الفهرس 
Phytoconstituents of the methanolic leaf extract of Araucaria bidwillii, family AraucariaceaeOnly 14 pages are availabe for public view
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Abstract
General Summary
Phytochemical and Biological Studies on Araucaria bidwillii, Family Araucariaceae, Cultivated in Egypt
Family Araucariaceae involves three genera Araucaria, Agathis and Wollemia. The three genera comprise approximately 41 species, 19 of these species belong to genus Araucaria. Our selected species is Araucaria bidwillii since few reports could be traced in literature concerning the chemical composition of this plant. John Carne Bidwill brought a live specimen to London where it was studied and named Araucaria bidwillii after him by the English botanist William Jackson Hooker in 1843.
The main objective of this study was the determination of the phytochemical composition of Araucaria bidwillii Hook methanol leaf extract and investigation of its biological activity.
This study was divided into three main chapters:
Chapter 1: Phytoconstituents of the methanol leaf extract of Araucaria bidwillii, family Araucariaceae
Chapter 2: Biological screening of Araucaria bidwillii leaves, family Araucariaceae
Chapter 3: Chemical composition and biological activity of the essential oil isolated from Araucaria bidwillii leaves, family Araucariaceae
Chapter 1: Phytoconstituents of the methanol leaf extract of Araucaria bidwillii, family Araucariaceae
This chapter included two parts:
a- Phytochemical screening of Araucaria bidwillii leaves
The results revealed the presence of flavonoids, sterols and/or triterpenes, carbohydrates and/or glycosides and tannins with absence of saponins, cardiac glycosides, alkaloids, anthraquinones and coumarins.
b- Phytochemical study of Araucaria bidwillii leaves:
Araucaria bidwillii methanol leaf extract was prepared and fractionated with petroleum ether, ethyl acetate and n-butanol respectively. Both the ethyl acetate and n-butanol fractions were subjected to further chromatographic fractionation. These fractions were manipulated through column chromatography for several times leading to the isolation of 13 compounds.
These compounds were identified by 1H NMR, 13C NMR, APT and 2D-NMR including 1H-1H COSY, HSQC, HMBC and ROESY spectra after comparison with previously reported data. Four of the isolated compounds were found to be new natural products which have not been reported before to occur in nature.
The new compounds were identified to be:
Compound (1): 7-Hydroxy-labda-8(17),13(16),14-trien-19-yl-7′-O-methyl-(E)-coumarate
Compound (2): 7-Hydroxy-labda-8(17),13(16),14-trien-19-yl-7′-O-methyl-(Z)-coumarate
Compound (3): 7-Hydroxy-labda-8(17),13(16),14-trien-19-yl-(E)-p-coumarate
Compound (4): 7-Hydroxy-labda-8(17),13(16),14-trien-19-yl-(Z)-p-coumarate
The other compounds were identified to be:
Compound (5): 7-Oxodehydroabietic acid
Compound (6): 3-α-Acetyl-11-keto-β-boswellic acid
Compound (7): Phloretic acid
Compound (8): Agathisflavone-4′,7,7′′-trimethyl ether
Compound (9): Cupressuflavone-4′,7,7′′-trimethyl ether
Compound (10): β-Sitosterol-3-O-glucopyranoside
Compound (11): Agathisflavone-4′,7′′-dimethyl ether
Compound (12): 7-O-Methyl-6-hydroxyapigenin
Compound (13): 4′,4′′′- Di-O-Methylamentoflavone
The first 10 compounds were isolated from the ethyl acetate fraction while the last 3 compounds were isolated from n-butanol fraction.
Chapter 2: Biological screening of Araucaria bidwillii leaves, family Araucariaceae
Biological screening of Araucaria bidwillii methanol leaf extract, its fractions and isolated compounds was done including anti-inflammatory, antimicrobial and cytotoxic activities.
a- In vitro anti-inflammatory activity:
The anti-inflammatory activity of A. bidwillii methanol leaf extract and n-butanol fraction was evaluated through measuring the levels of IL-1β, IL-6 and TNF-α in supernatant media of PHA-stimulated PBMCs.
The effect on IL-1β level: Cells challenged with PHA showed significant increase in IL-1β level about 5 fold compared with that of the untreated control cells. Indomethacin (10 µM) significantly decreased IL-1β level by about 68.7% compared to PHA-challenged group. Treatment of cells with A. bidwillii extract and n-butanol fraction resulted in concentration-related reduction of IL-1β level compared to PHA-challenged cells. At (100 µg/ml) concentration, the extract and its fraction significantly reduced IL-1β level from PHA-challenged cells by about 58.4% and 44.8%, respectively, however at the lower concentration (10 µg/ml) A. bidwillii extract has shown higher activity than that of the n-butanol fraction, being able to significantly decrease IL-1β level from PHA-stimulated group by about 43.7% compared to only 11.1% for the fraction.
The effect on IL-6 level: There was concentration-related decrease of IL-6 by treating the cells with A. bidwillii extract and its n-butanol fraction. Interestingly, the butanol fraction at the lower concentration (10 µg/ml) failed to show any significant difference from PHA-stimulated cells in contrast to 54.7% decrease of IL-6 upon treatment with the extract at the same concentration.
The effect on TNF-α level: Both the total extract and n-butanol fraction (at 100 µg/ml) significantly decreased TNF-α level from PHA-treated group by about 56.4% and 33.6%, respectively, however, the tested fraction at (10 µg/ml) has shown insignificant effect on lowering TNF-α level from PHA-stimulated group.
Results showed that the methanol leaf extract of Araucaria bidwillii revealed more potent in vitro anti-inflammatory activity compared to its phenolic-rich n-butanol fraction due to synergism.
In silico molecular modelling was carried out for the 3 isolated compounds from n-butanol fraction on the major enzymes responsible for the occurrence of inflammation which are TNF-α, COX-II and 5-LOX. It revealed that agathisflavone-4′,7′′-dimethyl ether (11) exhibited the fittest binding in TNF-α active sites with free binding energy (∆G) equals to -55.10 kcal/mol.
Meanwhile,7-O-methyl-6-hydroxyapigenin (12) exhibited an inhibitory activity against COX-II comparable to that of indomethacin with ∆G values of -50.46 and -48.74 kcal/mol, respectively. However, agathisflavone-4′,7′′-dimethyl ether (11) and 4′,4′′′-di-O-methylamentoflavone (13) exerted the most potent suppression to 5-LOX activity compared to 7-O-methyl-6-hydroxyapigenin (12) and indomethacin approaching that of nordihydroguaiaretic, the most potent well-known 5-LOX inhibitor
b- Antimicrobial activity:
Araucaria bidwillii methanol leaf extract, fractions and isolated compounds were tested for their antitubercular and antibacterial activities against Gram positive and Gram negative bacteria within a concentration range of 0-100 µM using agar well diffusion method and MICs were determined.
Results showed that none of the tested samples revealed significant activity within the tested concentration range. Araucaria bidwillii methanol leaf extract revealed moderate activity against (Staphylococcus aureus, ATCC 25923), also n-butanol fraction revealed moderate activity against (Staphylococcus aureus, ATCC 700699) while compound (6) (3-α-Acetyl-11-keto-β-boswellic acid) revealed moderate activity against (Enterococcus faecalis 51299).
c- Cytotoxic activity:
Araucaria bidwillii methanol leaf extract, fractions and isolated compounds were assessed for their antiproliferative activities against mouse lymphoma (L5178Y) cell line using the in vitro cytotoxicity (MTT) assay and kahalalide F as a standard antiproliferative agent.
Results revealed that the methanol leaf extract and ethyl acetate fraction showed moderate activity, compounds (3) and (4) revealed EC50 values of 2.22 and 1.42 µM, respectively, which were more potent than the standard drug kahalalide F with EC50 value of 4.30 µM although their respective methylated derivatives (1) and (2) exhibited weak to no activity.
Chapter 3: Chemical composition and biological activity of the essential oil isolated from Araucaria bidwillii leaves, family Araucariaceae
This chapter included two parts:
a- GC/MS analysis of the essential oil
A comprehensive study was done on the essential oil isolated from Araucaria bidwillii leaves to explore its chemical constituents. The essential oil is pale yellow with characteristic odor and lighter than water. The oil was subjected to high-resolution gas chromatographic analysis in order to determine its volatile constituents. One hundred and nine volatile compounds were identified by GC/MS.
Results of the GC/MS analysis of the essential oil showed that the percentage of the identified volatile constituents is 99.34% while that of the unidentified constituents is 0.66%. The major volatile compounds are (+)-beyerene (35.5%), hibaene (26%), (+/-)-(E)-nerolidol (11.6%), kaur-15-ene (3.14%), sclareol (2.34%), 2,3,3-trimethyl-2-(3-methyl-buta-1,3-dienyl)-cyclohexanone (2.27%), kaur-16-ene (2.24%), elixene (1.4%), spathulenol (1.31%), biformene (1.21%) and phyllocladene (1.01%).
b- Investigation of the biological activity of the essential oil
1- Antimicrobial activity
The essential oil isolated from Araucaria bidwillii leaves was tested for its antitubercular and antibacterial activities against Gram positive and Gram negative bacteria within a concentration range of 0-100 µM using the agar well diffusion method and MIC was determined. Results revealed that it showed no antimicrobial activity against the tested organisms.
2- Cytotoxic activity
A. bidwillii leaf essential oil was assessed for its antiproliferative activity against mouse lymphoma (L5178Y) cell line and HepG-2 cells using the in vitro cytotoxicity (MTT) assay. Results revealed that the essential oil showed no cytotoxic activity against the two cell lines.