الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 255 |  |  |
| | | from | 255 | | |
Abstract
Pomegranate is used in folk medicine for the treatment of different diseases such as ulcer, hepatic damage and snakebite, and recently considered as nature’s power fruit. Extracts of all parts of the fruit have therapeutic benefits and some studies proved that the peel, seed, bark, root, and leaves also have medicinal benefits. Fruits of pomegranate are widely consumed, fresh and in commercial products, such as juices, jam, jelly, vinegar oil, and wines while the rind extracts have potential use as natural food preservatives and nutraceuticals. Worldwide, there are thousands of accessions and more than 500 pomegranate cultivars with around 50 available commercially. It has been proved that a wide diversity in numerous physico-chemical and textural properties existed among cultivars of pomegranate grown in different countries. This diversity reflects also in the secondary metabolites. Based on that, the cultivars type is considered the prominent factor in the miscellany of the pomegranate activity and other physicochemical properties. A comprehensive description of chemo-diversity in pomegranate cultivars is a crucial step to select cultivar with enhanced biological, technological and nutritional properties. As metabolic profile is complex and requires more advanced calculations than regular statistics, a chemometric technique is required.
This study aims to establish and compare the metabolic profiles of seven pomegranate cultivars grown in Egypt and obtained from various geographical origins. These cultivars are Manfalouty, Assuity, H116, Black, Akka, Wonderful and Sweet. Phytochemical characterization was performed on leaves, seeds, and peels which are usually discarded and considered as industrial waste, Furthermore, different spectroscopic and chromatographic techniques coupled with chemometrics were utilized to develop an optimum model for discrimination between different cultivars. In addition, the insecticidal activity of the extracts obtained from different cultivars against mosquito larvae C. pipiens was assessed.
Chapter I: Pomegranate Leaves
1. Ultraviolet spectroscopic analysis
UV absorption spectra of pomegranate methanolic extracts were recorded in the range of 200-400 nm. UV spectroscopic profiling of different pomegranate cultivars in the specified wavelength range reflected to a large extent the phenolic composition of the samples. For indepth understanding of the similarities and differences among different pomegranate cultivars that cannot be detected by visual inspection of various UV spectra, the data was subjected to multivariate data analysis. Unsupervised chemometric analysis resulted in the production of PCA score plot and HCA dendrogram. SNV transformation method resulted in better classification and discrimination of all cultivars except for H116 and Manfalouty cultivars.
2. Fourier transform infrared (FTIR)-attenuated total reflection (ATR) spectroscopic analysis
FTIR-ATR spectra of 7 pomegranate leaf methanolic extracts were measured in the range of 400-4000 cm-1. For a better understanding of the variation among different cultivars, the data was analyzed by applying unsupervised recognition techniques including PCA and HCA. The results of PCA and HCA obtained from chemometric analysis better explained all cultivars segregation.
3. High performance liquid chromatography (HPLC) analysis
HPLC metabolic profiling for different cultivars of pomegranate laves were constructed utilizing the methanolic extracts. Multivariate analysis was applied to the HPLC data to investigate the ability of HPLC technique in combination with chemometrics to discriminate different pomegranate leaf cultivars. PCA score plot resulted in classification and discrimination of Manfalouty, Assuity and Black cultivars only.
4. UPLC-ESI-MS/MS based metabolic profiling and comparative untargeted metabolomic study
(LC/MS) is a powerful analytical technique used in the current work for:
4.1. Metabolic profiling of pomegranate leaves via UPLC-ESI-MS/MS both negative and positive ionization modes
Metabolite identification was based on comparing their retention time, full scan and MS2 data of each eluted compound with those reported in literature and databases. A total of 17 compounds were tentatively identified in the methanol leaf extracts of seven pomegranate cultivars. The negative ionization experiment revealed the different polyphenolic compounds. Whereas, the experiment performed in the positive mode revealed the alkaloid profile. The identified compounds in both positive and negative ionization mode were:
4.1.1. Ellagitannins and galloyl esters: Galloyl-HHDP-glucose, granatin A and its derivative, granatin B and its derivative and monogalloyl glucose.
4.1.2. Flavonoids and proanthocyanidines: Luteolin-O-hexoside, luteolin-O-pentoside, kaempferol-O-pentoside, apigenin-O-(rhamnosyl) hexoside and A-type procyanidin dimer.
4.1.3. Alkaloids: Pelletierine, N-methylpelletierine and N-acetyl-sedridine.
4.2. A comparative untargeted metabolomic study using multivariate data analysis to discriminate between different pomegranate leaf cultivars
The aligned peak list obtained from data processing of the leaf extracts of seven pomegranate cultivars in positive and negative ionization modes were obtained from MZmine software. Later, the data was imported to the Unscrambler for multivariate data analysis. The results of PCA and HCA obtained from positive ionization mode better explained all cultivars segregation, where Akka cultivar showed a unique chemical composition regarding their alkaloid content of N-acetyl-sedridine and N-methylpelletierine, while the two Iranian cultivars, Sweet and Black showed a characteristic composition of pelletierine. Whereas, in the negative ionization mode, Black cultivar showed a unique chemical composition of ellagitannins as granatin B and its derivative.
5. Investigation of larvicidal activity of pomegranate leaves against Culex pipiens
The current work investigated the possible larvicidal activity of the leaf methanol extracts of different pomegranate cultivars against the C. pipiens 3rd instar larvae according to the world health organization (WHO) guidliness. Black cultivar extract showed pronouncing insecticidal activities against C. pipiens mosquitoes, which can be related directly to their unique alkaloid and ellagitannin contents. It may be concluded that the Black cultivar extract is a promising natural insecticide against C. pipiens mosquitoes with a potential to control transmittance of malaria, dengue, yellow fever, and filariasis.
Chapter II: Pomegranate Peels
1. Ultraviolet Spectroscopic Analysis
UV absorption spectra of pomegranate methanolic extracts were recorded in the range of 200-400 nm. UV spectroscopic profiling of different pomegranate cultivars in the specified wavelength range will reflect to a large extent the phenolic composition of the samples. For indepth understanding of the similarities and differences among different pomegranate cultivars that cannot be detected by visual inspection of various UV spectra, the data was subjected to multivariate data analysis. Unsupervised chemometric analysis resulted in the production of PCA score plot and HCA dendrogram. The results of PCA and HCA obtained from chemometric analysis better explained all cultivars segregation.
2. Fourier transform infrared (FTIR)-attenuated total reflection (ATR) Spectroscopic Analysis
FTIR-ATR spectra of 7 pomegranate peel methanolic extracts were measured in the range of 400-4000 cm-1. For a better understanding of the variation among different cultivars, the data was analyzed by applying unsupervised recognition techniques including PCA and HCA. The results of PCA and HCA obtained from chemometric analysis better explained all cultivars segregation.
3. High performance liquid chromatography (HPLC) Analysis
HPLC metabolic profiling for different cultivars of pomegranate peels were constructed utilizing the methanolic extracts. Multivariate analysis was applied to the HPLC data to investigate the ability of HPLC technique in combination with chemometrics to discriminate different pomegranate leaf cultivars. PCA score plot resulted in classification and discrimination of all cultivars except Assuity cultivar
Validation of developed HPLC method was done according to ICH guidliness using punicalagin as standard. The calibration curve of caffeine exhibited good linearity (r2= 0.996) within test ranges. The LOD was 80.35274 µg/ml, while LOQ was 243.493152 µg/ml. In addition, the developed analytical method had good accuracy with overall recovery 100.17 ± 3.558%. Intra-day and Inter-day variability (%) were 3.89153 and 4.31515, respectively.
4. UPLC-ESI-MS/MS based metabolic profiling and comparative untargeted metabolomic study
(LC/MS) is a powerful analytical technique used in the current work for:
4.1. Metabolic profiling of pomegranate peels via UPLC-ESI-MS/MS both negative and positive ionization modes
Metabolite identification was based on comparing their retention time, full scan and MS2 data of each eluted compound with those reported in literature and databases. A total of 21 compounds were tentatively identified in the methanol peel extract of seven pomegranate cultivars. The negative ionization experiment revealed the different polyphenolic compounds. Whereas, the experiment performed in the positive mode revealed the alkaloid profile. The identified compounds in both positive and negative ionization mode were:
4.1.1. Ellagitannins and gallagyl esters: HHDP-glucose, galloyl-HHDP-glucose, bis-HHDP-glucose, digalloyl-HHDP-glucose, digalloyl-gallagyl-glucose, punicatannin A/B and punicalagin.
4.1.2. Flavonoids: (+)-Catechin/(−)-epicatechin, myricetin-O-hexoside, quercetin-O-hexoside, quercetin-O-pentoside, quercetin-O-rhamnoside, kaempferol-O-hexoside, luteolin-O-(acetyl-pentosyl) pentoside and quercetin-O-hexoside-O-acetylhexoside.
4.1.3. Organic acids: Citric acid
4.1.4. Alkaloids: Sedridine and N-acetyl-sedridine.
4.2. A comparative untargeted metabolomic study using multivariate data analysis to discriminate between different pomegranate peel cultivars
The aligned peak list obtained from data processing of the peel extracts of seven pomegranate cultivars in positive and negative ionization modes were obtained from MZmine software. Later, the data was imported to the Unscrambler for multivariate data analysis. The results of PCA and HCA obtained from both positive and negative ionization modes better explained all cultivars segregation, where the results of LC/MS in positive mode analysis are seemed to be in accordance and complementary to LC/MS in negative mode analysis. Akka cultivar showed a unique chemical composition regarding their alkaloid content of N-acetyl-sedridine (also as shown in leaves), while the two Iranian cultivars Sweet and Black showed a characteristic composition of sedridine (in positive mode), quercetin-O-pentoside, quercetin-O-rhamnoside and HHDP-glucose (in negative mode).
5. Investigation of larvicidal activity of pomegranate peels against Culex pipiens
The current work investigated the possible larvicidal activity of the peel methanol extracts of different pomegranate cultivars against the C. pipiens 3rd instar larvae according to the world health organization (WHO) guidliness. Black cultivar extract showed pronouncing insecticidal activities against C. pipiens mosquitoes, which can be related directly to their unique alkaloid and punicalagin contents. It may be concluded that the Black cultivar extract is a promising natural insecticide against C. pipiens mosquitoes with a potential to control transmittance of malaria, dengue, yellow fever, and filariasis
Chapter III: Pomegranate Seeds
1. GC/MS analysis of the n-hexane extract of the seeds of pomegranate
A total of 32 components were identified, accounting for 76.31% to 98.46% of Blk1 and Ast1 extracts composition, respectively. The compounds were identified according to RI identification based on a comparison of published Kovats retention indices and MS identification based on mass spectral data. The most abundant chemical classes were sterols (ranged between 25.62 and 45.49% in Aka1 and Swt2, respectively), followed by triterpenes (ranged between 25.77 and 36.06% in Aka2 and H116 1, respectively) and tocopherols (ranged between 2.67 and 24.04% in Blk1 and Aka2, respectively). Squalene was the major identified compound (representing from 25.77 to 36.06% of the total extract in Aka2 and H116 1, respectively), followed by γ-sitosterol (representing from 24.33 to 36.8% of the total extract in Aka2 and Swt2, respectively) and γ-tocopherol (representing from 1.58 to 23.12% of the total extract in Blk1 and Aka1, respectively).
2. Multivariate analysis for GC/MS results
In the current work, the metabolic profiles of seven pomegranate seed extracts obtained by GC/MS analysis were assessed to detect the variation in the secondary metabolites among the different studied cultivars in targeted method. Akka cultivar showed a unique clustering in PCA model with unique chemical composition of cis-9-Octadecenoic acid, TMS ester. Furthermore, Black and Sweet Iranian cultivars showed a certain degree of relatedness. H116 and Assuity, Manfalouty and Wonderful cultivars closely related to each other’s, where Squalene, β-Sitosterol TMS derivative and γ-Tocopherol characterize H116 and Assuity cultivars, and γ-Sitosterol characterizes Manfalouty and Wonderful.
3. Nutritional value of pomegranate seeds
In the current study, the pomegranate seeds of three cultivars of different geographical origin (Wnd: the United States industrial standard cultivar, Aka: the Palestinian cultivar and Ast: the Egyptian cultivar) were evaluated in terms of some quality properties including protein, fat, fiber, carbohydrate, ash, moisture and mineral contents. Carbohydrate was the highest constituent in all samples, ranging from 55 to 71.86% in Aka and Wnd, respectively. Whereas, ash presented the lowest values ranging between 1.45 and 2% in Wnd and Aka, respectively. Regarding minerals, phosphorus was the highest one followed by potassium, calcium, sodium and iron.