الفهرس 
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Abstract
Summary
Olive is an important crop native to Mediterranean countries whose cultivation covers about
8 million ha of the land. The world consumption of olive oil and olive fruits reached to
2,199,000 tons in 2010. It is worth mentioning that Egypt is among the ten countries with the
highest olive production around the world with a production of 332,321 metric tons of olives
annually. During the oil production large amounts of by-products as leaves are accumulated
with no particular use. However recently, several important biological activities of olive
leaves have been reported including cardio-protective activity; anti-diabetic and antimicrobial
properties in addition to their anticancer activities on various tumour types. Most of the
reported activities of the olive leaves are related to its chemistry as they are characterized by
the presence of oleuropein, a major secoiridoid, and its derivatives. The leaves are also
considered a rich source of polyphenols, flavonoids, lignans, triterpenes and fatty acids. Olive
tree is characterized by having different cultivars. Identification of olive cultivars is not only
important for the quality of oil production, but it also affects the composition of the leaf
extract.
Usually the differentiation between olive cultivars was based on morphology,
however this is not a reliable method and the use of DNA markers is a complicated and
expensive process. Therefore, the aim of our work is to create a model that can discriminate
between different olive cultivars based on chemical analysis of their leaf extracts and data
processing using Multivariate Data Analysis (MVA) techniques. Also, to assess the quality of
the leaf extracts from different cultivars collected in two seasons using different
phytochemical analysis with PCA instead of the use of the traditional method for the quality
control of the leaf extract depending only on quantitation of oleuropein.
Twelve different olive leaf cultivars were collected from HRI, Egypt in autumn (November
2015) and spring (April 2016). They include 6 Egyptian (ASH, AOK, MRK, HMD, TFH,
WAT), 3 Spanish (MAN, PIC, ABQ), 2 Greece (KOR, KAL) and 1 Italian cultivars (COR).
The UV screening, TF, PPh, oleuropein content were estimated for the leaf extract in both
seasons. All the data were exported to the Unscrambler software for PCA analysis in order to
create a model that can discriminate between the studied cultivars without previous
knowledge of the data.
The work performed in this thesis was divided in two parts:
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Part 1: Phytochemical Analysis
Chapter 1: Ultraviolet Spectroscopic Analysis of O. europaea L. Cultivars Leaf Extracts
The full absorption spectra over the range of 250 to 400 nm were acquired for both spring
and autumn extracts showing same spectral profile. Meanwhile, different absorbance
intensities were observed across the measured spectral range. Spring extracts were shown to
be considerably higher than those in autumn. This can give the first conclusion on the effect
of sampling time and type of cultivar on the secondary metabolites of the leaf. For better
visualization of the data and to understand the reason for this variation, UV data was
subjected to Principle Component Analysis (PCA). PCA model for autumn extracts showed
marked segregation among cultivars in relation to their botanical origin Figure (10). Also
ASH the Egyptian cultivar was clustered with the Spanish cultivars in the upper left quadrant
in close proximity to MAN. On the other hand, PCA model for spring samples Figure (11),
clearly marks a distinction of most Egyptian samples and again ASH was clustered with
MAN. This may infer the resemblance in their chemical composition. Also it showed the UV
spectroscopic analysis as a rapid readily available method for the discrimination of olive
cultivars using their leaf extracts.
Chapter 2: Quantitative Determination of Total Polyphenol Contents O. europaea L.
Cultivars Leaf Extracts in Autumn and Spring
The PPh in the twelve olive leaves cultivars was measured in the two seasons using Folin-
Ciocalteu’s reagent. The concentration of PPh for each extract was expressed as mg gallic
acid/g dried extract using the regression equation obtained from gallic acid standard
calibration curve. The leaves from different cultivars exhibited considerably high but varying
amounts of polyphenol content. In autumn, the PPh content ranged from 149.02 mg/g to
104.28 dried extract for MAN and MRK, respectively. However, in spring, a significant
increase in PPh was observed for most of the studied cultivars (p<0.001) except for HMD and
MAN, which showed no significant differences between the two seasons. It ranged from
113.53 to 199.01 mg/g dried extract for MRK and PIC, respectively. The results showed
higher PPh content for most of the studied cultivars during the flowering stage except for
Egyptian ASH and the Spanish ABQ whose PPh content decreased during this time.
Chapter 3: Quantitative Determination of Total Flavonoid Contents in O. europaea L.
Cultivars Leaf Extracts in Autumn and Spring
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The TF for each cultivar was measured and expressed as mg rutin equivalent /g dried leaf
extract using the regression equation obtained from rutin standard calibration curve. In
autumn, MRK, an Egyptian cultivar showed the highest TF content (55.06 mg/g dried leaf
extract) followed by COR and AGS, and lowest levels were detected in HMD (38.84 mg/g
dried extract). Meanwhile, in spring, KOR, showed the highest TF content (82.67 mg/g dried
extract) followed by COR and AGS, and lowest levels were observed again in HMD
(45.19mg/g dried extract). It is worth mentioning that for the same cultivar, the TF in the
leaves collected in spring were significantly higher than in autumn. PCA analysis was
performed using the TF and PPh contents as additional variables together with the UV scan
from (250-400 nm). A score plot for autumn extracts Figure (16A), showed the clustering of
Egyptian cultivars on the right side positive to PC1; except for ASH, which is located again
near the Spanish cultivars. All the three Spanish cultivars are located at the left side.
Examining the loading plot Figure (16B), the clustering of the Spanish cultivars was related
to their highest PPh contents. The addition of the new variables (TF and PPh) gave better
clustering that reveals the effect of the genotype on the leaves composition. The score plot of
spring extracts Figure (17) didn’t show good discrimination although it showed distribution
of cultivars relative to their PPh and TF contents.
Chapter 4: HPLC Assay of Oleuropein Content in O. europaea L. Cultivars Leaf Extracts
in Autumn and Spring
Oleuropein, a secoiridoid is present abundantly in olive leaves as a principle bioactive
component. Quantitative determination of oleuropein content in the leaf extract of each
cultivar was performed. The concentration of oleuropein was expressed as mg
oleuropein/100g dried extract using the regression equation obtained from oleuropein
standard calibration curve. Results showed marked variation in oleuropein content among
different cultivars. In spring, the Spanish cultivar, MAN, was found to have the highest
oleuropein content (218.94 mg/100g of dried leaf extract), followed by COR, and ASH. In
autumn, ASH, an Egyptian cultivar, was found to have the highest content (102.45 mg/100g
of dried leaf extract), followed by MAN, and TFH. Most of the studied cultivars showed
higher oleuropein content in spring than in autumn, suggesting that the flowering stage is the
time of choice for collecting the leaves in purpose of high oleuropein and PPh contents.
Although the quality control of olive leaf extract is usually performed by quantifying
oleuropein, it was found not to be the major constituent of the leaf in all cultivars. For autumn
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extracts (ABQ, KOR, WAT, HMD and KAL) oleuropein content was significantly low and
cannot be used as a marker for the quality control of these cultivars. Thus, the use of
multivariate data analysis becomes the best alternative for the quality assessment of their leaf
extracts.
Chapter 5: HPLC-PDA-ESI/MS/MS based metabolomics of O. europaea L. Cultivars Leaf
Extracts in Autumn and Spring
Olive leaf extracts were analysed in both positive and negative ionization modes. Metabolites
identification was based on comparing their retention time, ultraviolet and mass spectra of
each eluted compound with those reported in literature and databases. LC/MS data analysis
results in the identification of 49 compounds in the leaf extracts; belonging to different
phytochemical classes. This includes secoiridoids (oleoside, secologanoside, oleuropein-Ohexoside,
oleuropein), flavonoids (luteolin, luteolin-O-hexoside, luteolin-O-rutinoside,
quercetin, quercetin-O-hexosylrhamnoside), Pentacyclic triterpenes (oleanolic acid, betulinic
acid, uvaol) and various phenolics (hydroxytyrosol, ellagic acid, ethyl gallate). The analysis
resulted in the identification of a newly compound in nature; oleuropein-O-deoxyhexoside
(23). Its structure and MS2 spectrum is shown in Figure (21). Also, Dihydroxy-oxooleanenoic
acid (44), and Hydroxy-oxo-oleanenoic acid (45), were not previously reported
in olive leaf extract according to our knowledge. Studying the base peak chromatogram of all
extracts, Luteolin-O-hexoside was shown higher than oleuropein in HMD, WAT, ABQ, and
KOR during autumn; TFH, PIC during spring. In spring almost all the studied cultivars are
characterized by higher oleuropein, content; while in autumn the leaf extracts showed
relatively higher pentacyclic triterpenes than spring in most of the studied cultivars.
Non-targeted metabolic profiling was performed for the leaf extracts collected in autumn and
spring in negative ionization mode. MZmine software was used for data processing and
Unscrambler software for principle component analysis. PCA score plot for autumn extracts
Figure (28A) showed the clustering of cultivars based on their geographical origin.
Flavonoids and secoiridoids were found to contribute the most in species discrimination.
Concerning the identified metabolites, luteolin and diosmetin were found to be higher in
KOR, KAL, ABQ and TFH. On the other hand, the two Spanish cultivars MAN and PIC
were found to be rich in secologanoside, oleoside and oleuropein-O-hexoside; whereas
oleuropein, oleuropein aglycone and luteolin-O-hexoside were found to be higher in the
Egyptian cultivars ASH and AOK. PCA analysis for spring extracts showed the segregation
of cultivars based on their metabolic constituents. Here again, flavonoids and secoiridoids