الفهرس 
Reported phytochemical constituentsOnly 14 pages are availabe for public view
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Abstract
Leguminosae (Fabaceae) is commonly known as the legume, pea, or bean family of cosmopolitan distribution, with approximately 730 genera and around 19,700 species. This high species richness is reflected in great morphological and chemical diversity, from which multiple uses are derived. It includes trees, shrubs, and herbaceous plants perennials or annuals, which are easily recognized by their fruit (legume) and their compound, stipulated leaves.
The genus Pithecellobium possesses many biological and pharmacological activities such as antioxidant, antimicrobial, antidiabetic, cytotoxic, anti-inflammatory, anti-diarrheal, antifungal, and immunomodulatory activities.
Despite, several studies carried out on many species of this genus and have generated enormous data about the chemical composition and corresponding biological activity of extracts and their isolated compounds; there are still a lot to be explored. Upon surveying the current literature, few reports could be traced regarding the phytochemical constituents and the pharmacological actions of genus Pithecellobium. P. dulce offers an opportunity for bioprospection and need to be explored for potential biological activities and characterization of its bioactive constituents.
This initiated our interest to perform the present work which includes:
Chapter (I): Part I: A macro- and micro morphological study of the stems, leaves, petiole, seeds, and fruits of P. dulce was done.
Part II: A genetic study was done, in which the DNA of P. dulce was extracted from leaf samples and analysed using 13 decamer random primers which will help in further standardization and quality control studies.
Chapter (II): A comparative in- vitro biological investigation of P. dulce bark and leaves was carried out including, cytotoxic, antimicrobial and antioxidant activities.
Chapter (III): A comparative phytochemical investigation of P. dulce bark and leaves. Further chromatographic separation was carried out to characterize and isolate the relevant compounds that may be responsible for the biological activity. Moreover, complementing the comparison between the two organs, estimation of total phenolic and total flavonoid contents and investigation of lipoidal matter by GC-MS analysis were carried out. Additionally, UPLC-MS/MS analysis was carried out on the total methanolic fraction of P. dulce bark with the aim to identify its main active constituents.
Chapter (I)
Part I: Botanical overview and Genetic characterization of Pithecellobium dulce
I. Macromorphological study
The results of this study revealed that macromorphological features of P. dulce are like those of family Fabaceae, sub-family Mimosidae. They occur as a perennial evergreen shrub or rarely a small tree about10 to 15 meters in height.
Morphologically, the small branches of the stem have a yellowish-green color and some parts of it have white patches. The leaves of P. dulce are compound, bipinnate, stipulate, hairy, and have a dark green color. Also, the Fruits of P. dulce are very characteristic with their shape as they may be spirally curved or twisted into one or two circles. Fruits are green in color with a red tinge but when they ripen, they become with a bright red color. Additionally, seeds are characteristic with their shiny black color, while flowers are whitish green in color.
II. Micromorphological study
The micromorphological structure of the stem, leaf, petiole and fruit of P. dulce are similar as they could be differentiated by:
• The stem
It shows an epidermis, a narrow cortex, then a pericycle, formed of lignified fibres surrounding a continuous ring of vascular tissue with a wide parenchymatous pith in the centre that occupy about 2/3 the stem diameter.
• The leaf
A transverse section in the leaflet shows a planoconvex mid rib. It consists of upper and lower epidermises, a dorsiventral mesophyll having one row of palisade cells below the upper epidermis.
The upper and lower epidermises are formed of polygonal, isodiametric cells having straight anticlinal walls and covered with thick cuticle (upper epidermis) and smooth cuticle (lower epidermis). Stomata are present on both surfaces being more frequent on the lower epidermis. Mostly, they are of paracytic type. The trichomes are present on both surfaces, of non-glandular type, mainly unicellular, conical having blunt or acute apices and covered with warty cuticle.
• The petiole
A transverse section in the petiole is almost circular in outline. The epidermis is like that of the lamina, consisting of polygonal cells having straight anticlinal walls, showing stomata of paracytic type and numerous non-glandular trichomes. Also, it shows a parenchymatous cortex with an outer row of collenchyma cells. The vascular system is nearly like that of the mid rib of the leaf.
• The fruit
A transverse section in the pericarp shows an outer epidermis (epicarp) and inner epidermis (endocarp) enclosing in between a wide mesocarp, which is traversed by two big vascular bundles in both sides of suture and smaller one in between. Large continuous groups of crossed fibers are present in the middle of the mesocarp.
Part II: DNA fingerprinting of P. dulce using Random Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR) technique
Extraction of DNA from leaves of frozen plant then DNA amplification using RAPD analysis revealed that the total fragments was 181. The OPT-12 primer was found to be the most effective in generating polymorphic bands on application of the RAPD technique.
The principle of these markers is mainly depending on providing effective germplasm fingerprint and detecting DNA nucleotide sequence polymorphisms and genetic polymorphism using a single primer of arbitrary nucleotide sequence
Chapter (II)
In-vitro comparative biological study of P. dulce bark and leaves
This chapter focused on screening the following activities:
• In- vitro study
1. Cytotoxic activity
Cytotoxic activity of various extracts and fractions of P. dulce bark and leaves was carried out on HepG-2 and HCT-116 cell lines using MTT assay. All the bark extracts showed no significant cytotoxicity on HepG-2 cell line except the EtOAc extract showed the strongest and more significant cytotoxic activity with IC50 value of 6.73 μg/mL, while, the moderate activity was observed for the Aqueous extract with IC50 value of 13.3 μg/mL but the least cytotoxic activity was observed for the Total methanolic extract with IC50 value of 19.7 μg/mL. On the other hand, a strong cytotoxic activity against HCT-116 cell line was observed for only the EtOAc bark extract with IC50 value of 9.21 μg/mL, but no significant activity was observed for other extracts. For the leaves, no cytotoxic activity was observed for all extracts against HCT-116 cell line.
2. Antimicrobial activity
The antimicrobial activity of various extracts and fractions of P. dulce bark and leaves were assessed against certain microbial strains using agar well diffusion method.
All the bark extracts exhibited no antibacterial activity except the total methanolic and n-hexane and dichloromethane extracts. On the other hand, more antibacterial activity was observed for Gram-positive and Gram-negative bacterial strains of the P. dulce leaves extracts. Finally, no antifungal activity was observed for all various bark and leaves extracts against Aspergillus niger and Candida albicans fungal strains.
3. Antioxidant activity
Various bark and leaves extracts and fractions of P. dulce were evaluated for potential antioxidant activity using DPPH radical scavenging capacity assay.
All the bark extracts showed promising, strong, and significant antioxidant activity by exhibiting IC50 values of 15.6, 16.7, 19 and 19.5 μg/mL for EtOAc, Total methanolic, Aqueous, and n-butanol extracts, respectively. However, DCM bark extract showed moderate antioxidant activity by exhibiting IC50 value of 78.4 μg/mL, while, the lipohilic (n-hexane) extract showed mild activity with IC50 value of 573.5 μg/mL. On the other hand, all the leaf extracts possess mild antioxidant activity when compared with the bark extracts.
Chapter (III)
Phytochemical investigation of the bark and leaves extracts of P. dulce
1. Preliminary phytochemical screening
Comparative preliminary phytochemical screening of bark and leaves was done. In the bark, the results revealed the presence of carbohydrates and or/glycosides, sterols and/ or triterpenes, flavonoids, tannins, anthraquinones and saponins and the absence of volatile oil and alkaloids and/or nitrogenous bases.
On the other hand, in leaves, the results revealed the presence of carbohydrates and or/glycosides, sterols and/ or triterpenes, flavonoids and tannins and the absence of volatile oil, alkaloids and/or nitrogenous bases, saponins and anthraquinones.
2. Quantitative estimation of total phenolic and flavonoid content in the bark and leaves of P. dulce (Leguminosae)
• Spectrophotometric determination of total phenolic content
The total phenolic content of the bark and leaves of P. dulce was estimated using Folin-Ciocalteau spectrophotometric method showing that both organs containing valuable amount of phenolic acid equivalent to gallic acid, but the bark extract showed the highest total phenolic concentration than the leaves extract.
• Spectrophotometric determination of total flavonoid content
The total Flavonoid content of the bark and leaves of P. dulce was estimated using the aluminium chloride colorimetric method showing that P. dulce bark extract revealed the highest total flavonoid concentration (310.82 mg CE/g extract) than the leaves extract (45.36 mg CE/g extract).
3. Investigation of lipoidal matter of P. dulce bark and leaves
The saponifiable and unsaponifiable matters of the n-hexane fractions of both organs were prepared. The saponifiable matter was methylated and analysed using GC-MS, the unsaponifiable matter in P. dulce leaves was found to be 88.03% and 83.79% in P. dulce bark, while the saponifiable matter was 96.05% & 92.19% in leaves and bark, respectively.
4. UPLC-ESI-MS/MS analysis of P. dulce bark total methanolic extract
Tentative identification of 32 compounds from the total methanolic extract of the bark using the UPLC- ESI-MS/MS analysis was done which are:
- Dihydroxy benzoic acid
- Protocatechuic acid
- Caffeoyl-2-hydroxyethane-1,1,2-tricarboxylic acid
- Salicylic acid
- A-type procyanidin dimer
- Tetrahydroxypentanoic acid
- Hydroxybenzoic acid
- Luteolin
- Syringoylmalic acid
- Tectorigenin
- Galloyl-coumaric acid pentoside
- Tetrahydroxyisoflavone
- Fiestin
- Palmitic acid
- Oleic acid - Dihydroxy-dimethoxy isoflavone
- Piscidic Acid
- Daidzein
- 2-methyl anthraquinone
- Dihydroxy benzoic acid methyl ester hexoside
- Chlorogenic acid
- Caffeic Acid
- (epi)Catechin
- Gallic acid hexoside
- B-type procyanidin dimer
- 3,7,4’-trihydroxyflavone
- Dimethoxy benzoic acid
- B-type proanthocyanidin dimer
- B-type Proanthocyanidin trimer
- Genistein
5. Isolation and identification of the constituents of P. dulce bark
Pithecellobium dulce 100% methanolic extract was prepared and fractionated into four fractions, n-hexane, DCM, EtOAc and n-butanol fractions. N-hexane fraction was subjected for components identification using GC-Ms analysis while, DCM fraction was subjected for further chromatographic fractionation.
This fraction was manipulated through column chromatography leading to the isolation of the individual chemical constituents which were further purified using different chromatographic techniques.
The phytochemical investigation of the tested fractions resulted in the isolation and structural elucidation of 4 compounds: one anthraquinone, one flavonoid and two phenolic acids. Compounds were identified by 1H-NMR, 13C-NMR and 2D-NMR including 1H-1H COSY and HSQC spectroscopic data after comparison with previously reported data.
The isolated compounds include:
- Compound 1; 2-Methyl anthraquinone (Tectoquinone)
- Compound 2; 2,4-Dimethoxy benzoic acid
- Compound 3; 3,7,4’-Trihydroxyflavone (5-Deoxy kaempferol)
- Compound 4; Protocatechuic acid
from the isolated compounds, Tectoquinone, and 5-Deoxy kaempferol are the first time to be isolated from the species of Pithecellobium dulce.
6. In silico molecular docking analysis of 5 isolated compounds and UPLC-MS/MS identified compounds
The molecular docking for the isolated and identified compounds was performed against 5II2 receptor. Drug discovery studio using MOE is software used to dock the compounds. Daidzein showed the best scores compared to the native ligand orientation on the 5II2 anticancer receptor. However, compounds tectoquinone (2-methyl anthraquinone), Chlorogenic acid and Genistein revealed moderate anticancer activity when compared to the native ligand as their scores were less than Daidzein’s score. Also, 5-deoxy kaempferol (3,7,4’-trihydroxyflavone) compound showed the lowest interaction score as compared to the mentioned compounds scores. All other six compounds showed acceptable and suitable fitting on 5II2 receptor.