الفهرس 
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Abstract
GENERAL SUMMARY
Phytochemical and Biological Studies on Certain Plants Belonging to Family Crassulaceae
Family crassulaceae is a large family, widely distributed in cosmopolitan, especially southern Africa, except Australia and West pacific. It comprises about 35 genera and 1500 species.
In traditional medicine the juice of Kalanchoe is used for the local treatment of bruises, wounds, boils, insect bites, ear infection, dysentery fever, abscesses, coughs, skin diseases, cholera, urinary diseases, tissue injuries, arthritis and gastric ulcers.
In this thesis, two species that are presently cultivated in Egypt as ornamental plants have been selected. Kalanchoe thyrsiflora Harv. and Kalanchoe marmorata Baker were selected as plants for research as little could be traced in literature. Main goal of the thesis is phytochemical investigation of different extracts of both plants in a biologically guided manner in an effort to fractionate and separate hopefully new compounds of interesting chemical structure and effective biological activity.
This study is divided into three chapters:
The first chapter includes: DNA Profiling of Kalanchoe thyrsiflora Harv. and Kalanchoe marmorata Baker using Random Amplified Polymorphic-DNA (RADP-PCR) Technique.
Chapter two includes: Biological study of the different leaf extracts & subfractions of Kalanchoe thyrsiflora Harv. and Kalanchoe marmorata Baker viz. cytotoxic activity.
Chapter 3 comprises: Phytochemical Investigation of different leaf extracts of Kalanchoe thyrsiflora Harv. and Kalanchoe marmorata Baker.
CHAPTER 1
DNA Profiling of Kalanchoe thyrsiflora Harv. and Kalanchoe marmorata Baker. Using Random Amplified Polymorphic-DNA (RADP-PCR) Technique
The extracted DNA of the two Kalanchoe species was amplified using ten decamer primers to detect their genetic variability. Each of the ten primers had successfully directed the amplification of a genome-specific fingerprint of DNA fragments. Consequently this will serve to evaluate interspecific diversity between these species.
The ten primers were (OPB-12, OPB-18, OPB-09, OPB-15, OPO-09, OPB-01, OPO-02, OPA-02, OPA-06, OPA-11) of arbitrary sequences had generated 92 fragments in Kalanchoe thyrsiflora Harv., while 75 fragments were generated in Kalanchoe marmorata Baker.
The low percentage of similarity coefficients indicates that the two species are not closely related.
It can be concluded that the most relevant fragment resulting from the successful combination of template and primer was that produced by OPB-09, OPA-11 RAPD primers. Such primers could be used to discriminate between the two Kalanchoe species depending on their low values of similarity coefficients 0%, 16% respectively and high level of polymorphism. However OPA-02, OPB-18, which produces high values of similarity coefficient 60.86%, 53.33% respectively and low levels of polymorphism, could be used in the identification of different Kalanchoe species.
CHAPTER 2
Biological study of Different Leaf Extracts of Kalanchoe thyrsiflora Harv. and Kalanchoe marmorata Baker
The five extracts of both Kalanchoe species [aqueous extract, alcohol extract, dichloromethane fraction, ethyl acetate fraction and n-butanol fraction] at concentration of 0-50 µg/ml were tested for their cytotoxic activity using tumor cell line MCF7 (Breast carcinoma cell line). It was obvious that the most potent cytotoxic fraction was dichloromethane fraction of Kalanchoe thyrsiflora Harv. This active fraction had led to the isolation of two pure isolated compounds which were further tested for their cytotoxic activity on both breast carcinoma cell line and normal melanocyte cell line.
The two isolated pure compounds (-oxo-olean-12-ene and β-sitosterol) from the dichloromethane fraction of Kalanchoe thyrsiflora Harv. were also tested for their cytotoxic activity using both Breast carcinoma cell line (MCF7) and normal melanocyte cell line (HFB4)
The two Compounds showed similar moderate cytotoxic activity at IC50 17.4 and 17.6 µg/ml respectively. Comparing their activity on normal cell line, the two isolated compounds showed more selectivity on breast carcinoma cell line than normal melanocyte cell line.
By comparing dichloromethane fraction with the two isolated compounds, it was found that dichloromethane fraction exihibited higher cytotoxic activity than two isolated compounds. This is may be due to the synergistic activity of other components present in the dichloromethane fraction.
CHAPTER 3
Phytochemical Investigation of Different Leaf Extracts of Kalanchoe thyrsiflora Harv. and Kalanchoe marmorata Baker
Phytochemical screening of leaf extracts of both species revealed the presence of flavonoids, sterols and /or triterpenes, carbohydrates and or glycosides, tannins and cardiac glycosides which is typical to this genus; traces of coumarins & saponins and no anthraquinones nor alkaloids.
The aqueous extract of both species were fractionated using dichloromethane, ethyl acetate and n-butanol. The dichloromethane fraction of Kalanchoe thyrsiflora Harv. (5.27g) was fractionated over (silica gel) to yield the sum of (17) sufractions. The main fractions and their isolation were displayed in the scheme described in p. 134
from the (17) fractions, only two fractions IV, V were fully investigated. These fractions were manipulated through column chromatography (silica gel) leading to the isolation of the two individual chemical constituents. Isolated compounds were identified by IR, 1H-NMR, 13C-NMR after comparison with reported data.
1: 3-oxo-olean-12-ene
2: β-sitosterol
Ethyl acetate fraction (1.9 g) of Kalanchoe marmorata Baker was chromatographed on whatmann No. 3 MM for paper chromatography using BAW to yield 7 bands (A-G), each band dissolved in methanol HPLC grade then co-chromatographed using BAW and 6% acetic acid which showed major spots which were further subjected to isolation and purification using PPC and solvent 6% acetic acid leading to the isolation of the five pure compunds. The isolated compounds were identified by UV, 1H-NMR and 13C-NMR spectroscopic data after comparison with reported data.
3: Isorhamnetin-3-O-α-L-1C4-rhamnopyranoside
4: Quercetin
5: 4’-methoxy myricetin-3-O-α-L-1C4-rhamnopyranoside 6: Quercetin-3-O-β-D-4C1-glucopyranoside (isoquercitrin)
7: Protocatechuic-4-O-β-D-4C1-glucopyranoside
Leaves of Kalanchoe thyrsiflora Harv.
Dichloromethane fraction
• Silica gel
• Hexane: Acetone (increasing polarity)
1: 3-oxo-olean-12-ene
2: β-sitosterol
Leaves of Kalanchoe marmorata Baker
Fractionation
Ethyl acetate fraction
• whatmann No. 3 MM for paper chromatography
• BAW
3: Isorhamnetin-3-O-α-L-1C4-rhamnopyranoside
4: Quercetin
5: 4’-methoxy myricetin-3-O-α-L-1C4-rhamnopyranoside 6: Quercetin-3-O-β-D-4C1-glucopyranoside (isoquercitrin)
7: Protocatechuic-4-O-β-D-4C1-glucopyranoside.