Journal of Biotechnology Research Center

Background: Natural products of herbal medicines and their secondary metabolites have been suggested to treat T2DM and they may have the potential as DPP-4 inhibitors.
Objective: This study was conducted to estimate the phytochemical content , radical scavenging activity (RSA) and DPP-4 (dipeptidyl peptidase-4) relative inhibition activity (RIA) of different extracts of Punica granatum peel.
Material and Methods: Fruit peel of pomegranate plant Punica granatum were subjected to extraction with four solvents (distilled water(AQ), 80% methanol(ME), 80% acetone(AC) and a mixed solvent(MX) that included methanol, ethanol, acetone and n-butanol at proportions (7:1:1:1), respectively. Yielded extracts were phytochemically analyzed for total polyphenols (TP) , flavonoids (TF) and HPLC analysis. The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity (RSA) and DPP-4 (dipeptidyl peptidase-4) relative inhibition activity (RIA) were also assessed for each extract.
Results: The results showed that acetone solvent (AC) extract of fruit peel of Punica granatum recorded the highest content of TP and TF (159.9 ± 1.0 mg Gallic acid equivalent/g dry mass) and (7.6 ± 0.2 mg catechin equivalent/g dry mass, respectively) . Mixture extract (MX) was showed the highest antioxidant activity of (54.7 ± 0.9% RSA). While methanol extract (ME) recorded the highest DPP-4 RIA (47.1 ± 1.5%). HPLC analysis showed the presence of Gallic acid, Tannic acid, Ellagic acid and B carboxylic acid at concentrations of (94.61, 130.14, 528.46 and 159.64 ug/ml, respectively). Conclusion: The importance of natural products as radical scavengers and DPP-4 inhibitors is encouraged, and such biological effects were dependent on the plant species and the solvent of extraction. Punica granatum mixture extract was suggested to have a prominent RSA, while methanol extraction of Punica granatum peels are recommended to be a target for investigations involved in the development of anti-T2DM and anti-cancer therapies.

Background: Primary infection of maternal with toxoplasmosis during gestation and this infection transmission to the fetus continue to be the cause complex disease in offspring.
Objective: This study was conducted to test the utility of nested Polymerase Chain Reaction (nPCR) assay to detect recent infections with Toxoplasma in abortive women.
Material and methods: Toxoplasma gondii DNA was detected by using B1 gene as a target for amplification which was highly specific for T. gondii and is well conserved among all of the tested strains. Blood from 60 abortive women and 25 apparently healthy pregnant women with no history of abortion (as control group) were taken in this current study.
Results: The results revealed that nPCR was positive in 48(80%) subjects and negative in 12(20%), Chi-square- χ2 for patients and control was ( 13.82 , 15.75 ) respectively.
Conclusion: It can be concluded that nPCR assay in blood has advantage in detection of recent and active toxoplasmosis.

Background: Cutaneous leishmaniasis (CL) is a neglected disease in tropical countries, including Iraq. Several studies have sought to examine chemotherapies for leishmaniasis treatment but most of them are of toxic and/or undesirable side effect, therefore, the need for investigating new fewer toxic therapies is essential.
Aim of study: In this study, the cytotoxic effect of Artemisinin (ART), a novel herbal compound, was screened against the two forms, promastigotes and amastigotes, of the Iraqi isolate of Leishmania tropica, the causative agent of Baghdad boil.
Material and methods: Different concentrations (1000, 500, 250, 125, 62.5, 31.25, 15.6 and 7.8) µM of Artemisinin were screened to investigate the leishmanicidal activity of the herbal compound against the two forms of the parasite along three times of follow up (24, 48, 72) hour using MTT cytotoxicity assay.
Results: The results showed that growth rate and cell viability were significantly decreased at all studied concentrations. The IC50 was measured after 72 hours of follow up and was 2.625 µM and 2.636 µM for promastigotes and amastigotes, respectively.
Conclusion: These findings approved the leishmanicidal efficacy of Artemisinin against the of L. tropica and can be further studied to screen its effectiveness in vivo for exploring a safer herbal drug for treatment of cutaneous leishmaniasis.

Background: Forty isolates were obtained of Acinetobacter baumannii from (200) samples, the samples were collected from different cases including: - wounds, burns, Stool, Urinary tract infection urine.
Aim of study: The aim of study is to isolate and diagnose Acinetobacter baumanniii from different clinical cases and to investigate the fimH gene.
Objective: Respiratory tract infection and blood sample.For the period between 1/9/2016 to 30/11/2016.
Materials and methods: Identification of 40 isolates confirmed to be Acinetobacter baumannii included 9 isolates from blood, 1 isolate from urinary tract infections, 4 isolates from each of wound infections and Respiratory tract infection, 8 isolates from burns and 14 isolates from stool sample.
Results: The result revealed that the fimH gene was present in (20) isolates (50%) of Acinetobacter baumannii. The result Showed (7 (isolate (17.5%) of Stool and blood has fimH gene to each of them and Burns (3) isolates (7.5%) has fimH gene and (2) isolate (5%) of respiratory tract infection has fimH gene and finally only (1) isolate (2.5 %) of Wound infection. While all isolated for each of the urinary tract infections doesn't the fimH gene. The gel electrophoresis showed that the molecular weight of fimH gene was 508 bp.
Conclusion: Sequential analysis detection of fimH showed three silent mutations that did not affect the amino acid translation.

Background: Human Respiratory Syncytial virus (hRSV) is one of the major causes of viral respiratory tract infection in infants and young children.
Aim of study: The aim of this study was to determine the risk factors associated with hRSV infection.
Objective: This study included 100 hospitalized infants and young children with chest infection (39 female and 61 male) aged from (1) to (24) months, their mean age (6.87) months.
Material and methods: Nasopharyngeal/throat swabs specimens were collected over a three-month winter period from January to April, 2017. hRSV was determined by reverse transcription polymerase chain reaction (RT-PCR).
Results: The highest percentage of hRSV RNA (56.81%) was observed in the age group less than 6 months, followed by (27.27%) and (15.91%) in the age group 6-12 months and 12-24 months, respectively, which mean that (84%) of hRSV infections were under 12 months of age. Regarding the type of feeding, about (84%) of hRSV infections were positive in patients with bottle feeding which indicated that the role of breastfeeding in preventing hRSV infection and hospitalization is undisputed, Results showed that there was no significant association between these risk factors and the occurrence of hRSV infection (P˃ 0.05).
Conclusion: The hRSV-RNA is equally distributed between patients exposed and not exposed to smoking (50%).

Background: Polyphenols are considered an important compound with a wide range of medical activities. Several plant families are rich with phenolics. Attempts have been carried out to increase them at the intact and cellular levels.
Objective: The current study aimed to increase the production of polyphenols in Coleus blumei plant using two precursors at the whole and tissue culture levels.
Materials and methods: Two precursors namely Tyrosine and phenylalanine at the concentrations 0.20 , 0.25 or 0.30 g.l-1 were added either by spraying on the vegetative parts or supplemented to the tissue culture medium. Total phenols were estimated in the whole plant and compared with the total phenolic content in callus tissues. They were estimated by Folin-ciocalteu and spectrophotometric methods. Induction and maintenance of Coleus callus cultures were carried out on Gamborg medium (B5) for 21 days supplemented with the growth regulators 2,4 dichlorophenoxy acetic acid (2-4-D) at 0.8 mg.l-1 , 2 mg.l-1 Benzyl adenine (BA) and 0.5 mg.l-1 Naphthalene acetic acid (NAA). Stem explants were used as a source for callus induction.
Results: Results showed a significant increase in both fresh and dry weights of the fresh compared with control of intact plants in vivo after treatment with the two precursors. The fresh weight of shoots increased up to 15.9 g at 0.30 g.l-1 of Tyrosine treatment compared with the control which recorded 11.05 g. while the fresh weight of the shoots recorded 13.470 g at 0.30 g.l-1 after treatment with phenylalanine compared with the control which recorded 11.05 g. The dry weight of the shoots increased as well up to 3.127 g at 0.30 g.l-1 of Tyrosine treatment compared with control 1.837 g while the dry weight of shoot recorded 2.880 g at 0.30 g.l-1 phenylalanine treatment compared with the control 1.837g. Despite the slight increase in both root fresh and dry weights at 0.30 g.l-1 due to the treatment with Tyrosine or phenylalanine ,such increments were not significantly different with control. Total poly phenols in callus cultures accumulated more compared with mother plants after treatment with precursors. Phenols were also measured in both callus tissue and liquid medium then compared with the whole plant and control treatment. The highest value was recorded in callus cultures supplied with Tyrosine recording 348.36 µg.l-1 at 0.20 g.l-1 of Tyrosine while phenylalanine treatment recorded the highest weight 293.98 µg.l-1 when applied at 0.30 g.l-1 in both liquid medium and callus tissues. The intact plants however recorded the highest mean in total phenols in plants grown in vivo with mean values 68.58, 66.53 µg.l-1 for Tyrosine and phenylalanine respectively.
Conclusion: It is concluded that both precursors are good candidates for increasing total phenols in Coleus blumei. Plant tissue culture techniques can be utilized commercially for this purpose.

Background: The artificial induction of mutations represents an efficient tool in creating genetic variations. For this reason, the current study was carried out in the Plant Tissue Culture Laboratory/Genetic Engineering Institute to induce genetic diversity for salt tolerance in two local cultivars of bread wheat (Al-Iraq and Tamooz 2) using in vitro application of sodium azide (SA).
Objective: Immature seeds from both cultivars subjected to 0, 0.5, 1.0 and 2.0 Mm of SA to determine the optimal concentration for developing novel mutants.
Materials and methods: The optimal dose of SA mutagen was found to be 2.0 mM, resulting in a 42% reduction in callus fresh and dry weight. The developed callus was subjected to five different salinity levels using NaCl (6, 8, 10, 12 and 14 dS m-1).
Results: Mutants showed a significant decrease in the percentage of regenerated plants under salinity stress conditions. The used SSR markers approved the genetic diversity between the original and the mutants of the two cultivars (Al-Iraq and Tamooz 2) growing under normal and salinity stress. The presences and the absence of some fragments was prominent in plants derived from immature embryos of the two cultivars tested in salinity conditions. The used SSR markers (cfd 9, cfd4, cfd1 wmc405, PYL5, HKT1, HVA1 and htk1) were so efficient in distinguishing between the original and tissue culture-derived plants. Furthermore, the experienced SA levels induced a higher rate of mutant alleles in cv.
Conclusion: Al-Iraq with 19 mutant alleles than Tamooz 2 which showed only 13 mutant alleles. The current study represents an additional prove to the effectiveness of mutagens and tissue culture technique in developing novel variants with improved performance under stress conditions.

Abstract
Background: The research is involved development a new spectrophotometric method based on the oxidative coupling reactions for determination of important phenothiazine drug which is promethazine HCl in pure solutions and local pharmaceutical preparations.
Materials and methods: The Standard promethazine HCl was treated with organic reagent of P-Chloroaniline as a coupling reagent in the presence of oxidizing agent Ammonium Cerric (IV) Sulphate, the reaction leads to the formation a blue –greenish color product that has a maximum absorption at 603nm.
Results: The variables of reaction conditions including optimum volumes of both reagent and oxidizing agent, acidity of the reaction medium, order of addition and stability time were studied. The obtained results of the purposed method shows that a Beer law is obeyed in the range of 7-40ppm with a correlation coefficient (r2) of 0.9981. While the molar absorptivity (ξ) of 1.861x103 L.mol-1.cm-1, sandal sensitivity(s) of 0.172μg.cm-2, limit of detection (LOD) of 4.02ppm and limit of quantification (LOQ) of 13.39ppm were obtained. The developed method was compared with the standard method adopted by USP using F and t-tests and the results shows no significant deferent between both methods.
Conclusion: The analytical method was also applied successfully for pharmaceutical preparations containing promethazine HCl.

Background: Antibiotic resistance is a life threating problem and the need for an alternative is increasing worldwide.
Objective of this study is: Detecting the combination effect of AuNps with Amoxicillin/clavulanate (AMC) antibiotic against antibiotic resistant clinical bacterial isolates.
Materials and methods: Gold nanoparticles were biosynthesized using food origin Citrobacter freundii isolate (C2) which was isolated from chicken meat samples by pour plate method and identified using cultural characteristics, biochemical tests and the identification to the species level was completed by Vitek-2 system and this identification was confirmed by sequencing of the 16 s r RNA. The biosynthesis of gold nanoparticles was optimized in order to achieve the finest AuNps with a diameter range (30-60) nm. and the biosynthesized gold nanoparticles were characterized by visual observation and then characterized using various characterization techniques: Uv-vis spectroscopy, Fourier-transform infrared spectroscopy (FTIR) analysis, atomic force microscopy (AFM) analysis and scanning Electron Microscope (SEM). The combination effect of AuNps with AMC antibiotic was detected against clinical bacterial isolates.
Results: The results revealed the biosynthesized AuNPs were roughly spherical and poly-dispersed, and they were highly effective with concentration (62.5 µg/ml) that inhibit the bacterial growth, MIC values of AMC antibiotic against clinical isolates were determined as 500 µg/ml, while the combination of gold nanoparticles and AMC had wide spectrum of antibacterial activity against different isolates of the bacteria that used in this study.
Conclusion: There was a significant synergistic effect between the biosynthesized gold nanoparticles when used in combination with antibiotic where the minimum inhibitory concentrations of the combination (AuNps/AMC) were less than its concentration when each of them (AuNps) or (AMC) used separately.

Back ground: Leuconostoc is one of the species of lactic acid bacteria that produced biofilms.Probiotic bacteria that produced biofilm has been used as naturopathy against different microbial pathogens.
Objective: This study was conducted to determine the antimicrobial activity of Leuconostoc biofilm , against 24 isolates (4 of 6 different types) of food borne pathogens including Staphylococcus aureus , Salmonella spp, Escherichia coli , Klebsiella spp , Pseudomonas aeruginosa , Streptococcus mutans , Bacillus subtillus , Bacillus cereus, Bacillus sterothermophillus and Candida albicans.
Materials and methods: using various concentration in vitro by filter paper disk diffusion method .
Result: The present study showed the potent antimicrobial activity of the Leuconostoc mesenteroides biofilm against the all tested bacterial pathogens except Bacillus species and yeast Candida albicans. Biofilm produced by Leuconostoc mesenteroides showed highest zone of inhibition (13mm) against Escherichia coli and lowest zone of inhibition (7.0mm) against Streptococcus mutans .
Conclusion: Consequently, Leuconostoc mesenteroides biofilm may be used as an antimicrobial agent in food products to prevent spoilage.

Back ground: Cyproterone acetate (CPA) is a steroidal anti-androgen inhibits the testosterone and DHT action it is used as a medicine for prostate cancer by association with the androgen receptors located on the surface of prostate cells, thus preventing the association of testosterone with receptors.
Objective: Investigation of the anticancer activities of Cyproterone acetate against cancer cell lines testicular (Tera-1), macrophage (RAW 264.7) in comparison to non- tumorigenic fetal hepatic cell line (WRL-68).
Material and method: The cytotoxic effect of CPA was investigated according to selected parameters including: MTT assay as assay of cell function to determined cell viability, high content screening (HCS) technique for the apoptosis of cell. Only the most cytotoxic concentration of CPA and the most sensitive cells as assayed by MTT was selected to complete the other test: (HCS). (MTT) assay was carried out at the Centre of Biotechnology Research’s, Al- NahrainUniversityBaghdad,Iraq . The HCS assay was performed at the Centre for Natural Product Research and Drug Discovery, Department of Pharmacology,Faculty of Medicine, University of Malaya, KualaLumpur, Malaysia
Results: The most significant cytotoxic effect of Cyproterone acetate towards 2 cancer cell lines was obtained when its concentration was 1.25mg/mL. The Tera-1 Cells were more sensitive to Cyproterone acetate compared with RAW264.7 and WRL-68. cells. There was a significant decrease in valid cell count, nuclear intensity and mitochondrial membrane potential (MMP) when CPA (400 μg/mL)was used compared with doxorubicin (20 μg/mL) as a standard. Also, there was a significant increase in membrane permeability and cytochrome C releasing when CPA (400μg/ml) was used compared with positive control.
Conclusion: CPA showed cytotoxic effects against the Tera-1 and RAW264.7 cancer cell lines while the WRL-68. Cells was not affected as determined in-vitro by the MTT assay. The HCStechnique also showed toxic effect towards Tera-1.

Back ground: Microcins are ribosomal synthesized antimicrobial peptides produced principally by bacteria of the Enterobacteriaceae family that are active against other bacteria, either in the same species (narrow spectrum), or across genera (broad spectrum) that share the same ecological niche.
Objective: The ability of the Enterobacteriaceae to produce microsines.
Materials and methods: One hundred urine samples have been collected from patients suffering from UTI whom admitted to Al Hakim Hospital in AL-Najaf City during the period from 17October 2017 to 13 February 2018.
Results: The results of PCR technique for amplification show that all isolates were possess mic N by appearance of amplicon with molecular weight 938 bp while 46 (76%) isolates were possess mic N1and 24 (40%) isolates were possess mic N2 gene by appearance of amplicon with molecular weight 368 bp and 167 bp respectively.Cross-streak activity assay was used to detect the selected strains with antibacterial activities against pathogenic bacteria.
Conclusion: some isolates have one or more gene microcin encoded of , however, microcin N is present in all isolates.

Back ground: P.mirabilis is a gram negative bacterium, motile with its peritrichous flagella .Widely distributed in environment, especially in contaminated water and soil, Many virulence factors like LPS, urease, protease, hemolysin and biofilm formation play an important roles in the pathogenicity of P. mirabilis. Urease is a Nickel containing enzyme causes elevation of urine pH after hydrolyzing urea to ammonia and CO2 forming stones that blocks the urinary track.
Aims: The effect of tannic acid on the production of urease and protease.
Material and methods: Twenty one isolates of Proteus were collected from different sources, Clinical and animal sources all isolates were cultured on MacConkey and blood agar and identification of P. mirabilis by, Vitek -2 compact system. Determine the effect of tannic acid on the production of urease and protease.
Results: Twenty one isolates of Proteus were identified depending on Vitek-2 compact system, after identification, it turns out that only 18 isolate were P. mirabilis. All isolates were 100% able to produce urease and 72.2% isolate were able to produce protease. The addition of tannic acid showed an inhibitory effect on urease and protease production.
Conclusion: The effect of tannic acid on urease and protease depending on concentration, type of strain, incubation period, number of isolates and truculence of isolate.

Background: Silver nanoparticles are characterized with a high production techniques, in this study nanoparticles were manufactured using Origanum vulgare water extract and AgnO3.
Objective: Experimental factors such as morphology and optical properties of nanoparticles were studied through specific assay such as morphology, spectroscopy of UV rays (UV-Visible), FE-SEM, FT-IR, and Atomic Force Analysis (AFM).
Materials and Methods: showed that the spherical nanoparticles were found using TEM, with an average particle size distribution of 44.2 ± 88.1 nm with more than one active plant substance used within the Nano silver compound. In addition, the green nanoparticles have a response in treatment of damage in DNA based on the dose and structure of the nanoparticle in the lymphocytes of the patient with diabetic cells.
Results &conclusion: These nanoparticles can be considered healthy and can be used without harming healthy parts. The DNA rate in lymphocyte cells was calculated for patients with diabetes using the comet technique. The results showed that the rate of destruction of lymphocytes was reduced for diabetic patient.