elements,which not only provided new evaluating indicators for quality control of medicinal plants,but also provided scientific references for the market circulation and clinical application of L.japonicus.(4)The results of the optimization of extraction of L.japonicus:The experiment adopted the response surface methodology coupled with ultrasonic-assisted extraction to obtain the optimum extraction process of L.japonicus.Five factors including ethanol concentration,solid-liquid ratio,ultrasonic time,ultrasonic power and temperature were tested and the were chosen as evaluating indicators.The result indicated that the optimal technology was acquired as ultrasonic time of 38.2 min;temperature of 30.0℃;ethanol concentration of 48.9%(v/v);solid-liquid ratio of 30.0 mL·g-1 and ultrasonic power of 500.0 W.It displayed that the method of response surface methodology coupled with ultrasonic-assisted was valid and feasible for the extraction of natural antioxidants and anticoagulants from L.japonicus,and also provided theoretical basis for the particular development of L.japonicus,provided new direction and ideas for the clinical application.In the third chapter,the fingerprint and high quality specification of L.japonicus were established.And clear the best harvest time and the optimization of drying process of L.japonicus.(1)The establishment of the fingerprint of the L.japonicus:the chromatographic conditions of the HPLC fingerprint of the L.japonicus were as follows:Use the Waters SymmetryShieldTM Ce8(4.6×250 mm,5μm)column,mobile phase:acetonitrile(A)-0.1%formic acid(B),gradient elution(0-10%A,0-10 min;10-10%A,10-13 min;10-14%A,13-30 min;14-21%A,30-50 min;21-95%A,50-75 min;95-5%A,75-80 min).The flow rate is 0.40 mL·min-1,the column temperature is 30℃,the sample volume is 10 μL,and the detection wavelength is 277 nm.In results,12 peaks were determined and the three peaks among them were identitied as salidroside,leonurine hydrochloride and syringic acid.Meanwhile,set the leonurine hydrochloride as the reference,the ratio of leonurine hydrochloride and salidroside should in the range of 1.20～1.40.This method can be used for the overall evaluation of L.japonicus,and it was simple and rapid,stable and reliable,which can also be applied for the quality control of L.japonicus resources,and promoted the usage of L.japonicus,provided basis for the quality research of other Chinese medicine.(2)High quality specification of L.japonicus:The moisture content,ash content,acid-insoluble ash content,water soluble extractive,heavy metals and harmful elements,organic chlorine pesticide residues and thin-layer chromatography of L.japonicus were carried out to study the high quality standard of L.japonicus.The results showed that the samples from the base were better than that from market on the basis of the experiments above.In the TLC experiment,the samples from the base had more spots and matched with the standard better,while the market sample were irregular.stachydrine hydrochloride,salidroside,leonurine hydrochloride and syringic acid separated well by HPLC,and showed a good linear relationship in the linear range.The high quality specification of L.japonicus established in this part was a promotion of Chinese pharmacopoeia,provided new indicators and could reflect the holistic quality of the L.japonicus quickly and accurately,which also provided theoretical and scientific basis for the quality control and cyclic utilization of L.japonicus.(3)Clearing the best harvest time of L.japonicus:This experiment compared the content of stachydrine hydrochloride,salidroside,leonurine hydrochloride and syringic acid of the 13 samples collected from the beginning of July to the end of September.The content of the four compounds was selected as the evaluating indicator,and the result showed that the beginning of August was the best harvest time of L.japonicus.(4)Optimization of drying method for L.japonicus:This experiment compared modern processing and drying methods(hot air,infrared drying,microwave drying with traditional drying method(sun-drying,shade-drying)on the influence of the quality of the L.japonicus.The content of 24 ingredients including three alkaloids,four phenolic acids,five phenylpropanoids,eleven flavonoids and one iridoid glycoside was chosen as the standard.Based on the result of principal component analysis and technique for order performance by similarity to ideal solution,70℃ hot-air drying method was the optimum processing method for L.japonicus. |
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