| Chinese materia medica preparation is unlike traditional chemical pharmaceutical preparations, pretreatment in the process of the preparation is a key factor to the druggability and pharmacodynamics. "Chinese material basis - the overall efficacy - component structure preparations pretreatment" is a line that has a special intrinsic association. This line means to find a way to show the universality of the chemical composition in TCM component (limited "N" representative components) and establish ADME characterization methods to characterize traditional Chinese medicine ingredients. Component structural optimization and ADME characterization has a crucial role in the pretreatment of herbal preparations. It promotes the development of the formulation of traditional Chinese medicine component.The traditional Chinese medicine "herbal pair" with optimized combination of drugs effect in the process of traditional Chinese medicine syndrome differentiation. The "herbal pair" has a synergistic effect, corrects the bias and moderates the toxicity of each other to achieve complementary effects. The "herbal pair", ranges between compound of Chinese medicine and single herbs, occupies an important position in the prescription compatibility of traditional Chinese medicine which is Chinese clinical medicine with unique characteristics. The efficacy material base composition of traditional Chinese medicine is a complex entirety, there is a unique "structure" feature between their internal chemical composition/component, that contributes to the differences in efficacy. The compatibility of "herbal pair" changes the material basis of structure, corrects the bias. Material base structure changes inevitably have influence of drugs in vivo biopharmaceutical process. Therefore, illustrate the influence of the components structure change on pharmacodynamic and pharmacokinetic is the premise of elaborating scientific the drug use of "herbal pair" and also provides a scientific basis for the development of innovation composition of the structure of traditional Chinese medicine.Ligusticum chuanxiong Hort. and Paeonia lactiflora Pall, are the traditional medicines activating blood and eliminating stasis commonly used in the clinical, but now there is less of drug-depth study of the compatibility of Chuanxiong-chishao herbal pair. The essence of the compatibility of "herbal pair" is to change the actual structure of the components, "component structure theory" considers that the material basis of traditional Chinese medicine (compound) and herbs consists of a certain ratio relationship (structure), the stable component structure determines the efficacy of the different components of repeatability. Our study is based on the Chinese medicine "herbal pair" theory and material basis "component structure theory" validated of drug synergy through middle cerebral artery occlusion (MCAO) model of rats. We used Caco-2 model in vitro combined with the identification of blood components to rapid screen the main role in the "herbal pair" components of Chuanxiong-chishao herbal pair. The efficacy and ADME optimization process was carried out before and after the component structure optimization. We committed to provide a new idea and methods to clarify the scientific connotation of the drug. The main contents include the following sections:1. Study on the synergistic effects of Chuanxiong-chishao herbal pair on depressing cerebral ischemia injuryThis part adopts the middle cerebral artery occlusion (MCAO) model of rats to research the effect of Chuanxiong-chishao herbal pair on the depression of cerebral ischemia injury and explore the further possible mechanism. The results showed that compared with the single use of Chuanxiong or Chishao, Chuanxiong-chishao herbal pair had more stronger effect on depressing cerebral ischemia injury, namely, Chuanxiong-chishao herbal pair can significantly relieve neurologic impairment and reduce the cerebral infarction area of cerebral ischemia rats, at the same time, it can improve the oxidation index:elevated activities of SOD, CAT, reduced content of MDA and activity of LPO; increased content of PA, reduced the content of PAI-1 in rats plasma, improved the expression of VEGF as well as GRP78 and reduced the expression of p-p38MPAK, TLR-4, TLR-5, NFkBp65, PERK, CHOP, Caspase-3 and Caspase-12; improved the expression of hippocampal neurons-associated protein Akt, P-CREB, inhibited apoptosis of nerve cell and protect the blood brain barrier, reduced the phosphorylation of ERK protein, down-regulated TLR4, P-p38 lightning and NF-kBp65 protein; reduced the expression of NMDAR1 mRNA and increased the expression of VEGF mRNA; reduced the content of the evans blue (EB) which means improved the permeability of blood brain barrier. The results of this part showed that the joint use of Chuanxiong and Chishao has superior effect than the single use of the two medicines. And this prompted that Chuanxiong-chishao herbal pair have a synergistic effect on treatment of cerebral ischemia injury.2. The characterization and analysis of constituents/components of Chuanxiong and Chishao in rats blood.With previous efficacy assessments, we need a further reveal of components of herbal pair in the blood after compatibility. A Caco-2 monolayer model was used to study for the activity and mechanism of Chuanxiong and Chishao in the intestine in this part to provide the basis for the study of body process in vivo. It can promote monolayer permeability of total paeony glycoside and the amount of penetration was increased contemporarily after the combination of extractions of Chuanxiong and Chishao. LC/ESI/MS/MS technology was selected to find, identify and do attribution of the active ingredients of Caco-2. With the comprehensive analysis of the retention time, relative molecular mass fragment ion information of compounds, eight main components was identified, namely, protocatechuic, oxypaeoniflorin, catechin, albiflorin, caffeic acid, paeoniflorin, ferulic acid, and benzoyl paeoniflorin. Serum chemistry combined with UPLC/Q-TOF-MS showed the blood components of Chishao and Chuanxiong. Confirmed that their efficacy components in the blood. Four glycosides in Chishao:oxypaeoniflorin, albiflorin, paeoniflorin and benzoyl paeoniflorin; four phenolic acids in Chuanxiong:protocatechuic acid, catechin, caffeic acid and ferulic acid. This part of the study initially identified total paeony glycoside (TPG) and total phenolic acids (TPA) were the active constituents against cerebral ischemia in Chuanxiong-chishao herbal pair.3. The structural optimization and the preparation of anti-ischemic brain components TPG and TPA.Total paeony glycoside (TPG) and total phenolic acids (TPA) were the components into blood, but what is the relationship between optimal quantity and the work ratio? So, firstly we obtained the components which into the blood.A modern separation, analysis technology for separation and enrichment were used on TPG and TPA respectively to obtain components. Orthogonal design was chose to select the best extraction process, the resin adsorption and desorption properties of five kinds of macroporous were compared, finally D101 macroporous resin was screened for the separation and purification of TPG, AB-8 macroporous resin was for TPA isolated and purified. The total content of TPA and TPG finally obtained were 47.92% and 41.43%. Secondly, the oxygen/glucose deprivation (OGD) model of human brain microvascular endothelial cells (HBMEC) was established to optimize the activity against cerebral ischemia in vitro of different components of TPA and TPG structure components. By the related biochemical factors measuring, the results showed that the component structure of TPA-TPG (XS component) at 7:3 has the best effect on antagonizing against cerebral ischemia, the ability of reducing oxidative damage (SOD, MDA, NO and NOS) was the strongest, the ability of reducing expression of inflammatory cytokines like TNF-a, IL-6, sICAM-1 was the most obvious. Finally, the application of rat brain ischemia model in vitro model (MCAO) was used for verificationt the best structure XS component. Compared with the component structure not optimized, the structure and component ratio at 7:3 had the best protection of neural function defect, significantly reduced ischemic infarction area of rats, resisted free radical oxidation, significantly reduced expression of MMP-9 and elevated PA content in plasma serum, reduced the content of PA1-1, protected the brain blood-brain barrier in rats. To examine brain cell apoptosis of cerebral ischemia, the results of TEM, WB, Q-PCR, TUNEL and other technologies showed that TPA-TPG at 7:3 component structure has the strongest effect on brain cells of cerebral ischemia rats against apoptosis. The study in this part showed that the optimal treatment of cerebral ischemia was TPA-TPG component structure at 7:3.4. The bioavailability evaluation, transfer absorption and distribution in the brain tissue of Xiongshao component before and after component structure optimized.Can the optimized component structure improve the bioavailability of ingredients/components in vivo? This part conducted the study by using liquid chromatography mass spectrometry/mass spectrometry technique (HPLC/MS/MS) to analyze and compare the pharmacokinetic characteristics of protocatechuic acid, catechin, ferulic acid, coffee acid, albiflorin, paeoniflorin, oxypaeoniflorin and benzoyl paeoniflorin in rats by gastric perfusion of total paeony glycoside components (TPG), total phenolic acid of Chuanxiong component (TPA), Xiongshao component (XS) structure at 7:3. Through the key comparison of Cmax, t1/2 and AUC0～∞, the results showed that compared with the former structure optimization, components’ bioavailability in Xiongshao 7:3 component structure has been improved, and its half-life has also been significantly prolonged, which proved that Xiongshao component after structure optimization can improve the bioavailability of components, promote absorption, and also accordingly increase the effect. It also made a very good explanation for the difference of pharmacodynamic before and after optimization of component. After optimization of component structure, amount of paeoniflorin transferred from A side to B side increased significantly, the absorption coefficient of PA-B also significantly increased to 1.5 times than the original, and secretion of permeability coefficient of PB-A is also improved even though there is no significant difference. After optimized component structure of Xiongshao component, the existence of TPA component can promote the absorption of paeoniflorin in the intestinal tract. After structure optimization of component, transport quantity of ferulic acid from A side to B side increased significantly, the absorption coefficient of PA-B is also significantly improved, and apparent permeability (efflux rate) decreased from 0.86 to 0.68. After optimized component structure of Xiongshao component, the total paeony glycoside components can promote intestinal absorption of ferulic acid. The results showed that the optimized structure of Xiongshao component has a certain role in promoting the transportation of TPG and TPA. The brain has a unique physical structure, is the synergy of these components related to the changes of composition into the brain? This part, we also compared the distribution of XS compents in brain tissue before and after Xiongshao component structure optimization by UPLC. But for the micro content of component in the brain, some new technology like microdialysis will be designed to have depth study in the furture.5. Summarization.This study is based on the Chinese medicine "herbal pair" theory and material basis "component structure theory", optimizing the structure among components of Chuanxiong-chishao herbal pair and compared the efficacy and the physiological disposition before and after the optimization. The result showed that Xiongshao component had the best treatment of cerebral ischemia in rats and ADME/Eff in vivo process was promoted after optimization, which provided a theoretical basis for developing component innovation of traditional Chinese medicine. |
PDF Full Text Request