| OBJECTS:From the drug dosage, time of administration,sampling time, anticoagulants selection, donor selection, storage conditions and other research methods. comparison active ingredients of DCP and DCS, establishing and improving the traditional Chinese medicine plasma pharmacological methods (CPPM), a new standard experimental method to supplement semi-vivo tests of traditional Chinese medicine.METHODS:1.Through HPLC-MS method, rat DCP and DCS were detected to determine the active ingredients levels of Scutellaria Baicalensis and Buyang Huanwu Decoction. Through HPLC method, rat DCP and DCS were detected to determine the active ingredients levels and ionic strength from the samples of Radix Sophorae Flavescentis and Tiaowei Chengqi Decoction; The active ingredients content of Cassia Twig DCP and DCS in rats were determined by HPLC method, comparing the effects of different storage temperatures and times on the effective components of DCP and DCS. DCP from rat, rabbit and monkey were detected by HPLC to determine the active ingredients levels of Ginseng and Sijunzi Decoction.which could provide reference ranges (including drug dosage, time of administration and sampling time.) and evidence for the choice of anticoagulant and the basis for the selection of preservation conditions and evidence for the choice of donors for CPPM.2.The technology of 2-DE-MS was adpoted to study Ephedra Decoction containing medicine plasma and serum proteomics(peptide), which could investigate further the influences of coagulation and related processes on DCP and DCS.3.Through cryoscopy method and potentimetric method to Observed the effects of DCPs and DCSs coming from different traditional Chinese medicine sources (including Rhizoma Corydalis, Leech, Cinnabar, SiwuDecoction) on the DMEM osmotic pressure and PH value is to explore thefeasibility of traditional Chinese medicine plasma pharmacological methods in vitro experiment.RESULTS:1.Investigation of drug dosage, time of administration, sampling timeIn the group of Scutellaria Baicalensis, the baicalin level on DCP sampled of the higher doseafter administration 3 days was 5.48±2.51 ug/ml, while on DCS sampled after administration 5 days was 5.47±2.17 ug/ml. Besides, in the lower dose of Scutellaria Baicalensis group sampled after administration 5 days, the level of baicalin on DCP was 5.27±2.13 ug/ml and on DCS was 5.21±2.18 ug/ml. In the group of Buyang Huanwu Decoction, ferulic acid contents respectively were 0.386±0.12 ug/ml(from DCP in higher dose subgroup),0.384±0.116 ug/ml(from DCS in higher dose subgroup),0.383±0.127ug/ml(from DCP in lower dose subgroup) and 0.380±0.115 ug/ml(from DCS in lower dose subgroup) sampling after administration 3 days. All specimen were sampled at one hour after the last administration.2.1nvestigation of anticoagulants selectionOn the groups of Radix Sophorae Flavescentis, the content of matrine on different anticoagulant blood samples from large to small was DCP (heparin)> DCP (EDTA)>DCS (no anticoagulant)>DCS (citric acid), while rhubarb acid content of different anticoagulant blood samples from Tiaowei Chengqi Decocti on from high to low was:DCP (heparin)>DCS (no anticoagulant)>DCP (ED TA)>DCS (citric acid). There was no statistically significant difference betwee n the two groups of matrine and rhubarb acid (P<0.05). Compared with the co ntrol group, the ionic strength of DCS and DCP in Radix Sophorae Flavescenti s group were significantly lower (P< 0.01). It is same that ionic strength of DCP (heparin) and DCP (EDTA) in Tiaowei Chengqi Decoction group decrease d significantly (P< 0.01);Compared with the control serum,the plasma ionic s trength were increased, in which citrate anticoagulation plasma ionic strength in creased significantly(P< 0.01).3.Study on preservation condition and timeAfter saving 30d, compared with A group (no save), there were no significant differences (P>0.05)in the Cinnamic acid reduction rates in DCS and DCP between-20? group (12.9%,13.2%) and-80? group (11.4%,11.9%); Moreover, it is the same that the difference was not statistically significant in 60-day groups (-20 ?:18.0%,17.8%;-80 ?:14.8%,13.3%; P>0.05). After saving 180d, the reduction rate of Cinnamic acid in DCS and DCP from-20 ? group respectively were 39.9% and 38.3%, which had significant differences comparing with control group (P<0.05). However, there were no statistical significance under the condition of-80?(33.2%,32.6%, P>0.05).4.1nvestigation of plasma donorsThe levels of main active ingredients of ginseng in different animal donor DCPs is as follows:monkey> rabbit> rat, and the same situation also existed in Sijunzi Decoction (monkey> rabbit> rat).There was certain difference in levels of effective components coming from three different donors,but without significance(P>0.05).5.Plasma and serum proteomics researchCompared with BP, DCP had identified 32 differential protein spots, of which had 26 up-regulated expression and 6 down-regulated expression; Comparedwith BS, a total of Mahuang Decoction DCS identified 18 diverse protein spots. There were 14 points expressing upregulation and 4 points expressing downregulation; Compared with DCS, Mahuang Decoction DCP identified 26 various protein spots, of which 18 points were upregulated and 8 points were downregulate; Comparing with reserving and removing fibrinogen in plasma, Mahuang Decoction DCP identified 24 varying protein spots, including 20 upregulatedpoints and 4 downregulated points. On above basis, we successfully identified 7 highly-expressed different proteins in plasma an serum, which included ?-1-AT, ApoE, ApoA-I, ?-FIB, HP, FPA and molybdate ABC transporter, respectively.6.Effect of plasma on experimental system in vitroWith the prolongation of the culture time, the osmotic pressure and pH va lue of the culture medium increased gradually within each group. However, wh en comparing with the contemporaneous control group, the changes of osmotic pressure and PH value in adding containing medicine serum had no statisticaldi fferences(P>0.05), and the same situation existed in containing medicine plasma. Meanwhile, there was no significant difference on osmotic pressure and PH val ue between or containing medicine serum and or containing medicine plasma with the same drug and dose (P>0.05)CONCLUSIONS:l.The comparative study of Mahuang Decoction CPPM-DCP and CPPM-DCS s howed that the DCP in Mahuang Decoction can be more able to reflect the ph armacological effects of drugs, which could close to the real value of blood.Ti ps for using the DCP for semi-vivo tests of traditional Chinese medicine.2.The results suggest that there are difference compositions of DCP and DCS of Scutellaria Baicalensis and Buyang Huanwu Decoction groups. the desired CPPM of DCP preparation is adopting more than 2 times of clinical equivalent dose, administrating (i.g.) consecutively over 3 days and sampling DCP at 1H after the last administration.3.1n terms of ionic strength, the anticoagulation effects of EDTA on DCP were consistent with Heparin, but using citric acid to prevent DCP coagulation was best avoided in vitro study. On the whole, Heparin is the preferred anticoagulant during CPPM of preparing DCP. If Heparin have an effect on the experimental results, other anticoagulants could be chosen.4.The concentrations of cinnamic acid in DCS and DCP from Ramulus Cinnamomi groups decreased with prolonged preservation time and increased storage temperature. Based on the above research, the preservation condition of the traditional Chinese medicine plasma pharmacology method for Ramulus Cinnamomi is at-20 ? frozen storage no more than 60 days or at-80? ultra low temperature storage no more than 180 days.5.There was no significant difference in levels of effective components coming from three different donors (rat, rabbit and monkey). Based on the reliable quality, extensive use and convenient operation, rats are the first choice for the CPPM plasma donors.6. Effect of the DCP and DCS in Corydalis, leech, cinnabar and Siwu Decoction on DMEM osmotic medium pressure and PH value had no significant difference. |
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