Isoflavonoids from Astragalus mongholicus protect PC12 cells from toxicity induced by l-glutamate

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Abstract

lsoflavonoids, formononetin, 9,10-dimethoxypterocarpan 3-O-β-d-glucoside, ononin, calycosin 7-O-glc and calycosin, were isolated from the roots of Astragalus mongholicus Bunge (Leguminosae). The neuroprotective roles and direct antioxidant effects of these isoflavonoids were investigated by using PC12 cell model and DPPH (1,1-diphenyl-2-picrylhydrazyl) assay. Formononetin, ononin and calycosin were found inhibiting glutamate-induced cell injury, with an estimated 50% effective concentration (EC50) of 0.027 μg/ml, 0.047 μg/ml and 0.031 μg/ml, respectively. Pretreatment with them increased the activities of antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and prevented the release of lactate dehydrogenase (LDH) in glutamate-injured PC12 cells. On the other hand, calycosin 7-O-glc and calycosin showed more scavenging activity to DPPH radicals than formononetin in the cell-free system. The inconsistency between the neuroprotective capabilities of isoflavonoids and their directly scavenging activity to DPPH radicals indicated that formononetin, ononin and calycosin probably depended on increasing endogenous antioxidant and stabilizing the cells’ membrane structures to inhibit the cell damage induced by glutamate.

Introduction

Astragalus mongholicus Bunge (Leguminosae), known as Huangqi in China, has been used as one of the primary Chinese tonic herbs with a history of thousands of years (Roy and Cathiroe, 1999). In modern Chinese medicine, Huangqi is widely used as an immune modulator, especially to support immune health for various chronic degenerative diseases. It is also used as an adjunctive therapy to chemo- and radiation-therapy in cancer (Lei et al., 2003, Wang et al., 2003). Recently there are some reports on the neuroprotective activities of the crude extract from Huangqi (He et al., 1998, Yao et al., 2002). In this study we isolated and identified the active components in this plant and characterized their neuroprotective roles.

Glutamate is thought to be the major excitatory neurotransmitter in the central nervous system. Evidence is accumulating that glutamate and relative excitatory amino acid analogs cause a specific pattern of neurodegeneration in the brain of experiment animals, in primary culture of brain neurons and in some cultured neuron cell lines, including PC12 cell line (Atlante et al., 2001, Pereira and Oliveira, 1997, Seyfried et al., 2000). In PC12 cells, a rat pheochromocytoma cell line, lacking ionotropic glutamate receptors, the high concentration glutamate inhibits cystine uptake and depletes intracellular glutathione, which leads to the accumulation of reactive oxygen species (ROS) and ultimately causes cell death (Froissard and Duval, 1994). In the present study, after detecting the antioxidant activities of the five isoflavonoids isolated from Huangqi by DPPH assay in a cell-free system, we investigated whether these compounds were of neuroprotective activity in damaged PC12 induced by l-glutamate in vitro.

Section snippets

Chemicals

The plant materials used in this study were the dried roots of Astragalus mongholicus obtained from Dalian Pharmacy (Dalian, China) and authenticated by Dr. Xian Lan (Pharmacology College, Shandong University). Dulbecco's modified eagle's medium (DMEM) and fetal bovine serum were obtained from Gibco. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltera-zolium bromide (MTT), poly-l-lysine, propidium iodine (PI), N,N-dimethylformamide and sodiumdodecylsulfate (SDS), l-glutamate and DPPH were from Sigma

Radical scavenging activity of isoflavonoids from Huangqi

Formononetin, calycosin and calycosin 7-O-glc were found to scavenge free radicals generated by DPPH in a dose-dependent manner. Formononetin showed slightly scavenging effect, with 11% clearance rate at the concentration of 100 μg/ml.

Calycosin and calycosin 7-O-glc demonstrated more effective than formononetin, with 44.1% and 31.9% clearance rates at 100 μg/ml, respectively. 9,10-dimethoxypterocarpan 3-O-β-d-glucoside and ononin were detected to have no roles in scavenging DPPH free radicals at

Discussion

Increasing reports have provided evidences implicating oxidative stress as a major pathogenic mechanism in neurodegeneration such as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS) (Emerit et al., 2004, Sohal et al., 2002). Oxidant overproduction leads to oxidative molecular damage of the tissue (Zhu et al., 2004). Therefore, protecting neurons from oxidative injuries may provide useful therapeutic potentials for the prevention or treatment of

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