Derivative Analysis Of Biogenic Amines And Amino Acids In Traditional Chinese Medicine

Chemical study of Traditional Chinese Medicine (TCM) is an important part of modernization of TCM. It can clarify the pharmacological effects, mechanism of action and the material basis of TCM, and improve the preparation process and quality control standards of preparations. In recent decades, there were a systematic study of the chemical composition of TCM, and made a great achivement. However, the active constituents in most efficacy of TCM are not totally clarified. The water decoction is the most effective preparation in TCM. It is difficult to isolation and characterization of water-soluble components in decoction of TCM, especially those polar, volatile, no UV or fluorophore active structure. Chemical derivatization method can transform the constituents with polar, no UV or fluorophore active structure to the non-polar, UV or fluorophore active structure and improve the isolation property and detection sensitivity. This is an effective method to solve the problem in chemical study of TCM. The aim of this study is to use derivative method to study the chemical constituents in TCM, the main results obtained are as follows:A method was established by chemical derivatization, HPLC and MS for identifying and determining the biogenic amines (BAs) in TCM for the first time. The dansyl chloride derivatization conditions, chromatographic conditions, and sample pre-treatment method were optimized. The results showed that the pH of the buffer solution is a key factor for derivatizing. The purification is very important in sample pre-treatment. It should be combined with HPLC and MS method to identify the BAs in TCM. This method has good linearity, detection limit, precision and recovery. The established method can be used for identification and determination of BAs in TCM.From the structures of feruloyl histamine and ephedradine A-D and their possible biogenetic relationship in ephedra, it was speculated that histamine (HIS), putrescine (PUT), spermidine (SPD) and spermine (SPM) should be existed. The BAs in ephedra was determined. The ephedrae herba contains methylamine (MET), tryptamine (TRP), PUT, cadaverine(CAD), HIS, SPD, dopamine (DA) and SPM. The ephedrae radix et rhizome contains MET, PUT, CAD, HIS, SPD and SPM. This is the first reported that ephedra contains BAs. The results confirmed the speculation of existence of HIS, PUT, SPD and SPM in ephedra, and provided the experimental evidence of its biogenetic relationship. This methodology may be an effective way to study the chemical constituents in TCM and natural medicine.It is the first time to identify and determine the BAs in146commonly used TCM. In these TCM, PUT, SPD, SPM and CAD are contained in the majority of drugs; MET and phenylethylamine (PEA) are contained in part of TCM; only a small number of herbs contains HIS,5-hydroxytryptamine (5-HT), tyramine (TYR) and DA. BAs have higher contents in the flowers, leaves, fruits, seeds and some medicinal roots and tubers, especially in the protein-rich animal and pollen. There is some relationship with SPD and anti-aging effects of TCM. SPD could be one of the active constituents of TCM to produce anti-aging effects. It is important to study the BAs for its high content in drugs from animal source.This is the first time to study the distribution of BAs in different parts of the Lycium barbarum L.(Ningxia wolfberry). Ningxia wolfberry mainly contains PUT, HIS, SPD and SPM. SPD levels are high in leaves and flowers of Ningxia wolfberry. This result could provide foundation for development and utilization of Ningxia wolfberry.There are no reaction between TRP and cinnamic aldehyde in the boiling process of ephedra decoction (Ma huang tang), although the possiblity of Pictet-Spengler reaction is existed. The main possible reason is that the pH is not suitable. Other reasons may be the TRP is trace amount or too many constituents in the decoction.A reagent,4-bromine phenyl isothiocyanate, for derivatization of amino acid is designed and synthesized. It can be used for identifing and determing the amino acid by MS and HPLC. The amino acid derivative can be easily identified by twin peaks with2m/z difference in mass spectrum. The preliminary experimental results showed that the reagent could be used for the derivatization of amino acid, short peptides in TCM and promote the study of chemical constituents in TCM.