الفهرس 
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Abstract
Silybum marianum (L.) Gaernt (milk thistle) has been used for centuries as an herbal medicine for the treatment of liver disease. Milk thistle is an annual/biennial plant of the Asteraceae family, native of Mediterranean area and now growing and cultivated worldwide. S. marianum L. has been known since ancient times in traditional medicine, in Traditional Chinese Medicine seeds of milk thistle are used to increase bile secretion as well as for treatment of liver problems such as jaundice, hepatitis, gallstones and cirrhosis. Young fleshy stems and seeds of milk thistle are traditionally eaten by Arabs. In Europe, milk thistle was grown as a food source. In Sardinia (Italy), young heads and stems are eaten directly after peeling. In Spain, S. marianum L. has traditionally been used as a vegetable for salads and fried. Until now only silymarin from plant has market value. As the plant seeds contain a good amount of oil, this oil is still regarded as waste product. Besides, the plant produces a high yield of biomass, which is also considered as another waste product.
Therefore, the main objective of this work is to identify potential value in these waste products (oil and leaves). In addition, plant cultivation is essential in this work to ensure the continuous supply of the plant material.
This study includes the following.
• Cultivation of Silybum marianum L.
• Chemical investigation of both seeds oil and plant leaves.
• Improving yield of silymarin by different chemical and physical treatments.
• Biological investigation of plant oil and leaves including anti-inflammatory activity, cancer chemoprevention activity and antimicrobial screening.
• Molecular docking
Part 1: Cultivation of Silybum marianum L.
The cultivation of the plant was carried out at Wadi Elsheeh, Farm (about 50 Km south of Assiout city), Egypt. Cultivation practice, fertilization and harvest process are discussed in details in this work.
Part 2: Investigation of Silybum marianum L. Seeds
1. Extraction of Oil from Plant Seeds
A. Fatty Acid Composition
Three different methods; solvent extraction, cold pressing and ultrasonic-assisted extraction were used for the extraction of oil from plant seeds. Gas chromatography-mass spectrometry analysis of the fatty acids compostion of the prepared oils revealed variation in the percentage yield and chemical composition. The analysis showed the presence of nine fatty acids in all oil samples except nonanoic acid which was absent in the oil sample prepared by ultrasonic-assisted extraction. Also, linoleic acid was the most abundant in both cold pressed and ultrasound assisted oil samples (30.27, 46.69 %); respectively. where oleic acid was the major fatty acid in the oil sample prepared by organic solvent extraction (28.16 %). Ultrasonic-assisted extraction offered reduction in the extraction time (1 hr vs 6 hr) with oil yield (8 %) when compared to organic solvent (30 %).
B. Chemical Analysis of Silybum marianum L. Oil
Chemical analysis of S. marianum L. cold pressed oil was done. Acid, peroxide and iodine values were 4.49, 5.70 and 107; respectively.
2. Extraction of Silymarin from Silybum marianum L. Seeds
A. Effect of Extraction Solvent on the Yield and Quality of Silymarin
Three different solvent; acetonitrile, ethyl acetate and methanol were used for extraction of silymarin from plant seeds. The extract yields obtained with acetonitrile, ethyl acetate and methanol were 18, 16.5 and 13 gm; respectively. HPLC analysis of silymarin constituent’s in seed extracts varied according to the extraction solvent. Higher concentrations of taxifolin, silychristin, silydinin, isosilybin A and isosilybin B were recorded in methanol extract followed by acetonitrile extract while higher concentrations of silybin A and silybin B were recorded in acetonitrile extract followed by ethyl acetate extract.
B. Effect of Storage Time on Yield and Quality of Silymarin
The storage of the plant seeds decreased the concentration of silymarin as shown by HPLC analysis. The extract yields obtained by fresh sample and after storage sample were 20 and 16.5 gm; respectively. Higher concentrations of taxifolin, silychristin, silydinin and isosilybin B were recorded in the stored seeds sample while higher amounts of silybin A, silybin B and isosilybin A were found in fresh seeds sample.
C. Comparison of Silymarin Content in Egyptain and German
Silybum marianum L. Seeds
Two different seeds sources, seeds cultivated in Egypt and seeds purchased from Germany, were compared with respect to their silymarin content. No significant difference was observed in silymarin yield and composition as shown by HPLC analysis.
D. Effect of Particle Size on Total Extraction Yield and Silymarin Content of Silybum marianum L. Seeds
Nano-sized plant seeds gave higher amount of silymarin than that obtained from seeds ground by normal electric blender (4.36 and 3.64 gm; respectively). Also, silymarin components were higher in nano-sized particles.
3. Investigation of Silybum mariunm L. Leaves
A. Preliminary Phytochemical Screening
Preliminary phytochemical screening of the air dried powdered leaves of S. mariunm L. revealed the presence of carbohydrates and/or glycosides, sterols and/or triterpenes, flavonoids, tannins, and saponins and absence of alkaloids and cardiac glycosides.
B. Determination of Phenolic Acids and Flavonoids Content of Silybum mariunm L. Leaves
Phenolic acids and flavonoids content of plant leaves were estimated by HPLC. The most abundant phenolic acid was gallic acid (0.838 mg/g) while naringenin was the major flavonoid (0.955mg/g). Other compounds were identified as syringic acid, vanillin, caffeic acid, quercetin, coumaric acid and cinnamic acid (0.224, 0.110, 0.082, 0.038, 0.086, 0.042 mg/g; respectively) in the leaves extract.
C. Nutrient Compostion of Silybum mariunm L. Leaves
• Macro- and micro-elements of Plant Leaves
The analysis of macro- and micro-elements in the plant leaves revealed presence of iron, copper, zinc, calcium, nitrogen and potassium.
• Protein, Dry matter and Moisture Content of Silybum marianum L. Leaves
Leaves of S. marianum L are a rich source of protein (21.87 %) and moisture content of plant leaves was (4.14%) while the dry matter content was (95.85 %).
• Amino Acids Composition of Silybum marianum L. Leaves
Markedly amounts of essential amino acids as leucine (4.42%), arginine (2.05%) and lysine (2.53%) were detected. The plant leaves contains also non-essential amino acids as proline (12.87%), glutamic acid (7.86%), glycine (4.62%) and serine (1.34%).
D. Determination of Silymarin Content in the Leaves of Silybum marianum L.
During Different Growth Stages
HPLC analysis showed that silymarin components in plant leaves extracts varied during three different growth stages. The highest concentration of taxifolin was found in the pre-flowering stage while silychristin was found only in the pre-flowering stage and flowering stage. The highest amounts of silybin A found in fruiting stage then pre-flowering stage and flowering stage. Other components such as silydinin, silybin B, isosilybin A and isosilybin B were not detected in three different growth stages.
E. Isolation and characterization of the Main Constituents from Plant Leaves
S. mariunm L leaves were extracted with CH2Cl2/ MeOH (1:1) and 70% MeOH. The extracts were fractionated using column chromatography followed by semi-preparative HPLC to obtain six pure compounds. The isolated compounds were identified by 1H- and 13C- NMR and UV spectroscopy. The compounds were identified as cholesterol, stigmasterol, apigenin-7-O-β-D-glycoside, kaempferol, naringin and luteolin-7-O-β-7-rhamnoglucoside. This is the first isolation Silybum mariunm L. leaves.
Part 3: Biological Evaluation of Silybum mariunm L.
A. Evaluation of the Anti-inflammatory Activity
Anti-inflammatory activity of the total leaves extract (CH2Cl2/ MeOH (1:1)) and seed oil prepared by cold pressed, in comparison with silymarin, was estimated by inhibition of LPS-induced iNOS protein expression in RAW 264.7 macrophages. Western blotting analysis revealed the potency of silymarin to completely inhibit the LPS-induced iNOS protein expression at both tested concentrations (100 and 50 µg/ml). In addition, milk thistle leaves extract caused 41.5 and 48.5% inhibition of LPS-induced iNOS protein expression. This is the first report on the potential of milk thistle leaves as anti-inflammatory agents.
B. Evaluation of the Anti-cancer Activity
Anti-cancer activity of the total extract CH2Cl2/ MeOH (1:1) of leaves extract and seed oil prepared by cold pressed, in comparison with silymarin, was estimated by induction of the chemopreventive marker NQO1 using western blotting to assess the chemopreventive potential in murine Hepa-1c1c7 cells as inducers of the protein expression NQO1. Results revealed that the leaves extract caused a moderate NQO1 protein induction of 60% compared to the vehicle control. The induction shown was at 100µg/ml without apparent induction at 50µg/ml. This is also the first report on the potential of milk thistle leaves as anti-cancer agents.
D. Evaluation of the Anti-microbial Activity
Anti-microbial activity of the total extract CH2Cl2/ MeOH (1:1) of leaves extract, and seed oil prepared by cold pressed, in comparison with silymarin, was estimated using micro-dilution well plate method. The results revealed that the leaves extract and cold pressed oil gave minimum inhibitory concentration (MIC) at 153.8 and 178.7 µg/ ml; respectively against Staphylococcus epedrmidis while silymarin showed (MIC) at 298.1 µg/ ml. In addition, leaves extract showed MIC at 275.2 µg/ ml against Bacillus subtilis while silymarin and cold oil were inactive. Silymarin and leaves extract showed MIC at 257.8, 266 µg/ml and 410.2, 256 µg/ml against Salmonella typhimurium and Escherichia coli; respectively while all extracts were inactive against Staphylococcus aureus.
E. Docking Studies of Biologically Active Substances from Silybum marianum L. with Anti-oxidant and Anti-inflammatory Pathways
Molecular docking studies of major metabolites previously isolated from Silybum maraiunm L. suggested active hits for antioxidant and anti-inflammatory activities. Neosilyhermin B was found to interact with SER363, GLY364, LEU365, ALA366, GLY367, ASN414, ARG415, ILE416, GLY417, VAL418, GLY419, GLY462, VAL463, GLY464, VAL465, CYS513, THR560, GLY603 and VAL604 residues. These simultaneous binding of Neosilyhermin B with Keap1 and the strong binding energy suggested that the protein to be its direct binding target. Taxifolin was found to interact with LYS349, ARG351, GLY352, PHE353, ARG354, ARG356, HIS364, GLY365, GLY366, PRO368 and SER372 residues. These simultaneous binding of taxifolin with NFKB and the strong binding energy suggested that the protein to be its direct binding targe.