Studies On Quality Control And Effective Substance Basis Of Baihe Zhimu Tang

Baihe Zhimu Tang (BZT) comes from JinKuiYaoLue-a study on traditional Chinese medicine written by Zhang Zhongjing of Han Dynasty. This empirical formula is used as a treatment of Lily Disease and consists of Bulbus Lilii and Rhizoma Anemarrhenae. Lily Disease is a disease of functional disorder manifested by abnormality in diet habit, sleep and behavior, which is similar to menopause syndrome.Former research in our laboratory have revealed that Baihe Zhimu Tang is effective in rat model and can significantly adjust some important indexes. In this research, we want to explore the effective substances of BZT and set up methods to control the quality of BZT, and we also want to investigate the compatibility mechanism of BZT.Our studies were performed in five aspects as following, the results could provide scientific evidence for development of the new drug.1. High performance liquid chromatographic fingerprints of Baihe Zhimu Tang and its correlation to single herbsThe high performance liquid chromatographic (HPLC) fingerprints of Baihe Zhimu Tang were established for evaluating effective substance basis and compatibility regulation of Baihe Zhimu Tang. An Agela Venusil XBP-C18 column (4.6 mm×250 mm,5μm) was employed, and the gradient elution of acetonitrile and 0.1%formic acid was used as mobile phase, the detection wavelength was set at 315 nm, the column temperature was 25℃and the flow rate was 1 mL/min. By taking mangiferin as reference substance, the fingerprints of ten batches of Baihe Zhimu Tang, prepared by Bulbus Lilium and Rhizoma Anemarrhena from different producing areas, were analyzed under the same chromatographic condition, the results showed that there were sixteen common peaks contained in the tested samples. Five constituents were identified as 5-hydroxymethyl-2-furaldehyde (5-HMF), neomagiferin, mangiferin, isomangiferin and regaloside B by comparing with the peak retention time of reference substance and ultraviolet spectra. The HPLC fingerprints of Baihe Zhimu Tang established with above method show good characteristic and repeatability, and the method is stable and reliable, it can be used for the quality control of Baihe Zhimu Tang. The main chromatographic peaks of the HPLC fingerprints of Baihe Zhimu Tang are identified in the experiment and also 5-HMF is identified as the main constituent which changed significantly in decocting process.2. Infrared spectrum analysis of Baihe Zhimu Tang and its single herbsThe fourier transform infrared spectra (FTIR) of decoction of Bulbus Lilii, Rhizoma Anemarrhenae and Baihe Zhimu Tang (BZT) were tested. The change in the mixed decoction was discussed to study the relationship between Bulbus Lilii and Rhizoma Anemarrhenae after decoction. The results showed that some components of Bulbus Lilii and Rhizoma Anemarrhenae were retained in the mixed decoction of BZT, such as 2933 cm-1 and 1059 cm-1, but some components that never appeared in the two component spectra increased, such as 1724 cm-1 in Bulbus Lilii and 1453 cm-1 in Rhizoma Anemarrhenae. New absorption peaks were generated in the mixed decoction of BZT, such as 1871 cm-1 and 1522 cm-1. It can be showed that there are some differences in the chemical environment of the various chemical groups in BZT, and with the variation in absorption peak position, possibly some new chemical constituents were created. Pharmaceutical effective substances in BZT are not simply the addition of Bulbus Lilii and Rhizoma Anemarrhenae. This method can be used in the quiality control of BZT and proposed a new method in the compatibility research of traditional Chinese medicinal formulas.3. Analysis of chemical components in Baihe Zhimu Tang and its single herbs by high performance liquid chromatography-electrospray ionization-mass spectrometryA method was established for identifying the chemical components of a traditional Chinese medicinal formula Baihe Zhimu Tang and its single herbs by combining high performance liquid chromatography and electrospray ionization mass spectrometry (HPLC-ESI-MS). Samples were analyzed by a reversed-phase C18 column using a binary eluent of acetonitrile and water (with 0.05% formic acid) under gradient conditions, and analyzed by HPLC-MS techniques with an ESI source. The molecular ions of compounds in both negative and positive modes were observed for molecular mass information, and the potential structures were identified by attentive studying on the mass spectra of compounds and comparing with reference data and some of standards. The results showed that in MS detection, saponins in Baihe Zhimu Tang and its single herbs are easily to become positive ions in the electrospray ionization procedure, and they have strong responses, but the mass spectrometry signals of flavonoids and phenolic glucosides are week.38 compounds in Baihe Zhimu Tang including 3 flavonoids,4 phenolic glucosides and 31 saponins were identified through analyzing and comparing the total ion chromatograms (TIC) and mass spectra of Baihe Zhimu Tang and its single herbs. This method has the advantages of simple operation, rapid measurement and it is a powerful tool for identification of chemical components in Baihe Zhimu Tang.4. Screening and analysis of metabolic components in rat plasma, urine and feces after oral administration of Baihe Zhimu TangIn this part, high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) methods were developed for the analysis of metabolic components of BZT. A reversed-phase Shim-Pack VP-ODS C18 column with the column temperature at 35℃was used in the HPLC experiments, and the mobile phase system consists of aqueous formic acid (0.05%, v/v) and acetonitrile using a gradient elution at the flow rate of 0.2 mL/min. The ESI-MS was operated with a single-quadrupole mass spectrometer in both negative and positive ion modes. After oral administration of extracts of BZT, the rat's plasma, urine and feces were also analyzed; 37 metabolic components including 23 original components and 14 transformative components of BZT were detected. The analysis of metabolic components in BZT by HPLC-ESI-MS methods could be a useful means of identifying the multi-components of BZT and to hint at their possible metabolic mechanism of action in the body. The results also narrow the range of active compounds to be found in BZT, and pave the way for further investigations of pharmacology and active mechanism.5. Screening and analysis of metabolic components in rabbit plasma, urine and feces after oral administration of Baihe Zhimu TangIn this part, the same high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) methods were used for the analysis of metabolic components of BZT in rabbit. After oral administration of extracts of BZT, the rabbit's plasma, urine and feces were also analyzed; 25 metabolic components including 19 original components and 6 transformative components of BZT were detected. The analysis of metabolic components in BZT narrow the range of active compounds to be found in BZT, and pave the way for further investigations of pharmacology and active mechanism.