Saddeq Ayad Mohammed Al- rahimy
|Determination of Genetic Variation and Antimicrobial Activity of Conocarpus Plant in Kerbala Province||the Master Degree in Biology|
Conocarpus is one of dicot, multi branching and evergreen ornamental plant. Its native tropical regions of the world and widely distribute in all cities of Iraq due to fast growing in worm regions. Many parts of Conocarpus tree is used to treat many human infections due to its medical characteristics. Also, there is a lack of information about Conocarpus especially molecular studies, for these reasons current study was carried out.
Generally, thirty-two samples of leaves of Conocarpus were collected from different regions of holy Karbala city and they undergo to DNA extract to identify matK and 18S rRNA genes variations and mutations as well as to make phylogenic tree for both genes. So, the identification of these genes was done at first by polymerase chain reaction (PCR) then sequence for these genes was made. Also, the primers used to amplify 18S rRNA gene were designed in the current study by Primer Blast website software using sequence in genbank of Conocarpus erectus 18S rRNA as reference. In addition, cold aqueous leaf extract was prepared to determine antibacterial activity against six human pathogenic bacteria which were three of gram negative (Acinetobacter baumanni, Proteus mirabilis, Yersinia enterocolitica) and three positive bacteria (Enterococcus faecalis, Staphylococcus aureus, Streptococcus agalactiae).
Also, qualitative determination of bioactive compounds of aqueous leave extract was made by gas chromatography-mass Spectrophotometer (GC-MS) technique. Moreover, hemolytic cytotoxicity in vitro for aqueous Conocarpus extract was calculated using human blood of healthy non-smooker. Also, well diffusion method was used to show bacteria sensitivity to Conocarpus extract as well as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of extract were determined for each bacteria by serial dilution method. However, the concentrations of Conocarpus extract which
used in well diffusion method. In addition, genetic effect to the bacteria was monitored by random amplification of polymorphic DNA (RAPD) for all tested bacteria after and before the treatment with 1 µg/ml less than their own MBC to show maximum effect of extract.
However, all concentrations showed significant differences between them at probability >0.05 and >0.01. Also, the biggest inhibition zone was 22.5 mm for Yersinia enterocolitica at 200 µg/ml which it showed the highest sensitivity to extract. On the other hand, at the same concentration Acinetobacter baumanni, Proteus mirabilis, Enterococcus faecalis, Staphylococcus aureus and Streptococcus agalactiae showed 21.3mm, 22.0 mm, 15.3 mm, 19.0 mm and 16.2mm respectively. So, the lowest MIC was 2 µg/ml for Staphylococcus aureus and MBC was 8µg/ml for the same bacteria. Whereas, the MIC of Acinetobacter baumanni, Enterococcus faecalis, Streptococcus agalctiae, Proteus mirabilis and Yersinia enterocolitica was 20, 9, 7, 9 and 8 µg/ml respectively, while the minimum bactericidal concentration (MBC) for these bacteria was 36, 16, 14, 15 and 14 µg/ml respectively. However, hemolytic cytotoxicity of aqueus leaves extract of Conocorpus showed a little cytotoxicity, about 1.72% of hemolysis.
On the other hand, morphologic characteristic of all the 32 samples such as leaves shape, arrangement, stem shape and width, etc. were similar to Conocarpus lancifolius. However, the sequence of MatK gene showed complete matching (similarity) of MatK gene with Conocarpus erectus, while 18S rRNA showed presence of heterozygous point mutation of transition substitution purine to purine. The substitution of nitrogen base in mutation location was adenine(A) to guanine(G) in one chromosome, but another chromosome has adenine without exposed to such substitution. So, mutant chromosome showed 99.83% of matching (similarity) when it was compared with C. erectus while, non-mutant chromosome showed complete matching 100% of similarity with C. erectus.
On the other hand, GC-MS for the C. lancifolius extract showed nine alkaloid compounds and eight compounds of total phenols which they were 6 compounds for phenol derivatives and 2 compounds for flavonoids. In addition, 1 terpenoid compound and 3 alcohol compounds as well as 4 amino compounds were present. However, one amine compound was common with one of phenolic compound.