الفهرس 
Botanical and genetic characteristics of Pachira glabra (Pasq.) and Pachira aquatica (Aubl.)(Malvaceae)Only 14 pages are availabe for public view
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Abstract
General Summary
Family Malvaceae is one of the largest Plant families with almost 244 genus and 4,225 species. The family has become a part of human civilization due to its importance as producer of cotton, fiber yielding and many ornamental plants. Moreover, several Malvaceae species possess crucial medicinal value. Subfamily Bombacoideae (previously recognized as family Bombacaceae) comprises around 30 genera of tropical trees. The largest genera includes Bombax ,Ceiba, and Adansonia .
Genus Pachira, the largest genus in subfamily Bombacoideae, comprises more than 70 species of tropical evergreen trees, two of which are cultivated in Egypt namely Pachira aquatica, and Pachira glabra. Pachira species are popular as evergreen ornamental trees predominantly distributed in Central and South America, Africa and India. They are famous for their edible seeds that are consumed either raw or roasted in addition to the young leaves and flowers of some species that are consumed as vegetables.
Studies addressing the chemical composition and biological activities of the two Pachira species were found to be very scarce especially for P. glabra. Moreover, there is a lot to be explored about their botanical characteristics as well as their essential oils and their biological importance.
So, the aim of this study was directed to the following points:
1. Complete literature reviewing was presented concerning the phytochemistry and biology of subfamily Bombacoideae and genus Pachira.
2. Botanical characterization of macro-and micro-morphological features of Pachira glabra and Pachira aquatica leaves, petioles and stems was performed as well as DNA fingerprinting for both species.
3. Phytochemical screening of P. glabra leaves followed by in-depth phytochemical investigation of its methanol extract. Then, manipulating the various chromatographic techniques for isolation and characterization of the main active constituents. Moreover, LC-MS runs were done on the methanol extracts of Pachira glabra and Pachira aquatica leaves with the aim of tentative identification of the chief active constituents.
4. Biological investigation of cytotoxic activities of both Pachira glabra and Pachira aquatica methanol extracts. Besides, in vivo assessment of gastroprotective activity of P. glabra alcohol extract (PGE) and compound 1 was performed as well as evaluating their anti-H-pylori activity for better understanding of their underlying gastroprotective mechanisms.
5. Comparative investigation of the essential oil compositions of Pachira aquatica, and Pachira glabra leaves and stems was performed. Moreover, the antimicrobial activities of both leaf oils were evaluated against H. pylori and M. tuberculosis.
Chapter 1: Botanical and genetic characteristics of Pachira glabra (Pasq.) and Pachira aquatica (Aubl.) (Malvaceae)
The results of this study revealed that the morphological features of Pachira glabra and Pachira aquatica are highly similar to those of subfamily Bombacoideae. They occur as tropical trees of 6-15 m tall for Pachira glabra and about 25 m tall in case of Pachira aquatica. They are characterized by their erect cylindrical branches and their compound leaves. Leaflets are usually lanceolate demonstrating acute stipulate apex and entire margin with reticulate pinnate venation anastomosing near the margin. Flowers are solitary, actinomorphic and hermaphrodite, characterized by their showy appearance.
Morphologically, the two species could be differentiated by the color of the flower, where the white showy stamens are white in P. glabra and red in P. aquatica. They also differ in the texture and fracture of the stem and the length of the petiole being more rough and hard with short petioles in case of P. aquatica.
Microscopically, the abundance of some diagnostic elements like stellate hairs and calcium oxalate clusters as well as the dimensions of the microelements can aid in differentiation between the two Pachira spesies.
On the genetic level, it can be concluded that the most relevant fragment resulting from the successful combination of template and primer was that produced by B-02 and B-05 RAPD primers that exihibited minimum values of similarity coefficients and highest level of polymorphism. However, other RAPD primers that produced high values of similarity coefficient and low levels of polymorphism like G-14 RAPD primer could be used in the identification of different Pachira species.
Chapter 2: Chemical composition of the leaf methanol extract of Pachira glabra Pasq. (Malvaceae)
A. Phytochemical screening of P. glabra and P. aquatica leaves
Preliminary screening revealed the presence of flavonoids, tannins, sterols and/or triterpenes, carbohydrates and / or glycosides in both P. glabra and P. aquatica leaves. Besides, the absence of alkaloids, anthraquinones, cardiac glycosides or saponins was demonstrated in both leaves.
B. Estimation of total phenolic and total flavonoid contents
Results revealed higher total phenolic content in Pachira aquatica alcohol extract estimated as 92.34 ± 0.01 µg of gallic acid /mg of sample. On the other side, P. glabra alcohol extract demonstrated higher flavonoid content estimated as 108.32 ± 0.01 µg of quercetin /mg of sample.
C. Phytochemical study of P. glabra leaves
P. glabra methanol extract was prepared and fractionated using n-hexane, dichloromethane, ethyl acetate and butanol respectively. Both the ethyl acetate and butanol fractions were subjected to further chromatographic fractionation. Successive column chromatography was manipulated for the isolation of individual chemical constituents which were further purified using preparative high performance liquid chromatography or preparative thin layer chromatography.
The phytochemical study of the tested fractions resulted in the isolation and structure elucidation of eight pure compounds belonging mainly to 4-pyrones, cinnamic acid derivatives and flavonoids. Compounds were identified by 1H-NMR, APT, and 2D-NMR including HMBC, HSQC and 1H-1H COSY spectroscopic data after comparison with previously reported data.
The isolated compounds of P. glabra methanol extract include;
Compound (1): Ferulic acid
Compound (2): p-Coumaric acid
Compound (3): Quercetin 3-O-α-L-rhamnoside-3’-O-β-D-glucoside
Compound (4): Quercetin-3-O-α-L-rhamnosyl-(1’’’-6’’)-(4’’-O-acetyl)-β-D-galactoside
Compound (5): Quercetin-3-O-β-D-galactoside
Compound (6): 7-Hydroxy maltol-3-O-β-D-glucoside
Compound (7): Maltol-3-O-β-D-glucoside
Compound (8): Methyl coumarate
from the isolated compounds, compound 4 (Quercetin-3-O-α-L-rhamnosyl-(1’’’-6’’)-(4’’-O-acetyl)-β-D-galactoside) and compound 6 (7-Hydroxy maltol-3-O-β-D-glucoside) were first to be isolated from nature.
Compound 2 (p-Coumaric acid), compound 7 (Maltol-β-D-glucoside) and compound 8 (Methyl coumarate) were isolated for the first time from subfamily Bombacoideae;
The rest of compounds; Ferulic acid (1), Quercetin-3-O-α-L-rhamnoside-3’-O-β-D- glucoside (3) and Quercetin-3-O-β-D-galactoside (5) were isolated for the first time from genus Pachira.
D. LC-ESI -MS analysis of P. glabra and P. aquatica leaves methanol extracts
LC-ESI-MS analysis of the methanol extract of P. glabra leaves indicated the tentative identification of 15 compounds belonging to different classes of secondary metabolites; including benzoic acid derivatives, cinnamic acid derivatives, 4-pyrones, xanthones and flavonoids. However, LC-ESI-MS analysis of the methanol extract of P. aquatica leaves indicated the tentative identification of only 5 compounds belonging to cinnamic acid derivatives, xanthones, catechins, flavonoids, and biflavonoids.
Chapter 3: Biological investigation of the leaf methanol extract of Pachira glabra Pasq. and P. aquatica (Malvaceae)
A. In vitro assay for cytotoxic activities of Pachira glabra and P. aquatica total alcohol extracts
The Cytotoxicity of both Pachira glabra and P. aquatica total alcohol extracts were evaluated using MTT viability assay against three different cell lines. Both extracts of P. glabra and P. aquatica demonstrated moderate inhibitory activity against the viability of breast cancer cell line (MCF-7) with mean inhibitory concentration (IC50) of 6.93 and 6.20 μg/ml respectively. The alcohol extract of P. glabra demonstrated very weak cytotoxic activities against both liver hepatocellular carcinoma (HepG-2) and adenocarcinomic human lung cancer cells (A-549) exhibiting IC50 that exceeds 500 μg/ml against both cells.
On the other side, P. aquatica alcohol extract was slightly more potent in inhibiting the viability of HepG-2 and A-549 cells exhibiting IC50 of 280 and 383 μg/ml recpectively.
B. In vivo assessment of gastroprotective activity of P. glabra leaves methanol extract (PGE) and 7-hydroxy maltol-3-O-β-D-glucoside (HMG)
The anti-ulcer activities of P. glabra alcohol leaves methanol extract (PGE) and 7-hydroxy maltol-3-O-β-D-glucoside (HMG) were investigated by evaluating their in vivo gastroprotective activities in an experimental model of ethanol-induced gastric ulceration as well as their in vitro anti Helicobacter pylori activities.
Results showed that pretreatment with PGE (at doses 100 mg/kg, 200 mg/kg and 400 mg/kg) as well as HMG (25 mg/kg, 50 mg/kg and 100 mg/kg) markedly protected against the mucosal gastric lesions induced by ethanol administration and allowed to the reduction of the morphological observed signs as well as the histopathological injury caused by ethanol administration. The therapeutic effects, using PGE high dosage (400 mg/kg), as well as that of HMG (100 mg/kg), were the most effective for gastroprotection of the stomach mucosa comparable to reference omeprazole (20 mg/kg). So, those doses were selected for further immunohistochemical screening for better understanding of their underlying mechanisms. Pretreatment with PGE (400 mg/kg) and HMG (100mg/kg) significantly reduced the expression of NF-KB and COX-2 proteins supporting their anti-inflammatory properties. Besides, they exhibited a down regulation of the Bax protein expression in gastric mucosal tissue as shown by immunohistochemical staining supporting their anti-apoptotic properties.
C. In vitro anti Helicobacter pylori activities of P. glabra leaves methanol extract (PGE) and 7-hydroxy maltol-3-O-β-D-glucoside (HMG)
The antimicrobial activities of P. glabra methanol extract as well as compound1 were evaluated against Helicobacter pylori ATCC using MTT colorimetric assay. Both P. glabra methanol extract (PGE) and 7-hydroxy maltol-3-O-β-D-glucoside (HMG) demonstrated potent in vitro antimicrobial activity against Helicobacter pylori exhibiting minimum inhibitory concentrations of 0.125 and mg/ml and 0.0625 mg/ml respectively. Results are comparable to standard clarithromycin that exhibited MIC of 1.95µg/ml.
Chapter 4: Comparative analysis of volatile constituents of Pachira aquatica Aubl. and Pachira glabra Pasq., their anti-mycobacterial and anti- Helicobacter pylori activities and their metabolic discrimination using Chemometrics
The leaves and stems of the two Pachira species cultivated in Egypt, namely Pachira aquatica, and Pachira glabra were qualitatively and quantitatively examined for their volatile constituents employing GC/FID and GC/MS. Sixty compounds have been identified from P. aquatica leaves and stem representing 87.26 and 90.14%, respectively of their total oil content. However, fifty seven compounds were determined from P. glabra leaves and stems representing 89.67 and 94.69%, respectively.
Phytol represented the major component of P. glabra leaf oil with a content of 28.72% of the oil, however β-cadinene (15.46%), was the predominating compound in P. glabra stem oil. Palmitic acid methyl ester (21.08%) and 9,12-octadecadienoic acid ethyl ester (26.20%) were the prevailing constituents in Pachira aquatica leaves and stems oil, respectively. Hierarchical cluster analysis (HCA) and principle component analysis (PCA) as unsupervised chemometric technique were successfully employed to differentiate the two morphologically close related species based upon their GC chromatograms. Besides, P. aquatica leaf oil showed more effectiveness against both Mycobacterium tuberculosis and Helicobacter pylori infection showing MIC values of 50 and 20 µg/mL against them, respectively emphasizing its potent inhibitory activity against both hazardous infections.