Studies On Protective Effect Of Protocatechuic Acid From Alpinia Oxyphylla In PC12 Cells

Alpinia (A.) oxyphylla MIQUEL (Zingiberaceae) is an important traditional Chinese medicinal herb whose fruits are widely used as a tonic, aphrodisiac, anti-salivation, anti-polyuria, and anti-diarrhea according to the Chinese Pharmacopoeia. Recently, some novel pharmacological actions of A.oxyphylla were discovered, such as cardiotonic actions, anti-tumor effects, and anti-anaphylactic reactions. Especially, there have been growing evidences that the extract from the fruits of A.oxyphylla shows significant neuroprotective activity, although little is yet known about the pharmacological effects or active ingredients.Hydrogen peroxide (H₂O₂), glutamate (Glu) and 1-methyl-4-phenylpyridinium ion (MPP⁺), which are all inducers of oxidative stress, were used as neurotoxins to establish the oxidative stress models in cultured PC 12 cells that retain dopaminergic characteristics and have been widely used for neurobiological and neurochemical studies. Exposure of PC12 cells to above neurotoxins may cause viability loss and cell death in dose- and time-dependent manner. As a result, H₂O₂ at concentration of 0.4mM for 24h, Glu at 10mM for 48h and MPP⁺ at 1mM for 48h were respectively decided as the proper toxin concentration and time for all subsequent experiments.In our search for new active ingredients from the kernels of A.oxyphylla, an ethyl acetate extract was found to possess neuroprotective activity against oxidative stress-induced cell damage in cultured PC12 cells. From the extract, a phenolic compound was isolated through bioassay-guided fractionation and identified as protocatechuic acid (PCA) by IR, MS, and ¹H and ¹³C NMR spectroscopy. It was the first time which was isolated from the kernels of A.oxyphylla . At the concentration range of 0.06-1.2mM, PCA stimulated PC12 cellular proliferation and markedly attenuated oxidative stress-induced cell death in a dose-dependent manner. The inhibitions induced by PCA in the three oxidative stress-induced damage models were determined by estimating the IC50 values (the concentration of PCA inhibiting each oxidative damage by 50%). PCA showed the most active effect on MPP⁺-induced oxidative stress damage in PC12 cells. The result indicates that the cellular response to PCA may not be universal in different toxin.The neuroprotective effects of PCA on H₂O₂-induced direct oxidative stress in cultured PC12 cells were investigated. Exposure of PC12 cells to H₂O₂ induced a leakage of lactate dehydrogenase (LDH), which was accordant in cell viability denoted by MTT assay.Similarly, PCA decreased the leakage of LDH in a dose-dependent manner. By observing the nuclear morphological changes and flow cytometric analysis, PCA showed its significant effect on protecting PC 12 cells against H2C>2-induced apoptotic cell death. In these cells, the levels of glutathione (GSH) and activities of superoxide dismutase (SOD) and catalase (CAT) were augmented, while glutathione peroxidase (GSH-Px) activity remained unchanged. In addition, PCA also protected against cell damage induced by H2O2 and Fe2+, which generated hydroxyl radicals ("OH) by the Fenton reaction. These results suggest that PCA is very effective in preventing oxidative stress triggered by deterioration of cellular functions to reduce ROS levels. PCA can protect PC 12 cells against oxidative stress probably due to stimulating endogenous antioxidant defense mechanisms.The neuroprotective effects of PCA on MPP+-induced indirect oxidative stress in cultured PC 12 cells were investigated. LDH assay also revealed that application of MPP+ induced the leakage of LDH and PCA stabilized the cell membrane structure. Treatment with PCA significantly protected against MPP+-induced apoptosis/necrosis observed the nuclear morphological changes and measured using flow cytometric analysis. Both necrosis and apoptosis were precisely quantified using propidium iodide and annexin-V dual staining. The apoptosis in MPP+-induced PC 12 cells was associated with loss of mitochondrial membrane potential, the formation of ROS, GSH depletion, suppressions of SOD and CAT, activation of caspase-3 and down-regulation of Bcl-2. In contrast, treatment of PC 12 cells with PCA significantly prevented the above-mentioned mitochondrial dysfunction. These results suggest that PCA may have the capacity to counteract the toxicity of MPP+ by attenuating change of the mitochondrial membrane permeability that is associated with oxidative stress damage.In summary, the results suggest that PCA may be one of the primary active components in the kernels of A.oxyphylla and provide a useful therapeutic strategy for the treatment of oxidative stress-induced neurodegenerative disease such as Parkinson's disease.