| Platelet membrane immobilized chromatography(PMIC) is a new kind bio-chromatography, which combined the cytology and chromatography technique. According to the pharmacological theory, almost all effect /toxicity of drugs are through the binding of drug's molecular to the bio-targets such as receptors, channels and enzymes etc. This is background behind the bio-chromatography, for which can separate and analyze the effect substances by immobilizing them on the bio-targets.Platelet play a key factor in the thrombus diseases. There are receptors and other targets on it's membrane. And, Chinese Medicines, which active compounds are complicated system for their coming from nature products. So the idea raised: that the extract from herbs, if it had the effect on thrombus diseases, the effective chemicals in herbal extract should be conjugated to the receptors or the other targets on membrane of platelets. After washing out the non-conjugated substance, the effective chemicals in the herbs were isolated from the receptors or the other targets on membrane. The analysis of the effective chemicals in the herbs can be conducted via chromatography such as HPLC. This bio-chromotography had been named platelet membrane immobilized chromatography, PMIC.The first section was the study of anti-thrombus effective chemicals in the water extracts of Radix Salvia Milliorrhiza. Pharmacological studies shown that Radix Salvia Miltiorrhiza (Danshen) could improve the blood stasis via invigorate the circulation of blood. The chemicals of Radix Salvia Miltiorrhiza had been classified into two groups: Lipid dissolving dual- terpenes of quinone and water dissolving phenol acid. The platelet membrane immobilized chromatography had been used to analyze the effective chemicals.The fingerprint of water extracts of Radix Salvia Miltiorrhiza has been developed in the way of the linear gradient. The study showed the optimum detective wavelength is 278nm,the recording time is 70 min, The fingerprint obtained in this condition of water extract from Danshen is available as the background of PMIC. Thechemicals of water extracts of Radix Salvia were made fully binded on the targets of the platelet membrane under imitated physical environment. The unconjugated substances were washed down by the PBS of pH7.4, then the specific binding compounds were released out from the membrane of the platelet through the elution of the PBS of pH4. Finally, these elutions were analysed by HPLC. The results indicated that there were six characteristic active compounds binding to the membrane of the platelet. In order to trace the active compounds, we compared the chromatography characteristic of active compounds with serial organic liquid extracts. The results indicated that the chemicals, of which specific bind on the platelet membrane were mainly in the ethyl acetate extracts. So we focus the ethyl acetate extracts from the water extract of Danshen as an original part.The fingerprint of the mailuoning injection, a famous Chinese Medicine for treating thrombi disease,has been fulfiled.Selecting the peaks,of which are correspounding to the PMIC and make active chemicals in mailuoning injection with preparation HPLC.There are 8 peaks,of which are responsible for binding to platelet membrane targets,all them are from herbalk.Adopting preparing HPLC to prepare this 8 monomer chemical components.Further research shown that the characteristic compositions were chlorogenic acidsN caffeic acid and isochlorogenic acids by UV, MS and NMR techniques.All the isolated chemicals had been virified for their pharmacological effect through the antiplatelet aggregation action in vitro. The results shown that the herbal water extract is the powerfulest among the other herbs in the injection (ICso were 0.028mg/ml) and the chlorogenic acids and isochlorogenic acids are powerful separately.Since the fact that the endothelium and platelet share the same Fll receptor on their membrane, which are responsible for platelet aggregation secretion, it means that chemicals isolated from PM... |
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