Extraction, Separation And Pharmacological Study On The Active Components Of Tibetan Medicine Picrorhiza

Objective To extract and separate PicrosideⅡfrom Tibetan medicine Picrorhiza, and to explore the neuroprotective effect of picrosideⅡsample on experimental cerebral ischemia and its molecular mechanisms so as to provide some experimental evidences for the further development and utilization of Tibetan Picrorhiza and development innovative drug for cerebral ischemia.Methods (1) 500g Picrorhiza scrophulariiflora Pennell were extracted with 70% ethanol and obtained the total ethanol extract of Picrorhiza by using impregnation method.The macroporous resin and silica gel adsorption column chromatography method were used to separate ethanol extraction through the appropriate gradient elution. The content of picrosideⅡwas determined by HPLC. (2) Intraluminal thread methods were applied to establish the middle cerebral artery occlusion reperfusion model (MCAO/R) in rats.The model rats were randomly divided into four groups and exposed to different doses of PicrodideⅡthrough intravenous injection from trail vein (10,30 mg/kg), positive drug control group were given Extract of Ginkgo Biloba Leaves injection (10mg/kg). The neurological score and elevated body swing test (EBST) were conducted after the 24h-long ischemia reperfusion, Then pathological changes in lesions were observed, and a series of indicators including the activities of superoxide dismutase (SOD), nitric oxide synthase (NOS), content of malondialdehyde (MDA) and glutathione (GSH) in brain homogenate supernatant were determined, and then the cerebral infarction volume was observed with tetrazolium chloride (TTC) staining, which were measured with image-pro plus 6.0 software. Pathological changes in lesions were observed by hematoxylineosin (HE) staining.The expressions of bcl-2 and Caspase-3 gene at protein level were detected with immunohistochemical staining. Negative control group rats were injected with saline. (3) Antioxidant capacity of picrosideⅡwas determined using the DPPH assay in vitro taken Edaravone Injection as a positive drug. (4) Primary cultured cortical neuron damage induced by hydrogen peroxide (H2O2) was chosen to establish the model of neuron oxidative stress. The growth of neurons and cell morphology were observed by inverted microscope after administration. Then the effect of samples on the the activity of primary cultured neurons was detected by thiazolyl blue (MTT) assay.Results (1) The highest content of picrosideⅡwas found in F3 part by HPLC analysis in which the purity is about 91.60%. (2) The HPLC standard curve of picrosideⅡwas established with a correlation coefficient of 0.9999 in concentration range of 29.5～236μg/mL. (3) After cerebral ischemia reperfusion, neurological function deficit and cerebral infarction in ischemic hemisphere were oberserved. Compared with model group, The neurological score and the number of contralateral rotation of rats in PicrosideⅡtreatment group obviously decreased (P<0.05 or P<0.01). (4) Pathology results showed that swelling and necrosis in lesion of cortical neurons lessened, and GSH content and SOD activity significantly increased (P<0.01). In contrast, MDA content and NOS activity (which include these three subtypes T-NOS, cNOS iNOS) significantly decreased (P<0.01 or P<0.001). (5)The cerebral infarction volume and the expression of caspase-3 gene at protein level were lower than those in the picrosideⅡgroup, howerer the expressions of bcl-2 increased remarkably (P<0.05). (6) PicrosideⅡshowed antioxidant capacity of eliminating DPPH free radicals. (7) PicrosideⅡpromoted neuron growth and menefested as a longer neuron process and bigger neuron body in picrosideⅡexposure group than that in model group after three days exposure. This may suggust that picrosideⅡhas specicial effect of nerve nutrition.Conclusions (1) PicrosideⅡis main components of the picrosideⅡsample with its purity of about 91.65%. (2) PicrosideⅡcan remarkably improve the neural behavioral functions of model rats and pathological changes, it can also improve the antioxidation capacity of brain tissue and oxidation injury caused by cerebral ischemia reperfusion. PicrosideⅡcan reduce the cerebral infarction volume and upregulate bcl-2 protein expression, downregulate caspase-3 expression which may be an important mechanism of its protective effects on cerebral ischemia. (3) PicrosideⅡmaybe one of antioxidant active constituents of Picrorhiza scrophulariiflora. (4) Althrough PicrosideⅡcan not significantly increase the survival rate of neural cells it can improve the changes in cell morphology.