الفهرس 
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Abstract
28: Cytotoxicity of Nano- prebiotic EPS on WI-38 Cell Line
5. SUMMARY
The present study aims to use some of Lactobacillus sp. as natural source of therapeutic bacteria in protection or treatment of colon human cancer. On the other hand study of the active metabolite that can use for the protection or treatment of some human colon cancer. Extraction, purification, identification and characterization of the biologically active metabolic products produced by the selected Lactobacillus strain were conducted.
The results of the experimental studies which were conducted could be summarized in the following points:
Five samples were collected from the normal habitats of probiotics bacteria such as fresh raw animal milk, fermented foods cheese, infant feces. After isolation 26 pure Lactobacillus isolates were obtain. The pure cultures Gram-positive and catalase-negative were characterized cell morphology.
The pure 26 cultures were tested as producer of EPS. Only 8
Lactobacillus isolates were obtained as EPS producer.
The resulted sequence from PCR amplification process was aligned with the corresponding sequences of representive Lactobacillus species. The obtained results revealed that the sequence of the isolates showed highest similarity with Lactobacillus brevis.
The effect of eight EPS samples on the growth of colorectal adenocarcinoma ATCC ATB-37 (CACO2) carried out. The pure EPSs extracted from 8 producer Lactobacillus brevis strains were tested against CACO2 using MTT method mention in details at section Materials & Methods. Results revealed that EPSs inhibited the proliferation of the CACO2 cell lines in a dose- dependent manner. The IC50 values were found to be; 635, 338, 462, 436, 374, 349 and 716 ug/ml respectively. Results indicated that EPSs from Lactobacillus brevis strains have a
potent inhibitory effect on CACO2 cell line also, EPS from Lactobacillus brevis no. 2 considered the most potent activity with IC50 338 ug/ml.
Characterization of the EPS produced by Lactobacillus brevis no.2 done. The UV spectrum analysis of the purified EPS was revealed the detection of peak at 258 is characteristic of carbohydrates. The IR spectrum of the purified EPS was determined using a Fourier transform- infrared spectrophotometer (FTIR, Jasco 6100, and Model Japan). The IR (potassium bromide) spectrum illustrated in Fig. (22) exhibits absorbance peaks at: 3274 cm-1 (OH stretching vibration), and 1053 cm-1 (C-O stretching vibration). The broader peak at 1374 cm-1 is due to the stretching vibration of the hydroxyl groups (–OH). The stretching vibration of C-O groups was detected at 1053cm-1. On the other hands, the weak peak at 519 cm-1 was ascribed to that for the β-configuration of the sugar units in EPS. Each polysaccharide exhibited high absorbance in the region 1200–950 cm-1, which was within the so-called fingerprint region, where the position and intensity of the bands are specific for polysaccharide, allowing its possible identification, and this region was dominated by ring vibrations overlapped with stretching vibrations of (C– OH) side groups and the (C–O–C) glycosidic band vibration.
Preparation of Nano- prebiotic EPS to biosynthesize of nanocrystals that converted from extracellular biopolymer EPS from Lactobacillus brevis no.2 and characterization of these crystals using UV- vis spectroscopy, transmission electron microscopy (TEM) and Fourier transform-infrared spectroscopy (FTIR). This experiment was undertaken to know the size and shape of Nano- prebiotic EPS. Under magnification of 50 nm the size of Nano- prebiotic EPS were ranging from 5 to 17 nm. The results show that, the Nano- prebiotic EPS in the reaction mixture has a uniform spherical shape and showing varying sizes as observed.
Results explain how cell viability decreases when Nano- prebiotic EPS concentration increases. Nevertheless, five from six concentrations of Nano- prebiotic EPS recorded cytotoxicity effect as it is showed by the percentage of viability obtained. After 5 h exposure at 1000, 500, 250,
125, 62.5 and 31.25 ug/ml the percentages were 85, 83, 44, 3.9, 0.3 and
0.0 % respectively. These results suggest that Nano- prebiotic EPS cause more cytotoxicity with IC50 169.3 ug/ml than EPS in natural form with IC50 338 ug/ml.
Cytotoxicity of the EPS and Nano- prebiotic EPS on WI-38 Cell Line were carried out to determine the side effect of EPS and Nano- prebiotic EPS on normal cells. Results listed in table 4 indicated that IC50 of EPS and Nano- prebiotic EPS recorded 5036 and 2049 ug/ml. But, it should be noted that the IC50 on CACO recorded 338 and 169 ug/ml. The results showed that the natural EPS was less cytotoxic than Nano- prebiotic EPS and both were active and safe in recommended concentrations. Results showed that natural synthesis EPS was recommended.