التاثيرات المضادة للاكسدة والسمية الخلوية لمركب اللكنان المنقى من بذور نبات جوزة الطيب == Antioxidant And Cytotoxic Effects of Lignan Purified From Myristica Fragrans Seeds
Author name:
شیماء عصام عبد الوهاب البرزنجي
Supervisor name:
عصام فاضل علوان الجمیلي
Abstract:
In this study, natural lignan dimer was isolated from nutmeg seeds (Myristica fragrans) using organic solvent, partially purified using liquid/liquid partiation, purified using anion exchanger and chemically characterized using Benedict’s Reagent, Fehling’s Reagent and Molish’s Reagent. Then, by the aid of UPLC - PDA - IT - TOF - MS System, the molecular weight (626.221 Dalton) and the molecular formula (C39H45O7) of this dimer were determined. After that, the free radical scavenging activities were studied using stable free radical compound 1, 1 - Diphenyl - 2 - Picryl - hydrazil (DPPH). Results showed that 100, 10, 1 and 0.1 ?g/ml of purified lignan had 76.7 %, 65%, 28% and 8% scavenging activity respectively, while the same concentrations of partial purified lignan had 44.3%, 18.5%, 11% and 0% scavenging activity respectively.MTT(3 - (dimethylthiazol - 2 - yl) - 2, 5 - diphenyl tetrazolium bromide) assay was conducted to determined the IC50 for both purified and partial purified lignan using 4 different cell lines A549 (human lung adenocarcinoma epithelial cells), MCF7 (breast cancer cells), PC3(human prostate cancer cells), and HepG2(liver hepatocellular cancer cells), and to determine which cells type were be affected more by this natural lignan dimmer.The IC50 values for purified lignan were 85.17, 51.16, 108.4 and 60.21 ?g/ml while the IC50 values for partial purified one were 170.1, 84.14, 154.4 and 151.3 ?g/ml using A549, MCF - 7, PC - 3 and HePG2, respectively.The high content screening analysis (HCSA) and Cellomics Thermo Scientific maltiparametric Kits were used for the evaluation of cell - lignan interaction; 100, 50 and 25 ?g/ml of purified lignan caused 87.22, 69 and 53.36% reduction in MCF - 7cell count respectively and the same concentrations caused 98.1, 97.5 and 98.55% nuclear morphology changes. Results also revealed that these concentrations caused 7.7, 7.0 and 5.83% increase in MCF - 7 cells permeability respectively and they also caused 12.22, 11.15 and 0.2%decrease in mitochondrial membrane potential respectively, while these concentrations caused 11.12, 10.1 and 10% increase in Cytochrome C releasing from mitochondria to cytoplasim respectively.Reactive oxygen species (ROS) induction in MCF - 7 cells in the presence of 200, 100 and 50 ?g/ml of purified lignan caused 20, 11.48 and 9.61% ROS reduction respectively.MCF - 7 cell cycle was studied in the presence of 100, 50 and 25 ?g/ml of purified lignan, and results revealed that this compound blocked cell cycle at Sphase and the percentages of S - phase cells reduction were 74.33, 52.4 and 67%, respectively. This reduction was dose dependent while the same concentrations had no effect on MCF - 7 mitotic cells. Cell cycle arrest was detected immunofluorescently using BrdU antibodies (S - phase cell staining) and phosphor - Histone H3 antibodies (M - phase cells staining
