الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Teucrium polium (T.polium) commonly known as a Jaadah belongs to the family Lamiaceae and has been used since antiquity in tradition medicine.
The main objective of this study was to study the effect of gamma radiation as a decontamination tool on the chemical contents, antioxidant, antimicrobial, antibiofilm activity of T.polium and elucidate its mechanism of action.
Teucrium polium was collected from Adnaniah, Karak city (the south of Jordan) during May and June of 2017 when plants were at the late flowering stage. The collected plant was dried then grinded to fine powder.
Our studies were performed to find the effect of different doses of ionizing radiation on: microbial load, yield extract, phytochemical content, antioxidant activity as well as the antimicrobial, antibiofilm activity of T.polium ethyl acetate extract. To study the effect of extraction solvent on its activity, different solvents were tested: Hot water, methanol, ethyl acetate, ethanol, and hexane. Antimicrobial activity of different extracts against: two fungal strains (Candida albicans and Asperegillus fumigatus), three Gram-positive strains (Staphylococcus aureus, Bacillus subtilis and Enterococcus faecalis) and three Gram-negative strains (Pseudomonas aeruginosa, Escherichia coli and Actinobacter pneumonia) was performed, and results confirmed that ethyl acetate T.polium extract is the good antibacterial activity and shown no activity against fungi compared to other extracts. T.polium powder was irradiated using gamma radiation at doses of (5, 10, 15, and 20 KGy). Antimicrobial analysis of irradiated plant extracts of different doses shown higher activity of irradiated plant extract of 20 KGy compared to other doses and the control, for that, 20 KGy dose was chosen for more studies.
T.polium extract (control and 20KGy) were analyzed for chemical components by GC/MS, fractionated by column chromatography and thin layer chromatography. Non-irradiated and irradiated subfractions were evaluated for antibacterial activities against P.aeroginosa and S.aureus. The chemical components of the subfractions which have high antimicrobial activity were analyzed by GC/MS.
Investigation of the modes of action of T.polium against P.aeruginosa and S.aureus was determined using growth inhibition curve, DNA fragmentation, scanning electron microscope (SEM) and total protein, catalase activity, lipid peroxidation, and reduced GSH. Antibiofilm mode of action was determined on the level of biofilm formation related gene expression under the effect of the extract. The results showed that, Exposing plant powder to different doses of gamma radiation efficiently reduced total microbial load. While, 5 kGy showed reduction of bacterial count by 99.50% and no fungal counts as compared to the control (0 KGy). A complete sterility was achieved at a dose level of 10 KGy. Ethyl acetate extract proved to be of high antibacterial activity among other extracts.
Plant exposed to 20 KGy showed higher antibacterial activity compared to other doses. Also significantly increased total phenol contents, total flavonoid contents, tannins content and antioxidant activity compared to the control.
GC/MS analysis of 20 KGy and non-irradiated T.polium crude ethyl acetate extract revealed that gamma radiation markedly affected plant content of chemical components, as some new compounds appeared and some were disappeared. In addition, compounds percentages were markedly influenced as some were increased, while others decreased. Generally, 4’,6-Dimethoxyisoflavone-7-O-β-D-glucopyranoside, Phytol and Farnesol were the main constituents of non-irradiated and irradiated T. polium extract, and there are no previous studies isolated these compounds from T. polium.
The minimum inhibitory effect (MIC) of non-irradiated T. polium extract against S. aureus and P. aeruginosa were 8 mg/ml and 2 mg/ml respectively, while, it was for 20KGy irradiated plant 4 mg/ml for both bacterial strains. Column chromatography resulted fractions were combined together into subfractions according to their TLC profile similarity. Subfractions F5 and R3 of non-irradiated (F) and irradiated (R) T.polium respectively have high antibacterial activity against P.aeruginosa and S.aureus, than other subfraction showed more inhibitory effect by irradiated plant extract compared to the non-irradiated. GC/MS analysis of Subfractions F5 and R3 revealed that both subfractions (F5 & R3) are composed of the same major components with variable in concentrations which are the following Isolongifolol, 3’,7-Dimethoxyflavonol and Levomenthol.
T. polium antibacterial mechanism of action against S. aureus and P. aeruginosa was evaluated using SEM photos, as appeared irregular with changes in the cell size and shape compared to the control cells (untreated). Also plant extract induced (non-irradiated and irradiated) a marked DNA fragmentation. And, oxidative stress in P.aeruginosa and S.aureus confirmed by a significant decreases of catalase (CAT), glutathione (GSH) content associated with an increases in lipid peroxidation product malondialdehyde.
The results showed that plant extracts (non-irradiated and irradiated) have antibiofilm activity for P.aeroginosa and S.aureu with higher effect on P.aeruginosa, with higher inhibitory effect induced by irradiated plant extract. Analyzing plant extract effect on biofilm formation related genes expression (PelA and PslA) using QRT PCR in P.aeruginosa cells, results showed down regulation of genes expression after treatment with plant extract compared to the control, worth to mansion that irradiated plant extract has more inhibitory effect on biofilm formation and on PsLA gene expression than PeLA.