الفهرس 
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Abstract
Endophytic fungi are well recognized for their production of a wide range of secondary metabolites beneficial to people, plants, and the environment. Fungal bioactive substances play an important role in a number of industries and professions, including bioremediation, agriculture, pharmacy, and medicine.
The purpose of this study was to isolate fungal endophytes from seven medicinal plant samples, Moringa oleifera, Thymus vulgaris, Foeniculum vulgare, Rosmarinus officinalis, Azadirachta indica, Olea europaea and Simmondsia chinensis that were collected from the Botanical Garden, Faculty of Science, Ain Shams University. Focusing on secondary metabolites production by endophytic fungi and their applications in several biological sectors was also aimed.
The endophytic fungi were isolated and purified on different media and applying the law of the colonization frequency (CF), then extracting the secondary fungal metabolites with petroleum ether and ethyl acetate solvents, followed by examining the antibacterial activity against two types of Gram-positive bacteria Staphylococcus aureus, Bacillus subtilies and two types of Gram-negative bacteria Escherichia coli, Proteus vulgaris .
The antifungal activity against three fungal strains Candida albicans ,Aspergillus fumigatus and Aspergillus flavus was also examined .All tested microorganisms were obtained from Regional Center of Mycology and Biotechnology (RCMB),to identify the most potent endophytic fungi, followed by morphological identification by microscopic examination at the Regional Center of Mycology and Biotechnology, Identification Unit, Al-Azhar University, and then confirmation of the identification by Molecular Genetics based on the 18S rRNA analysis.
The minimum inhibitory concentration (MIC) of two ethyl acetate crude extract from Moringa oleifera was also evaluated.
The antiviral activity was evaluated using a cytopathic effect inhibition assay, then evaluation of antioxidant activity by DPPH Radical Scavenging Activity, and cytotoxicity and antitumor activity were evaluated using viability assay. chromatographic separation of the major components using LC/MS chromatographic separation technique was carried out. Analytical statistics; one-way analysis of variance (ANOVA 1) was used.
The obtained results revealed that:
A total of fifteen endophytic fungal strains were isolated from leaves and stems of four out of seven medicinal plant samples. These four plants were Thymus vulgaris, Rosmarinus officinalis, Foeniculum vulgare and Moringa oleifera. It was remarkable that the number of fungal endophytic isolates was more in the stems of Moringa oleifera, Thymus vulgaris, and Rosmarinus officinalis than that in the plant leaves; meanwhile, the number of Moringa oleifera endophytic isolates was the largest among. It was noted that Potato Dextrose Agar (PDA) is the best substrate for fungal isolation and thrive followed by Malt Extract Agar (MEA). The colonization frequency (CF) of endophytic fungi found in Moringa oleifera stems was 60% exceptional when compared to those found in the leaves (40%).
The two highly bioactive Chaetomium isolates from Moringa oleifera were subjected to the morphological identification and Molecular Genetics based on the 18S rRNA analysis. The two fungal species were deposited in Gene Bank under accession number (ON782293) and (ON778729) respectively. Accordingly, the fungus isolated from Moringa oleifera leaves had a resemblance of 96.4 % to Chaetomium laterale LC4146, where as the fungus isolated from Moringa oleifera stem revealed a higher homology of 99.6% with sequences of Chaetomium interruptum CBS126660.
The crude metabolites extracted from Chaetomium sp, that isolated from Moringa oleifera leaves and stem recorded antimicrobial activities against most test microorganisms (A. fumigatus, S. aureus, B. subtilis, E. coli and P. vulgaris), while lack fungal activities against the resistant A. flavus and C. albicans. The other endophytic isolates showed weak to non-effective antimicrobial activities against the test organisms. Regarding to these results the two isolates of Chaetomium were chosen as the most antimicrobial potent isolates and subjected to the further studies. These remarkable results were achieved when compared to petroleum ether extract, residual aqueous broth, and the +ve control. Statistical analysis (ANOVA-1) revealed that the responses of each microbe to the varied extracts were extremely significant.
The minimum inhibitory concentration (MIC) of ethyl acetate crude extract of Chaetomium laterale was found ranged from 12.5 to 0.39 mg/ml showing its high activity against Proteus vulgaris, followed by both Staphylococcus aureus and Bacillus subtilis, then Aspergillus fumigatus and Escherichia coli. However, Chaetomium interruptum ethyl acetate crude extract, showed MIC ranged from 12.5 to 0.781 mg/ml against Proteus vulgaris followed by Staphylococcus aureus and Aspergillus fumigatus then Escherichia coli, and finally Bacillus subtilis
The present work reports evaluation of the in vitro antiviral activity of the prepared metabolites against Herpes Simplex type-1 virus (HSV-1) by means of a cytopathic effect inhibition assay. from the obtained results, it was noticed that the ethyl acetate and petroleum ether extracts possess weak to moderate anti-HSV activity.
The in vitro results of the tested samples against HSV-1 recorded that ethyl acetate metabolites of Chaetomium laterale and ethyl acetate metabolites of Chaetomium interruptum show good activities against HSV-1, with EC50 values of 83.6 and 317.8 μg/mL, respectively, with respect to acyclovir (3.2 μg/mL). The ethyl acetate metabolites of Chaetomium laterale yielded the highest effects against HSV-1, in vitro at non-cytotoxic concentrations with respect to their EC50and SI values. Furthermore, petroleum ether metabolites of Chaetomium laterale and petroleum ether metabolites of Chaetomium interruptum exhibited weak activity against HSV-1.
The antioxidant activities of the tested fungal extracts were analyzed by DPPH radical scavenging assay. DPPH method is widely used to measure the ability of compounds to act as hydrogen donors and radical scavengers. The IC50 for Chaetomium extracts and ascorbic acid was 315.8 ± 16.7, 209.1 ± 14.2, 373.4 ± 21.9, 96.3 ± 4.8 and 13.9 ± 0.43μg/mL, respectively.
The antitumor activities of the fungal extracts were evaluated against three tumor cell lines. The fungal extracts exhibited a potential anticancer effect on the tested three cancer cell lines in a dose dependent manner with inhibition percentage ranging between 83 to 97.5% at the highest tested conc. 1000 µg/mL. The highest inhibitory activity of the tested fungal extracts was measured against hepatocellular carcinoma (HepG2 cell line). Also, these results indicate that the three carcinoma cells were less susceptible to the cytotoxic effect of the fungal extracts compared with Cisplatin reference drug.
The potent inhibitory activity (EC50) was reported for the fungal extract; ethyl acetate metabolites of Chaetomium interruptum against HepG-2, PC3 and HCT-116 carcinoma cells was 25.8±2.4, 53.2±2.9 and 37.9±2.3 µg/mL, respectively
The fungal extracts exert a low toxicity to Vero African Green Monkey normal cells (CC50= 128.4± 8.8; 148.1± 9.5; 436.3± 39.1 and 372.8± 23.4 µg/mL for Chaetomium extracts respectively). However, the selective index (the ratio between CC50 value for normal cells and EC50 value for cancer cells) of ethyl acetate metabolites of Chaetomium interruptum towards HepG-2, PC3 and HCT-116 was 16.91, 11.51 and 8.2, respectively.
The chromatographic separation by LC/MS reported that, Tributyl acetyl citrate, Hexanedioic acid, 2,4-ditert-butylphenol,1,2-benzene dicarboxylic acid and 1-Hexadecanol recorded the highest content of separated compounds respectively.