الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 215 |  |  |
| | | from | 215 | | |
Abstract
Summary
The incidence of viral infection increases world wide either
with novel strains of viruses or with drug resistant strains. In this
context, the production of antiviral drugs from natural sources
such as marine microorganisms is a vital demand.
The present study aimed to maximize the antiviral potential
of newly discovered Streptomyces isolate. The obtained results
can be summarized as the following:
1. Seventeen sediment samples, collected from Qarun Lake and
Hurghada, Egypt during 2012, were used to isolate
actinobacteria using 5 different isolation media.
2. Starch casein agar medium was proved to provide the highest
count of the actinobacteria comparing with the other tested
media.
3. Eighty two actinobacterial isolate were screened for their
antimicrobial activities using well diffusion method on 6
bacterial including Escherichia coli (ATCC 10536),
Staphylococcus aureus (ATCC 6538), Pseudomonas
aeruginosa ATCC 9027, Bacillus subtlis (ATCC 6633),
Bacillus cereus (ATCC 10876) and Salmonella typhimurium
(ATCC 14028) and 4 fungal strain; Aspergillus
niger111EMCC, Aspergillus flavus ATCC 204304,
147
Penicillium chrysogenum ATCC 10106 and Candida albicans
105EMCC.
4. Eighteen actinobacterial isolate were found to have
antimicrobial activities and further screened for their antiviral
activities against RNA virus model (VSV) and DNA virus
model (HSV-2) using CPE inhibition assay.
5. Of 18 isolates, nine isolates proved to have antiviral activities
against VSV and HSV-2. Q3 isolate was found to be the most
bioactive.
6. Q3 isolate as the most potent antiviral producer was identified
based on its morphological, cultural, physiological and
molecular studies of 16S rRNA to be a novel strain for which
the name S. qarunensis sp. nov. is proposed. This isolate was
recorded in the GenBank under accession number of
KT748525
7. Classical medium optimization was performed. It has been
found that starch (20 g/l) as a carbon source, soybean meal (3
g/l) as nitrogen source , Incubation temperature (35°C),
incubation period of 3 days and pH of 8.5 give the highest
antiviral activities and growth rate of S. qarunensis sp. nov.
148
8. Plackett-Burman statistical design was used to determine the
significant factors affecting antiviral activities. It was found
to be soybean, CaCO3 and ZnSO4.
9. Central Composite design was used to determine the optimum
level of each significant factor giving the highest antiviral
potential. The results revealed that using soybean (4 g/l),
CaCO3 (0.01g/l) and ZnSO4 (0.01 mg/l) increased the
antiviral activity by 2.22 fold compared to the basal medium.
10.Ethyl acetate was the most powerful solvent in the extraction
of the antiviral metabolites which reduced the VSV titer by
41.67% and 45.45% for HSV-2.
11.TLC plate developed using n-hexane: ethyl acetate (9:1) as
eluting agent provides a well separation of 7 pure compounds
coded A-G so this system was used for the column
chromatography.
12.Among eight compounds recovered from preparative TLC,
compound A was the most active against VSV and HSV-2
with EC50 of 96 and 90μg/ml, respectively so it was
identified.
13.Compound A was the safest antiviral compound with
selectivity index of 9.8 for VSV and 10.5 for HSV-2.
149
14.By studying the mechanism by which compound A affect on
the tested viruses, the results indicated that it inhibits
attachment stage of virus replication cycle.
15.The physical properties of the compound A was as the
following:
v Light yellow in color.
v Oily form.
v Soluble in ethanol, hexane, ethyl acetate,
chloroform as organic solvents.
v Insoluble in water.
16.The chemical structure elucidation of the produced antiviral
compound A was based on detailed spectroscopic analysis
including IR, UV, 1H NMR, 13CNMR, mass spectrum and
elemental analysis. Such techniques indicated that the target
compound is Di (2-ethyl hexyl) phthalate with molecular
formula; C24H38O4 which have potent antiviral activities
against the two tested RNA (VSV) and DNA (HSV-2) viruses.
150
Conclusion
from the present study, it is concluded the following;
1. The sediment sample of Qarun Lake is a good source of
biomaterial for isolation of antimicrobial potential new
actinobacteria.
2. 16S rRNA analysis is a powerful method to confirm the
identity of actinobacteria.
3. The statistical approach using RSM showed significant
results for optimizing the process parameters for maximal
antiviral activities using Streptomyces qarunensis sp. nov.
4. The optimized medium developed to obtain the highest
antiviral potentials has the following compositions (g/l);
starch,20; casein, 0.3; soybean, 4; K2HPO4, 2; NaCl, 2;
MgSO4,0.05, CaCO3,0.01and ZnSO4(0.01mg/l) with 500ml
natural sea water and 500ml distilled water with pH of 8.5
and incubation temperature of 35ºC at 200 rpm for three
days.
5. N-hexane: ethyl acetate (9:1) solvent mixture is a powerful
to isolate non polar compounds.
6. The molecular formula of compound A, di-(2- ethyl hexyl
phthalate), was C24H38O4.
151