الفهرس 
Introduction
- Chapter I: Review of literature sponges (Porifera) of the order DictyoceratidaOnly 14 pages are availabe for public view
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Abstract
Spongia irregularis is a marine sponge belonginig to family Spongiidae, order Dictyoceratida. It is variable in shape characterized with black surface and brown internal tissues with some reddish darker patches. S. irregularis distributed in many locations in the Red Sea. Also, it has been reported in Eritrea and the Indian and Pacific oceans.
This work consists of two parts:
Part I: Chemical and biological investigations of the marine sponge Spongia irregularis including:
• Preliminary phytochemical screening of the marine sponge, Spongia irregularis.
• Extraction, fractionation and isolation of secondary metabolites of Spongia irregularis.
• Identification of the isolated compounds from Spongia irregularis.
• Cytotoxic potential of Spongia irregularis supported by metabolomics analysis and molecular docking studies.
• Anti-HCV potential of Spongia irregularis supported by molecular docking studies.
• Anti-covid -19 molecular docking studies of the marine sponge Spongia irregularis.
Part II: Chemical and biological investigations of the marine sponge Spongia irregularis-associated actinomycetes including:
• Metabolomics analysis of the marine sponge Spongia irregularis-associated actinomycetes.
• Multivariate data analysis of the marine sponge Spongia irregularis-associated actinomycetes.
• Evaluation of the cytotoxic activities of the marine sponge Spongia irregularis-associated actinomycetes.
• Evaluation of the anti-HCV activities of the marine sponge Spongia irregularis-associated actinomycetes.
Part I
Chapter I
Preliminary phytochemical screening of the marine sponge Spongia irregularis
The air-dried powdered sponge Spongia irregularis was subjected to preliminary phytochemical screening for their metabolites. The results revealed that the powdered marine sponge Spongia irregularis contains carbohydrates, nitrogenous compounds and sterols and/or triterpenes. On the other hand, it is free from crystalline sublimate substances, flavonoids, saponins, tannins and cyanogenic glycosides.
Chapter II
Extraction, fractionation and isolation of secondary metabolites of the marine sponge Spongia irregularis
The sponge body was air dried and reduced to fine powder producing 200 g weight. It was extracted using 95% ethanol, then the dried ethanolic extract (15.3 g) was further suspended in the least amount of distilled H2O, transferred to a separating funnel, and fractionated using different organic solvents (petroleum ether, ethyl acetate (EtOAc) and n-butanol) to afford three successive fractions, petroleum ether fraction (3.8 g), EtOAc fraction (1.06 g) and n-butanol fraction (3.41 g).
1- Column chromatographic fractionation of the petroleum ether fraction:
The petroleum ether fraction (3.8 g) was gross fractionated using silica gel column chromatography. Firstly, petroleum ether was used as an eluent followed by petroleum ether-ethyl acetate gradient elution. Similar fractions were combined together and concentrated affording six subfractions (I-VI). The subfractions II (820 mg), III (460 mg), IV (420 mg) and V (1.8 g) were separately subjected to further chromatographic purification on silica gel affording thirteen compounds (1-13).
2- Column chromatographic fractionation of the ethyl acetate fraction:
The ethyl acetate fraction (1.06 g) was fractionated using size exclusion CC using methanol as the eluting solvent. Similar fractions were combined together and concentrated resulting in three subfractions (I - III). The subfractions I (480 mg) and II (210 mg) were further chromatographed using a silica gel CC and dichloromethane-methanol as a mobile phase (gradient elution) resulting in isolation of five compounds (14-18). In addition, the subfraction III (100 mg) was further purified on Sephadex LH-20 CC using methanol as the eluting solvent, resulting in the isolation of compound 19.
Chapter III
Identification of the isolated compounds from the marine sponge Spongia irregularis
The structure elucidation of the isolated compounds was performed on the basis of various spectroscopic methods including: (1H-NMR, 13C-NMR, DEPT, APT, HR-ESI-MS-MS and EI-MS), in addition to comparison of the physical, chemical, and chromatographic characters of the isolated compounds with those of the available literature.
Chapter IV
Cytotoxic potential of the marine sponge Spongia irregularis supported by metabolomics analysis and molecular docking studies
Metabolomic profiling of the marine sponge Spongia irregularis using LC-HR-ESI-MS for dereplication purposes was carried out. The LC-HR-ESI-MS profiling of S. irregularis revealed the presence of different types of terpenes, nitrogenous compounds, and other miscellaneous compounds. Also, evaluation of the cytotoxic activities of the total extract and different fractions were performed against three different cancer cell lines. Results showed that the highest cytotoxic activities against HepG-2, MCF-7, and CACO-2 cell lines were displayed by the n-butanol fraction with IC50 values of 9.6±0.02, 4.3±0.10 and 5.6±0.03 µg/mL, respectively. The identified compounds were docked for binding affinity to MSK1. Results showed that methyl 2-(palmitamidomethyl) acrylate, spongiacysteine and Spongiain E displayed moderate to high binding affinity to MSK1.
Chapter V
Anti-HCV potential of the marine sponge Spongia irregularis supported by molecular docking studies
Evaluation of the anti-HCV activity of the total extract and its different fractions were performed. The results demonstrated that the n-butanol and ethyl acetate fractions exhibited high anti-HCV activity with IC50 of 3.3±0.08 and 12.6±0.05 µM, respectively. Molecular docking calculations revealed the high binding affinity of nakijiquinone F against the allosteric pocket of HCV NS3–NS4A and the active site of HCV NS3 helicase. Molecular dynamics simulations, followed by molecular mechanics-generalized born surface area energy calculations, demonstrated the structural and energetic stability of nakijiquinone F in complex with HCV targets.
Chapter VI
Anti-covid-19 metabolites from the marine sponge Spongia irregularis supported by molecular docking studies
The compounds, which were isolated from petroleum ether fraction, were identified using different NMR spectroscopic techniques and mass analysis and were docked against two viral proteases 3CLpro and PLpro, which are promising targets in discovery of anti-COVID 19 drug candidates. The results revealed that compounds 3 (palmitic acid), 4 (octadeca-9-enoic acid), 5 (N-(2-hydroxyhexadecanoyl)-2-(aminomethyl)-2-propenoic acid methyl ester), 6 (N-pentadecanoyl-2-(aminomethyl)-2-propenoic acid) and 12 (mooloolabene C) exhibited reasonable binding affinities with different amino acid residues via forming hydrogen bonds or hydrophobic interactions. Thus, natural products derived from marine sponges can be effective candidates and new agents against COVID-19 that require further in vitro and in vivo studies.
Part II
Chapter I
Metabolomics analysis of the marine sponge Spongia irregularis-associated actinomycetes
Three actinomycetes belonging to the genera Rhodococcus, Streptomyces and Micromonospora were recovered from the marine sponge Spongia irregularis. Applying OSMAC approach, each strain was allowed to grow on four different media (M1, ISP2, OLIGO and MA) resulting in twelve extracts. All extracts were subjected to metabolomics analysis using LC-HR-ESI-MS for dereplication purposes. Results revealed the presence of a wide range of metabolites within different extracts using the four mentioned media.
Chapter II
Multivariate data analysis of the marine sponge Spongia irregularis-associated actinomycetes
Multivariate data statistical analysis was carried out using MetaboAnalyst 4.0 web server for the differentiation between extracts. The variations detected between extracts could be attributed to the great effect of growth media in induction of variable metabolites; highlighting the power of OSMAC approach in the discovery of microbial secondary metabolites.
Chapter III
Evaluation of the cytotoxic activities of the marine sponge Spongia irregularis-associated actinomycetes
The cytotoxic activity of the 12 total extracts of S. irregularis-associated actinomycetes were evaluated against three different cell lines (HepG-2, CACO-2 and MCF-7 cell lines). The results showed that the total extract of Micromonospora sp. UR44 using ISP2 medium exhibited high cytotoxicity against HepG-2, CACO-2 and MCF-7 cell lines with IC50 of 5.6 ± 0.09, 7.9 ± 0.12 and 8.9 ± 0.01 µg/mL, respectively, while the extracts of the same strain using M1, OLIGO or MA media were inactive. On the other hand, the total extracts of Streptomyces sp. UR32 and Rhodococcus sp UR21 using M1 medium were inactive while extracts of other media showed different cytotoxicities against the three cell lines. The various results for each strain clarify that using different media consequently induces production of different metabolites.
Chapter IV
Evaluation of the anti-HCV activities of the marine sponge Spongia irregularis-associated actinomycetes
The anti-HCV activity of the 12 total extracts of S. irregularis-associated actinomycetes were performed. The results showed that the total extracts of Micromonospora sp. UR44 using ISP2 and OLIGO media exhibited anti-HCV activity with IC50 of 4.5 ± 0.22 µM and 3.8 ± 0.18 µM, respectively. Also, the total extract of Streptomyces sp. UR32 using ISP2 medium exhibited anti-HCV activity with IC50 of 5.7 ± 0.15 µM. On the other hand, the other nine extracts were inactive.