الفهرس 
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Abstract
Family Fabaceae is one the largest plant families comprising more than 780 accepted genera and over 19,500 species ranging from giant trees to small herbs. Erythrina is one of the genera of family Fabaceae. It embraces 152 accepted species that are widely distributed in tropical and subtropical regions. Erythrina was traditionally used in folk medicine for treatment of different diseases, like skin sores and wounds, trachoma, arthritis, rheumatic disorders, respiratory disorders, tuberculosis, CNS disorders and insomnia. In addition, many studies have reported the cytotoxic activities of different Erythrina extracts and isolated compounds. Erythrina is known for its distinctive alkaloids which are characterized by a unique nucleus, many of which have displayed promising neurological activities.
The work performed in this study aimed at preliminary phytochemical and biological screening of the leaves of different Erythrina plants followed by in-depth phytochemical investigation of the plant extract showing the most promising biological activities using suitable extraction and fractionation approaches to separate its major components and further explore their biological activities.
This thesis includes 31 tables, 43 figures, and 13 photos, along with 286 references to previous studies related to the research topic and conducted work.
The present study included two parts:
1. Phytochemical screening on certain Erythrina species with in-depth phytochemical studies on the alkaloid-rich fraction obtained from the acid base fractionation of the methanol leaf extract of E. corallodendron.
2. Biological investigation on different Erythrina leaf extracts, fractions and isolated compounds.
Part I: Phytochemical investigation on certain Erythrina species
Chapter I: Preliminary phytochemical screening on E. berenices, E. vespertilio and E. corallodendron leaves
1. Phytochemical screening of E. berenices, E. vespertilio and E. corallodendron leaves
The preliminary phytochemical screening revealed that the tested plants’ leaves contain carbohydrates, glycosides, alkaloids, flavonoids, saponins, sterols and triterpenes while both tannins and anthraquinones were absent.
2. Solvent fractionation of the methanol leaf extracts of E. berenices, E. vespertilio and E. corallodendron
E. berenices, E. vespertilio and E. corallodendron methanol leaf extracts were fractionated with n-hexane, dichloromethane, ethyl acetate. TLC analysis of the fractions showed that the dichloromethane fraction of E. corallodendron contains more alkaloids than the other tested fractions.
3. LC-ESI-MS analysis of the dichloromethane fraction of E. corallodendron leaves
The dichloromethane fraction of E. corallodendron was subjected to LC-ESI-MS analysis to determine the nature of its major constituents. The analysis confirmed that the fraction is rich in alkaloids.
Chapter II: Phytochemical studies on E. corallodendron leaves
1. Acid-base fractionation of the methanol leaf extract of E. corallodendron
The concentrated methanol leaf extract of E. corallodendron (172 g) was subjected to acid base fractionation from which two fractions were obtained, a fraction containing the non-polar components of the extract (DCM-I) weighting 150 g, and an alkaloid-rich fraction (AF) weighting 5.32 g.
2. LC-ESI-MS/MS analysis of the alkaloid-rich fraction (AF)
The alkaloid rich fraction (AF) was subjected to chromatographic analysis using UPLC-ESI-MS/MS technique in both ESI +ve and -ve modes. Thirteen different compounds were tentatively identified in AF. All the identified compounds were alkaloids, except for one dihydroxy flavanone dihexoside.
3. Compounds isolation from the Alkaloid-rich fraction (AF)
The alkaloid rich fraction (AF) was subjected to further fractionation and purification which resulted in isolation and structural elucidation of five dienoid erythrinan alkaloids, among which erysovine N-oxide was reported for the second time from nature. The chemical structures of these compounds were identified based on their 1H, 13C-NMR and 2D-NMR (HSQC and HMBC) spectroscopic data after comparison with their previously reported data.
Part II: Biological investigation on certain Erythrina species
Chapter I: Preliminary biological screening on n-hexane, dichloromethane, ethyl acetate and aqueous fractions obtained from the methanol leaf extract of E. berenices, E. vespertilio and E. corallodendron
This chapter includes screening the following activities:
1. Cytotoxicity against Vero and Molt-4 cell lines
The cytotoxicity of the fractions of E. berenices, E. vespertilio and E. corallodendron were evaluated against Vero and Molt-4 cell lines using MTT assay. The aqueous fraction of E. corallodendron presented the lowest toxicity (CC50 = 713.22 μg/mL) on Vero cells compared to the dichloromethane fraction of E. vespertilio which showed the highest toxicity (CC50 value = 92.40 μg/mL) on the same cell line. Regarding Molt-4 cell line, dichloromethane fractions of E. vespertilio and E. corallodendron displayed potent cytotoxicity to these cells with IC50 values of 10.37 and 13.14 μg/mL, respectively.
2. Antiviral activity against HSV 1
The fractions of E. berenices, E. vespertilio and E. corallodendron were tested for their ability to protect Vero cells from HSV 1 infection using MTT assay. Each fraction was tested at its maximum nontoxic concentration on Vero cells. The ethyl acetate fraction of E. vespertilio showed the highest activity where it displayed an antiviral effect of 43.41% at concentration of 62.5 μg/mL, whereas the n-hexane fraction of E. berenices displayed the lowest activity with an antiviral effect of 9.27% at concentration of 125 μg/mL.
3. Anti-inflammatory activity
The fractions of E. berenices, E. vespertilio and E. corallodendron were tested for their ability to inhibit superoxide anion generation and elastase release by the fMLF/CB-stimulated human neutrophils. The n-hexane, dichloromethane fractions of E. corallodendron and E. vespertilio displayed highly potent activity against superoxide anion generation with IC50 values of 0.03, 0.02, 0.41 and 0.07 μg/mL, respectively. Regarding the inhibition of neutrophilic elastase release, the dichloromethane fractions of E. corallodendron and E. vespertilio displayed the highest inhibition of elastase release with IC50 values of 2.58 and 3.38 μg/mL, respectively.
Chapter II: Biological investigations on the methanol leaf extract of E. corallodendron, the fractions obtained from its acid base fractionation and isolated compounds
This chapter includes screening the following activities:
1. Anti-angiogenic activity
The methanol leaf extract of E. corallodendron (TE), the fractions obtained from its acid base fractionation (the non-polar fraction (DCM-I) and the alkaloid rich fraction (AF)), and the isolated compounds from AF were assayed for their inhibitory effect on endothelial progenitor cells (EPCs) growth using SRB assay. AF limited EPCs cells survival to 32% compared to the DMSO control group. Among the tested compounds, erythrinine N¬-oxide displayed the highest percent inhibition for EPCs cells survival with a value of 50% compared to DMSO control group.
2. Anti-inflammatory assay of the isolated compounds
The compounds isolated from the alkaloid rich fraction (AF) were investigated for their effect against superoxide anion generation and elastase release by the fMLF/CB-stimulated human neutrophils. Among the tested compounds, erythrinine N-oxide displayed the highest activity with mean percent inhibition values of 29 and 36.18% for superoxide anion generation and elastase release, respectively.
3. Antiviral activity against SARS-CoV-2 pseudovirus
The methanol leaf extract of E. corallodendron (TE), the non-polar fraction (DCM-I), the alkaloid rich fraction (AF), and the compounds isolated from AF were assayed for their ability to neutralize SARS-CoV-2 pseudovirus infection using luciferase activity assay. TE displayed the highest mean percent inhibition for viral replication in the infected cells with value of 19.03%. Among the tested compounds, erythrinine N-oxide displayed the highest mean percent inhibition for viral replication with a value of 18.20%.
4. In vitro cholinesterase inhibitory activity
The alkaloid rich fraction (AF) was assayed for its inhibitory effect on both AChE and BuChE enzymes. In addition, the compounds isolated from AF were assayed for BuChE enzyme inhibition. The assays were done using modified Ellman’s method. AF displayed relatively higher inhibition for BuChE enzyme compared to AChE enzyme with an IC50 value of 23.71 and 30.31 µg/mL, respectively. Among the tested compounds, erysovine N-oxide (100 µM) displayed the highest mean percent inhibition for BuChE enzyme with a value of 29.56 % compared to DMSO control.
5. In silico computational studies on the isolated compounds
The chemical structures of the compounds isolated from AF were docked into the binding sites of the crystal structure of recombinant human AChE and BuChE enzymes. Erythrinine displayed the best fit with the key residues of both enzymes. Additionally, erythrinine was subjected to molecular dynamics studies to validate its stability in complex with AChE and BuChE which revealed the higher stability of its complex with the latter enzyme. In silico ADME and toxicity studies were performed on all isolated compounds in comparison with donepezil as reference drug. from these studies it was noticed that 8-oxoerythrinine display the safest profile among the tested compounds, however it lacks BBB permeability in contrast to erysovine N-oxide which displays the best ADME profile but high predicted carcinogenicity and oral toxicity.