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Studies On Constituents From Astragalus Mongholicus And Their Neuroprotective Activities

Posted on:2007-01-31Degree:DoctorType:Dissertation
Country:ChinaCandidate:D H YuFull Text:PDF
GTID:1104360182982457Subject:Biochemical Engineering
Abstract/Summary:PDF Full Text Request
Astragalus membranaceus (Fish.) Bge. var. mongholicus, a member of the Leguminosae, has been used as one of the primary Chinese tonic herbs with a history of thousands of years. It can strengthen the superficial resistance, discharge of pus and benefit the generation of the new tissues. Its active agents and mechanisms of astraglus in neuron system have not been systematically studied. This work aims at isolation and identification of active compounds in this plant and to elucidate the mechanisms of their protection to injured PC 12 neuron cells.Through bioassay-guided fractionation way, twenty constituents were isolated from 95% ethanol extract of astragalus roots. Based on chemical and spectroscopic data (IR, UR, NMR, MS), ten of them were identified as formononetin (I), ononin (II), calycosin (III), calycosin 7-O-β-D-glucoside (IV), 9,10-dimethoxypterocarpan 3-O-β-D-glucoside (V), adenosine (VI), pinitol (VII), daucosterol (VIII), β-sitosterol (IX) and saccharose (X), respectively. The compound VI and VII were isolated from this plant for the first time.PC 12 cells attacked by xanthine (XA)/xanthine oxidase (XO) were designated as the model by which the protective effects of these isoflavonoids (Compounds I-V) on the cell damages from reactive oxygen species (ROS) were investigated. Fluorescence staining and lactate dehydrogenase (LDH) assay were used to investigate the effects of isoflavonoids on cell membrane structures. To elucidate the mechanism of neuroprotection activity, the content of some antioxidant agents and activity of related enzymes in PC 12 cells were assayed, including glutathione (GSH), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). The experiment results indicated that formononetin (0.05 μg/ml) and calycosin (0.1 μg/ml) protected PC12 cells against oxidative toxicity significantly, which may be mediated by increasing the intracellular antioxidant and stabilizing the cell membrane structures.To clarify the mechanisms of their neuroprotection activity, we studied the antioxidant activities of these five isoflavonoids in the cell-free system. They had slight or no scavenging activities to DPPH ftee radicals. They also failed to scavenge superoxide in the xanthine (XA)/xanthine oxidase (XO) system. Calycosin and calycosin 7-O-glucoside showed the capability of inhibiting XO activities and 50% inhibition concentrations (IC50) were more than 40 μM. These results indicated that the probable mechanisms behind these isoflavonoids' protective effects on the model cells couldn't result from their direct antioxidant activities.The indirect oxidative stress model was established by using PC12 cells damaged by high concentration L-glutamate and used to further investigate the neuroprotective effects of the five isoflavonoids. Formononetin and calycosin protected damaged PC12 induced by glutamate at the concentration 0.1 μg/ml, respectively. Based on the experiment results, it can be concluded that formononetin and calycosin may be through increasing the intracellular antioxidant, stabilizing the cell membrane structure and increasing the Bcl-2 protein expression to decrease the cell injuries.In conclusion, isoflavonoids from astragalus protect PC12 cells against oxidative toxicity by improving intracellular antioxidant functions, rather than scavenging free radicals directly. The bioactivities of these isoflavonoids depend on the number and position of hydroxyl groups and the permeability to cell membranes. Moreover aglycone is more effective than its derivative glycoside.
Keywords/Search Tags:Astragalus mongholicus, Constituents, Neuroprotective, Antioxidant, PC12 cells
PDF Full Text Request
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