| Modern research suggests that nerve cells protection is the most important mechanism in preventing and treating the neurodegenerative diseases, such as Alzheimer’s disease, major depression, Parkinson’s disease and stroke.Pigeonpea [Cajanus cajan (L.) Millsp.] belongs to Leguminosae and Cajanus, leaves of which is widely used as a traditional medicine in the folk. Recently, studies demonstrated that stilbenes containing extract-fraction from Cajanus cajan (L.) have protective effect on Abeta(25-35)-induced cognitive deficits in mice. However, the active ingredients and underlying mechanism of neuroprotection is still unknown. In present study, the isolation and identification of neuroprotective compounds of Cajanus cajan leaves were conducted under the guidance of the screening activity, and the mechanism research of strong neuroprotective compounds were investigated continually.Firstly, four fractions of Cajanus cajan leaves ethanol extract are prepared. Using PC 12 cells damage induced by corticosterone as in vitro model, the protective effects of the four fractions are determined through MTT assay. The result shows that neuroprotective compounds are mainly gathered in low polarity, including petroleum ether extract, dichloromethane extract and ethyl acetate extract. Whereafter, by using a variety of chromatographic separation technologies and spectrum technologies,23 compounds are separated and identified from Cajanus cajan leaves ethanol extract, including nine flavonoids, four stilbenes and ten other kinds of compounds, namely Cajaninstilbene acid (CSA), Longistylin A, Longistylin C, Cajanolactone A, Pinostrobin, Orientin, Isovitexin, Vitexin, Cajanol, Cajanin, Prunetin, Pratensein, (2R,3R)-2,3-dihydro-5-hydroxy-7,4’-dimethoxyflavone, Ethyl 10’,16’-dihydroxy hexadecanoate, Vanillic acid, Ethyl heptadecanoate,2-Oquebrachitol, 2,3,4-trihydroxy-isovaleric acid, Stigmasterol, Betulinic acid, Heptadecanoic acid, β-sitosterol and β-daucosterol.In order to further clarify the chemical compositions in ethanol extract of Cajanus cajan leaves, we use UPLC-QTof-MS technology to do qualitative analysis in its ethanol extract, and finally identified 18 compounds, including fourteen flavonoids and four stilbenes. Four stilbene compounds were all isolated, but eight flavonoids are not isolated yet, they are Quercitrin, Carlinoside, Isoquercitrin, Rutin, Quercetin, Luteolin, Apigenin and Cajaflavanone, respectively. The analysis results of UPLC-QTof-MS show that the stilbenes and flavonoids are main compounds in Cajanus cajan leaves; stilbenes mainly existed in a form of aglycones, flavonoids are both in form of aglycones and glycosides.By establishing three in vitro screening models which exposures PC 12 cells to corticosterone, glutamate and hydrogen peroxide, respectively, eight major compounds (including four flavones:Vitexin, Orientin, Pinostrobin and Cajanol; four stilbenes:Cajaninstilbene acid, Longistylin A, Longistylin C and Cajanolactone A) were chosen to investigate the neuroprotective effect. The results indicate that stilbenes possess better cytoprotection than flavonoids. In addition, CSA has stronger protective activity towards corticosterone-induced damage, while Longistylin C shows rather high protective effect against glutamate-induced injury.In order to discuss the relationship between neuroprotection of stilbenes and antioxidant activity, in vitro antioxidant evaluation methods (chemical and biological evaluation) are employed to assess the antioxidant activity of stilbene compounds. The result based on chemical evaluation shows that CSA has strong free radical scavenging activities, while Longistylin A possesses rather good reductive activity. Biological evaluation shows that four stilbenes all have different antioxidant activity, through reducing intracellular reactive oxygen species (ROS) and Methane Dicarboxylic Aldehyde (MDA) contents, as well as increasing the activity of Superoxide dismutase (SOD) and Catalase (CAT) in cell. Among them, CSA possesses the strongest cytoprotection, Longistylin A takes second place. The experiments turns out that chemical evaluation and biological evaluation have certain relevance. To illustrate the possible neuroprotective mechanisms of stilbenes, CSA and Longistylin C are selected to reseatch the protective effect against corticosterone and glutamate in PC 12 cells, respectively.Neuroprotective effect of Longistylin C is investigated by MTT colorimetric assay, LDH release assay, Hoechst 33342 staining assay and flow cytometry apoptosis detection, and the results prove that Longistylin C has significant resistance to glutamate-induced injury in PC 12 cells. Besides, western blot analysis demonstrates that Longistylin C could down-regulate the protein expression of N-methyl-D-aspartate receptor (NMDAR/NR2B), calcium/calmodulin dependent protein kinase Ⅱ (CaMKII), GRP78, GADD153, XBP-1, Caspase-12 and up-regulate p-ERK1/2, p-CREB, resulting in relieving endoplasmic reticulum stress and oxidative stress, as well as restoring mitochondrial dysfunction induced by glutamate.Neuroprotective effect of CSA against high corticosterone concentration in PC12 cells is investigated by MTT colorimetric assay, LDH release assay, Hoechst 33342-PI double staining assay and flow cytometry apoptosis detection, and the result indicates that CSA could protect cells from the damage exposed by corticosterone. The protective mechanism research of CSA against corticosterone shows that CSA could be an inhibitor of histone deacetylase-6 (HDAC-6), which could induce hyperacetylation of the heat shock protein 90 (HSP90) proteins, the latter dissociates from its essential cochaperone p23, and loss chaperone activity toward glucocorticoid receptor (GR). On the other hand, the western blot analysis demonstrate that CSA has estrogen-like effect, it can combine with estrogen receptor (ER), and activate MAPK signal pathway through up-regulating protein expression of p-MEK, p-ERK1/2 and p-CREB which is proved to be involved in regulation of neurons and synaptic plasticity. Simultaneously, it could induce the expression of p-Akt and p-Bad which activates mitochondrial apoptotic pathway. In addition, CSA is revealed to protect cells from oxidative damage and endoplasmic reticulum stress-induced apoptosis via decreasing intracellular ROS content and calcium ion concentration.In present study, stilbenes are proved to be the main neuroprotective compounds in ethanol extract of Cajanus cajan leaves by the research of combining chemical separation, activity screening and molecular mechanism. In terms of anti-apoptotic mechanism, one of which was realized by inhibiting the downstream oxidative and endoplasmic reticulum stress, restoring mitochondrial functions, and adjusting the upstream receptors as well.Moreover, the main apoptotic mechanisms and in vitro damage models of nerve cells, chemical constituents and pharmacological activities of Cajanus cajan leaves are also reviewed in this thesis, which provides scientific basis for researching and developing neuroprotective compounds, and supplies references for exploiting preventive drugs from Cajanus cajan leaves to cure neurodegenerative diseases. |
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