Study On The Substantial Basis Of Antithrombotic Activity And The Mechanism Of Action Of Salvia Deserta Schang

Salvia deserta schang is a plant grown in Xinjiang province in China, and its dried roots are called xinjiang-danshen. This plant and danshen (roots of S.miltiorrhizae Bunge) which is one of the most versatile Chinese herbal drugs are all belong to the same family of Labiatae. Salvia deserta schang is commonly used for the treatment of cardiovascular diseases such as angina pectoris, myocardial infarction and stroke. The previous research found that the lipid-soluble compound6,7-Dehydroroyleanone could protect myocardial ischemia and inhibit platelet aggregation, while it is still unknown for the substantial basis and the mechanism of action of its antithrombotic effect. For the further development of this plant, this paper mainly focused the research for screening the aetive part of antithrombotic activity, its substantial basis and the mechanism of actions.Objective:1) Screening the active components from Salvia deserta schang (SDS) for antithrombotic effect;2) Analysis of the substantial basis of antithrombotic effects from Salvia deserta schang;3) To establish the methods of quality control of the active part of Salvia deserta schang;4) To investigate the antithrombotic mechanism of Salvia deserta schang. Methods:1) The turbidimetic method was used for determining the effect of different extractions by water,95%ethanol and EtOAc from Salvia deserta schang root, stem, leaf and flower on inhibiting the platelet aggregation induced by denosine diphosphate (ADP); the more effective parts of different extractions were screened within the effect on maximum platelet aggregation rate as index; The model of FeCl3induced arterial thrombosis in rats was used to examine the antithrombotic activity of the extracts by water,95%ethanol and EtOAc from Salvia deserta schang root; The antithrombotic activity of different groups was measured by examining the changes of thrombotic morphology, weight and thrombosis inhibition rate, together with the activity of TXB2, 6-Keto-PGF1α and ET in plasma were determined by radioimmunoassay to evaluate the effects of antithrombotic effect;2) The chemical constituents of the active part of Salvia deserta schang were researched by the system solvent extraction, silica gel separation chromatogram technology and the chemical structures of which were characterized by means of physico-chemical methods and spectroscopic techniques (such as MS,1H-NMR,13C-NMR, HMBC and HMQC);3) The methods of quality control of Salvia deserta schang and its active part were established by the methodology validation;4) The model of FeCl3induced arterial thrombosis in rats was used to examine the antithrombotic activity of the active part from Salvia deserta schang root; The antithrombotic activity of different groups was measured by examining the changes of thrombotic morphology, weight and thrombosis inhibition rate, furthermore, the activity of TXB2,6-Keto-PGFia and ET in plasma were determined by radioimmunoassay and AT-Ⅲ, PC, PLG, PAI-1, vWF and t-PA to evaluate the effects of antithrombotic effect. Results:1) EtOAc extraction from Salvia deserta schang showed significant platelet aggregation inhibition on rabbit induced by ADP (P<0.01); EtOAc extraction could markedly decrease the weight of thrombotus (P<0.01). The concentration of TXB2H1plasma was significantly decreased (P<0.05) and the concentration of6-keto-PGF1α was increased (P<0.05), the ratio of TXB2/6-keto-PGF1α also decreased (P<0.05);2) Ten compounds were isolated from EtOAc extract and were elucidated on the basis of the physico-chemical properties and spectra analysis. Among them,1compound was isolated from the genus Salvia for the first time,3compounds were diterpenoid quinones,3compounds were triterpenoid,2compounds were steroids,1compound was organic acids;3) Three effective constituents were separated clearly and respectively in30min, the linear ranges of Horminone,7-O-acetylhorminone,6,7-dehydroroyleanone were0.31～4.56μg (r=0.9996),0.01-0.24μg (r=0.9994),0.40～6.00μg (r=0.9995), respectively; The average recoveries (n=9) were100.3%,100.6%,102.%, respectively;4) It was shown that each EtOAc extract group had significantly inhibited the weight of thrombosis (P<0.01, P<0.01, P<0.01) and the effect of high dose group was especially markedly better than compound danshen dripping pills; Low, middle and high dose groups could obviously decrease the concentrations of TXB2and vWF (P<0.01, P<0.01, P<0.01). The ratio of TXB2/6-Keto-PGF1α was significantly decreased (P<0.01) in middle group. The concentrations of t-PA in middle and high dose groups were increased obviously (P<0.05, P<0.01); The concentration of PAI-1were significantly decreased (P<0.05, P<0.01, P <0.01) and the ratio of t-PA/PAI-1were significantly increased (P<0.05, P<0.01, P< 0.01) in all dosage group while the concentrations of PLG and PC showed no increase, concentration of AT-Ⅲ showed no decrease. Conclusions:1) The EtOAc extract of Salvia deserta schang root has anti-thrombotic effect;2) Substantial basis of antithrombotic effect of Salvia deserta schang root were the total terpene substances which contained diterpenoid quinones and triterpenoid compounds;3) The inhibition of platelet adhesion and aggregation is one of the mechanisms of its anti thrombotic effect;4) The mechanism of action of anti thrombotic effect of Salvia deserta schang total terpene substances may have the correlation with the enhance in the activity of fibrinolytic system;5) Antithrombotic effect of the active part has nothing to do with anticoagulant activity;6) The HPLC method was established for simultaneous detection of3diterpenoid quinones which was simple and accurate method with nice recovery rates and good repetition, this could be used to control the quality of Salvia deserta schang extract and Salvia deserta schang.