Discussion On The Mechanism Of Peganum Seedling Processing Based On The Transformation And Activity Of Harmonia Cephalospora And Harmonia

Peganum harmala L. which is perennial herbs belongs to genus Peganum of Zygophyllaceae. Its seeds and whole plant can be used as medicine. It is used to treat coughs, asthma, rheumatic pain, nameless pain, and it is used as ethnic herbs for many years in Uygur, Mongolian, Kazakh. Peganum harmala L. is one of our rich resources and widely distributed as a great value medicinal resource in China. The seeds medicinal ingredients of Peganum harmala L. is higher. Currently, the pills of Peganum harmala L. prepared from the Second Affiliated Hospital of Xinjiang Medical University are to be used in clinical treatment of psoriasis. It was showed that the formulation is safe, reliable and effective over the years by the clinical practice. The alkaloids is the main active ingredient in the seeds of Peganum harmala L.. The alkaloids of Peganum harmala L. has a wide range of pharmacological activity, in particular, has significant anti-cancer effect. The previous studies have shown that Harmaline and Harmine are the highest concentrations and the main active ingredient of the alkaloids of Peganum harmala L.The plant of Peganum harmala L. is less poisonous, so before the traditional use of Peganum harmala L., the seeds were processed. The doctors of Uygur, Kazak and Mongolian use the seeds which is in wine soaked for clinical treatmeat to ensure the safety and effectiveness. It is reported that seeds of Peganum harmala L. need fry before using in a few literature.At present, the research on the processing of seeds of Peganum harmala L. is few. The change of chemical composition before and after processing is not clear. So this thesis is based on the main chemical components harmaline and harmine of the seeds of Peganum harmala L., studying the content changes of harmaline and harmine before and after frying and exploring the possibility of conversion between the two alkaloids by plant cell suspension culture system. Selecting LC-MS to study the metabolism of liver microsome of conversion component. At the same time, comparing the anti-cancer activity in vitro of harmaline and harmine and inhibition of harmine on cell migration, speculating the processing mechanism of the seeds of Peganum harmala L.First, study on extraction and separation process of two kinds of alkaloids and the fragmentation pathways from the seeds of Peganum harmala L.After the seeds of Peganum harmala L. were crushed, passing through a 40 mesh screen, with petroleum ether stripped of fat-soluble substances. The method of total alkaloids obtained is acid solution to dissolve before alkaline substances precipitation. Total alkaloids immediately followed by silica gel column chromatography, eluted with a solution of methylene chloride:methanol. We can collecte the same ingredients and give the crude compound by crystallization in methanol, and then let the crude compound repeatedly through the column chromatography. So we can get the compounds 1 and 2 of recrystallized from methanol. Compound 1 was identified as harmine, and compound 2 was identified as harmaline.To study the fragmentation pathways of harmaline and harmine by HPLC-MS. Harmaline and harmine were analyzed by ESI-MSn in positive ion mode. The first and multi-stage mass spectrum diagrams were obtained. To analyze mass spectrometry fragments of the harmaline and harmine, under the positive mode, RDA decomposition occurred mainly, and -CH3、-OH were lost.In this part, D101 was choosed to separate and purify the harmaline and harmine of Peganum harmala L. PH, extract concentration, flow rate, extraction volume, eluants of different ethanol concentration and other factors were investigated. The adsorption and desorption crafts of D101 macroporous resin were optimized. The appropriate adsorption conditions are:pH9, the extract concentration 0.6g/mL, flow rate 2BV/h, The appropriate desorption conditions are:70% ethanol used as eluent, flow rate 1.5BV/h. The harmaline and harmine of Peganum harmala L. transfer rate can reach 70% and the process is stable.Second, the processing mechanism research of harmaline and harmine.The content of harmaline and harmine in the seeds of Peganum harmala L. before and after frying. Take the seeds clean, put them in the good warm-up flying container, then let the pot slowly warming up. When we can see the seeds slightly muster and smell the aroma,we can fill them out and let them cool. So we get the seeds fried products of Peganum harmala L. by frying and determinate the content of harmaline and harmine by HPLC. It was found that the content of harmine of the original product was 72μg/g, and the content of harmaline of the original product was 219μg/g; The content of harmine of the fried products was 125μg/g, and the content of harmaline of the fried products was 193μg/g. After frying harmaline content decreased and the content of harmine increased.The biotransformation of harmaline by plant cell suspension system. In order to study the conversion of harmaline and harmine, the paper study the bioconversion of harmaline by plant cell suspension culture system. Cultivating plant cell suspension culture systems of the periwinkle, securinine and tobacco, and adding ethanol solution of harmine to continue to get harvest. Applicating TLC and MS to identify transformation products. It is showed that biotransformation of harmaline occurs in three kinds of plant cell suspension culture system, and the product was harmine.The metabolites of harmine in human liver microsomes. Studying the metabolites of harmine in human liver microsomes by HPLC-MS. Establishing two parts hepatic microsomal enzyme incubation system of harmine parallelly. Detecting the metabolites after the reaction by LC-MS-MS. The results showed that harmine peak, impurity peak and metabolite peaks achieve a good separation by MS. The metabolites of harmine is Harmol.The experimental study of harmine and harmaline on anti-cancer activity in vitro. We can observe the inhibition of cell proliferation of harmine and harmaline by CCK-8 experimente and the cell migration rate on Hela of harmine by cell migration assay. It was found that median lethal dose IC50 of harmine was about 7.549μg·mL-1 and when the concentration of harmine is 20μg·mL-1, the inhibition rate on Hela cells was the biggest. The median lethal dose IC50 of harmaline was approximately 38.67μg·mL-1 and when the concentration of harmaline is 80μg·mL-1, the inhibition rate on Hela cells was the highest. The cell migration Experimental results show that:harmine could significantly inhibit the proliferation of Hela cells. When the concentration in the 0-2.5μg·mL-1 range, the inhibition is obvious, and the higher the drug concentration, the more significant inhibition.Third, the change of the content of harmaline and harmine in different processing methodsWe can get the seeds wine products from the seeds in wine and determinate the content of harmine and harmaline by HPLC. The content of harmine was 115μg/g, and harmaline was 199μg/g in the seeds wine products.we can find that the content of harmine in different processing methods is fried products> wine Products> original products.