الفهرس 
Introduction
CHAPTER I: Extraction, fractionation and isolation of constituents of Nephthea mollisOnly 14 pages are availabe for public view
 |  | from | 170 |  |  |
| | | from | 170 | | |
Abstract
This is the first metabolomic analysis of the crude ethanol extract of Nephthea mollis. The chemical profiling of dereplicated compounds was consistent with literature data on secondary metabolites of N. mollis. The dereplicated compounds were identified using online databases. Twenty two compounds were identified based on their mass fragments. Most of the identified compounds (17 compounds) were identified as sesquiterpenes compounds, in addition to few diterpenes and terpenoid-related carboxylic acid.
Identification of the isolated compounds from petroleum ether fraction of the total ethanol extract of Nephthea mollis
Eight compounds were isolated from petroleum ether fraction of the total ethanol extract. The structure elucidation was performed on the basis of various spectroscopic methods and the isolated compounds are listed in (Table 22).
This is the first metabolomic analysis of the crude ethanol extract of Nephthea mollis. The chemical profiling of dereplicated compounds was consistent with literature data on secondary metabolites of N. mollis. The dereplicated compounds were identified using online databases. Twenty two compounds were identified based on their mass fragments. Most of the identified compounds (17 compounds) were identified as sesquiterpenes compounds, in addition to few diterpenes and terpenoid-related carboxylic acid.
Identification of the isolated compounds from petroleum ether fraction of the total ethanol extract of Nephthea mollis
Eight compounds were isolated from petroleum ether fraction of the total ethanol extract. The structure elucidation was performed on the basis of various spectroscopic methods and the isolated compounds are listed in (Table 22).
Part II
Biological and Docking studies of Nephthea mollis
Chapter I
Evaluation of antitrypanosomal activity and in silico docking study of dereplicated compounds of metabolomics analysis of total ethanol extract of Nephthea mollis
A- Antitrypanosomal activity:
Using the protocol of Huber and Koella (Huber and Koella, 1993), the crude ethanol extract and its derived fractions; petroleum ether and ethyl acetate of Nephthea mollis were tested for their in vitro antitrypanosomal effects against Trypanosoma brucei. The results indicated that the crude ethanol extract and ethyl acetate fraction exhibited remarkable activities with IC50 values of 6.4 and 3.7 µg/mL, respectively. However, the petroleum ether fraction showed weak antitrypanosomal activity, with an IC50 value of (>50 µg/mL).
B- In silico molecular docking study for antitrypanosomal activity:
Twenty two dereplicated compounds of Nephthea mollis were docked using in silico MD simulations within ODC (PDB ID: 1NJJ) active sites. Docking of dereplicated compounds within ODC active sites revealed a number of interesting observations, which can explain the antitrypanosomal activities of crude ethanol extract and ethyl acetate fraction; first of all, binding free energy for five of the identified compounds (2-deoxy-12-ethoxy-7-O-methyl lemnacarnol, nephthenol, 4α-O-acetyl-selin-11-en, eudesma-4,7(11)-diene-8β-ol, and chabrolidione A) were better than ORX. Additionally, as found with ORX, most of the test compounds showed either H-bonding and/or H-pi binding interactions with key amino acid residues: GLU 274, ARG 277, and HIS 197. Overall, the docking results demonstrated how well the overlay of the dereplicated compounds of Nephthea mollis with ORX within ODC active sites, which could be used for explaining their antitrypanosomal activity.
Chapter II
Evaluation of cytotoxic activity and in silico docking study of dereplicated compounds of metabolomics analysis of total ethanol extract of Nephthea mollis
A) Cytotoxic activity:
The in vitro tumor cell growth inhibitory activity of the crude ethanol extract and its derived fractions, petroleum ether and ethyl acetate, obtained from N. mollis, was evaluated against three human cancer cell lines (HePG-2, MCF-7, and CACO-2) using the MTT assay. The results showed that the ethyl acetate fraction of the soft coral N. mollis was the most potent cytotoxic agent against all tested cell lines, with the maximum activity observed against HePG-2 followed by MCF-7 and CACO-2, with IC50 values of 5.3, 6.7, and 7.4 µg/mL, respectively. In comparison, the IC50 value of doxorubicin was 2.6, 2.2, and 3.2 µg/mL against HePG-2, MCF-7, and CACO-2 cell lines, respectively. In addition to, the crude ethanol extract obtained from N. mollis also exhibited strong activity against all tested cell lines, with IC50 values of 11.6, 14.2, and 13.4 µg/mL against HePG-2, MCF-7, and CACO-2 cell lines, respectively. However, the petroleum ether fraction showed weak activities against all tested cell lines, with IC50 values (>50 µg/mL).
B- In silico molecular docking study for cytotoxic activities
MD simulations were performed on dereplicated compounds of the total ethanol extract of N. mollis to explore their possible mechanism as cytotoxic agents. MD simulations were performed within 3 active sites (1M17, 3RCD, and 2R4B), which are commonly found within HepG-2, MCF-7 and CACO-2 cancer cell lines, Overall, all the dereplicated compounds showed good interactions profile within 3 active sites used for MD simulations especially compounds; 1S,4R,5S-guia-6,9-dien-4-ol, chabrolidione A, nephthediol and nephtheoxydiol.
Chapter III
Evaluation of anti-HCV activity and in silico docking study of dereplicated compounds of metabolomics analysis of total ethanol extract of Nephthea mollis
A- Anti-HCV activity:
The anti-HCV NS3 helicase and protease activities of the crude ethanol extract and its derived fractions, petroleum ether and ethyl acetate, obtained from N. mollis were evaluated in vitro. The results showed that the petroleum ether fraction exhibited the highest inhibitory action against HCV NS3 helicase, with an IC50 value of 1.32, and protease, with an IC50 value of 4.32. The crude ethanol extract exhibited moderate inhibitory activities against HCV NS3 helicase and protease, with IC50 values of 2.24 and 16.76, respectively. The ethyl acetate fraction exhibited weaker inhibitory activities against HCV NS3 helicase and protease compared to the petroleum ether fraction and crude ethanol extract.
B- In silico molecular docking study for anti-HCV activity
MD simulations were performed on dereplicated compounds of N. mollis to explore their possible mechanism as anti-HCV agents. The dereplicated compounds of the N. mollis were docked within 2 active sites; HCV NS3-4A protease-helicase with a co-crystallized ligand, F9K (PDB ID: 4A92) and HCV H184N mutant of pentaerythritol tetranitrate reductase with a co-crystallized ligand, FMN (PDB ID: 3P82). Most of the dereplicated compounds showed medium binding score (S) within both 2 active sites.
Chapter IV
In silico molecular docking study of some of the isolated compounds from petroleum ether fraction of the total ethanol extract of Nephthea mollis for antitrypanosomal, cytotoxic and anti-HCV activities
Running MD simulations within active site of ornithine decarboxylase (1NJJ), epidermal growth factor receptor (1M17), and hepatitis C virus protease (4A92) for 5 isolated compounds revealed interesting results that worth mentioning; 4 of them have better docking score than co-crystallized ligand within ornithine decarboxylase active site. Also, two compounds; 24-methylenecholesterol, and litosterol showed moderate and comparable docking score to co-crystallized ligand within epidermal growth factor receptor active site. However, all the compounds exhibited moderate docking scores within the active site of the hepatitis C virus protease.