Antioxidant activity, induction of apoptosis and anti-inflammatory action of Alpinia pricei Hayata

• Main Authors: Yu-Shan Yu, 尤譽姍
• Other Authors: 顏國欽
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/49263750883570848773

碩士 === 國立中興大學 === 食品暨應用生物科技學系 === 96 === Alpinia pricei Hayata (Zingiberaceae) is cultivated throughout Asia and is an endemic plant in Taiwan. Alpinia species have been widely used as a medicine for antihypertension, antiallergy, antinociception, antiplatelet and antispasm. However, limited scientifically confirmed information is available on other biological functions of A. pricei Hayata. The aims of this study were to evaluate antioxidant activity, apoptosis inducing activity and anti-inflammatory properties of Alpinia pricei Hayata. There are three topics included in this study: (1) Antioxidant activity and bioactive constituents of Alpinia pricei Hayata; (2) Induction of apoptosis by Alpinia pricei Hayata in human leukemia cells via a Fas- and mitochondria-mediated pathway; (3) Anti-inflammatory effect of Alpinia pricei Hayata and its active compounds in lipopolysaccharide-stimulated RAW 264.7 macrophages. The antioxidant activity and bioactive compounds of 70% ethanol extracts from Alpinia pricei Hayata (EEAP) were investigated. The results of total phenolics content, trolox equivalent antioxidant capacity (TEAC), oxygen-radical absorbance capacity (ORAC) and the cellular antioxidant activity (CAA) indicated revealed that EEAP had good antioxidant activity. From the formation of conjugated diene and thiobarbituric acid reactive substances (TBARS), EEAP (2.5 μg/mL) was found to possess inhibitory effect on the oxidative modification of low-density lipoprotein (LDL) induced by Cu2+. H2O2-induced DNA damage in human lymphocytes was significantly decreased by EEAP as determined by comet assay. An HPLC analysis revealed that EEAP contained phenolic compounds such as chlorogenic acid (48.4 mg/g extract), pinocembrin (12.3 mg/g extract), caffeic acid (3.1 mg/g extract), ferulic acid (1.2 mg/g extract), rutin (0.9 mg/g extract), p-hydroxybenzoic acid (0.56 mg/g extract), apigenin (4.6 μg/g extract) and curcumin (4.5 μg/g extract). These findings suggest that phenolics and flavonoids in EEAP may directly contribute to the antioxidant activity, the inhibitory effect on LDL oxidative modification and protection of DNA damage of EEAP. The anti-cancer activity and their molecular mechanism of EEAP were also investigated. First, the cytotoxicity effect of EEAP on various human cancer cells was studied. The results indicated that EEAP had relatively high inhibitory activities in CH27, HL-60 and A549 cells with IC50 values of 49, 59 and 89.9 μg/mL, respectively. Treatment of CH27, HL-60 and A549 cells with EEAP markedly increased accumulation of the sub-G1 phase (apoptotic cells). The results also indicated that EEAP had the highest pro-apoptotic activities in HL-60 human leukemia cells among the tested cancer cells. Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis flow cytometric assay showed that EEAP increased the number of early apoptotic and late apoptotic cells. PI and DAPI staining showed that apoptotic bodies appeared when cells were treated with 25 μg/mL of EEAP for 48 h. Treatment of HL-60 cells with EEAP caused the loss of mitochondria membrane potential (ΔΨm). Western blot data revealed that EEAP induced apoptosis in HL-60 cells through the Fas- and mitochondria-pathway. The results further showed that the active phenolic compounds in EEAP, including caffeic acid, apigenin, curcumin and pinocembrin, decrease HL-60 cell viability with IC50 values of 84.2, 7.5, 5.5 and 10.2 μM, respectively. Caffeic aicd, apigenin, curcumin and pinocembrin also caused a marked alteration in ΔΨm. These results demonstrate that EEAP-induced apoptotic ability in HL-60 cells can be related to its phenolic compounds, including caffeic aicd, rutin, apigenin, curcumin and pinocembrin. Finally, the molecular mechanism underlying the anti-inflammatory properties of EEAP and its active compounds was studied using RAW 264.7 macrophages. Treatment with EEAP inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) and prostaglandin E2 (PGE2) production in RAW 264.7 macrophages. Western blotting and real-time RT-PCR analysis showed that EEAP treatment decreased protein and mRNA expression of LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in RAW 264.7 macrophages, respectively. In EEAP, only apigenin, curcumin and pinocembrin could inhibited LPS-stimulated NO and PGE2 production. Furthermore, apigenin, curcumin and pinocembrin decreased LPS-induced iNOS and COX-2 protein and mRNA expression in RAW 264.7 macrophages, respectively. In addition, EEAP and pinocembrin inhibited LPS-induced nuclear translocation of nuclear factor-κB (NF-κB) and NF-κB-mediated reporter gene expression. EEAP and pinocembrin also significantly inhibited LPS-induced intracellular reactive oxygen species (ROS) production and p47phox protein expression in RAW 264.7 macrophages. Pinocembrin inhibited phosphorylation of mitogen-activated protein kinase (MAPK) in LPS-stimulated RAW 264.7 macrophages. Taken together, these results indicate that EEAP and its active compound pinocembrin suppresses the LPS-induced production of NO and PGE2 by inhibiting NF-κB activation, suppression of ROS generation and inhibiting phosphorylation of MAPK. In conclusion, the results indicate that phenolics and flavonoids in EEAP may directly contribute to the antioxidant activity, the inhibitory effect on LDL oxidative modification and protection of DNA damage of EEAP. EEAP induces apoptosis in HL-60 cells through the Fas- and mitochondria-pathway. EEAP and its active compound pinocembrin suppresses the LPS-induced production of NO and PGE2 by inhibiting NF-κB activation, suppression of ROS generation and inhibiting phosphorylation of MAPK.