Studies On Pharmacological Effects Of Effective Components Group Of Xiaoshuan Tongluo Prescription

Effective components Group (ECG) is a new concept in traditional Chinese medicine (TCM) research. It is defined as rational combination of all the effective components contained in a TCM recipe according to its clinical indication, and as a prerequisite of course, the recipe must show good clinical efficacy. This combination, on one hand, is the rational combination of active ingredients. On the other hand, is the combination of the targets in biosystem. The mechanisms of TCM actually involve interactions of two complex systems, which are complicated material system composed of various active substances and sophisticated biological system composed of drug targets in pathological conditions. The interaction between the two complex systems contributes to the treatment of disease. ECG better describes the interaction of these two complicated systems, which makes the study of TCM more scientific and rational.The main contents of ECG study are as follows:1. Selection of TCM prescription and establishement of high-throughput screening models. The identification of efficacy of TCM prescription on a specific disease is the first thing first in ECG study. Then several reliable high-througnput screening models which are related to the indications of TCM prescription should be established on the basis of principles of modern pharmacology in order to evaluate the bioactivities of constituents in TCM prescription.2. Sample preparation and activities assessment. The components in TCM prescription are extracted and separated by chemical methods such as column chromatography which is used to afford successive samples. The key step is to obtain the samples with confirmative constituents and bioactivities on account of further combination of different compounds for activity test.3. Activity screening and comprehensive evaluation. Activity screening should be based on various models and the result of one sample on different models should be evaluated comprehensively, which is the foundation for determination of the composition of ECG.4. Identification of the ECG composition and study of ECG preparation technology. The keynote of study of preparation technology is the establishment of suitable technics for production of ECG in a large scale, which can lead to rational combination of effective ingredients consistent with the results of experimental and theoretical analysis. The suitable preparation technology should be established by validation of drug efficacy and verification of active compounds to gain ECG of TCM prescription accord with the original purpose.5. Quality standardization of ECG. The quality control of ECG should be carried out to validate the efficacy of ECG in different batches.6. Preclinical research of ECG. The pharmacokinetics, pharmacodynamics and mechanisms of drug actions are studied for further development of TCM prescription.This paper deals with the study of the ECG of Xiaoshuan Tongluo (XECG), an important multiherbal formula in TCM extensively used to improve the sequelae of blood stasis in China, and focuses on the quality control, pharmacokinetics, pharmadynamics, main material basis and possible mechanism of XECG.I. Quality control of XECG and pharmacokinetics studyAn UPLC-MS/MS method was established for efficient identification and quantification of18chemical constituents simultaneously in the extract of XSTL and XECG. The chemical compounds detected included protocatechualdehyde, ligustrazine, ferulic acid, vitexin, hyperin, rutin, quercetin, notoginsenoside R1, ginsenoside Re, ginsenoside Rgl,costunolide, curcumin, formononetin, ginsenoside Rbl, astragaloside, tanshinone I, cryptotanshinone and tanshinone Ⅱ A. The separation was performed on a Waters ACQUITY UPLCTM BEH C18(2.1mm×100mm,1.7μm) column with a gradient mobile phase of methanol-0.1%formic acid at a flow rate of0.2ml/min. Ion mode:ESI+, ESI". The standard curves of most components showed good linearity over the concentration range of1.0-500.0ng/ml with r=0.9896-0.9996. The RSD of intra-day and inter-day precisions were lower than15%at three different levels. The RSD of stability analysis of components in24hours were all lower than15%except notoginsenoside R1(17.41%). After the identification, four different brands of commercial XSTL products in various dosage forms and XECG were evaluated.Pharmacokinetic research of XSTL conducted in eight healthy volunteers showed that only ginsenoside Rgl, notoginsenoside R1, ginsenoside Re, ginsenoside Rb1were detectable in plasma with extremely low concentrations, and the other fourteen compounds could not be detected at any time after administration.Pharmacokinetic research of XSTL and XECG conducted in rats showed that the absorption and bioavailability of hyperin, rutin, ginsenoside Rgl, notoginsenoside R1, ginsenoside Rbl and ferulic acid increased significantly in XECG compared with XSTL, but for formononetin, it was inversed. The results showed that the absorbance of ECG was much better than the pills used clinically, which suggesting that ECG might advance the absorbance of other components.Ⅱ. Antithrombotic effect and mechanism of XECGIn this part, we evaluated the effect of XECG in the treatment of thrombotic diseases. Four animal models were introduced, which were ferric chloride-induced carotid artery thrombosis model, venous thrombosis rat model induced by ligation of the inferior vena cava, acute blood stasis model induced by epinephrine conbined with ice-water bath and the collagen-induced acute pulmonary embolism model in mice. In these models, XECG exhibited therapeutic potential on activation of blood circulation and removal stasis at different degrees, with better effects than XSTL in most of the models.In ferric chloride-induced carotid artery thrombosis model, XECG significantly prolonged time to occlusion (TTO) and then delayed thrombosis. In venous thrombosis model induced by ligation of the inferior vena cava, the wet weight of thrombus reduced significantly after administration of XECG. XECG didn't influence blood viscosity, plasma viscosity and erythrocyte aggregation in acute blood stasis model, but platelet aggregation, as well as the contents of TXA2and D-dimer in plasma. In acute pulmonary embolism model, the time to lose righting reflex significantly prolonged with the treatment of XECG, but the mortality time was not affected, probably due to low XECG dose.XECG inhibited ADP-induced platelet aggregation, which might be one of the mechanisms of its antithrombotic activity. In vitro data further confirmed the antithrombotic role of XECG. Among the active ingredients contained in XECG, ginsenoside Re, ginsenoside Rgl, tanshinone II A, salvianolic acid B and rutin might be he main components to exert anti-platelet aggregation activity of XECG. APTT, PT and TT were prolonged, which suggested that both endogenous and exogenous coagulation systems could be inhibited by XECG XECG also had a notable impact on redox system. SOD activities in brain tissue and plasma significantly increased with decreased MDA levels after XECG treatment. In addition, NO content and LDH activity in plasma significantly decreased too, while GSH levels in brain tissue and plasma increased slightly. To illustrate the antioxidant activity of XECG and its material base, we carried out several in vitro experiments and the results indicated that XECG had excellent activities of DPPH free radical scavenging, mitochondrial protection from lipid peroxidation and inhibition of AGEs formation. In DPPH scavenging test, quercetin, protocatechualdehyde, rutin, ferulic acid, curcumin and salvianolic acid B showed strong radical scavenging capacity. In mitochondrial lipid peroxidation experiments, protocatechualdehyde, quercetin, curcumin and salvianolic acid B presented strong protective effect. Similarly, salvianolic acid B, quercetin, rutin and protocatechualdehyde showed excellent activities in AGEs formation inhibition tests. Ginsenoside Re, ginsenoside Rgl, tanshinone Ⅱ A, salvianolic acid B and rutin demonstrated strong anti-platelet aggregation effect, which indicated that these compounds might be the material basis of XECG for anti-platelet aggregation activity.These results suggested that XECG played an important role in prevention even reversion of thrombosis through interacting with multiple targets in the different phases of disease. The antithrombotic mechanism of XECG might be ascribed to its potential on anti-platelet aggregation, coagulation system inhibition and oxidative stress reduction.Ⅲ. Effect of XECG on vascular function and its mechanismAbnormal vascular function triggered by different causes played a key role in the development of thrombotic diseases. In this part, three in vitro models were used to illustrate the effect of XECG on vascular function and its mechanism, including relaxation of isolated rat thoracic aorta, protection of endothelial cells and inhibition of vascular smooth muscle cell proliferation. The results showed that XECG relaxed the aorta pre-contracted by NE and high K+in a dose dependent manner, stronger than XSTL. The role of XECG was partly endothelium-dependenet with activating NO-cGMP pathway and partly endothelium-independent by directly blocking calcium channels in vascular smooth muscle cells. XECG also inhibited VSMC phenotype transformation, leading to reduction of its contractile function. Moreover, XECG pre-incubation could improve blood vessel sensitivity to NE at low concentration. Interestingly, however, XECG pre-incubation decreased maximum contraction caused by high concentration of NE, which might be associated with increase in expression and activity of eNOS. To conclude, The influences of XECG on vascular function focused on regulation of blood vessel homeostasis, rather than simply inhibited contraction stimulated by different factors. Further study should be performed to illustrate the specific mechanism For single compound, tanshinone IIA, tanshinone I, cryptotanshinone, cinnamaldehyde, cinnamic acid, astragaloside, formononetin showed strong relaxative effect on NE pre-contracted vessels, while for high K+pre-contracted blood vessels, only formononetin, quercetin, ferulic acid and curcumin showed strong vasorelaxation activity. These compounds might be the material basis of XECG for vasorelaxation. The vasorelaxant effect of formononetin showed that the relaxation by formononetin was partly endothelium-dependent with activition of NO pathway; partly endothelium-independent by directly opening the potassium channels and blocking the calcium channels on vascular smooth muscle cells. Additionally, formononetin could inhibit VSMC phenotype and reduced the contractile ability of VSMC.Oxidative injury experiments conducted in endothelial cells showed that XECG could protect endothelial cells from oxidative damage, inhibit endothelial cell apoptosis and reduce its adhesion to neutrophil. Ferulic acid, quercetin, cinnamic acid and astragaloside exerted strong protective effect, and might be the main material basis of XECG for protecting endothelium against oxidative damage. XECG could inhibit VSMC proliferation. Quercetin, salvianolic acid B, protocatechualdehyde, rutin, ginsenoside Rbl, ginsenoside Re, ginsenoside Rgl, astragaloside, gigustrazine,, vitexin, cinnamic aldehyde and cinnamic acid might account for this effect due to their higher inhibition rate on VSMC proliferation.In summary, we concluded that XECG could inhibit thrombosis through regulating vasomotor function, protecting endothelial cell from oxdative stress and inhibiting vascular smooth muscle cell proliferation.