Study Of Vitis Quinquangularis Rehd Extract (VRE) And Its Function And Mechanisms On Antithrombotic Effects

Objective:To provide an experimental support of the anti-thrombotic drug development, Vitis quinquangularis Rehd (VRE) effective sites was extracted and investigated its function and mechanisms on antithrombotic effects.Methods:(1) Extraction of VRE effective sites and its chemical components identification:Chemical components of VRE effective sites were analyzed using modern medicine separation process and liquid mass spectrometry.(2) Effect on thrombosis in rat model:The rats were randomly divided into five groups:Control group:with distilled water intragastric infusion; VRE high dose group (VRE-HD); VRE medium dose group (VRE-MD); VRE low dose group (VRE-LD); ASP group. After giving prophylactic intragastric infusion once a day for three continuous days, rats were anaesthetized by chloral hydrate at30minutes after the last administration in each group. The right external jugular vein we separated was ligatured at the distal end and in which a narrow opening was cut. Then the previously prepared polyethylene pipe, was inserted toward the proximal in the narrow opening followed by ligation fixed. At the common carotid artery end, the left common carotid artery we separated was clamped with an arterial clip at the proximal. Then the other side of polyethylepe was inserted toward the proximal in the narrow opening which was previously cut in the left common carotid artery followed by ligation fixed. Timing when the left common carotid artery was unclamped and the blood flow keep unobstructed for15minutes. The cotton was taken out to be measured the thrombus weight (either wet or dry). At last, The blood was sampled from the abdominal aorta and centrifuged at room temperature. Then the plasma left was used to determine the content of TXB2and6-keto-PGF1α by radio immunoassay.(3) Platelet aggregation assay of VRE effective sites:The rats were randomly divided into five groups:Control group:with distilled water intragastric infusion; VRE high dose group (VRE-HD); VRE medium dose group (VRE-MD); VRE low dose group (VRE-LD); ASP group. After giving prophylactic intragastric infusion once a day for three continuous days, rats were anaesthetized by chloral hydrate at60minutes after the last administration in each group. The blood was sampled from the abdominal aorta and centrifuged to obtain platelet-rich plasma. Then the platelet-rich plasma was used to determine the maximum platelet aggregation of control group, VRE-MD group and ASP group in the same time span by platelet aggregation device using ADP and collagen as inducers.(4) Platelet aggregation in vitro experiments:The blood was sampled from the human vein; PRP was got after blood was centrifuged. PRP was added with Apyrase and PGE1, and then centrifuged. Platelets remains after supernatant discarded were added with Tyrode’s buffer, from which washed platelets were got. Washed platelets in all groups were used to determine the maximum platelet aggregation of different dose groups in the same time span by platelet aggregation device using collagen, CVX and Thrombin as inducers.(5) The system for live-cell assays under shear-flow:Calcein AM was added into rat blood and human blood separately, its final concentration was at4μM.After one hour of time incubation at room temperature (RT), water solution of VRE effective sites was mixed and incubated for10minutes.20uL collagen I (160ug/mL) was poured into Bioflux microchannel with shear force at5dyn/cm2, keep static1hour at RT. The fluorescence labeled blood with VRE was delivered into hole A, The fluorescence labeled blood without VRE was delivered into hole B as negative control. With amount of50uL each hole, the shear force at10dyn/cm2was applied to them, images were collected up to expectation while thrombin turned into collagen protein by FITC fluorescence microscope.Results:(1) Extraction of VRE effective sites and its chemical components identification:Dihydromyricetin, Quercetin and Apigenin were identified in VRE effective sites using liquid mass spectrometry.(2) Effect on thrombosis in rat model:It was shown that each VRE group had significantly inhibited thrombosis and down regulate the level of TXB2and up regulate the level of6-keto-PGF1α in plasma compared with the control group.The effect of VRE-MD group was especially markedly, better than that of ASP group too.(3) Effect on platelets aggregation:Platelets aggregation inhibition of VRE effective sites was more significant than Aspirin on rat and mouse induced by ADP and collagen. Platelets aggregation inhibition induced by collagen, CVX and Thrombin in human blood was found in VRE-LD, VRE-MD, VRE-HD groups within tested dose range in dose dependent pattern. The result of live cell analysis experiments showed VRE effective sites have inhibited platelets aggregation induced by collagen on rat and human blood.Conclusions:1. Three ingredients including Dihydromyricetin, Quercetin and Apigenin were identified in VRE effective sites using liquid mass spectrometry.2. VRE effective sites had significantly the anti-thrombotic function, and this effect may be related TXB2and6-keto-PGFi1α3. VRE effective sites had inhibited platelets aggregation induced by ADP and collagen on blood of rat and mouse and platelets aggregation induced by collagen, CVX and Thrombin on blood of human, This effect may be the antithrombotic mechanism of VRE effective sites.