الفهرس 
Chapter 1 : IntroductionOnly 14 pages are availabe for public view
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Abstract
The augmentation of resistant bacteria impedance to traditional antibacterial, the enduring infections concomitant to biofilms, and the low accessibility of new antibacterial have made the exploration of new therapeutic alternates requisite. Consequently, finding out natural components with new scaffolds that have improved chances of acquiring new binding modes or addressing novel targets is one strategy to combat microbial pathogens.
It is important to identify curative measures which hold no loopholes and act effectively and specifically to diminish cancer. Cancer is also a huge public health problem around the globe. It is the second-largest reason of death in the World. Of these reasons, growing focus is attracted by research and development of new groups of anticancer agents that demonstrate effective and selective toxicity in tumor cells.
The interest in developing anticancer drugs has increased significantly. Medicinal plants are a unique source of novel drugs and remedial compounds. Therefore, many medicinal plants are yet to be investigated regarding their production of natural products from endophytic microbes, and endophytic fungi are an important source for new compounds with antibacterial, antifungal, anticancer properties and antibiofilm properties. Artemisia spp. is considered as one of the most important medicinal plants. Such a plant has been traditionally used to treat gastrointestinal disorders, heart diseases, stomachache, diabetes, and wound healing. The phytochemical evaluation of A. judaica proves to be a rich source of flavonoids, tannins and phenolics .
Production of valuable compounds by microorganisms usually performed in bioreactors controlled systems called fermentation process. Filamentous fungi may produce several forms of secondary metabolite having essential medicinal activities such as antibiotics, anti-inflammatory and anticancer activities. It is known that fungal secondary metabolism is impaired by several environmental influences, such as light, medium structure, pH, temperature and humidity, and agitation intensity,in addition carbon catabolite repression reduces the production of enzymes and secondary metabolites only in SmC. Changing these factors during cultivation can shock or stress the organism leading to increased or sometimes decreased production of metabolites.
One important tool in the synthetic biology toolbox is metabolomic which catalogues the entire complement of small metabolites in a biological sample, Metabolomic is a newly developed technique in the field of systematic biology following genomics and proteomics, and it explores the metabolic mechanism of the entire organism by detecting the changes in metabolites. Moreover, the metabolomics approach based on high-performance liquid chromatography high-resolution mass spectrometry (HPLC-HRMS) has improved the prospects for identification of known (untargeted) and unknown (targeted) compounds in complex mixtures obtained from plants, animals, and microorganisms origin, thus it provides full chemical profiling of such complex mixtures.
In this study, segments of roots, stems and leaves of Artemisia spp. were screened for isolation of endophytic fungi. A total of 10 endophytic fungi were isolated and identified as Fusarium oxysporum, Aspergillus terreus (1), Aspergillus oryzae from A. arborescens root segments, Aspergillus flavus and Aspergillus terreus (2) from A. arborescens leaves segments. Whereas, Penicillium chrysogenum (1) was isolated from roots segments of A. judaica, in addition, Talaromyces pinophilus (1) which isolated from leaves segments. Finally, Pleosporales sp was isolated from A. annua stem segments and one species of Penicillium chrysogenum (2), and another one species of Talaromyces pinophilus (2) were isolated from A. annua root segments. Most of these genera were common endophytic fungi. All of the 10 endophytes isolated from Artemisia are belonged to the phylum Ascomycota, except Fusarium oxysporum and Pleosporales sp. which isolated were types of sterile fungi.
The main purpose of this study was planned to examine the isolated fungal endophytes in their antimicrobial activity, antibiofilm and cytotoxicity activity 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.
So the following points were studied:
A) Isolation and identification of endophytic fungi from segments of Artemisia spp.
B) Screening for antimicrobial activity.
C) Evaluation of fungal endophytes for production of oxidative enzymes.
Production of oxidative enzymes
D) Fermentation assay in liquid medium
Preparation of ethyl acetate extract from fermentation assay in liquid medium (MPDB)
Determination of minimum inhibitory concentrations (MIC)
Antibiofilm assay
Cytotoxic assay
E) effect of elicitors on secondary metabolites of isolated endophytic fungi .
F) Metabolic analysis
G) Docking study
The results obtained can be summarized as the following:
10 endophytic fungi were isolated and identified (from 3 Artemisia spp.).
A whole of ten fungal endophytic isolates were undergone to a preliminary examination for antimicrobial potentials on agar solid medium (PDA) by plugs method (dual culture method) against two bacterial strains (Bacillus cereus, E. coli) and three isolates of filamentous fungi (phytopathogens for tomato fruits; Aspergillus flavus, Aspergillus niger and Fusarium oxysporum).
All the isolated endophytic fungi showed activity against at least two of the tested microbes, thus 100% (10/10) of the isolates were found to be active. Only one 10% (1/10) of the endophytic fungal isolates which identified as Pleosporales sp. AAnEF1 showed activity against the tested Gram negative bacterium E. coli, moreover, the same isolated (Pleosporales sp.) represented significant antifungal effects against all the tested fungal pathogens. On the other hand, A. terreus (1) AArEF4 showed the most potential as antifungal against the all tested fungal pathogens, in addition, its activity as antibacterial against Bacillus cereus (Gram positive) was shown. Penicillum chrysogenum (1) AJEF2 was one of the first antimicrobial levels when tested against microbes. Generally, A. terreus (2), Penicillum chrysogenum (1)and Pleosporales sp. could be considered the most potential fungal endophytes as antifungal and antibacterial in this study.
A total of eight isolates (80%) of the isolated fungal endophytes showed positive for catalase and peroxidase (antioxidant enzymes) that were observed for immediate bubbling in the test tube in presence of H2O2, whereas, laccase only represented by Fusarium oxysporum (1/10, 10%) in testing enzyme activity qualitatively on agar plates. ABTS oxidation test qualitatively with and without H2O2 exhibited that peroxidase and laccase activity was diminished, use of ABTS as substrate for laccase provided for the rapid visual expression of the enzyme positives. On solid PDA media supplemented with ABTS substrate, the organism formed pale green up to dark purple zone (leave for overnight) due to the oxidative enzyme activity. Endophytes in laccase ranged between negative (-) and positive activity (+) in presence of ABTS on agar plates.
The antimicrobial potential of the most active strain, A. terreus (2) AArEF4, P. chrysogenum AJEF2 and Pleosporales sp. AAnEF1 were explored using different growth media in liquid fermentation assays. The obtained results demonstrated that there were three media types (REB, MREB and MPDB) exhibited high varied growth inhibitory activities towards the tested microbes, as evidenced by the recorded inhibition zones, when culture filtrate of these media were tested.
Result of MIC values of the crude EtOAc extract varied between extracts of different isolates as well as different media types, in addition, MIC ranged between 31.25 to 1000 µg ml-1 against the tested bacterial/fungal human pathogens. In generally, bioactive metabolites in extracts of endophyte static cultures showed more antimicrobial activity than the other shaked cultures. Moreover, MPDB cultures were the most active as antimicrobial effect than the two examined liquid media when MIC was tested.
Extract concentrations in MBC test represented high values in relation to values of the mostly tested MIC. Extracts exhibited bactericidal effect (MBC) against the tested gram negative bacteria with lower concentrations needed when compared with the other tested gram positive (S. aures, S. epidermidis, S. pyogene, Bacillus sp.) that needed high concentrations.
Generally, modified media extracts (MREB and MPDB) of A. terreus had high level of activities either in BIC or MIC compared with un-modified Rice medium. On the other hand, extracts of Pleosporales sp. were the lower in its inhibition activities of the measured MIC/BIC, where no activity (NA) was shown mostly against the tested microbe species; in addition, high concentrations of extract more than 1000 µg ml-1 for inhibition were used in the test. Interestingly, shaked cultures showed lower activities in MIC/BIC than static for all tested culture media
In vitro cytotoxicity by measuring half maximal inhibitory concentration (IC50) of endophytic fungal extracts were determined. Among EtOAc extract of endophytic fungi, extract of Pleosporales sp. had the lowest value of IC50 (4.12 µg mL-1) using extract of MPDB culture against MCF-7 human breast cancer cells line which represented the highest cytotoxic activity. A. terreus EtOAc extract against the same cells line came in the next level of cytotoxic activity which gave values of IC50 as 6.94 and 8.53 µg mL-1, for cultures of MPDB and Rice media, respectively.
In MTT assay for these three potential fungal extracts against HEPG-2 (human hepatocellular carcinoma) cancer cell line, P. chrysogenum extract exhibited high cytotoxic activity with IC50 value of 4.77 µg mL-1 when rice broth extract was used. In addition, level of cytotoxic activity by A. terreus EtOAc extracts came in the next level.
Generally, MPDB culture medium mostly represented the best extracts in cytotoxicity of MCF-7 human breast cancer cells line, on the other hand, cytotoxicity of HEPG-2 (human hepatocellular carcinoma) cancer cell line more affected by extracts of Rice media.
Result of screening artemisinin production revealed that there is no artemisinin production in TLC test by fungal endophytes compared with TLC of pure artemisinin.
Metabolomic profiling of the ethyl acetate extract recovered from MPDB culture broth of both A. terreus , P. chrysogenum and Pleosporales sp. was 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.
The effect of elicitation was displayed by comparing the concentration of the dereplicated metabolites in the MPDB medium with and without adding nalidixic acid. The obtained results of culture filterate of A.terreus (2) AArEF4 revealed that nalidixic acid induced isochroman derivative pseudodeflectusin (2), benzofuran derivative; ustusorane A (3), phenalenone derivative; funalenone (5), and polyketide derivative; atrochrysone carboxylic acid (9), as well as betulinan A (10), while the other identified compounds were remarkably suppressed.
Nalidixic acid effect on the dereplicated metabolites characterized from P. chrysogenum AJEF2 upon cultivation using MPDB culture medium revealed that nalixadic acid could induce the steroidal compounds, including citreoanthrasteroid B (13), citreoanthrasteroid A (14), secocitreoanthrasteroid (16), and citreospirosteroid (17) as well as gladiolic acid, while the other identified compounds were remarkably suppressed except 4,7-Dihydroxymellein showed no effect with nalidixic acid.
The obtained results of Pleosporales sp. revealed that all dereplicated compounds were suppressed using nalidixic acid except asterric acid (11) which was induced and Alternaric acid; 10,11-Dideoxy, 6,19-dihydro which showed no effect with nalidixic acid elicitation effect..
The antibiofilm forming potential against P. aeruginosa clinical isolates might be attributed to its content of gladiolic acid (5), 8-hydroxy-6-methylxanthone-1-carboxylic acid methyl ester (10), cyclo (L-tryptophanyl-L-tryptophanyl) (12), and citreoanthrasteroid A (14), which are hypothesized to possess a potent anti-biofilm formation, postulated by docking studies.
All compounds were tentatively identified for the first time from P. chrysogenum, except, 2’,3’-Dihydro sorbicillin and Penicitide B were earlier reported from this species, but only nine were tentatively identified for the first time from A. terreus, while the others, including terrecyclic acid A, 5(6)-dihydro-6- hydroxyterrecyclic acid A, atrochrysone carboxylic acid, betulinan A, asterriquinone D, and 12,15,25,26-Tetrahydroxyergosta-4,6,8(14),22-tetraen-3- one were earlier reported from this species.
The metabolic diversity of bioactive leads produced by endophytic microbes, particularly fungi, play an important role in protection the plant against pathogens. The inhibition of a wide range of plant and animal pathogens has been previously reported by bioactive leads synthesized by endophytic fungi. Consequently, the isolation and identification of endophytes become extremely important, whereas, the biological activities of medicinal plants can be a result of the capability of their endophytes to produce biologically new active extrolites that would be recommended in medicine and in pharmaceutical industries.