الفهرس 
Chapter 1 : IntroductionOnly 14 pages are availabe for public view
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Abstract
Multidrug resistance (MDR) has recently been linked to an alarming increase in infections. Microorganisms can become resistant to several different classes of antimicrobials, a condition known as MDR. Due to the approximately 7 million deaths and over USD 20 billion in economic losses caused by MDR and cancer each year, they pose a danger to both human health and the global economy. Additionally, one of the major issues that contribute to the high mortality rates in the world is cancer. The World Health Organization (WHO) on the other hand believes that antimicrobial resistance (AMR) is a growing and serious threat to global public health that endangers the ability to effectively prevent and cure a wide range of diseases brought on by bacteria, parasites, viruses, and fungi. WHO states that due to the fact that AMR occurs everywhere in the world, worldwide efforts are required to combat this issue since novel resistance mechanisms develop and proliferate everywhere.
It has become necessary to investigate a number of medicinal herbs for their possible antimicrobial activity because to the growing failure of chemotherapeutics and antibiotic resistance demonstrated by pathogenic microbial infectious agents. Secondary plant metabolites (phytochemicals), previously unknown for their pharmacological effects, have undergone substantial research in recent years as potential sources of therapeutic medicines. Natural goods offer countless prospects for new drug leads due to their unequalled abundance of chemical diversity, whether as pure chemicals or as plant extracts.
For new and reemerging infectious illnesses, significant efforts are required to find new antimicrobial agents with diverse chemical structures and unique mechanisms of action. Endophytes have been linked to plants for more than 400 million years, and by encouraging plant growth, they are crucial components of natural ecosystems. It appears that endophyte-infected plants are frequently healthier than endophyte-free ones.
These protective mechanisms, which result in a complex interaction between the endophyte and the host, affect chemical alterations by enhancing the production of endophytic secondary metabolites in response to a pathogenic invasion. Endophytes are a huge source of biodiversity that contain a variety of as-yet-unidentified bioactive substances that are a quickly replenishable, repeatable, and endless source of novel structures with medicinal promise. It is evident that the pharmaceutical industry’s ability to succeed in the future rests on the successful integration of a number of complementary technologies, including metabolic engineering, combinatorial biosynthesis, and combinatorial chemistry. There have been numerous reports of functional metabolites that were derived from endophytes, which are biological factories for generating metabolites.
This study’s main objective was to investigate at isolated fungal endophytes from Nigella sativa seeds in their antimicrobial activity and cytotoxicity activities in order to provide additional data for the utilization of bioactive metabolites from these endophytic fungi, moreover, determine their potential in production of some important oxidative enzymes.
Therefore, the following points were studied:
1) Isolation and identification of endophytic fungi from Nigella sativa seeds using diverse culture media
2) Cultivation of pure endophytic strains in a large scale using different culture media.
3) Extraction of extra and intra-cellular metabolites from endophyte cultures.
4) Investigation of the obtained extracts for their antimicrobial and anticancer potentials.
5) Qualitative and quantitative determination of total antioxidant activity and antioxidant enzymes produced by the isolated endophytes.
6) Metabolomic analyses for the extracts, which displayed the promising antimicrobial and anticancer activities in attempt to characterization of the extracted metabolites, attributed for these activities.
The results obtained can be summarized as the following:
• 10 endophytic fungi were isolated and identified from Nigella sativa seeds.
• All of the endophytic fungi isolated from Nigella sativa seeds are belonged to the phylum Ascomycetes, except for Fusarium species, which belong to Deutromycetes.
• The shape, structure, and color of colonies, conidiophores, conidia, and/or sexual fruiting bodies and spores were used as the basis for the first identification. A few illustrations of how scanning light microscopy (SLM) can be used to discover morphological characteristics of fungi.
• In plug diffusion screening, most of the isolated endophytic fungi showed activity against at least two of the tested microbes, thus 80% (8/10) of the isolates were found to be active.
• Results of antibacterial effect of the isolated endophytic fungi represented 80% active against the bacterium Staphylococcus aureus, showing high levels of zone of inhibition reached to 11.5 mm, on the other hand, Escherichia coli was most resistant to these endophytic fungi except against one isolate (Aspergillus glaucus, SA9), which gave low zone of inhibition (2.5 mm).
• Two fungal endophytes (Aspergillus flavus SA8, Aspergillus rubr SA10) were active against Klebsiella pneumonia and gave 4-4.5 mm zone of inhibition.
• In disc diffusion screening assay, all extracts of the isolated endophytic fungi showed variation in antimicrobial activity against the tested microbes by measuring the inhibition zone and calculating Antimicrobial Index (AI). The higher activity of fungal extract was shown against Candida albicans by metabolites of SA4 (Aspergillus nomius) which gave 25 mm inhibition zone with Antimicrobial Index value of 1.
• No antibacterial activity was shown against E. coli by extracts of the all tested endophytic fungi, which reflects the same results of primary screening experiment by plug diffusion method.
• Qualitatively, seven isolates (70%) of the tested fungal endophytes showed varied ranges of positive activities for peroxidase, which were observed for immediate bubbling in the test tube in presence of H2O2. On the other hand, the use of ABTS as substrate for laccase provided for the rapid visual expression of the enzyme positives, endophytes isolates in laccase were negative (-) when ABTS was added to agar plates.
• A considerable total antioxidant capacity was observed in EtOAc of only three endophytic fungal isolates (Aspergillus amstelodami SA3, Aspergillus nomius SA4, and Fusarium oxysporium SA6), and the reduction power assay showed reduction capacity at considerable values of activity when the standard ascorbic acid was used.
• The antimicrobial IC50 was measured to extract of the most active fungal endophytes (Penicililum oxalicum SA1, Aspergillus chevalieri SA2, Aspergillus amstelodami SA3, Aspergillus nomius SA4, Penicililum oxalicum SA5 and Fusarium oxysporium SA6) were performed for measuring IC50 using the colormetric microdilution assay (ELISA reader). IC50 values of the crude extracts varied between extracts of different endophytic isolates. IC50 gave ranged values between 1.1 to 437.5 µg/mL when tested against the bacterial/fungal human pathogens.
• Moreover, the results revealed that IC50 values of 1.1, 1.4, 1.5, 1.5 and 1.6 μg/mL by Fusarium oxysporium SA6 extracts showed the highest antimicrobial activity (Antifungal/antibacterial) against Staphylococcus aureus, Pseudomonas aeruginosa, MRSA, Klebsiella pneumonia and Candida albicans, respectively. Also, the extracts of SA3, SA4, SA5 and SA6 showed high activity (SA3, 2.5; SA4, 4.3; SA5, 4.2; SA6 1.5) against MRSA when IC50 was measured compared with the standard (Ciprofloxacin, IC50 25.7).
• Cytotoxic activities of ethyl acetate extracts of the most active fungal endophytes (Penicililum oxalicum SA1, Aspergillus chevalieri SA2, Aspergillus amstelodami SA3, Aspergillus nomius SA4, Penicililum oxalicum SA5 and Fusarium oxysporium SA6) were evaluated against three different cell lines (HepG-2 human hepatocellular carcinoma, MCF-7 human breast cancer, and Caco-2 human colon cancer) to check their bioactivity against these cell lines. The current study showed secondary metabolites effects of these endophytic fungi on the cell viability for HepG-2, MCF-7 and Caco-2 cancer cell lines, whereas, in some cases of extracts showed higher cytotoxic potentials as compared to the positive control.
• In TLC, mycelium extracts in PDA culture medium of the most potent six isolates endophytic fungi (SA1, SA2, SA3, SA4, SA5 and SA6) (six isolates of eight tested) of Nigella sativa were the most convenient isolates to produce numerous metabolites either in short or long wave length UV detection.
• Metabolomic profiling of the ethyl acetate extracts recovered from most active isolates were accessed via LC-HR-MS by employing macros and algorithms that coupled MZmine with databases, specially, the DNP database where, high resolution mass spectra and retention times were used for the identification of dereplicated compounds in good agreement with previously isolated and identified compounds from Aspergillus, Penicillium and Fusarium species.
The metabolic diversity of bioactive leads produced by endophytic microbes, particularly fungi, play an important role in protection the plant against pathogens. Several plant and animal diseases have been discovered to be inhibited by bioactive leads produced by endophytic fungus. The ability of medicinal plants’ endophytes to produce biologically novel active extrolites that would be recommended in medicine and the pharmaceutical industries makes the isolation and identification of endophytes extremely important, whereas the biological activities of medicinal plants can result from this ability.