الفهرس 
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Abstract
Phytochemical and Biological Study on Certain Plants belonging to
Family Myrtaceae
Family Myrtaceae comprises 3000 species distributed in 100 genera, including Eucalyptus, Clove, and Guava. They have a wide distribution in tropical and sub-tropical regions of the world (Evan, 2002). The family yields some economic products including essential oils used for perfumery and medicinal purposes (Evans, 2002). Eucalyptus is a diverse genus of trees comprising 800 species (Coppen, 2002). The leaves of some species are used for the production of essential oil. The major constituent of the oil is eucalyptol (cineol), which has expectorant and antiseptic properties (Low et al., 1974; Hasegawa et al., 2008). E. gomphocephala DC. is an evergreen tree, indigenous to the southwest of western Australia (Hillis, 1967). E. camaldulentis Dehnh., known as red river gum is an evergreen tree native to Australia (Quattrocchi, 2000).
The scope of the work presented in this thesis aims at investigating promising biological activity of two Eucalyptus species (E. gomphocephala, E. camaldulensis) and to study their phytochemical composition, in order to identify the compounds responsible for bioactivity. Though many phytochemical constituents and valuable medicinal uses were reported from different plants belonging to this genus, yet only one report concerning the phenolic composition of E. gomphocephala was traced in the available literature (Hillis, 1967). This necessitated an in-depth biological and phytochemical study on this selected plant.
The present study comprised two parts:
1. Phytochemical investigation of the aqueous acetone extracts of E. gomphocephala and E. camaldulensis.
2. Biological screening of the extracts, fractions and isolated compounds.
PART I
The phytochemical study of E. gomphocephala and E. camaldulensis included preliminary phytochemical screening, fractionation of aqueous acetone extracts using different chromatographic techniques and structure characterization of the isolated components using different spectroscopic analyses. This can be summarized as follows:
1. Preliminary phytochemical screening
The leaves of E. gomphocephala and E. camaldulensis revealed the presence of the following constituents, flavonoids, hydrolysable tannins, carbohydrates and/ or glycosides, sterols and/ or triterpenes.
2. Phytochemical investigation of the aqueous acetone extracts
The 70% aqueous acetone extract of E. gomphocephala was fractionated using solvent-solvent partitioning to obtain three fractions. The EtOAc fraction was shown to have a variety of phenolic compounds in quite high concentration. The EtOAc fraction also exhibited the highest antioxidant activity when compared to the other fractions. Based on these findings, it was felt reasonable to carry out further phytochemical and biological studies on the EtOAc fraction. A sample of the EtOAc part was fractionated on a column packed with Sephadex LH-20. The elution process was started with water, followed by H2O-MeOH mixtures of decreasing polarities to yield 8 sub-fractions (I-VIII). In the same way, the 70% aqueous acetone extract of E. camaldulensis was fractionated to yield 4 sub-fractions. The fractions were subjected to high performance liquid chromatography, coupled to photodiode-array and electrospray ionization mass spectrometric analysis (HPLC–PDA–ESI/MS/MS) in order to obtain a tentative identification of their components. The employed method was optimized for separation and identification of different compounds including ellagitannins, flavonoids, phloroglucinol derivatives and galloyl esters. The PDA data were useful for the identification of the class of the phenolic compounds; whereas the MS data provided further structural characterization. This work represents the first study that utilizes the HPLC–PDA–ESI/MS/MS technique for in-depth identification of the phenolic constituents of both plants.
Fractionation of the EtOAc soluble part of E. gomphocephala using different chromatographic techniques resulted in the isolation of ten phenolic compounds, including two new natural compounds and one compound reported for the second time from nature. It is worth noting that it is the first study demonstrating the isolation and structure elucidation of these ten compounds from E. gomphocephala. The structures of the phenolic compounds were elucidated on the basis of 1D and 2D NMR (1H, 13C, 1D-TOCSY, DQF-COSY, HSQC, HMBC) and ESI-MS/MS.
The two new compounds were identified as:
Compound (3): 2,4,6-trihydroxy-5-methyl-acetophenone 2-O-β-D-glucopyranoside
Compound (8): benzyl 3’,4’,6’-tri-O-galloyl-β-D-glucopyranoside
Compound (1) was identified as 2,4,6-trihydroxy-3-methyl-benzaldehyde 2-O-β-D-glucopyranoside, which represents the second report of this naturally occurring compound.
Compound (10): camptothin B, an ellagitannin dimer, and compound (2): brevifolincarboxylic acid were reported for the first time from the genus Eucalyptus.
Compound (5): benzyl 6’-O-galloyl-β-D-glucopyranoside and compound (6): quercetin 3-O-(2’’-O-galloyl)-α-L-arabinopyranoside, were reported for the second time from the genus.
The other compounds; methyl gallate (4), tellimagrandin I (7) and 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (9) were isolated for the first time from the selected plant E. gomphocephala.
PART II
Biological activity of the aqueous acetone extracts, fractions obtained from the crude extracts as well as the isolated pure compounds of E. gomphocephala was investigated regarding:
1. Determination of the antioxidant activity using different assays:
1,1-diphenyl-2-picrylhydrazyl (DPPH•), hydroxyl radical, superoxide anion radical scavenging assays and determination of the total phenol and flavonoid contents.
2. Cytotoxicity of the different samples was tested against cancer cell lines using the sulphorhodamine-B (SRB) to assess cell viability.
3. Determination of the anti-biofilm activity of the different samples of E. gomphocephala against Staphylococcus aureus biofilms.
4. Determination of the acetylcholinesterase inhibitory activity of E. gomphocephala aqueous acetone extract.
This work represents the first study that tests biological activities of E. gomphocephala. Also, the antioxidant activity and the cytotoxicity of most of the isolated compounds were tested for the first time. Moreover, the cytotoxic effect and the antioxidant activity of E. camaledulensis were tested and compared to those of E. gomphocephala.
The total extracts, all the obtained fractions and the isolated compounds are tested for their antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH•), hydroxyl radical and superoxide anion radical scavenging assays. The EtOAc fraction exhibited strong hydroxyl radical and DPPH• radical scavenging activities (IC50 values of 2.78, and 10.77 µg/ml respectively) when compared to the total extract and the other fractions, which could be attributed to its high phenol content. Based on these findings, we have attempted to carry out further phytochemical and biological studies on the EtOAc fraction.
The cytotoxicity of the EtOAc fraction of E. gomphocephala and total aqueous acetone extract of E. camaledulensis was evaluated on MCF-7, Hep-2, HepG-2, HeLa, HCT-116 and Caco-2 cell lines. The results indicated that the EtOAc fraction reduced viability of all cell lines in a dose-dependent manner after a continuous exposure during a 48 h period. The cytotoxic effect was greater on MCF-7 and HepG2 cell lines (IC50: 38.8 and 28.0 µg/ml). Therefore, these two cell lines were selected for further testing of the cytotoxic effect of the different fractions and the isolated compounds obtained from the EtOAc fraction implementing the bioactivity-guided fractionation approach. The fractions exhibited more cytotoxicity when compared with their individual components, which was attributed to the presence of a combination of different phytochemicals in these fractions which have synergestic action. The results indicated that the aqueous acetone extract of E. camaledulensis reduced viability of all cell lines in a dose-dependent manner. The cytotoxic effect of the total extract was greater on MCF-7 and HCT-116 cell lines (IC50: 36.5 and 33.3 µg/ml).
The tested samples of E. gomphocephala showed no effect against Staphylococcus aureus biofilms. Also, the aqueous acetone extract of E. gomphocephala exhibited weak acetylcholinesterase inhibitory activity.