الفهرس 
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Abstract
Dioon spinulosum Dyer ex Eichler is one of the cycads that belongs to Zamiaceae family. Superficially it resembles palm. This plant has reported cytotoxic activity against Hep-2 cell line and used in folk medicine by ancient people in Mexico for treatment of ulcers and cancer (Mena-Rejon et al., 2009), the popular use is for gastric cancer and gastrointestinal disorders (Alonso-Castro et al., 2011).
THIS STUDY INCLUDES TWO PARTS:
PART I: Chemical Study
Includes six chapters:
Chapter 1: DNA fingerprinting of Dioon spinulosum.
Chapter 2: Preliminary phytochemical screening of Dioon spinulosum.
Chapter 3: Investigation of the lipid content of the leaves of Dioon spinulosum by GC/MS.
Chapter 4: Metabolomic profiling using LC/HR MS analysis.
Chapter 5: Extraction and chromatographic investigation of the major constituents
of Dioon spinulosum Dyer leaves.
Chapter 6: Identification and structure elucidation of the isolated compounds of Dioon spinulosum leaves.
PART II: Biological Study
Chapter 1: DNA fingerprinting of Dioon spinulosum
The analysis of the amplified fragments generated by SCoT and ISSR reactions revealed that: SCoT 4, SCoT 6, SCoT 8, HB-9 and HB-12 can be used for the identification of D. spinulosum Dyer at the genetic level since they generated the highest number of bands with wide molecular size, and the proportion of the polymorphism indicates that D. spinulosum and D. edule exhibit moderate level of polymorphism.
Chapter 2: Preliminary phytochemical screening of Dioon spinulosum
The preliminary phytochemical screening of D. spinulosum leaves revealed:
Presence of flavonoids, sterols, carbohydrates and/or glycosides and/or triterpenes in the leaves of D. spinulosum Dyer and absence of steam volatile substances, tannin, saponin, alkaloids, anthraquinones and cardiac glycosides.
Chapter 3: Investigation of the lipid content of the leaves of Dioon spinulosum
The lipoidal matter of the leaves of D. spinulosum obtained by fractionation of alcohol 70% extract with n-hexane. The n-hexane fraction was subjected to saponification procedure. Then GC analysis is performed for unsaponifiable fraction and fatty acid methyl ester (FAME) in order to identify their constituents.
GC analysis of the unsaponifiable matter:
The unsaponifiable matters of D. spinulosum dyer ex. were dominated by high amount of hydrocarbons; 18 compounds and represent (75.02%) including 1-butylhexyl Benzene (4.42%), 1-propylheptyl Benzene (3.63%), 1-ethyloctyl Benzene 2.49%, 1-methylnonyl Benzene (2.54%), 1-propyloctyl Benzene (7.25%), 1-ethylnonyl Benzene (5.8%), 1-methyldecyl Benzene (7.18%), 1-butyloctyl Benzene (4.54%), 1-propylnonyl Benzene (6.02%), 1-ethyldecyl Benzene (4.34%), 1-methylundecyl Benzene (5.94%), 1-pentyloctyl Benzene (5.98%), 1-butylnonyl Benzene (4.17%), 1-propyldecyl Benzene 4.13%, 1-ethylundecyl Benzene (2.30%), 1-methyldodecyl Benzene (3.51%), Heneicosane (0.2%), Pentatriacontane (0.6%).
Sterols was 6 compounds represent(12.21%), β-sitosterol (3.32%), stigmastan-3,5-diene 2.44%, Pregnan-20-one,3-(acetyloxy)-5,6:16,17diepoxy (2.79%), androstane-11,17-dione,3-[(trimethylsilyl)oxy]-17-[O-(phenylmethyl)oxime] (3.62%), stigmastan-6,22-dien,3,5-dedihydro 0.02%, 4,6-cholestadienol (0.02%). Terpenes represented by four compounds (12.53 %), represented by squalene (3.32%), nerolidol (5.13%), Cis-farnesol( 4.08%) and phytol (0.16%).
GC analysis of fatty acid methyl esters (FAME):
The total identified FAME 23 compounds (81.03 %). Methyl 23-hydroxycholate tetratms (10.32 %) was the major identified saturated fatty acid, 9,12,15-octadecatrienoic acid,2-[(trimethylsilyl)oxy]-1-(trimethylsilyl)oxy]methyl]ethyl ester (Z,Z,Z) (6.6 %) and 9,12-octadecadienoic acid (Z,Z)-2,3-bis[(trimethylsilyl)oxy] propyl ester (5.66 %) are the major identified unsaturated fatty acids.
Chapter 4: Metabolomic profiling using LC/HRMS analysis
The dereplication of aqueous of total ethanol extract 70% of D. spinulosum Dyer was identified according to Dictionary of Natural Products (DNP 23.1, 2015 on DVD) and Reaxys online database, and revealed the identification of one flavanonol (Aromadendrin), one diterpene alcohol (Trans-Phytol), six biflavones: Amentoflavone, Amentoflavone-4’-Me ether (Bilobetin), Amentoflavone - 7-Me ether (Sequoiaflavone), Amentoflavone-4’,4’’’-Me ether (Isogenkgetin), 4’,4’’’,7-O-tri methyl amentoflavone (Sciadopitysin) and Amentoflavone-7,7’’,4’,4’’’-Me ether (7,7’’,4’,4’’’-O-tetra methyl amentoflavone).
Chapter 5: Extraction and chromatographic investigation of the major constituents of Dioon spinulosum Dyer leaves
(A) chromatographic investigation of the n-hexane fraction:
A weighed amount of unsaponifiable matter (3 g) was subjected to fractionation by a Silica gel column (150 g silica, 5 cm x 70 cm). Elution was performed starting with 100% n-hexane and the polarity gradually increased with EtOAc in 10% stepwise increment till 100% EtOAc. Fractions (25ml) each were collected, concentrated and monitored by TLC. Similar together to give four main fractions:
Fraction A: (1-16) eluted with 10% EtOAc in n-hexane.
Fraction B: (0.7 g, 18-30, eluted with 20-30% EtOAc in n-hexane) was further purified by rechromatography on Sephadex LH-20 column (2 cm x 75 cm), Isocratic elution DCM/MeOH (95:5) to yield compound D1 (200 mg).
Fraction C: (31-48) eluted with 40% EtOAc in n-hexane.
Fraction D: (49-60) eluted with 100% EtOAc.
(B) chromatographic investigation of fraction the dichloromethane fraction:
DCM extract of D. spinulosum Dyer Ex. leaves (8 g) was chromatographed on silica gel column (5 cm x75 cm) using 100% DCM and the polarity gradually increased with MeOH in 5% increment, fraction (25 ml) each were collected and monitored by TLC. Similar fractions were pooled together to give four main fractions:
Fraction A: (1-13) eluted with 100% DCM.
Fraction B: (0.5 g, 14-22, eluted with 5% MeOH in DCM) was further purified by rechromatography on silica gel column (1 cm x 60 cm) eluted isocratically with DCM: MeOH (95:5) to yield compound D2 (8 mg).
Fraction C: (0.5 g, 23-39, eluted with10-15% MeOH in DCM) was further purified by rechromatography on silica gel column (1 cm x 70 cm) eluted with 100% DCM then increase the polarity with 1% MeOH to yield compound D3 (5 mg).
Fraction D: (2 g, 40-52, eluted with 20% MeOH in DCM) was further purified by rechromatography twice using sephadex LH-20 (2x60 cm) eluted with DCM: MeOH (80:20) to yield compound D4 (300 mg).
(C) chromatographic investigation of the ethyl acetate fraction:
The EtOAc extract of D. spinulosum Dyer leaves (5 g) was chromatographed on a polyamide column (5cm x70 g) eluted with 100% H2O and the polarity gradually decreased with MeOH in 20% stepwise increment till 100% MeOH. Fractions (25ml) each were collected and monitored by TLC.
Similar fractions were pooled together to give four main fractions:
Fraction A: (1-19) eluted with 10 % MeOH in H2O.
Fraction B: (0.7 g, 20-25, eluted with 20 % MeOH in H2O) was further purified by rechromatography on sephadex LH-20 column (2 cm x75 cm), eluted with 20% H2O in MeOH to yield compound D5 (10 mg).
Fraction C: (26- 36) eluted with 50 % MeOH in H2O.
Fraction D: (37-52) eluted with 100 % MeOH.
Chapter 6: Identification and structure elucidation of the isolated compounds of Dioon spinulosum leaves
The structure of D1 which isolated from n-hexane fraction was identified by 1HNMR and 13C NMR as well as comparison with the available literature.
Compound D1: β-sitosterol (white needle crystals).
The structures of D2, D3, D4 which isolated from DCM fraction were identified by 1HNMR and Dept Q NMR as well as comparison with the available literature.
Compound D2: 7,7’’,4’,4’’’-O-tetramethylamentoflavone (pale yellow powder)
Compound D3: Sciadopitysin (pale yellow powder)
Compound D4: Amentoflavone (yellow powder)
The structures of D5 which isolated from EtOAc fraction was identified by 1HNMR as well as comparison with the available literature.
Compound D5: Aromadendrin ( white powder)
The structures of identified compounds are presented in Figure (30).
Figure (30): Structure of compounds isolated from Dioon spinulosum leaves
Part II: In vitro cytotoxic activity of Dioon spinulosum on MCF-7 and HELA cell lines
In vitro cytotoxic activity of the Dioon spinulosum total ethanol 70% extract and successive fractions by SRB assay against MCF-7 and HELA cell lines. D. spinulosum shows a good cytotoxic activity against MCF-7 cell line as the total ethanol 70% extract (IC50=22.5 µg/ml), EtOAc fraction of D. spinulosum (IC50=18 µg/ml) and Aromadendrin compound D5 separated from EtOAc fraction shows (IC50=12.5 µg/ml) as shown in Table (22) and Figure (28), also it exhibits a good cytotoxic activity against HELA cell line as the total ethanol 70% extract (IC50= 21.8 µg/ml), the n-hexane fraction showed (IC50= 21.1 µg/ml), EtOAc fraction showed (IC50 = 26.5 µg/ml) and the DCM fraction showed (IC50 = 24.5 µg/ml) and n-butanol fraction showed (IC50= 21.4 µg/ml) as presented in Table (22) and Figure (29).
So D. spinulosum can be potentially used as adjunctive therapy for breast and adenocervix carcinoma.depending on its secondary metabolites content.