Nose-to-brain delivery of chrysin transfersomal and composite vesicles in doxorubicin-induced cognitive impairment in rats: Insights on formulation, oxidative stress and TLR4/NF-kB/NLRP3 pathways.

Many cancer survivors suffer from chemotherapy-induced cognitive impairment known as 'Chemobrain'. Doxorubicin -topoisomerase II inhibitor- is widely used in breast cancer, hematological cancers and other neoplasms. However, it is reported to precipitate cognitive impairment in cancer patients via inducing oxidative stress and inflammatory response. Chrysin -5,7 dihydroxyflavone- has promising antioxidant, anti-inflammatory and anticancer properties, but suffers low bioavailability owing to its poor solubility and extensive metabolism. In the present study, chrysin was successfully formulated as transfersomal lipid vesicles and chitosan composite vesicles (CCV) exhibiting a nanometric size range, high drug entrapment efficiency, and controlled release over a 72h period. Intranasal administration of optimized chrysin formulations at a reduced dose of 0.5 mg/kg improved doxorubicin-induced memory impairment in rats evidenced by behavioral testing, inhibition of acetylcholinesterase activity and oxidative stress markers; catalase, reduced glutathione, lipid peroxidation and hydrogen peroxide. This could reduce caspase-3 expression inhibiting apoptosis. Moreover, chrysin formulations were able to inhibit doxorubicin-induced Tol-like receptor 4 (TLR4) and p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) protein expression which in turn, reduced procaspase-1, Cysteinyl Aspartate Protease-1 (caspase-1) and Interleukin-1ß (IL-1ß) protein expression via inhibiting Nod-like receptor pyrin containing 3 (NLRP3) inflammasome. Collectively, our findings suggest the enhanced therapeutic potential of chrysin when formulated as transfersomes and CCV against chemotherapy-induced chemobrain via hindering acetylcholinesterase, oxidative stress and TLR4-NF-kB(p65)-NLRP3 pathways.

Molecular identification, genotyping of virulence-associated genes, and pathogenicity of cellulitis-derived Escherichia coli.

BACKGROUND AND AIM: Avian colibacillosis, which is caused by avian pathogenic Escherichia coli (APEC), is a major bacterial disease that affects birds of all ages worldwide, causing significant economic losses. APEC manifests in several clinical forms, including cellulitis, and its high pathogenicity is attributed to harboring numerous virulence-associated genes (VGs). This study evaluated the pathogenicity of the cellulitis-derived E. coli (O78) strain through molecular identification of genes coding for seven virulence factors and by conducting an in vivo assessment of capability for cellulitis induction in broiler chickens. MATERIALS AND METHODS: This study was performed using a previously isolated and identified cellulitis-derived E. coli (O78), which was screened for seven VGs using molecular detection and identification through polymerase chain reaction followed by nucleotide sequencing and phylogenetic analysis. Experimental infection by subcutaneous (SC) inoculation in broilers and its pathogenicity was confirmed in vivo by cellulitis induction. The impact of cellulitis on broiler performance was assessed. RESULTS: Molecular genotyping proved that the isolate harbored five virulence genes (iroN, iutA, tsh, iss, and papC) and was negative for stx1 and hly genes. The amplified products for iroN, iss, and iutA were subjected to sequencing and phylogenetic analysis, and the results indicate the highest similarity and matching with E. coli submitted to the National Center for Biotechnology Information GenBank. SC inoculation of bacteria in broiler chickens resulted in cellulitis, as indicated by thick red edematous skin with yellowish-white material in the SC tissue at the inoculation site, and the abdominal muscle showed redness and increased vacuolization. Histopathological examination revealed moderate-to-severe caseous inflammatory reaction with a marked accumulation of heterophils and mononuclear cells in the SC fatty tissue. The average feed intake, body weight gain (BWG), and feed conversion ratio (FCR) were lower in infected chickens in comparison with those of the control non-infected chickens. CONCLUSION: This study proves that molecular techniques are accurate for pathogenicity determination in virulent bacteria, with the advantages of being rapid, time-saving, and economical. Cellulitis is associated with economic losses that are represented by a lower BWG and FCR.

Molecular identification of Mycoplasma synoviae from breeder chicken flock showing arthritis in Egypt.

AIM: Arthritis is one of the most economic problems facing poultry industry worldwide. The study was done to detect possible causes of arthritis in breeder chicken flock with emphasis on molecular identification of Mycoplasma synoviae (MS). MATERIALS AND METHODS: This study was carried on chicken from broiler breeder flock of 57 weeks' age in Dakahlia, Egypt, suffered from arthritis with frequently 5-7% decrease in egg production, reduced fertility, and hatchability. Forty blood samples were randomly collected from individual birds in sterile tubes and used for serum separation. Serum samples were tested using serum plate agglutination (SPA) test against colored antigens for Mycoplasma gallisepticum (MG), MS, and Salmonella gallinarum-pullorum (SGP). On the other hand, 24 joint samples were collected. Of those 24 samples, 12 joint samples were subjected to bacteriological examination, while the other 12 were utilized for molecular diagnosis by polymerase chain reaction (PCR) for MS and avian reovirus (ARV). RESULTS: SPA test results revealed the presence of antibodies against MG, MS, and SGP in tested sera in rates of 14/40 (35%), 35/40 (87.5%), and 9/40 (22.5%), respectively. Furthermore, 19 bacterial isolates were recognized from joint samples and identified as five Staphylococcus spp., nine Escherichia coli, three SGP, one Citrobacter, and one Proteus. The identified Staphylococcal isolates were three coagulase-positive staphylococci (two Staphylococcus aureus and one Staphylococcus hyicus) and two coagulase-negative staphylococci (one Staphylococcus epidermidis and one Staphylococcus lentus), while E. coli isolate serotypes were 1 O11, 2 O55, 3 O78, 1 O124, 1 O125, and 1 untyped. PCR proved that 12/12 (100%) samples were positive for MS variable lipoprotein hemagglutinin A (vlhA) gene, while ARV was not diagnosed in any of the examined samples. Four amplified vlhA gene of MS isolates (named MS-2018D1, MS-2018D2, MS-2018D3, and MS-2018D4) was successfully sequenced.Analysis of phylogenetic tree revealed the presence of 100% identity between each two sequenced isolates(isolates MS-2018D1 and MS-2018D4 and also isolates 2018D2 and MS-2018D3). However, the nucleotide similarity between four isolates was 88.6%. On the other hand, our field isolates MS-2018D1, MS-2018D4, MS-2018D2, and MS-2018D3 showed nucleotide identity with vaccine strain MS-H 98.4%, 98.4%, 88.1%, and 88.1%, respectively. Furthermore, the nucleotide similarities with field strains from Argentina ranged between 87.8% and 98.6%. CONCLUSION: Four field isolates of MS were identified in examined broiler breeder flock. A phylogenetic study of these isolates revealed the variation between isolated MS strains and vaccine strain. Therefore, further studies are required for evaluating the vaccine efficacy against the present field isolates of MS. In addition, application of MS immunization of breeder flocks is necessary for proper control of the disease.

Antimicrobial resistance genes in pathogenic Escherichia coli isolated from diseased broiler chickens in Egypt and their relationship with the phenotypic resistance characteristics.

AIM: The aim of this study was to determine the relationship between phenotypic resistance and genotypic resistance of isolated serotyped pathogenic Escherichia coli isolates from the clinically diseased broiler. MATERIALS AND METHODS: A total of 160 samples (heart, liver, kidney, and lung) were collected from 18 to 34 days old clinically diseased broiler from 40 broiler farms (3-5 birds/farm) reared in Giza and Kaluobaia Governorates for the isolation of pathogenic E. coli. Various E. coli isolates were tested for the pathogenicity based on Congo red (CR) dye binding assay. The obtained CR-positive E. coli isolates were subjected to serological identification using slide agglutination test. Disc diffusion test was used to study the sensitivity pattern of E. coli isolates to available 12 antibiotics. Polymerase chain reaction was performed for the detection of antimicrobial resistance genes in the studied pathogenic E. coli isolates. RESULTS: The results revealed that 56 samples (35 %) were positive for E. coli. The results of the CR assay indicates that 20 isolates of 56 (35.7%) were positive and 36 isolates (64.3%) were negative. Identified E. coli serotypes of CR-positive isolates were 1 (O24), 2 (O44), 2 (O55), 5 (O78), 2 (O86), 1 (124), 3 (O127), 1 (O158), and 3 untyped. Resistance rate in disc diffusion test was 85% to oxytetracycline and kanamycin; 80% to ampicillin (AMP), clindamycin, and streptomycin (S); 75% to enrofloxacin; 65% to chloramphenicol; 55% to cefotaxime and gentamicin (CN); 45% to trimethoprim+sulfamethoxazole; 35% to erythromycin (ERI); and 30% to oxacillin. All strains are multidrug-resistant (MDR). Antibacterial resistance genes CITM, ere, aac (3)-(IV), tet(A), tet(B), dfr(A1), and aad(A1) were detected in 14 (70%), 12 (60%), 12 (60%), 8 (40%), 11 (55%), 8 (40%), and 9 (45%) of tested 20 isolates, respectively. Multidrug resistance was detected in the form of resistance to 42%-83.3% of tested 12 antibiotics. Three isolates (15%) of 20 tested isolates showed a relationship between phenotype and genotype and 17 (85%) showed irregular relation. Strains are sensitive and show resistant gene (P-G+) presented in three isolates for AMP (beta-lactam), one for ERI (Macrolide), as well as five isolates for trimethoprim (pyrimidine inhibitor). E. coli isolates had resistance and lacked gene (P+ G-) reported meanly in one isolate for CN (aminoglycoside), two isolates for tetracycline, four isolates for ERI, seven isolates for trimethoprim, and eight isolates for S (aminoglycoside). CONCLUSION: The study demonstrates that E. coli is still a major pathogen responsible for disease conditions in broiler. E. coli isolates are pathogenic and MDR. Responsible gene was detected for six antibiotics in most of the isolates, but some do not show gene expression, this may be due to few numbers of resistance genes tested or other resistance factors not included in this study.

Treatment of Vinca minor Leaves with Methyl Jasmonate Extensively Alters the Pattern and Composition of Indole Alkaloids.

Alkaloids extracted from mature Vinca minor leaves were fractionated by preparative HPLC. By means of HRMS and NMR data, the main alkaloids were identified as vincamine, strictamine, 10-hydroxycathofoline, and vincadifformine. Upon treatment with methyl jasmonate (MeJA), the pattern and composition of the indole alkaloids changed extensively. While 10-hydroxycathofoline and strictamine concentrations remained unaltered, vincamine and vincadifformine levels showed a dramatic reduction. Upon MeJA treatment, four other indole alkaloids were detected in high quantities. Three of these alkaloids have been identified as minovincinine, minovincine, and 9-methoxyvincamine. Whereas minovincinine and minovincine are known to occur in trace amounts in V. minor, 9-methoxyvincamine represents a novel natural product. Based on the high similarities of vincamine and 9-methoxyvincamine and their inverse changes in concentrations, it is postulated that vincamine is a precursor of 9-methoxyvincamine. Similarly, vincadifformine seems to be converted first to minovincinine and finally to minovincine. Because MeJA treatment greatly altered the alkaloidal composition of V. minor, it could be used as a potential elicitor of alkaloids that are not produced under normal conditions.

Synthesis, docking, cytotoxicity, and LTA4H inhibitory activity of new gingerol derivatives as potential colorectal cancer therapy.

Leukotriene A4 hydrolase (LTA4H) is a proinflammatory enzyme that generates the inflammatory mediator leukotriene which may play an important role in chronic inflammation associated carcinogenesis. [6]-gingerol, the major bioactive compound of Zingiber officinale, is a potential inhibitor of LTA4H, a highly expressed enzyme in colorectal carcinoma. Eighteen compounds; seven of natural origin (including [4]-, [6]-, [8]-, and [10]-gingerol), five new and six known semi-synthesized [6]-gingerol derivatives were examined using docking, in vitro cytotoxicity against human colon cancer cells (HCT-116) and LTA4H aminopeptidase and epoxide hydrolase inhibitory studies. Methyl shogoal (D8) showed to be the most potent compound against HCT-116 cells (IC50; 1.54µM). Remarkably, D8 proved to be non-cytotoxic to normal cells; (TIG-1) and (HF-19) with high selective index (SI; 52.3). Furthermore [6]-gingerol derivatives showed potent LTA4H inhibitory activities in comparison to the universal positive controls (bestatin and 4BSA). Among the natural gingerols, [10]-gingerol (N3) exhibited the highest LTA4H aminopeptidase and epoxide hydrolase inhibitory activities with IC50; 21.59 and 15.24µM, respectively. Meanwhile, methyl shogoal (D8) and 4'-O-prenyl-[6]-gingerol (D10) retained the highest inhibition with IC50; 4.92 and 3.01µM, for aminopeptidase, and 11.27 and 7.25µM for epoxide hydrolase activities, respectively.

Cytotoxic constituents of Alocasia macrorrhiza.

An indole alkaloid, 2-(5-hydroxy-1H-indol-3-yl)-2-oxo-acetic acid (1) isolated for the first time from nature, in addition to the nine known compounds 5-hydroxy-1H-indole-3-carboxylic acid methyl ester (2), alocasin B (3), hyrtiosin B (4), α-monopalmitin (5), 1-O-ß-D-glucopyranosyl-(2S, 3R, 4E, 8Z)-2-[(2(R)-hydroctadecanoyl) amido]-4,8-octadecadiene-1,3-diol (6), 3-epi-betulinic acid (7), 3-epi-ursolic acid (8), ß-sitosterol (9) and ß-sitosterol 3-O-ß-D-glucoside (10) were isolated from the rhizomes of Alocasia macrorrhiza (Araceae). Their structures were elucidated by 1D and 2D NMR spectroscopic data. Of these compounds, 6 exhibited the strongest cytotoxicity against the four tested human cancer cell lines (IC50 of about 10 µM against Hep-2 larynx cancer cells).

Evidence-based medicinal value of Rudbeckia hirta L. flowers.

A phytochemical investigation on the 5-lipoxygenase (5-LOX) inhibitory methanolic extract of Rudbeckia hirta L. flowers yielded 10 phenolic metabolites, including three phenolic acids, two phenolic acid esters, four flavonol glycosides and a trimethylated flavonol. The structures of the isolated metabolites were determined on the basis of spectroscopic analyses and by comparison with the literature data. Seven of these metabolites were isolated for the first time from the genus Rudbeckia. The in vitro 5-LOX inhibitory, immunomodulatory and antioxidant (oxygen radical absorbance capacity) activities of the isolated compounds were evaluated, and the results provided a new scientific evidence for the ethnopharmacological use of the herb in inflammatory conditions.

A new highly oxygenated pseudoguaianolide with 5-LOX inhibitory activity from Rudbeckia hirta L. flowers.

A new highly oxygenated pseudoguaianolide, rudbeckolide (1), was isolated from Rudbeckia hirta L. flowers. The structure of this terpenoid lactone was established on the basis of extensive 1D and 2D NMR spectroscopic analyses. The compound exhibited strong 5-lipoxygenase inhibitory activity (84.9% inhibition at 10 µg/mL) in vitro and the result provided partial evidence for the usage of the plant as traditional medicine.

A novel antioxidant phenanthrenoid dimer from Juncus acutus L.

A novel phenanthrenoid symmetrical dimer 8,8'-bidehydrojuncusol [1,1',6,6'-tetramethyl-5,5'-divinyl-8,8'-biphenanthrene-2,2',7,7'-tetraol], a related phenanthrenoid monomer, a phenolic chromone, and five flavonoids derivatives have been isolated from the halophyte Juncus acutus L., Juncaceae. The structure of the dimeric phenanthrenoid was determined on the basis of spectroscopic analyses, including 2D NMR spectroscopy, and by spectral correlations. The new dimer and the other isolated compounds bearing four phenolic hydroxy groups showed the significant in vitro antioxidant activity comparable with that of ascorbic acid using 2,2'-azino-bis[3-ethylbenzothiazoline-6-sulphonate] (ABTS) radical cation decolourisation assay. On the basis of the results from an in vitro anti-inflammatory assay using lipopolysaccharide-stimulated RAW264.7 macrophage cells linked with immunoblot analysis, it was found that dimerisation of dehydrojuncusol [1,6-dimethyl-5-vinyl-8-phenanthrene-2,7-diol] molecule nearly nullified its inhibitory effect on the expression of the pro-inflammatory inducible nitric oxide synthase (iNOS) protein.

Determination of tetraconazole and diniconazole fungicide residues in tomatoes and green beans by capillary gas chromatography.

A sensitive gas chromatographic method using an electron-capture detector (ECD) has been developed for the determination of tetraconazole and diniconazole fungicide residues in tomatoes and green beans. The developed method consists of extraction with methanol, partition with methylene chloride, and column chromatographic clean-up, followed by capillary gas chromatographic determination. The recoveries of both fungicides were greater than 90% for both plant samples. The limits of determination of the method were 0.001 ppm for both fungicides. The method was applied to determine residues and the rate of disappearance of tetraconazole and diniconazole from tomatoes and green beans [open field treatment, 50 cc of Domark 10% EC (emulsifiable concentrate), and 35 cc of Sumi-eight 5% EC; both for 100 l of water]. The fungicides incorporated into the plants decreased rapidly with a half-life around 3 days for diniconazole and from 4.5 to 6.5 days for tetraconazole. No residues could be detected in the plants during the period of study of 21 days after field application. Hence, the plants could be used safely after that period of time.

Antioxidant and immunomodulatory constituents of henna leaves.

The immunomodulatory bioassay-guided fractionation of the methanolic extract of henna (Lawsonia inermis L.; syn. Lawsonia alba L.) leaves resulted in the isolation of seven compounds; three have been isolated for the first time from the genus, namely p-coumaric acid, 2-methoxy-3-methyl-1,4-naphthoquinone and apiin, along with the previously isolated compounds: lawsone, apigenin, luteolin, and cosmosiin. Structural elucidation of the isolated compounds was based upon their physical, chemical as well as spectroscopic characters. Their immuomodulatory profile was studied using an in vitro immunoassay, the lymphocyte transformation assay. The ABTS [2,2'-azino-bis (3-ethyl benzthiazoline-6-sulfonic acid)], free radical scavenging assay depicted that all isolated compounds exhibited antioxidant activity comparable to that of ascorbic acid.

Immunomodulatory triterpenoids from the oleogum resin of Boswellia carterii Birdwood.

The immunomodulatory bioassay-guided fractionation of the oleogum resin of frankincense (Boswellia carterii Birdwood) resulted in the isolation and identification of 9 compounds; palmitic acid and eight triterpenoids belonging to lupane, ursane, oleanane, and tirucallane skeleta were isolated form the resin. These triterpenoids are lupeol, beta-boswellic acid, 11-keto-beta-boswellic acid, acetyl beta-boswellic acid, acetyl 11-keto-beta-boswellic acid, acetyl-alpha-boswellic acid, 3-oxo-tirucallic acid, and 3-hydroxy-tirucallic acid. The structures of the isolated compounds were deduced based on spectroscopic evidences. The lymphocyte transformation assay of the isolated compounds proved that the total extract retained more activity than that of any of the purified compounds.

Chemistry and immunomodulatory activity of frankincense oil.

The yield of steam distillation of frankincense essential oil (3%); and its physicochemical constants were determined. Capillary GC/MS technique was used for the analysis of the oil. Several oil components were identified based upon comparison of their mass spectral data with those of reference compounds published in literature or stored in a computer library. The oil was found to contain monoterpenes (13.1%), sesquiterpenes (1%), and diterpenes (42.5%). The major components of the oil were duva-3,9,13-trien-1,5alpha-diol-1-acetate (21.4%), octyl acetate (13.4%), o-methyl anisole (7.6%), naphthalene decahydro-1,1,4a-trimethyl-6-methylene-5-(3-methyl-2-pentenyl) (5.7%), thunbergol (4.1%), phenanthrene-7-ethenyl-1,2,3,4,4a,5,6,7,8,9,10,10a-dodecahydro-1,1,4a,7-tetramethyl (4.1%), alpha-pinene (3.1%), sclarene (2.9%), 9-cis-retinal (2.8%), octyl formate (1.4%), verticiol (1.2%) decyl acetate (1.2%), n-octanol (1.1%). The chemical profile of the oil is considered as a chemotaxonomical marker that confirmed the botanical and geographical source of the resin. Biologically, the oil exhibited a strong immunostimulant activity (90% lymphocyte transformation) when assessed by a lymphocyte proliferation assay.