الفهرس 
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Abstract
Compositae (Asteraceae) is the largest family of vascular plants, their greater proportion are herbaceous, and about 2 percent are trees or shrubs.
The most common species reported in Egypt are Conyza dioscoridis (L.) Desf. and Conyza bonariensis (L.) Cronquist localized in Beni Suef and Fayoum.
Farmers in Egypt called C. dioscoridis (L.) Desf. species as mosquito tree as it acts as insect repellent. C. bonariensis (L.) Cronquist, its popular name is ”Buva” or ”margaridinha-do-campo”.
The literature on Conyza species was concerning their chemical contents, biological activities and their folk medicinal uses.
The present study includes four parts:
Part I: DNA Profiling of the Selected Species
The extracted DNA of each of the two Conyza species was amplified using ten decamer primers to detect their genetic variability. The ten primers of arbitrary sequences generated a total of 50 fragments in Conyza dioscoridis (L.) Desf. while only 48 fragments were generated in Conyza bonariensis (L.) Cronquist.
The study revealed that the pattern obtained using (OPB-14, OPB-15, OPB-19, OPB-20, OPG-12 and OPO-17) was almost identical in the two species; this was supported by their respective similarity coefficients (100%). These primers could be used as indicators for obtaining genetic markers as well as in the identification of different Conyza species, The OPB-07 primer was found the most effective in generating polymorphic bands with the least similarity coefficient, 44.45% and thus could be used to differentiate between the two species. The high percentage of similarity coefficients 89.8% indicates that the two species are closely related.
Part II: Comparative Phytochemical Study
Chapter I: Proximate analysis
Results of determination of the pharmacopoeial constants of the whole plant of the two species showed that the percentage of most of the constants (total ash, acid insoluble ash, water soluble ash and crude fibers) exceeded in Conyza bonariensis than Conyza dioscoridis. Only the percentage of moisture content of Conyza dioscoridis was much greater than that of Conyza bonariensis.
Chapter II: Preliminary phytochemical screening
Results of the phytochemical screening of total ethanol 70% of the whole plants of C. dioscoidis (L.)Desf. and C. bonariensis (L.) Cronquist and the three organs of C. dioscoidis (leaves, flowers and roots) revealed the presence of :
1. flavonoids, tannins, carbohydrates and/or glycosides, sterols and/or triterpenes in all organs of the two plants.
2. Steam volatile substances and coumarins were identified in all the tested samples except the roots of Conyza dioscoridis (L.) Desf. being localized in the leaves and flowers of the cited plant.
3. Alkaloids, saponins, crystalline sublimate, anthraquinones and cardiac glycosides are absent from the two plants.
Chapter III: Successive extraction and TLC examination of extractives
Results of TLC screening revealed the presnce of:
terpenes and sterols in all organs (leaf, flower and root) of C. dioscoridis.
flavonoids in the ethyl acetate and ethanol extracts of the root.
major spots of the flavonoids in the ethanol extract of the root.
Chapter IV: Spectrophotometric determination of phenolics
Results of determination of total phenolic contents in both species revealed that total contents (phenolic, flavonoids and tannins) were higher (261; 42.27 and 158.87 mg/g) in C. dioscoridis than C. bonariensis (207. 5; 18.08 and 121.37 mg/g) respectively.
Part III: Biological Study of the selected species
Chapter I: Comparative Evaluation of The Biological Activities of the whole plants of Conyza dioscoridis (L.) Desf. and Conyza bonariensis (L.) Cronquist
A. In-vitro biological studies
Evaluation of antimicrobial activity
The total ethanol 70% extract of C. dioscoridis and C. bonariensis were tested against different strains of bacteria and fungi and the study revealed that:
The total ethanol 70% extract of Conyza dioscoridis showed prominent activity (200µg/ml) against Mycobacterium pheli and moderate activity (400µg/ml) against three types of strains: Bacillus subtilis, Listeria innocua and Candida albicans.
The total ethanol 70% extract of C. bonariensis was inactive against all the strains.
2. Evaluation of the free radical scavenging activity
Free radical-scavenging activity of the alcoholic extract of C. dioscoridis showed higher % inhibition (73.5%) when compared to all C. bonariensis (69.5%) and the control rutin (88.2%).
3. In-vitro evaluation of cytotoxic activity
Three human cancer cell lines were chosen for testing the activity of the total ethanol 70% extracts of C. dioscoridis and C. bonariensis.
Results revealed the following:
1. The extract of C. dioscoridis showed higher activity in MCF7 (IC50 = 2.97) than C. bonariensis (IC50 = 17.5).
2. The extract of C. bonariensis was more active in HCT116 (IC50 = 21) and HELA (IC50 = 5.4) than C. dioscoridis (IC50 = 34.6) for HCT116 and (IC50 = 18) for HELA.
B. In-vivo biological studies
Acute toxicity study (determination of LD50)
Results of LD50 for both total ethanol 70% extracts of C. dioscoridis and C. bonariensis revealed that both extracts were safe up to 50mg/kg b.w and showed no signs of toxicity or mortality.
. Evaluation of anti-inflammatory activity 2
Results of the anti-inflammatory activity of total ethanol 70% of whole plant of C. dioscoridis and whole plant of C. bonariensis revealed a significant inhibition for C. dioscoridis (74.2%) when compared to C. bonariensis (59%) and the Indomethacin standard (57.51%).
3. Evaluation of antihyperglycemic activity
Results revealed the following:
a. Determination of blood Glutathione (GSH) level assay showed that the total ethanol 70% of C. dioscoridis exhibited an activity (45.04 ± 3.44 mg %) higher than C. bonariensis (40.78 ± 3.71 mg %), (P<0.05).
b. Determination of Serum Malondialdehyde (MDA) level assay showed that the total ethanol 70% of C. dioscoridis exhibited an activity (2.04 ± 0.3 nmol/ml) higher than C. bonariensis (3.03 ± 0.53 nmol/ml), (P<0.05).
c. Determination of serum glucose level assay showed that the total ethanol 70% of C. bonariensis exhibited an activity (104.63±7.38 nmol/ml) higher than C. dioscoridis (109.6±8.83 nmol/ml), (P<0.05).
d. Determination of serum insulin level assay showed that the total ethanol 70% of C. bonariensis exhibited an activity (6.13±0.70 ng/ml) higher than C. dioscoridis (6.10±0.40 ng/ml), (P<0.05).
All the previous results were comparable to the standard.
4. In-vivo evaluation of cytotoxic activity
Results of brine shrimp activity of total ethanol 70% extracts of C. dioscoridis and C. bonariensis at concentration of 50 mg/ml showed that the extracts are non-toxic to the tested organism at the level of the tested doses and suggest trials of higher doses which might be more effective. These findings appear to be contradictory to those obtained for the In-vitro evaluation of the cytotoxic potential against selected human cell lines; yet, this could be attributed to the low parallelism of the tested cell lines with brine shrimp lethality assay which has been reported to correlate only reasonably well with cytotoxic and other biological properties
Chapter II: Comparative Evaluation of the Biological Activities of the different organs of Conyza dioscoridis (L.) Desf.
A. In-vitro biological studies
Evaluation of antimicrobial activity
The successive extracts of leaves, flowers and roots of C. dioscoridis were tested against different strains of bacteria and fungi and the study revealed that:
The total ethanol 70% extract of roots and the ethyl acetate extract of leaves of C. dioscoridis showed prominent activity (50µg/ml) against Mycobacterium pheli followed by both ethyl acetate and petroleum ether extracts of flowers (100µg/ml) and chloroform and ethyl acetate fractions of roots (100µg/ml), petroleum ether of roots and chloroform extracts of flowers (200µg/ml) and showed moderate activity (400µg/ml) of petroleum ether, chloroform and ethanol of leaves. The ethanolic extract of roots showed prominent activity (100µg/ml) against Bacillus subtili followed by chloroform extract of flowers, ethyl acetate extract of leaves, chloroform and ethyl acetate extracts of roots (200µg/ml) and showed moderate activity (400µg/ml) of ethyl acetate extract of flowers, chloroform extract of leaves and petroleum ether, chloroform and ethanol extracts of leaves, flowers and roots. The chloroformic fraction of flowers showed prominent activity (200µg/ml) against Listeria innocua and moderate activity (400µg/ml) of ethyl acetate and ethanol extracts of root. The ethyl acetate and ethanolic fractions of roots showed prominent activity (200µg/ml) against Candida albicans and moderate activity (400µg/ml) of petroleum ether and chloroform extracts of flowers and chloroform extract of roots.
The chloroform extract of flowers and roots showed prominent activity (200µg/ml) against Staphylococcus aureus ”Non-pathogenic LMG 3242”.
No activity was shown against the following strains: Escherichia coli, Enterococcus faecalis, Staphylococcus aureus ”pathogenic LMG 3242” and Staphylococcus aureus ”Lab. Strain” for all extracts.
2. Evaluation of the free radical scavenging activity
Free radical-scavenging activity of the alcoholic extract of C. dioscoridis flowers showed the best inhibition (87.5%) when compared to the other tested samples: root (76.5%), leaf (70.5%) and the control rutin (88.2%).
3. In-vitro evaluation of cytotoxic activity
Three types of human cancer cell lines were selected for evaluation of the cytotoxic activity of the alcoholic extracts of C. dioscoridis three organs.
Results revealed that the flower extract of C. dioscoridis was the most significant organ (IC50=2.67) compared to leaf and root extracts against MCF7 cell line.
B. In-vivo biological studies
1. Acute toxicity study (determination of LD50)
Results showed the safety of all the tested extracts up to 0.5g/kg b.wt.
. Evaluation of anti-inflammatory activity 2
Results revealed that the total ethanol 70% extract of C. dioscoridis leaves was the most significant (76.2%) followed by flowers (72.6%) then root (66.6%) when compared to Indomethacin (78.5%).
3. Evaluation of antihyperglycemic activity
Results revealed the following:
a. Blood Glutathione (GSH) level of the total ethanol 70% extract of leaves of C. dioscoridis was the most significant (45.14 ± 3.24 mg %) when compared to other organs, (44.25 ± 4.58 mg %) for roots and (44.14 ± 4.11 mg %) for flowers, (P<0.05).
b. Determination of Serum Malondialdehyde (MDA) level assay showed that the total ethanol 70% extract of leaves of C. dioscoridis was the most significant organ (2.02 ± 0.31nmol/ml) when compared to other organs, (2.20 ± 0.38 nmol/ml) for roots and (5.43 ± 1.07 nmol/ml) for flowers, (P<0.05).
c. Determination of serum glucose level assay showed that the total ethanol 70% extract of roots of C. dioscoridis was the most significant organ (106.45±8.25 nmol/ml) when compared to other organs, (109.36 ± 9.83 nmol/ml) for leaves and (284.81 ± 39.82 nmol/ml) for flowers, (P<0.05).
d. Determination of serum insulin level assay showed that the total ethanol 70% extract of roots of C. dioscoridis was the most significant organ (6.53±0.83 ng/ml) when compared to other organs, (6.17 ± 0.68 ng/ml) for leaves and (2.55 ± 0.57 ng/ml) for flowers, (P<0.05).
All previous results were comparable to the standard.
4. In-vivo evaluation of cytotoxic activity
Results showed that the ethanol extract of the flower of C. dioscoridis exhibited the highest cytotoxic effect on brine shrimp nauplii (100% lethality) followed by that of the leaf (64.28%); the least activity being observed for the root extract (46.66%). In addition, despite the inefficiency of the total ethanol extract of the whole plant, when similarly estimated, separate examination of the different organs, under the same experimental conditions, confirms our suggestion that higher doses (>10mg/ml) of the whole plant should be tried.
Part IV: Phytochemical Study of the Root of Conyza dioscoridis (L.) Desf.
C. dioscoridis (L.) Desf. roots were chosen for the phytochemical study and isolation of the compounds based on their best results in antihyperglycemic activity when compared to the other organs of the same species and the whole plant of C. bonariensis (L.) Cronquist.
Also, little or no previous studies were reported on this organ.
Chapter I: Investigation of the Petroleum ether extract of the root
Study of lipoidal matters of the root extract of C. dioscoridis (L.) Desf.
The petroleum ether extract was fractionated into unsaponifiable and saponifiable fractions which were qualitatively and quantitatively analyzed using GLC apparatus and results are summarized into the following:
1. Identification of twenty one components representing 67.04% (hydrocarbons: 39.47% and sterols and triterpenes: 27.30%).
2. The major hydrocarbon was identified as n- Nonacosane (9.83%); the major sterol was stigmasterol (5.71%) and the major triterpene was Lupeol (9.29%).
3. Identification of sixteen components of fatty acids representing 86.44%. (saturated fatty acids 82.1% and unsaturated fatty acids 4.34 %).
4. The major saturated fatty acid was identified as octacosanoic acid (montanic acid) (70.99%) and Erucic acid was the major unsaturated fatty acid (1.84%).
USM of the roots of C. dioscoridis was subjected to a silica gel VLC using eluents of increasing polarities, where three compounds were isolated and identified as C1, C2, and C3.
Chapter II: Investigation of the chloroform extract
The chloroform extract was subjected to VLC using eluents of increasing polarities. Three compounds were isolated and identified as C4, C5 and C6. The isolated compounds were further purified using different chromatographic techniques.
Chapter III: Isolation and Identification of Phenolics from the Root
The ethanol extract was subjected to CC using eluents of increasing polarities and seven compounds were isolated and identified as C7, C8, C9, C10, C11, C12 and C13.
The isolated compounds of all the extracts were characterized by their m.p, chromatographic behavior and different spectral data. Structure elucidation and identification of the compounds was carried out by interpretation of their spectral data and comparison with the available literature. The isolated compounds from Conyza dioscoridis (L.)Desf.