الفهرس 
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Abstract
General summary
Genus Bauhinia (Family: Fabaceae or Leguminosae) sub-family: Caesalpinioideae. It comprises 300–350 species of trees and shrubs distributed mostly in tropical regions. Various species of the genus Bauhinia is widely used in traditional medicine for the treatment of various diseases mainly type 2 diabetes mellitus. The plant under study, Bauhinia retusa Roxb is a deciduous tree cultivated in Egypt and the few literature concerning it stimulated us to carry out this study.
This study includes:
• DNA fingerprinting of the plant.
• Botanical study of the organs of the plant.
• Biological screening of the different organs of the plant. Including: In-vitro antioxidant effect as well as, in-vivo antidiabetic activity, anti-hepatotoxicity, anti-hyperlipidemic, anti-nephrotoxicity and antioxidant. Furthermore, the antimicrobial activity of the fractions of the leaves and for the compounds isolated from the hexane fraction.
• The chemical study including: a preliminary phytochemical screening; HPLC/MS analysis; GLC/MS analysis of the lipoidal matter and the essential oil composition; Extraction, isolation and identification of the major constituents of the leaf fractions.
Part I: DNA fingerprinting and Botanical study of the organs of Bauhinia retusa Roxb.
Chapter 1: DNA fingerprinting of Bauhinia retusa Roxb.
The plant DNA is extracted and amplified using RAPD technique with the ten decamer primers showed characteristic bands and generated 22 fragments pattern. In conclusion, it was declared that The primer OPC-19 produced the highest number of rapid amplified polymorphic DNA (RAPD) fragments (4 fragments), while the lowest number of fragments was produced by OPA-18, OPB-2 and OPB-9 (1 fragment). The maximum size was 1380 base pairs after using primer OPC-19 and OPO-11 which produced RAPD fragments. However, the minimum molecular size was 220 base pairs when OPA-07, OPB-02 and OPC-04 primers were used. Thus, DNA fingerprinting can be considered as the identifying parameters to authenticate and recognize B. retusa Roxb.at genetic level.
Chapter 2: Botanical study of the organs of B. retusa Roxb.
(A) Macromorphological study:
The whole tree B. retusa Roxb is large and semi-deciduous; grows up to 10-15 m in height with branches spread 3–6 m outwards which droop at the ends. The main stem (trunk) is huge measures about 100-150 cm in diameter, very hard, thick and cylindrical with rough surface covered by a thick brown cork showing irregular longitudinal fissures and cracks. Branching is monopodial and the branches hold pairs of alternate leaves and/or inflorescences. The young branches are green, flexible, glabrous or slightly hairy and cylindrical with about 0.5-1cm diameter. While, the old branches are brown, woody, rough and cylindrical with diameter 1-1.5 cm.
Leaves are simple, petiolate, exstipulate, and alternate. The mature leaves measures 17-18 cm length and about 10-15 cm width, slightly broader than long. While, the young leaves have a length of about 14-15cm length and 8-9 cm width with lobed margin at the base and cordate shape. The lamina is subcoriaceous, glabrous measures 13-14cm length and 15cm width. With entire margin, apex entire or notched, base cordate or truncate. The lower surface is green hairy compared to the dark green glabrous upper surface. The midrib is more prominent in the lower surface and the venation is palmate reticulate usually 8-10 nerved. The petiole is green, cylindrical, 6-7 cm length, swollen at both ends (pulvinate).
Inflorescence is compound raceme (panicle) composed of many florets most are in the bud form with or more developed flowers. Each Floret is very small 3 cm length, 2cm width, pedicellate, and off-white in color with dark spots, small bracts, hermaphrodite, zygomorphic and covered with soft hairs. Calyx 5-7.5 mm is concave, silky pubescent and with white color. Petals 1cm long, bented, obovate, hairy, white spotted with purple. Androecium consists of 3 stamens. Gynoecium consists of an ovary that is densely hairy on the margins. The style is elongated and sticky stigma with red color.
The fruits are twisted woody, very hard legumes 10-17cm long, 4-5 cm width, oblong and dehiscent. Each legume encloses 5-7 Seed.
The seeds are flat, orbicular, dark-brown, hard and smooth measures 1.5-2 cm long and 1.5-1.8 cm width.
(B) Micromorphological study:
In the leaf transverse section, the mesophyll is dorsiventral; the palisade layer is discontinuous. Several layers of the spongy tissue are present. The midrib is more prominent in the lower surface. The vascular tissue consists of collateral arc-shaped vascular bundle divided into two uneven parts, the phloem and the xylem which is endarch.
The petiole, a transverse section in the leaf petiole shows that it has a circular outline with slight projection from one side; the section showed that the petiole consists of the following layers; the epidermis consists of one layer of tangential elongated cells; then the cortex, the pericycle, The vascular tissue which contains a collateral vascular bundle divided to 2 unequal parts: the phloem and the xylem and finally the pith. A small central vascular bundle appears in the center of the pith and contains an outer layer of inner xylem followed by inner phloem layer and enclosing narrow central pith.
The stem, a transverse section shows that the outline is four armed or quadrilateral shaped and consists of a layer of epidermis of tangential elongated cells followed by the cortex layer, next to cortex the pericyclic layer takes place and the vascular tissue consists of a continuous layer of open collateral vascular bundle and finally the pith takes a large portion of the total stem diameter.
The flowers, a transverse section in the flower bud was taken and showed that it’s with nearly more or less circular outline and consists of consecutive layers of calyx, corolla, androecium and gynoecium respectively. The androecium consists of three stamens. Each stamen is contains a filament and anther. The anther shows two lobes attached by a connective tissue. Each anther lobe has 2 pollen sacs containing numerous smooth, spherical pollengrains with three germ pores. The ovary in transverse section appears oval in outline, unilocular with ovule attached to a marginal placenta.
Part II: Biological screening
Chapter 1: In-vitro study of the antioxidative potential of the leaves, fruits and seeds of B. retusa Roxb using DPPH assay
The anti-oxidative potential of the leaves, seeds and fruits ethanolic and aqueous extracts were evaluated using the In-vitro 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. The average IC50 of each extract is calculated. The test revealed that the aqueous extract of B. retusa leaves is the most effective anti-oxidant followed by B. retusa leaves ethanolic extract. While, the ethanolic extract of the fruits is the least activity as compared to Ascorbic acid as a reference antioxidant drug.
Chapter 2: In-vivo comparative biological study of the alcoholic and aqueous extract of the leaves, fruits and seeds of B. retusa Roxb.
• Acute oral toxicity study of the leaves, seeds and fruits of the alcoholic and aqueous extracts of Bauhinia retusa:
Safety of B. retusa Roxb leaves, seeds and fruits different extracts was investigated through determination of their LD50, which is was found to be (5 g/kg B.wt.).
• Induction of diabetes mellitus:
Type 2 diabetes mellitus and the oxidative effect on the liver and the kidney were induced in female albino rats of the Sprague Dawely strain (150-170 gm) by streptozocin(STZ) injected intra-peritoneal by a single injection of (50mg/kg B.W) dissolved in 0.1M citrate buffer (PH 4.5). Then, after seven days the rats were investigated for fasting blood glucose level, the rats with blood glucose level of 250-300mg/dl were taken for study. Furthermore, their oxidative parameters were monitored.
A. Anti-diabetic effect:
The blood glucose level and glycosylated haemoglobin were significantly decreased in treated diabetic rats compared to those non-treated diabetic rats. where all the tested treatments improved the blood glucose level and glycosylated haemoglobin specially the alcoholic extracts of the leaves, fruits and seeds respectively followed by the aqueous extract of the leaves, fruits and seeds respectively. The alcoholic extract of the leaves decreased the blood glucose level to more than the half (from 261.6 mg/dl to 108.9 mg/dl) compared to non-treated diabetic rats and also decreased HbA1C % from 9.88 to 6.71.
B. Anti-hepatotoxicity:
All the liver functions parameters improved upon treating the STZ- diabetic rats with alcoholic and aqueous extracts of the leaves, fruits and seeds. Serum level of AST, ALP and total bilirubin showed a significant decrease in diabetic rats treated with the alcoholic extract of the leaves, fruits and seeds respectively followed by the aqueous extracts of the leaves, fruits and seeds.
C. Anti-hyperlipidemic activity:
The alcoholic and aqueous treatments may consider as moderate anti-hyperlipidemic drugs, as they improved serum lipids and lipoproteins (T.C, T.G, HDLc, LDLc, and VLDLc). Also, there was a significant decrease in risk 1 ratio (T.C/HDLc) and in risk 2 ratios (LDLc/HDLc) in diabetic rats treated with the alcoholic extracts of the leaves, fruits and seeds followed by the aqueous extracts of the leaves, fruits and seeds.
D. Nephroprotective activity:
All the kidney functions parameters decreased in the diabetic treated rats compared to non-treated diabetic rats. Serum creatinine level, urea and uric acid decreased in diabetic rats treated with the alcoholic and aqueous extracts of the leaves, fruits and seeds. The effect of the alcoholic extract of the leaves in nearly equal to gliclazide.
E. Antioxidant activity:
The oxidative stress decreased in treated diabetic rats compared to non-treated rats. The total antioxidant capacity (TAC) increased in diabetic rats treated with the alcoholic extract of the leaves, fruits and seeds followed by the aqueous extract of the leaves, fruits and seeds. While glutathione (GSH) level increased in diabetic rats treated with the alcoholic extracts of the leaves then the seeds and finally fruits followed by the aqueous extracts of leaves, fruits and seeds. On the other hand, Malondialdehyde (MDA) level significantly decreased in the diabetic rats treated with the alcoholic extract of the leaves ,fruits and seeds followed by the aqueous extract of the leaves ,fruits and seeds respectively.
Chapter 3: In-vitro antimicrobial screening of the leaves fractions of B. retusa Roxb.
The antifungal activity of the leaves fractions evaluated against 2 pathogenic fungi using Sabouraud dextrose agar medium and ketoconazole as standard antifungal drug. The four fractions have no activity against the two fungi, Aspergillus flavus and Candida albicans.
The antibacterial activity of the leaves fractions against two gram positive and two gram negative activity was evaluated using the diffusion agar technique with Gentamycin as standard broad spectrum antibacterial drug. The ethylacetate fraction was the most active fraction against Staphylococcus aureus and Klebsiella pneumonia. On the other hand, the dichloromethane fraction is the only active fraction against Bacillus subtilis and the most active against Escherichia coli.
In general, the leaves fractions have no antifungal activity and are mild antibacterial agents.
Part III: Phytochemical study
Chapter 1: Preliminary phytochemical screening of the leaves, fruits and seeds.
The preliminary phytochemical screening of the different organs revealed the absence of Alkaloids and cardiac glycosides in all tested organs. Flavonoids and Sterol/triterpenes is the main class found in all tested organs. Saponins were detected in Leaves and Fruits. But, traces in the seeds. Tannins were present in Leaves and Fruits. But, traces in the seeds. The presence of Anthraquinone glycosides in the fruits and it’s found in trace amount in the leaves and absent in the seeds.
Chapter 2: Investigation of the essential oil of the flowers of B. retusa Roxb.
Freshly collected flowers were subjected to hydrodistillation using a Clevenger’s apparatus. The obtained oil was subjected to GLC/MS analysis.
GLC/MS analysis of the essential oil:
It was revealed that Carbonyl hydrocarbons and oxygenated monoterpenes constituted the predominant class in the essential oil of B. retusa Roxb flowers. The major constituents are Artemisia ketone (18.1%), camphor (10.39%), cyclotetradecane (9%), hexyl isopropyl sulfite (6.55%), oleamide (5.05%), isopulegol (4.82%), palmitic amide (4.14%), linalool (3.76%) and eucalyptol (2.41%).
Chapter 3: Identification of the active constituents of the leaves and leaf fractions of B. retusa Roxb using HPLC/DAD/ESI-HRMS.
Analysis of the methanolic extract take place lead to identification of total 45 compound in the leaves, 54 in the pericarp and 43 in the seeds.
Chapter 4: Investigation of the lipoidal matter of leaves of B. retusa Roxb.
The lipoidal matter of the leaves of B. retusa Roxb obtained from the alcoholic extract by fractionation with n-hexane. The n-hexane fraction was subjected to saponification procedure. Then GLC analysis is performed for the unsaponifiable fraction, as well as the fatty acid methyl ester (FAME) in order to identify their constituents.
GLC analysis of the unsaponifiable matter:
11 known compounds were identified with phytol, a diterpene (4.2 %) is the major component.
GLC analysis of fatty acid methyl esters (FAME):
A total of 23 known compounds were identified; The saturated fatty acid palmitic acid constituted the highest relative percentage (19.2 %) followed by linolenic acid (13.9%) .
The presence of the unsaturated fatty acids is very important as they act as potent anti-inflammatory agents. Therefore, the plant can be used as a remedy for the treatment of inflammatory related diseases.
Chapter 5: Extraction, isolation and identification of the major compounds of the leaves of B. retusa Roxb.
(A) Isolation and structure elucidation of the major constituents of the dichloromethane fraction:
Isolation of the compounds of the dichloromethane fraction was performed by fractionation on VLC column using dichloromethane and dichloromethane-methanol mixture followed by successive chromatographic techniques for further isolation and purification. 2 compounds were isolated (D1 and D2). The structures of the isolated compounds were identified by 1HNMR, Dept Q NMR as well as comparison with the available literature.
Compound 1: Methylgallate, a white needle crystals.
Compound 2: Quercetin, yellow powder .
(B) Isolation and structure elucidation of the major constituents of the ethylacetate fraction:
The ethylacetate residue was fractioned with polyamide column using distilled water 100% , water-methanol mixture and methanol 100% as eluents , the obtained fractions were rechromatographed on sephadex LH-20 using (methanol: water 80:20%) as eluent for further purification lead to isolation of three Compounds (E1, E2 and E3). The structures of the isolated compounds were identified by 1HNMR, Dept Q NMR as well as comparison with the available literature.
Compound 3: Methylgallate (white needles)
Compound 4: Quercetrin (yellow powder)
Compound 5: Isoquercetrin (yellow powder)
(C) Isolation and structure elucidation of the major constituents of the n-butanol fraction:
The n-butanol residue was fractioned with polyamide column using distilled water 100% , water-methanol mixture and methanol 100% as eluents , the obtained fractions were rechromatographed on sephadex LH-20 using (methanol: water 80:20%) as eluent for further purification. Lead to isolation of three Compounds (B1, B2 and B3). The structures of the isolated compounds were identified by 1HNMR, Dept Q NMR as well as comparison with the available literature.
Compound 6: gallic acid, white crystals.
Compound 7: Afzelin, yellow powder.
Compound 8: Tellimoside, yellow powder.
The identified compounds are presented in Table 35.