الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 178 |  |  |
| | | from | 178 | | |
Abstract
General Summary
A Comparative study on the Chemical and Biological profiling of Acacia species growing in Egypt.
A widespread interest in herbal drugs is witnessed lately because of its safety, being inexpensive and having minimal adverse effects, however there is a need for documentation of research work carried out on traditional medicines. The folk remedies from plants have always showed the path to the scientists to search for new medications and newer drug molecules in order to maintain and promote healthy life.
Acacia is the second largest genus in the Leguminosae family, comprising more than 1350 species worldwide with members found in almost all habitats. This genus possesses diverse biological activities as well as a group of important phytochemical constituents. The three species Acacia longifolia, Acacia tortilis and Acacia farnesiana were chosen for this study since no compiled information representing the effect of extracting solvent and seasonal variation on the chemical composition and consequently the anti-inflammatory activity were traced in literature.
The present study is divided into four Chapters:
1. Chemical profiling of the leaves and bark of Acacia farensiana , Acacia tortilis and Acacia longifolia
2. Biological investigation of Acacia farnesiana, Acacia tortilis and Acacia longifolia
3. Optimization modeling
4. Chemometric data anlaysis on the Acacia leaves
Chapter 1: Chemical profiling of the leaves and bark of Acacia farensiana ,Acacia tortilis and Acacia longifolia
This includes:
1. Phenolic characterization of the Acacia species
TLC Screening
There was a noticeable difference in the number of spots and the colour among the three species. The colour intensity of the spots in each species varied according to the season and solvent used for extraction showing which indicates variation in concentrations.
Identification of Phenolic compounds
• LC-MS Chemical Profiling
The analysis showed that the leaves of the three species varied in composition when compared to each other; however there was no variation in the compounds with change of season or extracting solvent used for each species. Using HRESIMS and UV data from literature the constiuents of the extracts were identified. Rutin was the common constituent found in the leaves extract of the three sepcies. Vicenin 2, isoquercetrin, kaempferol O-rhamno-O-heoxoside, and isorhamnetin O-rutinoside were identified in the leaves of Acacia farnesiana. In Acacia longifolia leaves catechin, myricetin hexoside, myricetin 3-rhamnoside, kaempferol 3-O glucoside, quercitrin, kaempferol hexoside and luteolin were identified while in Acacia tortilis kaempferol 3-rutinoside, myricetin-3-O rutinoside and kaempferol 3-rutinoside were detected.
The peak area% of each peak in the chromatogram was calculated as a percentage of the total area of all peaks to provide a suitable approximation of the relative amounts of components in each extract. The concentration of each compound in the extracts showed a significant difference with change of season and extraction solvent.
• Characterization and identification of rutin
Compound 11 was isolated using column chromatography and was confirmed to be rutin using NMR data.
2. Phenolic Quantification of the Acacia species
Quantitative determination of total polyphenolic content, rutin and catechin.
Rutin concentration in the leaves of A.tortilis was the highest when compared to the other two species in both seasons and using 50% ethanol and 70% ethanol as extraction solvents. In regards to collection season, rutin was found highest in winter using 70% ethanol in A. tortilis (245.42±6.82 ug/mg extract) and A.longifolia (3.00±0.00 ug/mg extract) while rutin was highest in spring in A. farnesiana (60.87±0.43ug/mg extract). Based on the gallic acid calibration curve, total polyphenol content (TPC) was determined to be highest in the leaves of all species when collected in spring and extracted with 70% ethanol. A. longifolia showed the highest TPC regardless of collection season and extraction solvent. Regarding the bark extracts, the concentration of catechin and the total polyphenolic content was highest in A. tortilis regardless of the collection season and extraction solvent. Catechin concentration was found particularly high in spring using 50% ethanol as an extraction solvent in all species.
Chapter 2: Biological investigation of Acacia farnesiana, Acacia tortilis and Acacia longifolia
This includes:
Anti-inflammatory activity of Acacia extracts
Evaluation of the in vitro anti-inflammatory activity
Higher inhibition percentage of COX-1 and COX-2 enzymes was observed when compared to indomethacin and NS-398 respectively as positive controls. A.tortilis leaves showed highest inhibition of COX-1 enzyme when compared to the other two species which corresponds to the rutin concentration. A. farnesiana showed a higher inhibition on COX-1 (70.31 ± 8.86%) in spring with 70% ethanol than winter (61.63 ± 8.05%) which is also a reflection of the rutin concentration and total polyphenolic content. A. farnesiana showed highest inhibition on COX-2 enzymes. The bark extracts of the three species collected in spring and extracted with 50% ethanol showed a slightly higher inhibition percentage of COX-1 which is in coherence with the catechin concentration.
Evaluation of the in vivo anti-inflammatory activity:
A. Carrageenan paw edema evaluation
50 % AF bark (s) was evaluated for its ability to inhibited carrageena paw edema The anti-inflammatory action was exerted during the second phase of inflammation with a maximum swelling inhibition of 34% at 120min and 37.8% at 240min for doses of 100 and 200 mg/kg, respectively. Compared to the control group, Diclofenac sodium at 10 mg/kg, the bark extract at both doses appeared to have a similar inhibitory activity on carrageenan-induced paw edema.
B. Histological Examination of paw tissues
Histological images showed decreased edema, congestion and cell infiterationwhen treated with both doses of the extract 50% AF bark(s) when compared to the control.
Chapter 3: Optimization modeling
For better visualization and utilization of the results, an optimization model using the findings of the chemical and biological data was developed.
1. The model showed that collection of the three species in the spring season and the use of the 70% solvent system resulted in a synergistic effect on the total phenolics content.
2. A. farnesiana scored the highest COX2 inhibition specifically when collected in winter and extracted using 50% solvent system. The collection season interacted with the solvent system, where changing the solvent system from 50% to 70 % in winter increased the mean inhibition of COX2 enzyme, and an opposite result was obtained in spring where a decrease in COX2 inhibition resulted.
3. On the other hand regarding the barks extracts, optimum results were obtained when A. farnesiana was collected in spring and extracted using 50 % ethanol.
4. A strong inverse correlation between TPC and the mean inhibition of COX2 in case of the leaves was observed (r = 0.8) and a medium inverse correlation (r = 0.55) between the same two responses in case of the barks.
Chapter 4: Chemometricdata analysis of Acacia leaves
When Cluster analysis was performed on the leaf samples data (TPC, rutin concentration, and COX 1 and COX 2 inhibition) it showed discrimination that was in coherence with the taxonomical classification of the species. Two main clusters were revealed; that of A. farnesiana and A. tortilis on one hand and that of A. longifolia in another class. This is in coherence with the grouping of the former speci in the Vachellia taxon as mentioned previously while that of A. longifolia in the Acacia taxon.No meaningful clustering was observed with the data obtained for the bark samples due to the complexity of the bark tissue in regards to phytoconstituents and tissue variability.
Examination of the PCA score plot described by PC1 and PC2 and accounting for 72% and 27% of the variance respectively revealed that the species are differentiated with each one in a separate quadrant. Rutin is the discriminating factor of A. tortilis while myricetin is the component responsible for A. longifolia segregation. A. farnesiana is segregated as a result of high content in vicenin and isorhamnetin. These results are in coherence with the taxonomical classification mentioned previously.