الفهرس 
Review of LiteratureOnly 14 pages are availabe for public view
Abstract
Family Fabaceae, the third-largest angiosperm family, is comprising about 751 genera and
19,500 species. It presents great morphological and life history diversity and shows a
significant increase in species diversification rate over the last 60 million years compared to
angiosperms as a whole. Genus Albizia is one of the most remarkable lineages of family
fabaceae regarding its variable phytochemical constituents including saponins, phenolics,
alkaloids and terpenes besides its wide range of biological activities including (cytotoxic,
antimicrobial, anti-malarial, antioxidant, antidiabetic, hepatoprotective, antidepressant,
immunomodulatory, and anthelmintic activities). It encompasses approximately 150 species,
typically trees and shrubs native to tropical and subtropical regions of Asia and Africa.
This genus, belonging to family Fabaceae, subfamily mimosoideae, tribe Ingeae, was
subjected to frequent taxonomic changes due to its complex and unstable taxonomic history.
Therefore, accurate identification of closely related species belonging to genus Albizia is a
priority because it plays an important role in the quality control as well as the medcinal
exploitation of the genus. Classification of plant species can be achieved using a variety of
methods including analysis of morphological characteristics, chemical constituents, and
molecular genetic structure variations. Phylogenetic investigations of some Albizia species has
been undertaken by means of multivariate analyses of morphological traits combined to
different DNA molecular analysis techniques. Nevertheless, no reports document the use of
molecular genetic classification tools with chemotaxonomic analysis. The present work
discusses a chemometric comparative study between nine Albizia species cultivated in Egypt;
Albizia lebbeck, A. julibrissin, A. odoratissima, A. procera, A. anthelmintica, A. guachapele,
A. myriophylla, A. richardiana and A. lucidior utilizing the strengths of; Molecular taxonomy
via ISSR and SCoT techniques, chemotaxonomy via selected spectroscopic and
chromatographic techniques and chemometrics for the first time to resolve phylogenetic
relationship among genus Albizia and provide baseline for future breeding programs and can
be similarly applied in other studies. The leaves of different species under investigation were
collected in summer 2017 and subjected to different assays as mentioned in the thesis different
chapters that were divided into:
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Chapter 1: DNA Fingerprinting via ISSR and SCoT Techniques for Resolution of
Phylogenetic Relationship Among selected Albizia Species
The nine Albizia species under investigation were selected to assess their genetic
relationship by using ISSR and SCoT markers. Ten primers, five for each technique, were used
in this assessment. Both techniques exhibited higher percentage of polymorphism of 64.28%
and 67.5% for ISSR and SCoT, respectively compared to the percentage of genetic similarity.
Therefore, both techniques can be used to assess the genetic discrimination among studied
species. The results of both techniques were combined and the resultant dendrogram was
compared to the previous reports in the literature of using different DNA barcoding and
fingerprinting techniques to obtain a reliable taxonomic classification. A. anthelmintica (5)
formed a separate cluster as a monotypic species while remaining eight species exhibited a
certain degree of genetic relatedness depending on their relative distance on dendrogram and
this is in line with previous literature. Closer genetic relatedness can be observed between each
of the following pairs of species; A. myriophylla (7) and A. richardiana (8), A. odoratissima
(3) and A. lebbeck (1), A. lucidior (9) and A. julibrissin (2), finally A. guachapele (6) and A.
procera (4).
Chapter 2: Spectroscopic Analysis of the Leaf Extracts of selected Albizia Species
Coupled to Unsupervised Chemometric Techniques
2.1. UV-Vis Spectroscopic Analysis
The absorption spectra over the range of (250-400 nm) were obtained from triplicate
analysis of the methanolic leaf extracts of selected Albizia species. UV spectroscopic profiling
of selected Albizia species in the specified wavelength range will reflect to a large extent the
phenolic composition of the samples. The obtained data was subjected to multivariate analysis
for better resolution and exploration of the chemical relatedness among different species.
Unsupervised chemometric analysis resulted in the production of PCA score plot and
HCA dendrogram. Each of A. anthelmintica (AA) and A. myriophylla (AM) was forming a
separate cluster positive to PC1. This was in accordance with UV analysis HCA dendrogram
and combined ISSR and SCoT dendrogram that showed A. anthelmintica (5) as monotypic
species arising from separate branch and A. myriophylla (7) in the closest position adjacent to
each others. Moreover, both of A. guachapele (AG) and A. procera (AP) were forming the
same cluster on PCA score plot and this is in accordance with combined ISSR and SCoT
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dendrogram. Finally, A. odoratissima (AO), A. lebbeck (AL), A. lucidior (AU) and A.
julibrissin (AJ) were clustered together at the negative side of PC1 and this was in accordance
with combined ISSR and SCoT dendrogram.
2.2. Quantitative Determination of the Total Phenolic Content of the Leaf Extracts of
Selected Albizia Species
Total phenolic content was calculated according to Folin ciocalteu assay using Gallic
acid as a standard whose calibration curve and linear regression equation used to calculate TPC
among different species expressed as (mg GAE/ g dried extract). TPC values were ranging
from 273.32 to 37.3 (mg GAE/ g dried extract). The highest TPC was exhibited by A.
myriophylla (AM) followed by A. anthelmintica (AA) of values equal 273.32 and 226.49 (mg
GAE/ g dried extract) respectively and this finding is in line with with previously mentioned
UV spectroscopic profile PCA that displays each of them forming a separate cluster
differentiating from other species at the positive side of PC1. In contrast, A. richardiana (AR)
showed the lowest TPC of 37.30 (mg of GAE/g of dried extract) which clustered at the negative
side of PC1 in UV spectroscopic profile PCA.
2.3. Quantitative Determination of the Total Flavonoid Content of the Leaf Extracts of
Selected Albizia Species
Total flavonoid content was calculated according to AlCl3 colorimetric assay using
quercetin as a standard whose calibration curve and linear regression equation used to calculate
TFC among different species expressed as (mg QE/ g dried extract). TFC values were ranging
from 78.91 to 27.32 (mg QE/ g dried extract). The highest TFC was exhibited by A. guachapele
(AG) followed by A. anthelmintica (AA) and A. richardiana (AR) of values equal 78.91, 70.36
and 59.01 (mg QE/ g dried extract) respectively, whereas A. myriophylla (AM) exhibited the
lowest TFC equals 27.32 (mg QE/ g dried extract).
2.4. Multivariate analysis of UV spectroscopic data, TPC and TFC of selected Albizia
Species
Total phenolic and total flavonoid contents expressed in (mg/g) of each extract were
added to the UV scan points from (250-400 nm) as additional variables to generate a new PCA
score plot able to produce more reliable chemical profiling among different Albizia species.
Results can be discussed by observing both of PCA score plot and loading plot.
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Each of A. anthelmintica (AA) and A. myriophylla (AM) is forming a separate cluster
at the negative side of PC1 discriminated from the rest of the species and this is can be
explained according to TPC from loading plot as both species exhibited the highest TPC among
other species. These findings are in accordance with combined ISSR and SCoT dendrogram
which displays a clear genetic relatedness between these two species.
Both of A. guachapele (AG) and A. richardiana (AR) are forming the same cluster at
the positive side of PC1. This clustering can be justified according to TFC on loading plot as
both species exhibited the highest TFC values among other species. A. procera (AP), A.
lucidior (AU), A. julibrissin (AJ), A. lebbeck (AL) and A. odoratissima (AO) are clustered
together at the positive side of PC1 and negative side of PC2 which indicate a strong chemical
proximity among them and this finding can be explained according to UV spectroscopic profile
from the loading plot.
Chapter 3: chromatographic Analysis of the Leaf Extracts of selected Albizia Species
Coupled to Unsupervised Chemometric Techniques
3.1. High-Performance Thin-Layer chromatographic Analysis
HPTLC was utilized to produce an alkaloidal profile of the methanol leaf extracts of
nine species of Albizia as a chemotaxonomical tool in the plant systematic. Samples were
scanned under UV at 254 and 365 nm and the resulted data were subjected to multivariate
analysis. A. procera and A. lucidior showed a strong chemical proximity as they formed the
same cluster at both scanning wavelengths (254 and 365 nm). A. guachapele displayed a
separate cluster at both scanning wavelengths (254 and 365 nm) that indicates its unique
alkaloidal composition. A. julibrissin, A. odoratissima, A. anthelmintica and A. myriophylla
showed certain degree of chemical relatedness as they clustered together at both scanning
wavelengths (254 and 365 nm). Subsequent spraying with dragendorff’s reagent and scanning
in visible region at 550 nm and under UV at 254 nm confirmed the previous findings and
showed the highest alkaloidal content exclusively in A. procera and A. lucidior. HPTLC
analysis results were in accordance with phylogenetic analysis and UV, TPC and TFC
chemometric analysis mentioned in previous chapters.
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3.2. UPLC-ESI/MS/MS Based Metabolic Profiling and Comparative Untargeted
Metabolomic Study of selected Albizia Species
Recent approaches in untargeted metabolomics aiming to analyze, compare and
classify samples with respect to their detected compounds (either known or unknown
compounds). The differences and similarities in phytochemical composition will
necessarily reflect the genetic variability primarily. (LC/MS) is a powerful analytical
technique used in the current work for:
3.2.1. Metabolic Profiling via Identification of Different Compounds in both Negative and
Positive Ionization Modes
Metabolites tentative identification was based on comparing their retention time,
ultraviolet absorption and mass spectra of each eluted compound. A total of 65 compounds
that belong to different classes of secondary metabolites including:
3.2.1.1. Flavonoids: 39 compounds were tentatively identified as:
3.2.1.1.1. Flavonol-O- glycoside, Flavonol 3,7 di-O- glycoside or Flavonol acyl
glycoside: of kaempferol, quercetin and myricetin aglycones.
3.2.1.1.2. Flavan-3-ols: gallocatechin and epigallocatechin.
3.2.1.1.3. Flavone-O or C-glycosides: of apigenin and diosmetin aglycones.
3.2.1.2. Organic, phenolic acids and their derivatives: 20 compounds were tentatively
identified as quinic, caffeic, piscidic, eucomic and ferulic acids or their derivatives.
3.2.1.3. Saponins: 1 compound was tentatively identified as oleanolic acid derivative.
3.2.1.4. Alkaloids: 4 compounds were tentatively identified as budmunchiamine alkaloids.
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Some compounds were reported in genus Albizia for the first time that can be used
as chemotaxonomic markers for a specific species which reported to contain including:
Apigenin 7-O-dihydroferuloyl galactoside and glucoside isomers which were
identified in A.myriophylla and A. lebbeck.
Apigenin 7-O-pentosyl (12) hexoside which was identified in A.myriophylla and
A. lebbeck.
Quercetin 3-O-rutinoside 7-O-deoxyhexoside which was identified in A. procera
and A. lucidior.
Quercetin 3, 7-di-O-hexoside deoxyhexoside which was identified in A. procera
and A. julibrissin.
Quercetin 7-O-feruloyl hexoside which was identified in A. anthelmintica.
Methyl myricetin 7-O-deoxyhexoside which was identified in A.odoratissima
Kaempferol di-7-O-hexoside 3-O-di-deoxyhexoside which was identified in A.
lucidior.
Kaempferol 3-O-neohesperidoside 7-O- hexoside which was identified in A.
lebbeck.
Kaempferol 7-O-neohesperidoside which was identified in A. lebbeck, A.
odoratissima, A. anthelmintica and A. richardiana.
3.2.2. Comparative Untargeted Metabolomic Study Using Multivariate Data Analysis to
Discriminate Between Albizia Species
Combining plant metabolomics with one or more tools of multivariate data analysis
(PCA or HCA) is capable of detecting and comparing the distribution of phytochemical
constituents among different samples. The aligned peak list was obtained from data
processing of the leaf extracts of nine Albizia species in negative ionization mode using
MZmine software. After that, the data was imported to the Unscrambler software for
multivariate data analysis. The resultant PCA score plot can successively discriminate
between different species and the clustering pattern can be justified by the presence of
different compounds displayed in the loading plot. PC1 and PC2 together were
corresponding to 89% of variations between samples.
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A. lebbeck (AL), A. odoratissima (AO) and A. myriophylla (AM) were clustered
together at the negative side of PC1 and positive side of PC2. This is in accordance with
combined ISSR and SCoT dendrogram that showed a strong genetic relatedness between
A. lebbeck (1) and A. odoratissima (3). A. procera (AP), A. lucidior (AU), A. julibrissin
(AJ), A. guachapele (AG) and A.anthelmintica (AA) were clustered together at the positive
side of PC1 and negative side of PC2. This is in accordance with combined ISSR and SCoT
dendrogram that showed a strong genetic relatedness between each pair of A. lucidior (9)
and A. julibrissin (2), and A. procera (4) and A. guachapele (6). Moreover, A. procera (AP)
and A. lucidior (AU) were found to possess strong chemical proximity in HPTLC
fingerprint analysis. Finally, A. richardiana (AR) was formed a separate cluster at the
negative side of both PC1 and PC2 discriminated from the rest of species.
Chapter 4: Investigation of In Vitro Antiplasmodial Activity of the Leaf Extracts of
selected Albizia Species
Malaria is a serious risk to humanity and the major cause of mortality and morbidity
in the malaria- endemic countries. The absence of effective malaria vaccines emerges the
need of discovery of new bioactive constituents as an effective chemotherapeutic agent in
malaria control. The in-vitro antimalarial activity was performed on the selected nine
Albizia species according to the quantitative microdilution technique against CQ-resistant
P. falciparum strain (FcB1) using chloroquine as reference standard (IC50= 20 ng/mL).
The highest percentage of inhibition equal to 86.52% and 89.31% were exhibited
by A. procera and A. lucidior, respectively. Moreover, the calculated IC50 values of A.
procera and A. lucidior were 3.4 µg/mL and 3 µg/mL, respectively compared to
chloroquine standard (IC50 = 20 ng/mL) which indicate that both species possess the most
potent antiplasmodial activity among other species. That crucial finding can be necessarily
correlated to HPTLC analysis results which showed that both species contain the highest
alkaloids content among other species, LC/MS results and comparative untargeted
metabolomics study which showed that there are some alkaloids exclusively isolated from
these two species and strong chemical proximity does exist.