Piloquinone, potent cytotoxic compound from Egyptian Streptomyces pilosus SBG-NRC-216 Mohamed I Abo-Alkasem, Ahmed A El-Beih, Tarek E Mazeed, Enas M Mostafa, Hassan M Awad, Samia A Ahmed, Saadia M Easa, Fawkia M El-Beih, Ahmed I El-Diwany Egyptian Pharmaceutical Journal 2019 18(3):179-187 Background and objective Piloquinone shows biological activity as an anticancer and antitrypanosomal agent, and it acts as an inhibitor for monoamine oxidase, the main causative factor of Alzheimer’s disease. The aim of this study was to produce Piloquinone as a bioactive compound from a local Streptomyces isolate for many medical purposes. Materials and methods Fifty Streptomyces isolates for their capacity of Piloquinone production were tested. The potent Piloquinone producer, SBG-NRC-216 isolate, was identified by conventional and genetic methods. The produced compound was purified and identified on the basis of the spectroscopic analysis. A series of experiments were conducted to investigate efficacy of the compound. Results and conclusion The producer isolate was identified by phenotypic and genotypic methods. These methods confirmed that the strain name was Streptomyces pilosus SBG-NRC-216. The produced compound was purified and identified as Piloquinone. It showed a potent anticancer activity against five different human tumor cell lines: breast cancer cell line MCF-7, human liver cancer cell line HepG2, human lung cancer cell line A549 and human colon cancer cell lines Caco-2 and HCT-116. The results also indicated that Piloquinone had a weak antiviral activity against the H5N1 virus. The results proved that Piloquinone can be used as a bioactive compound that has many industrial and medical purposes.

Identification of a novel anticancer compound through screening of a drug library on multicellular spheroids Walid Fayad Egyptian Pharmaceutical Journal 2019 18(3):188-200 Background and objectives A multicellular cancer spheroid model has proven to mimic the in-vivo tumors more closely compared with the conventionally used monolayer model. Thus, the spheroid model estimates the in-vivo activity more accurately than its counterpart the monolayer model. Accordingly, a library of 320 chemically diverse compounds was screened for their cytotoxicity against MCF7 human breast carcinoma spheroids, aiming for identification of novel compounds active against this type of solid tumor. Materials and methods MCF7 spheroids were generated in 96-well plates by a centrifugation method. The spheroids took 5 days to reach ∼500-μm diameter and were ready for treatment. The initial screen was performed at 50 μM in triplicates. A dose–response study followed the initial screen. A counterscreen was carried out using RPE1 normal cell spheroids to identify the selectivity of active compounds. The acid phosphatase method was applied to measure the cytotoxicity of compounds. A clonogenic assay was used to investigate the viability of remaining cells after treatment with test compounds. Results and conclusion The compound (4,5-dibromo-6-oxo-1(6H)-pyridazinyl)methyl 3-chlorophenylcarbamate was identified in this study for the first time with reasonable toxicity on MCF7 cancer spheroids. This compound is suggested as a lead compound for the development of more active derivatives against solid tumors. Additionally, the multicellular spheroid model was proved as a useful and applicable platform for identification of novel compounds for the treatment of solid tumors.

Biosynthesis of alkaline protease by alkaliphilic Bacillus sp. NPST-AK15 cells immobilized in gel matrices Dina A Many, Abdelnasser S.S Ibrahim, Ahmed I El-Diwany Egyptian Pharmaceutical Journal 2019 18(3):201-207 Background and objectives Proteases are an important group of hydrolytic enzymes catalyzing the hydrolysis of various proteins by cleavage of the peptide bonds between the amino acids residues. Proteases have applications in several fields including medical and pharmaceuticals industries. Bacterial cell immobilization by entrapment techniques is one of the most effective approaches used in biotechnology at laboratory and industrial scale. Herein, we report the production of alkaline protease by immobilized halotolerant alkaliphilic Bacillus sp. strain NPST-AK15 cells in batch and repeated batch fermentation. Materials and methods Alkaline proteases-producing halotolerant alkaliphilic Bacillus sp. strain NPST-AK15 (accession no. KP295749) was previously isolated from hypersaline soda lakes, located at Wadi El- Natrun Valley (Egypt). Three different matrices were tested for immobilization of Bacillus sp. strain NPST-AK15 whole cells by entrapment technique including alginate, gelatin, and agar gel. Results and discussion Among various matrices tested for whole cell immobilization of Bacillus sp. NPST-AK15, alginate was found to be the best matrix for cell entrapment and alkaline protease production, showing the highest specific productivity (3214.34 U/g wet cells/h) and enzyme production (923.4 U/ml), followed by cells immobilized in agar and gelatin. Furthermore, the production of alkaline protease by Bacillus sp. NPST-AK15 immobilized in alginate gel was enhanced by investigation of the influence of various parameters on alginate beads preparation including alginate concentration, bead size, and biomass loading. Maximum enzyme production (1020.1 U/ml) and specific productivity (4086.9 U/g wet cells/h) were achieved using alginate concentration of 3.0% (w/v), bead diameter of 3.5 mm, and cell loading of 0.50 g wet weight of cell biomass per 0.3 g of sodium alginate. The immobilized Bacillus sp. NPST-AK15 cells exhibited operation stability in repeated batch fermentation, retaining 89.1 and 61.3% of its productivity after five (120 h) cycles and 10 (240 h) cycles, respectively.

Does glycogen synthase kinase-3 β signaling pathway has a significant role in date palm pollen cancer therapy? Mai O Kadry, Rehab M Abdel Megeed, Hassan Z Ghanem, Ahmed S Abdoon, Abdel-Hamid Z Abdel-Hamid Egyptian Pharmaceutical Journal 2019 18(3):208-215 Background and objective Palm pollen (PP) has attracted much attention for its wide applications as an anticancer natural product due to their high phenolic and flavonoid contents. In the current of PP to inhibit the progression of hepatocellular carcinoma was assessed in a rat model. Materials and methods First, high-performance liquid chromatography analysis was performed to identify the active constituents in PP. Diethyl nitrosamine as a hepatocarcinogenic agent was administered at a dose of 4 gm/kg body weight intraperitoneally for 4 months sequenced by PP treatment orally (200 mg/kg) daily for 3 weeks. Biochemical and molecular analyses were estimated. Results and discussion HPLC analysis showed the presence of chlorogenic acid, quercetin, coumaric acid, caffeine, vanillin, and ferulic acid in PP. Diethyl nitrosamine significantly elevated serum lipid peroxide, nitrite/nitrate levels, and decreased glutathione level. In addition to an obvious alternation of tumor necrosis factor-α1, nuclear factor kappa-β, cellular oncogene-Fos, and glycogen synthase kinase-3β genes expression. Meanwhile, PP improved all the previously deviated biochemical parameters reflecting great antioxidant, anticancer, and anti-inflammatory index. These findings were confirmed histopathologically.

In-vitro adventitious root production of Cichorium endivia L. and antioxidants, total phenolic, and total flavonoids assessments Mona M Ibrahim, Mohamed K El-Bahr, Mohamed R Rady Egyptian Pharmaceutical Journal 2019 18(3):216-227 Background and objectives Chicory plant serves as a vegetable with higher nutritional value, having major antioxidant properties. The aim of this work was in-vitro production of adventitious roots from Cichorium endivia subsp., pumelum L. and exploring the capacity of adventitious roots for antioxidant activities as well as determine their contents of total phenolic and flavonoids compounds compared with different parts of C. endivia. Materials and methods For in-vitro adventitious root induction, root segments were cultured on half-strength Murashige and Skoog solid medium supplemented with different concentrations of indole-3-butyric acid and 0.1 mg/l α-Naphthalene acetic acid. The cultures were incubated under total darkness and or 16/8 h (light/dark) photoperiod. Murashige and Skoog liquid medium with different carbon sources was used for adventitious root production. Two different solvents (aqueous ethanol and chloroform) were used for bioactive components extraction process. 2, 2′‐diphenyl 1‐Picryl-hydrazyl radical scavenging capacity (RSC) as well as total phenolic and flavonoides contents were estimated in produced adventitious roots compared with different plant parts (seeds, leaves, and roots). Results and conclusion Medium supplemented with 0.1 mg/l α-Naphthalene acetic acid and 1.0 mg/l indole-3-butyric acid recorded maximum adventitious root induction percentage (100%) in the dark condition. High-yield production of adventitious roots (6.60±0.5 g) was found in the liquid medium that contains sucrose as the carbon source. The aqueous ethanol extracts recorded higher RSC% values than chloroform extracts in all plant parts. Aqueous ethanol extract of seeds recorded maximum RSC% (92.8%) at 2.5 mg/ml of extract. Total phenolic contents showed maximum value with aqueous ethanol extract of seeds (18.17±0.40 mg/g of extract), whereas minimum value recorded with chloroform extract of seeds (0.28±0.05 mg/g of extract). The flavonoids contents showed maximum value also with aqueous ethanol extract of seeds (94.43±1.00 mg/g of extract), followed by aqueous ethanol extract of leaves (93.68±0.1 mg/g of extract), and minimum value with chloroform extract of leaves (2.60±0.18 mg/g of extract).

Potential effects of silver nanoparticles, synthesized from Streptomyces clavuligerus, for controlling of wilt disease caused by Fusarium oxysporum Amr A El-Waseif, Mohamed S Attia, Dina E El-Ghwas Egyptian Pharmaceutical Journal 2019 18(3):228-235 Background Fusarium oxysporum causes wilt disease, which is considered a destructive disease, leading to decreased growth and death of most infected plants. Materials and methods After 7 days of incubation of Streptomyces clavuligerus on starch nitrate medium, the synthesis of silver nanoparticles (AgNPs) was done by using the supernatant from the microorganism. The color changed to dark brown, proving the formation of AgNPs. The size of AgNPs was analyzed using transmission electron microscope. Various concentrations of AgNPs (20, 40, 60, 80, and 100 μl) were investigated against F.‏oxysporum by using agar well diffusion method. Disease symptoms, disease index percent, phytochemicals, and metabolic indicators of resistance in plant, such as the reaction to induction of systemic resistance, were recorded in tomato plants. Results and conclusion The resultant AgNPs had size from 4 to 38 nm and were oval to spherical in shape. The observed inhibition zones were 12, 18, 19, 23, and 27 mm in diameter correspondingly. The growth of Fusarium has been reduced by 60, 40 ppm, and followed by 20 ppm. Treatment with different concentration of nanoparticles resulted in different responses regarding the total phenol content, proline content, and total protein of Fusarium-infected plants. Applications of 60 ppm by foliar shoot+root immersion and root immersion methods were the best treatments and reduced percent disease indexes by 8 and 11%, respectively. Therefore, it could be suggested that the application of tested treatments could be commercially used for controlling Fusarium wilt disease of vegetable plants, as they are effective against this disease, are less expensive, and are safe.

Molecular characterization of the microbiota in patients with ulcerative colitis in the Kingdom of Saudi Arabia Azzam Abdulmohsen Al-Quait, Essam Jamel Al-Yamani, Dina A Abdelfattah, Abdelnasser Salah Shebl Ibrahim, Ali Abduallah Al-Salamah Egyptian Pharmaceutical Journal 2019 18(3):236-244 Background Ulcerative colitis (UC) is a chronic inflammatory disorder of the gastrointestinal tract. Herein, we report a comparative analysis of intestinal microbiota in Saudi patients with UC and healthy individuals using a culture-independent approach. Materials and methods Intestinal biopsies of the five Saudi patients with UC and five healthy citizens were collected, homogenized, and DNA extracted. Genomic libraries of 16S rDNA were constructed using these biopsies. Results and discussion Among the 96 clones analyzed, 39 distinct bacterial strains were found to belong to two main genera: Bacteroides (46%) and clostridium (26%). Levels of uncultured bacteria and uncultured Bacteroidetes were higher in patients with UC than in healthy individuals, and there was a marked decrease in bacterial diversity and evenness in patients with UC relative to healthy individuals. A group of bacteria in healthy individuals was absent in the microbiome of patients with UC, including Bacteroides fragilis, Bacteroides vulgatus, Prevotella spp., Bacteroides coprocola, Escherichia coli, and Streptococcus thermophiles, whereas another group of bacteria found in Saudi patients with UC was not detected in healthy individuals, including Staphylococcus warneri, Bacteruim LF48, Weissella confusa, and enterococci. The results confirm that UC is a multifactorial disease in origin, and some specific bacteria act as etiological agents of UC. Conclusion UC is a multifactorial illness, expressed not only by the dysbiosis of the intestinal microbial flora but also is referred to other causes like the type of diet of each patient, his/her immunity, and genetics.

Dimeric naphtho-γ-pyrones and further diverse bioactive metabolites from the marine-derived Aspergillus flavus Af/MMA 2018 Mohamed E El Awady, Ann G Boulis, Attia R Attia, Mohamed Shaaban Egyptian Pharmaceutical Journal 2019 18(3):245-253 Background and objective Sponge-associated fungi are known for their production of structurally diverse secondary metabolites, many of which exhibit different pharmacological activities. Materials and methods Isolation and identification of fungal isolate from the marine sponge Echinodictyum flabelliforme collected from the Red Sea coast of Hurghada, Egypt, was done. Working up and purification with the ethyl acetate extract produced by the marine-derived Aspergillus flavus Af/MMA 2018 afforded nine bioactive compounds. Structure of the isolated compounds was determined on the basis of NMR (1D and 2D) and mass (EI, ESI, HRESI MS) spectra and by comparison with the corresponding literature studies. Biologically, the antimicrobial, antioxidant, and antitumor activities (using Ehrlich cells) of compounds were studied in comparison with the original extract. Results and conclusion Working up and purification of the ethyl acetate-extracted residue produced by the marine-derived A. flavus Af/MMA 2018 afforded nine diverse bioactive compounds: five dimeric naphtho-γ-pyrones, that is, aurasperone A (1), aurasperone B (2), aurasperone D (3), aurasperone F (4), and aurasperone E (5), along with β-sitosterol glucoside (6), cerebroside C (7), glyceryl linoleate (8), and linoleic acid (9). Cerebroside C showed strong antimicrobial activity against different test organisms, whereas aurasperone E showed maximum DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) scavenging activity (67.41%) after 1 h, and by using different concentrations, giving 98.99% at 1000 μg/ml. The maximum antitumor activity against Ehrlich ascites carcinoma cells (70.9%) was attributed to the dimeric naphtho-γ-pyrone aurasperone E.

In-vitro propagation of the multipurpose Egyptian medicinal plant Pimpinella anisum Ahmed Amer, Hanaa Omar Egyptian Pharmaceutical Journal 2019 18(3):254-262 Background and objective Plant tissue culture technology offers a solution for meeting the increasing commercial demand for medicinally important plants, especially cross-pollinating species, whose genetic heterogeneity presents difficulties when using traditional propagation methods. Herein, we describe an effective, rapid, andq simple protocol for the micropropagation of anise (Pimpinella anisum). Materials and methods We investigated the effect of the type of explant and the type and concentration of plant growth regulator, either individually or in combination, on plant micropropagation. Results and conclusion Although multiple shoot formation rate was higher for nodal than for shoot tip explants, there was no significant difference in rooting response between shoots arising from either of them. Maximum shoot response, number of shoots per explant, and shoot length were observed in nodal explants grown on Murashige and Skoog medium supplemented with 5 μmol/l 6-benzylaminopurine, 1 μmol/l kinetin, and 0.5 μmol/l naphthalene acetic acid. The most effective medium for root regeneration contained 3 μmol/l of either indole-3-butyric acid or naphthalene acetic acid. Interestingly, there was no evidence for hyperhydricity, which is commonly found in cultured anise, using our method. Plantlets were successfully hardened and transferred to the greenhouse, with an 85% survival rate. This protocol provides an efficient means for large-scale production of anise, as well a basis for further research aimed at genetic improvement of this plant.

Influence of foliar fertilization on the growth and yield of chia (Salvia hispanica) plant Ahmed M Salman, Elsayed A Omer, Mohamed S Hussein, Eman Sewedan, Amira R Osman Egyptian Pharmaceutical Journal 2019 18(3):263-275 Background Salvia hispanica plant is a new introduced crop to the Egyptian cultivation system to enrich it with new species or varieties of medicinal and aromatic plants. S. hispanica commonly known as chia is an annual herbaceous plant belongs to the mint family (Lamiaceae) and is native to southern Mexico and Northern Guatemala. Chia seeds are a promising source of antioxidants owing to their content of omega-3 and the presence of polyphenols, chlorogenic acid, caffeic acids, myricetin, quercetin, and kaempferol. This study was carried out to evaluate the effect of different doses and portions of NPK and/or mixture of biofertilizer (Azotobacter chroococcum+Bacillus megaterium+Bacillus subtilis) on the growth and yield of chia (S. hispanica) plant. Materials and methods This investigation was carried out in the Hawareya location, Beheira Governorate, Egypt (North West of the Nile Delta), during the two successive seasons 2016/2017 and 2017/2018. The experimental layout was randomly distributed in a split-plot design with three replicates. The 10 treatments of NPK (1.5, 3, and 4.5 g/l in 1, 2, or three portions and control) were randomly distributed in the main plots, whereas the two foliar applications, namely, control and biofertilizers, were randomly distributed in the subplots to study the influence of foliar application treatments, Nitrophoska foliar fertilizer (1.5, 3, and 4.5 g/l) in 1, 2, or three portions (as control) and a mixture of A. chroococcum 10 g/l+B. megaterium 10 g/l+B. subtilis 10 g/l on the growth and yield of chia plant. Data were recorded for the plant height (cm), number of branches/plant, number of inflorescence/plant, herb fresh weight (g/plant and ton/fed), herb dry weight (g/plant and ton/fed), and seeds weight (g/plant and kg/fed). Results The results showed that different doses and portions of NPK and/or mixture of biofertilizer significantly increased the vegetative growth and yield of chia plant. The results of the 2 years indicated that the highest values of plant height (199.33 cm/plant), number of branches (22.56 branches/plant), number of inflorescence (58.89 inflorescence/plant), fresh herb weight (1053.33 g/plant and 24.58 ton/fed), dry herb weight (352 g/plant and 8.22 ton/fed), seeds weight (24.22 g/plant), and seeds yield (565 kg/fed) of S. hispanica were recorded with NPK 3 g/l (two portions)+biofertilizer. Conclusion From these results, we may conclude that the recommended treatment to obtain the best growth characteristics and yield of S. hispanica are the application of NPK 3 g/l (two portions)+biofertilizer.