Separation, Purification And Bioactivities Of Principal Alkaloids From Macleaya Cordata (Willd) R.Br.

Macleaya cordata (Willd) R.Br. is a perennial plant of the Papaveraceae family, which contains many isoquinoline alkaloids, including sanguinarine (SA), chelerythrine (CHE), protopine (PRO) allocryptopine(ALL), and so on. They exhibit a great variety of biological and pharmacological activities such as antitumour, antibacterial, killing snail and treating liver disease. On the basis of the previous research, the alakloids from Macleaya cordata (Willd) R. Br were selected as research object in the dissertation. Firstly, four of alkaloids with high purity from M. cordata were separated and purified. And then the bioactivities of the alkaloids were researched. Finally, CHE-loaded microspheres with sustained-release properties were prepared under the optimized conditions.In order to research the physical and chemical properties of alkaloids from M.cordata, a proposal of separation scheme to obtain alkaloids with high purity from the plant based on column chromatography and solvent method was presented. Eight of isoquinoline alkaloids were separated from the fruits of M.cordata and identified by the methods of 1H-NMR,13C-NMR and ESI-MS. Also, the technical scheme for extraction and purification of PRO, ALL, SA and CHE were preliminarily developed which is based on solvent method and pH regulation. It affords useful inference for the application of the alkaloids from M.cordata.Chromatographic analysis method can be used for the quality control of raw medicinal herb of M.cordata. Therefore, Ultra performance liquid chromatography-electrosprayionization-mass spectrometry (UPLC-ESI-MS) couplet technology was used to set up a rapid method to analyze alkaloids in M.cordata. The whole analysis time for the plant sample (11 min) was 1/3 of that of conventional HPLC (45 min). The proposed method was applied to quantify the eight of alakloids in nine samples of M.cordata in the paper. Significant variations of alkaloid contents were exhibited in the samples from different habitats. The results showed that eight of alkaloids had higher content in M.cordata and SA is the highest among them. It was also found that different sources of M.cordata had larger difference in alkaloid content, suggesting that the variation in alkaloid content may lead to different clinical efficacy for M.cordata. Finally, the analysis methods of UPLC-ESI-MS and HPLC were comparatively investigated.The bioactivities of bisulfates of sanguinarine (BSAN), bisulfates of chelerythrine (BCHE) and the proportional mixture of them (BTA) against the snail of Oncomlania hupensis were investigated. The effect of killing snail is BSAN>BTA>BCHE. And their bioactivities exhibited both time-and concentration-dependent toxicity against the snails. The damage of cell ultrastructure of liver and genital system induced by BAT was studies by transmission electron microscopy (TEM). It was found that both liver and gonads are the target organs damaged by BTA which suggested that BTA was potency molluscicide since it exhibited higher molluscicidal activity to kill O. hupensis with lower LC50 of BTA(0.69 mg/1,48h; 0.40 mg/1,72 h) and it was easy to be obtained with low-cost.The pharmacological activities of ALL against schistosomal liver fibrosis were studied. The LD50 of ALL was 562.4 mg/kg determinated by the acute toxicity tests in mice. And the animal model of schistosomal liver fibrosis in mice was successfully established. The pathological observation from optical microscopy showed that the fiber spacing of mice liver cells in the groups with ALL treatment in middle and high doses decreased more significantly than that in model group. At the same time, granuloma mitigated and the fibrosis degree was from I to II level. The pathological ultrastructure observed by TEM had shown that the fiber distribution in mice liver cells in high dose group of ALL has been reduced. The degree and extent of pathological damage were lower obviously than the model group and the praziquantel group, but the inflammation was still visible. Mice serological index of liver fibrosis with liver tissue immunohistochemical indexes showed that ALL for schistosomal liver fibrosis in mice have certain curative effect.The binding constants, binding sites, effective distance, interaction thermodynamic parameters between SA and bovine serum albumin (BSA) was obtained by the method of fluorescence spectroscopy. So did the CHE. Also the influence of the two alkaloids on the molecular conformation of BSA was discussed. Both the charges and frontier orbital rail of SA and CHE were analyzed using B3LYP/6-31G method of density functional theory. Moreover, the spatial structure of BSA molecule was obtained by homology modeling method. The spatial conformation and the binding sites for CHE molecule interacting with the BSA was simulated by molecular docking techniques. So did the SA. The thermodynamic parameters (ΔG) were-30.29 kJ·mol-1 for SA molecule interaction with BSA. So did CHE molecular with BSA (AG,-29.45 kJ·mol-1). The conclusions of simulation experiment of molecular docking were consistent with the fluorescence experimental results.Carboxymethylchitosan (CMCS) microspheres containing the antitumor drug CHE·HC1 have been successfully prepared by emulsion cross-linking method. The prescription procedure was designed and analyzed by response surface methodology (RSM) with a three-level, three-factor Box-Behnken design (BBD). The desirability function was employed to optimize the drug-loading rate, entrapment rate and other technological parameters. When CMCS concentration is 2.30%, CMCS: CHEC1 ratio is 20:1, O/W phase ratio is 3.37:1, the entrapment rate and drug-loading rate are 57.4% and 4.16%, respectively. The structure and surface features of the obtained microspheres were characterized by scanning electron micrograph (SEM), X-ray diffraction(XRD), Fourier transform infrared (FTIR) spectra and differential scanning calorimetry analysis (DSC). Moreover, the drug release behavior in vitro has also been investigated in simulated gastric fluid (pH 1.0) and intestinal fluid (pH 7.4). The results revealed that the CHE-loaded microspheres had spherical, rough morphology with a narrow size distribution. The CHE-loaded microspheres showed sustained-release property at pH 7.4. The CHE release from CMCS microspheres followed the Higuchi matrix model.