الفهرس 
introduction
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Abstract
General summary
Lobularia libyca (Viv.) C.F.W. Meissn. . is a herb belongs to family Brassicaceae and known in Arabic as Khurm-El-ibra. It is an annual shrub, grows in coastal lands, native to North Africa from Canary Islands to Iran.
The present study includes the following:
Part 1: Botanical study and DNA fingerprint of L. libyca.
Part 2: Phytochemical study of L. libyca.
Part 3: Biological study of the different extractives from the whole plant and seeds of L. libyca.
Part 1: Botanical study and DNA fingerprint of Lobularia libyca (Viv.) C.F.W. Meissn.
Chapter 1: Macromorphology
I- The root
The root is bright yellow to yellowish brown in color. The surface is usually rough showing numerous wrinkles and longitudinal fissures. The fracture of the root is fibrous on in the inner part and smooth on the outer part.
II- The young stem
The young stem is erect, herbaceous and cylindrical. It is green in color, sometimes with violet tinge. The surface is rough and covered with appressed whitish hairs.
III- The old stem
The old stem is greenish purple in colour, hard, rough with sympodial branching. It is also covered with appressed whitish hairs. It breaks with a fibrous fracture. There is no cork.
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IV- The leaf
Leaves are alternate, sessile and exstipulate. The lamina is simple, linear oblong in shape with obtuse sometimes acute apex.
The leaves are dark green to grayish green. The surface is rough and covered with longitudinally appressed whitish hairs. The midrib is prominent on the lower surface. The leaf has a slightly fleshy texture.
V- The flower
Flower is very small, bisexual, actinomorphic and hypogenous. The calyx composed of four sepals, while the corolla has four free white petals. The androecium has six tetradynamous stamens, while the gynaecium shows superior ovary of two united carpels, short style and capitate stigma. The inflorescence is raceme type.
VI- The fruit
The fruit is true, simple, dry and dehiscent siliqula which opens with 2 flat or convex valves. The surface of the fruit is rough and covered with closely appressed fine whitish hairs. The pericarp is pale green to green, sometimes with a violet tinge. It is derived from superior bicarpellary bilocular ovary and contains 4-5 seeds.
VII- The seed
The seed is circular in shape, flattened, winged with a transparent membranous wing. The testa is thin and brittle. The seed is albuminous, derived from anatropus ovule, with an accumbent embryo.
Chapter II: Micromorphology
I- The old root
A transverse section of the old root is more or less circular in outline. The cork cells are tangentially elongated tabular cells with cellulosic walls. The cortex consists of several rows of somewhat tangentially elongated cells
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with thickened cellulosic walls showing few scattered myrosin cells. The endodermis is indistinct. The phloem is formed of continuous band of several rows of delicate elements traversed by narrow, non-lignified medullary rays. The cambium is distinct and formed of 2-3rows of thin walled radially arranged cambiform cells. The xylem is wide, constituting the main part of the root. It consists of lignified radially arranged elements. The vessels are mostly pitted. Also there are few tracheids that are fusiform, having blunt apices and pitted walls.
II- The young stem
A transverse section in the young stem is more or less circular in outline, showing 6-8 ridges. It is formed of an epidermis which is composed of one row of polygonal cells with straight beaded anticlinal walls and covered with thick smooth cuticle. Hairs are abundant, non-glandular branched unicellular. The cortex is formed of chlorenchymatous tissue followed by 2-4 layers of parenchymatous cells showing few scattered myrosin cells. The pericycle is parenchymatous interrupted by few groups of fibers. The vascular tissue is relatively wide forming a ring traversed by lignified medullary rays. The phloem consists of thin-walled phloem parenchyma, sieve tubes, and companion cells. The xylem is formed of spiral and pitted lignified vessels accompanied by wood fibers and wood parenchyma. The pith is wide and parenchymatous.
III- The old stem
A transverse section of the old stem is more or less similar to that of the young stem except that it is more circular and the ridges are more prominent. The vascular tissue is wider with a narrower pith. The endodermis is more indistinct and pith cells are more lignified near the vascular bundles.
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IV- The leaf
A transverse section of the leaf shows upper and lower epidermises, enclosing an isobilateral mesophyll. The upper and lower epidermises are nearly similar, they show polygonal isodiametric or slightly axially elongated cells with straight anticlinal walls in the upper epidermis and slightly wavy anticlinal walls in the lower epidermis. They are covered with thin smooth cuticle and show anisocytic stomata. The upper and lower epidermal cells contain mucilage.
Hairs are abundant covering both surfaces. They are non-glandular branched unicellular, as well as, glandular hairs with unicellular stalk and unicellular head. The mesophyll is composed of the palisade tissue which consists of 2-3 layers of columnar closely packed cells extending continuously on the upper surface only. The spongy tissue is narrow formed of irregular shaped parenchyma cells. The cortical tissue of the midrib is parenchymatous. The pericylce is formed of fibers with relatively thin lignified walls and wide lumina. The vascular tissue is composed of arches of xylem vessels and phloem patches beneath them forming a collateral vascular bundle. The xylem vessels show spiral thickening. The cambium is formed of 2 rows of radially arranged small thin cellulosic cambiform cells. The phloem tissue consists of sieve elements and phloem parenchyma.
V- The flower
The petal has a pappilosed epidermis while, the epidermis of sepal is densely covered wih non-glandular unicellular branched hairs. The stigma is pappilosed. The pollen grains are spherical and smooth with three germ pores.
VI- The fruit
The epicarp is composed of green polygonal cells and covered with non-glandular hairs. The innermost layer of mesocarp shows fibrosclerides.
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The septum is composed of large lignified parenchyma overlapped with elongated thin walled cellulosic parenchymatous cells.
VII- The seed
A transverse section of the seed shows the testa followed by the endosperm and the embryo. The testa starts with the epidermis which is formed of one row of large polygonal isodiametric cells covered with thick smooth cuticle contining mucilage. The hypodermis is formed of one row of tabular cells. The endosperm starts with an aleurone layer. The remainder of the endosperm is formed of thin-walled cellulosic polygonal cells. The embryo is formed of isodiametric or slightly elongated thin-walled parenchyma cells filled with oil droplets and protein masses showing few well distinct myrosin cells.
Chapter III: DNA fingerprint of L. libyca
The DNA fingerprinting of L. libyca was done to help the identification and characterization of the plant. The DNA of L. libyca was amplified using eleven decamer primers to reveal 89 different RAPD fragments. According to the RAPD technique, we could suggest the use of primers C-1, Z-19 and C-16 for the selective discrimination of Lobularia libyca (Viv.) C.F.W. Meissn.
Part 2: Phytochemical study of Lobularia libyca (Viv.) C.F.W. Meissn.
Chapter I: Preliminary phytochemical screening
Preliminary phytochemical screening of the different organs of the plant under investigation revealed the presence of carbohydrates and/or glycosides, sterols and/or triterpenes, and free or combined flavonoids.
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Spectrophotometric determination of the total phenolics content was carried out using the Folin-Ciocalteu colorimetric method. The total phenolics content of the total alcoholic extract, ethyl acetate fraction and n-butanol fraction were 25.26, 0.38 and 8.23 mg GAE/g fresh plant respectively.
Chapter III: Quantitative estimation of total flavonoids content.
Spectrophotometric determination of the total flavonoids content was carried out using the aluminum chloride colorimetric method. The total flavonoids content of the total alcoholic extract, ethyl acetate fraction and n-butanol fraction were 3.56, 0.1 and 1.05 mg quercetin equivalent/g fresh plant and 4.33, 0.121 and 2.36 mg rutin equivalent/g fresh plant respectively.
Chapter IV: HPLC analysis of phenolic compounds of Lobularia libyca (Viv.) C.F.W. Meissn.
The HPLC analysis of the total alcoholic extract, ethyl acetate fraction and n-butanol fraction revealed that dihydroxy benzoic acid and pyrogallol are the major phenolic compounds present in the total alcoholic extract, while vanillic acid is the major phenolic compound found in the ethyl acetate fraction. Moreover, chlorogenic acid is the major phenolic compound present in the n-butanol fraction.
Chapter V&VI: Isolation and characterization of compounds from the alcoholic extract of Lobularia libyca (Viv.) C.F.W. Meissn.
By fractionation of the total alcoholic extract and estimation of the total phenolics and flavonoids contents of the fractions, it was found that the highest yield was the n-butanol fraction (19 g). So the n-Butanol fraction was subjected for more fractionation by polyamide column chromatography. Fraction 2 was subjected to by polyamide column chromatography to isolate compound 3. Moreover, by comparing the weight of the ethyl acetate fraction (1 g) to its contents of phenolics and flavonoids, it is found that it contains a
Chapter II: Quantitative estimation of total phenolics content.
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reasonable amount of them, so the fraction is subjected to descending preparative paper chromatography to isolate its phenolic compounds which are compound 1 and 2.
The identification of isolated compounds was carried out on the basis of UV spectral analysis, 1D (1H and 13C) and 2D (HMBC) NMR and high resolution mass (HRMS) spectroscopic data and ESI-MS spectrometry. The compounds (1-3) were to be kaempferol, quecetin and quercetin 3-O-α-L-rhamnopyranosyl (1’’’→4’’)-β-D-galactopyranoside-7-O-α-L-rhamnopyranoside respectively.
Chapter VII: Investigation of the glucosinolate hydrolysis products and other volatile compounds of Lobularia libyca (Viv.) C.F.W. Meissn.
Three different glucosinolates detected in seeds which are glucoiberverin, glucoiberin and glucoerucin. These GLS were first time to be observed in the genus. Moreover, in the leaves only one GLS is detected which is glucoiberverin and in roots no GLS were detected under the same experimental conditions. Some other minor volatile constituents were detected in seeds such as ethyl benzene, menthone and β-pinene.
Part 3: Biological study of Lobularia libyca (Viv.) C.F.W. Meissn.
Chapter I: The in-vitro antioxidant and hepatoprotectctive activity.
The antioxidant activity of Lobularia libyca (Viv.) C.F.W. Meissn. was investigated using DPPH method and expressed as IC50. The activity of the total alcoholic extract and n-butanol fraction were assayed and were found to be 140 and 10.49 μg/ml respectively. The activity of the l-ascorbic acid that was used as standard was 1.26 μg/ml.
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It was found that the n-butanol fraction and the novel compound (quercetin 3-O-α-L-rhamnopyranosyl (1’’’→4’’)-β-D-galactopyranoside-7-O-α-L-rhamnopyranoside) show promising hepatoprotective activity comparable to silymarin which was used as standard. The n-butanol fraction shows higher hepatoprotective activity than the isolated compound 3.
Chapter II: The in-vitro antimicrobial and cytotoxic activity of glucosinolates hydrolysis products.
1- Antimicrobial activity
The antimicrobial activity of the GLS hydrolysis products obtained from the seeds was investigated using disc diffusion method against eight different organisms. The greatest activity was on Candida albicans and pseudomonas aeruginosa, while aspergillus flavus shows complete resistance to the extract.
2- Cytotoxic activity
The cytotoxic activity of GLS hydrolysis products obtained from the seeds were studied on different human carcinoma cell lines which were breast (MCF-7), hepatic (HUH-7), lung (A-549) and colorectal (HCT-116) carcinoma cell lines. The seed hydrolysate shows a great selectivity to the type of the cancer cells where colorectal HCT-116 cells are the most sensitive to the plant extract (IC50 = 0.31 μg/ml) followed the hepatic HUH-7 cells (IC50 = 2.25 μg/ml). Breast MCF-7 shows moderate sensitivity to the plant extract (IC50 = 37 μg/ml), while lung A549 cells shows a great resistance to the plant extract (IC50 320 μg/ml).