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Exploration of Antimicrobial, Antioxidant, Anticancer and Biosafety of Selected Medicinal Plants

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Exploration of Antimicrobial, Antioxidant, Anticancer and Biosafety of Selected Medicinal Plants Exploration of Antimicrobial, Antioxidant, Anticancer and Biosafety of Selected Medicinal Plants A Thesis Submitted for full requirement for the Degree of Doctor of Philosophy in Pharmacy Submitted By SalwaIbraheimAhmeida Abdulla Supervisor: Prof. Mohamed Elfatih Ahmed Omer Co-supervisor: Prof. Aisha Zoheir Al Magboul 1 December (2018) 2 DEDICATION TO THE SCEINCE TO SPIRITS OF My PARENTS TO MY SISTER, A MAM AFTER MAM TO MY WOUNDERFUL AMAZING BROTHERS TO MY LOVELY KIND SONS, NEPHEWS AND NIECES TO MY TEACHERS AND EVERY ONE TAUGHT ME A WORD TO ALL MY DEAREST FRIENDS, NICE RELATIVES AND NABOURES TO THE WOUNDERFUL HOME COUNTRY, LIBYA,THE ETERNAL LOVE Salwa 3 ACKNOWLEDGEMENTS First and forever thanks to ALLAH and his Messenger Muhammad peace be upon him Thanks, appreciation and respect to my supervisors, Prof. Mohamed El-Fatih Ahmed Omer (Department of Pharmaceutics and Microbiology, Faculty of Pharmacy, Al-Neelain University, Khartoum, Sudan) and Prof. Aisha Zoheir Al Magboul (Department of Microbiology and Phytochemistry, Medicinal and Aromatic Plants and Traditional Medicine Research Institute, National Centre for Research, Sudan) for their guidance, supervision, advices, support and unlimited encouragement. A lot of thanks and appreciation to Prof. Faiza Ahmed Omer (Department of Histopathology, veterinary research Organization, Soba, Sudan), Prof. AmnaHamadElhassan (Department of pharmacology, National Centre for Research,Khartoum, Sudan), Prof. Tarig El-Hadiya (Department of toxicology and dean of faculty of pharmacy, International Africa University, Khartoum, Sudan) for their help and advices in their field of specialization. My gratitude to Prof. SalhaFarage Ben Jwairef, my supervisor inMaster degree, who encouraged and helped me build the foundation stone for my Ph.D (Head of Department of Microbiology, Libyan Academy, Libya). Also my gratitude to Dr.Osama Kherret and his laboratory staff members (Department of Pharmacology, Faculty of Veterinary, Omar Al-Mukhtar University, Libya). I thank the Graduate College and the Faculty of Pharmacy, Al-Neelain University, Khartoum, Sudan. Also I thank the staff members and technicians of the Departments of Microbiology & Parasitology, Pharmacology, Phytochemistry and Biochemistry in Medicinal and Aromatic Plants and Traditional Medicine Research Institute, National Centre for Research, Sudan. Thanks and appreciation to the Faculty of Pharmacy and Omar Al-Mukhtar University, Al- Bayda city, Libya for their candidacy and thanks to the beloved Libya for dispatching me for the PhD Degree. 4 Many thanks and appreciation to the Libyan embassy in Sudan specially Dr.Salama Mohammed Salama, Dr.FathiaAbdulhamedDallaf, Mr.Almahdi Hassan Al-TayabIdress and Mr.Abdulgader Mohammed Blegasimfor theircooperation andsupport. Furthermore, I would also like to thank everyone who has had a positive impact on my scientific career both in my beloved home country Libya and in Sudan. ABSTRACT The use of plants for treating diseases is well clear and even if their chemical constituents are not completely known but their use is well observed as widely disseminate. This is due to its clear efficacy which attributed to the disclosure of their therapeutic properties. This study was aimed to screen the antimicrobial activity of the chloroform, methanol, ethanol and aqueous extracts of nine Libyan medicinal plants (Myrtuscommunis, Pistacialentiscus, Capparisspinosa, Salvia fruticosa, Artmesiaherba Alba, Juniperusphonicea, Rosmarinusofficinalis, Citrus aurantiumandMarrubiumvulgare) against standard Gram positive bacteria (Bacillus subtilis NCTC 8236, Staphylococcus aureus ATCC 25923) and Gram negative bacteria (Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC 27853) and fungi (Cadidaalbicans ATCC7596, Aspirogellusniger ATCC9763 ), the activities of these plants extracts were compared with commonly used antibiotics. Some of these plants were further screened for their antimicrobial activity against clinically isolated bacteria (methicillin resistant Staphylococcus aureus, Echerichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Enterobacter cloacae, Acinetobacterbaumanii and Klebsiellapneumoniae). In vivo investigation in rats for healing of wound that infected with either Staphylococcus aureus or Pseudomonas aeruginosa was done using the methanol leaves extract of the Myrtuscommunis . Phytochemical screening, study of the antioxidant activity, study of the cytotoxicity (Antitumor), study of the safety of the most promising biologically active plants extracts were done. Screening of the methanol extracts of leaves of Myrtuscommunis, Capparisspinosa and fruits of Pistacialentiscus for their toxicity (acute and sub-acute) through evaluation of liver function, kidney function, blood glucose, lipid profile, hematology parameters and histopathology profile via rat animal model was done. Screening of pharmacological effect of the three tested extracts on animal isolated vital organs (rabbit aortic strip, rabbit jejunum, rat uterus and frog abdominal muscle) were done with use of standard methods. Disk diffusion method was used to determine the susceptibility of the tested 5 microorganisms towards plant extracts. The minimum inhibitory concentration of the most active plants against standard bacteria was determined using the agar plate dilution method, the extract was tested on three types of wounded experimental rats, comparison was made between standard antibiotics treated wounds (3% Tetracycline for Pseudomonas aeruginosa infected wounds and 2% Fucidin for infected Staphylococcus aureus wounds). Standard basic phytochemical analysis and Gas chromatography/Mass Specrta, DPPH Scavenging assay and Sulfo-Rhodamine-B (SRB) assays were methods used in this study to identify the constituents and evaluate the antioxidant and antitumor activities of methanol extracts of leaves of Myrtuscommunis ,Capparisspinosa and fruits of Pistacialenticus. HEPG2 (human liver cell carcinoma), HCT (human colon cell carcinoma), MCF7 (human breast carcinoma) and PC3 (human prostatic cell carcinoma) cells lines were used to examine the antitumor activities of the tested extracts. Fifty eight (72.5%) of these extracts showed antimicrobial activity against one or more of the tested organisms. Out of 80 investigated extracts 92% showed active inhibition of the growth against standards Gram positive bacteria, 56.2% against Gram negative bacteria and 35% against the fungi. Bacillus subtilis NCTC 8236, Staphylococcus aureusATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosaATCC 27853 and Candida albicansATCC 7596 were inhibited with percentages of 47.5%, 45%, 27.5%, 28.75% and 35% respectively. The highest growth inhibition activity against Bacillus subtilisand Staphylococcus aureus was showed with chloroform and methanol extracts of Myrtuscommunis leaves and the highest growth inhibition activity against Escherichia coli and Pseudomonas aeruginosa shown with methanol and ethanol extracts of Myrtuscommunis leaves respectively. The highest growth inhibition activity against Candida albicansshown in this study was observed with ethanol extract of Capparisspinosa bark and methanol extracts of Salvia fruitosa bark and Myrtuscommunis leaves, while the highest inhibition activity against the Aspergillusnigerwas shown with methanol extract of Pistacialentiscus fruits. The minimum inhibitory concentration shown in this study was 3.125 mg/ml from methanol extract of Myrtuscommunis leaves against Pseudomonas aeruginosa followed by 6.25 mg/ml by methanol extract of fruits of Pistacialentiscusagainst Candida albicans and methanol extract of leaves of Myrtuscommunis against Staphylococcus aureus and Candida albicans. 12.5 mg/mlwas the minimum inhibition concentration against Escherichia coli and Pseudomonas 6 aeruginosa in this study seen with methanol extract of bark of Salvia fruticosa. Other remained extracts showed minimum inhibitory concentrations ranged 50 – 100 mg/ml. Among the clinical bacterial isolates the results revealed that the methanol extracts of Myrtuscommunis leaves, Pistacislentiscus fruits and Salvia fruticosa bark showed antibacterial activity against all tested isolates. The methanol extract of Capparisspinosa leaves in this study showed clear antibacterial activity against both standard Staphylococcus aureus and clinically isolated methicillin resistant Staphylococcus aureus. The antibacterial activity of six reference antibiotics was determined against clinical isolates and their activities were compared with the activity of plants extracts. This screening clarified that except of Ciprofloxacin 30µg, Myrtuscommunis considered the best more active choice followed with Salvia fruticosa for treating infection caused by methicillin resistant Staphylococcus aureus, Enterobacter cloacae, Escherichia coli, and Pseudomonas aeruginosa compared to other tested antibiotics. Furthermore, Methanol extracts of Myrtuscommunis leaves, Pistacialentiscus fruits and Salvia frutisoa bark in this study had clear activity against clinical Acinetobacterbaumanii isolates compared with all tested antibiotics. Also the chloroform bark extract of Capparisspinosa in this study ranks the fourth level among the tested extracts and appeared as the only one inhibited the growth of methicillin resistant Staphylococcus aureus compared with tested extended beta- lactam antibiotics (Ceftazidime 30µg, Ceftriaxone 30µg). The results proved that Myrtuscommunis leaves extract accelerating wound healing process without any significant differences between
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