Hossam M. Hassan

Dual Induction of New Microbial Secondary Metabolites by Fungal Bacterial Co-cultivation

The frequent re-isolation of known compounds is one of the major challenges in drug discovery. Many biosynthetic genes are not expressed under standard culture conditions, thus limiting the chemical diversity of microbial compounds that can be obtained through fermentation. On the other hand, the competition during co-cultivation of two or more different microorganisms in most cases leads to an enhanced production of constitutively present compounds or an accumulation of cryptic compounds that are not detected in axenic cultures of the producing strain under different fermentation conditions. Herein, we report the dual induction of newly detected bacterial and fungal metabolites by the co-cultivation of the marine-derived fungal isolate Aspergillus fumigatus MR2012 and two hyper-arid desert bacterial isolates Streptomyces leeuwenhoekii strain C34 and strain C58. Co-cultivation of the fungal isolate MR2012 with the bacterial strain C34 led to the production of luteoride D, a new luteoride derivative and pseurotin G, a new pseurotin derivative in addition to the production of terezine D and 11-O-methylpseurotin A which were not traced before from this fungal strain under different fermentation conditions. In addition to the previously detected metabolites in strain C34, the lasso peptide chaxapeptin was isolated under co-culture conditions. The gene cluster for the latter compound had been identified through genome scanning, but it had never been detected before in the axenic culture of strain C34. Furthermore, when the fungus MR2012 was co-cultivated with the bacterial strain C58, the main producer of chaxapeptin, the titre of this metabolite was doubled, while additionally the bacterial metabolite pentalenic acid was detected and isolated for the first time from this strain, whereas the major fungal metabolites that were produced under axenic culture were suppressed. Finally, fermentation of the MR2012 by itself led to the isolation of the new diketopiperazine metabolite named brevianamide X.

Exploring the Antimicrobial and Antitumor Potentials of Streptomyces sp. AGM12-1 Isolated from Egyptian Soil

The occurrence of extensive antibiotics resistant bacteria increased the demands for mining out new sources of antimicrobial agents. Actinomycetes, especially Streptomyces sp. have grasped considerable attention worldwide due to production of many useful bioactive metabolites. In the present study, a total of 52 actinomycetes were isolated from agricultural soil samples in Beni-Suef, Egypt. All isolates were characterized based on colony morphology, mycelium coloration, and pigment diffusion. They were screened for their capabilities to show antimicrobial activities against different indicator microorganisms, and only 20 isolates have shown significant antimicrobial activities against at least one of the tested indicator microorganisms. The isolate AGM12-1 was active against all tested microorganisms and showed a marked antitumor activity with IC50 3.3 and 1.1 μg/ml against HCT-116 and HepG-2 cell lines respectively. It was genotypically characterized as Streptomyces sp. with the presence of PKS Π biosynthetic gene cluster. Mannitol, ammonium sulfate, pH 7, 2% inoculum size and incubation for 11 days at 30°C were the optimum conditions that used to maximize the production and hence allowed purification of one active antimicrobial compound to homogeneity using high performance liquid chromatography with a molecular mass of m/z 488.05. Nuclear magnetic resonance structural elucidation showed that this compound was a diketopiperazine derivative.

Biologically-guided isolation of leishmanicidal secondary metabolites from Euphorbia peplus L.

Leishmaniasis is a worldwide health problem, highly endemic in developing countries. Moreover, the severe side effects and the reported drug resistance make it an urgent need to search for effective drugs that can replace or supplement those currently used. In a research program designed to investigate the antileishmanial activity of plants collected from the Egyptian flora, twenty extracts from fifteen plants growing in Egypt have been investigated for in vitro leishmanicidal activity against Leishmania donovani promastigotes. Among the tested extracts, the methanol extract of Euphorbia peplus aerial parts exhibited a significant antileishmanial activity as it produced 100% inhibition of growth with activity similar to amphotericin B. The total extract was subjected to liquid-liquid fractionation using solvents of different polarities, followed by testing the antileishmanial activity of the successive fractions. Phytochemical exploration of the active n-hexane fraction (which produced 75% inhibition of growth) led to isolation of four compounds: simiarenol (1), 1-hexacosanol (2), β-sitosterol (3), and β-sitosterol-3-O-glucoside (4) from the biologically active sub-fractions. Structure elucidation was aided by 1D and 2D NMR techniques. In conclusion, E. peplus plant has many non-polar secondary metabolites that can be used as drug leads for treatment of leishmaniasis.

Comparative DNA profiling, phytochemical investigation, and biological evaluation of two Ficus species growing in Egypt.

Aim and Background: A comparison between two Ficus species, cultivated in Egypt, was carried out in this study. Their DNA analysis revealed that they are not closely related. Materials and Methods: The pharmacopoeial constants of the leaves showed higher total ash and acid insoluble ash in F. lyrata than in F. platypoda. The other parameters were close in both species. Preliminary phytochemical screening revealed the presence of carbohydrate and/or glycosides, tannins, flavonoids, sterols, and triterpenes in their leaves and was detected in traces in their stems. Results: Saponification of n-hexane extract of the leaves yielded 46% and 74.8% for the unsaponifiable matters and 20% and 15% for the fatty acids for F. platypoda and F. lyrata, respectively. n-Docosane (21.69%) and n-heptacosane (33.77%) were the major hydrocarbons in F. platypoda and F. lyrata, respectively. b-Sitosterol was the main sterol, palmitic (22.07%) and carboceric (35.72%) acids were the major identified saturated fatty acids in both species, while linoleic acid was the main unsaturated fatty acid (18.66% and 16.7%) in both species, respectively. The acute toxicity study revealed that the two species were safe up to 2 g/kg. The antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and pyrogallol as the standard was more significant for F. platypoda (232.6 μg/ml) than for F. lyrata, (790.9 μg/ml). The oral antihyperglycemic activity in diabetic rats using alloxan revealed that the 80% ethanolic extract of the leaves of F. platypoda was more active than that of the leaves of F. lyrata in decreasing the blood glucose level at 200 mg/kg/day (107.9 ± 5.817, 127.2 ± 4.359) and 400 mg/kg/day (64.11 ± 4.358, 127.7 ± 6.889), respectively, when compared with the diabetic control gliclazide (172.3 ± 2.089). Conclusion: The results of this study provide evidence that the two Ficus species have antioxidant and antihyperglycemic activity, in the order F. platypoda and then F. lyrata

Epigenetic Modifiers Induce Bioactive Phenolic Metabolites in the Marine-Derived Fungus Penicillium brevicompactum

Fungi usually contain gene clusters that are silent or cryptic under normal laboratory culture conditions. These cryptic genes could be expressed for a wide variety of bioactive compounds. One of the recent approaches to induce production of such cryptic fungal metabolites is to use histone deacetylases (HDACs) inhibitors. In the present study, the cultures of the marine-derived fungus Penicillium brevicompactum treated with nicotinamide and sodium butyrate were found to produce a lot of phenolic compounds. Nicotinamide treatment resulted in the isolation and identification of nine compounds 1–9. Sodium butyrate also enhanced the productivity of anthranilic acid (10) and ergosterol peroxide (11). The antioxidant as well as the antiproliferative activities of each metabolite were determined. Syringic acid (4), sinapic acid (5), and acetosyringone (6) exhibited potent in vitro free radical scavenging, (IC50 20 to 30 µg/mL) and antiproliferative activities (IC50 1.14 to 1.71 µM) against HepG2 cancer cell line. Furthermore, a pharmacophore model of the active compounds was generated to build up a structure-activity relationship.

Diketopiperazine derivatives from Enterobacter cloacae isolated from the Red Sea alga Cystoseira myrica

Aim This study is an attempt to explore the biological activities of isolated endophytic bacteria from marine sources that were coded A1, A2, and A3 (Padina pavonica), A4 (Cystoseira myrica), A5 (Acanthophora dendroides), and A6 (Sargassum sabrepandum). The bacteria coded C1, C2, and C3 were isolated from the soft coral Nephthea mollis and S1 and S2 were isolates from the sponge Hymedesmia spp. The primary aim of the study was the identification of active compounds. Materials and methods The bioactive compounds were extracted using ethyl acetate from nutrient broth media; biological activities of the extracted metabolites and 16S rDNA identification of the most promising isolate were studied. The eight major fractions of the extract showed different composition patterns when identified by liquid chromatography/mass spectrometry analyses. Results and conclusion Agar diffusion assay showed inhibitory activities of A4 extracts against the growth of most pathogenic microorganisms. Identification using PCR 16S rDNA and electrophoresis confirmed 98% identity to the Enterobacter cloacae strain GH1 (ac: JF261136.1). Eight compounds out of fifteen in the extract were identified as diketopiperazine derivatives. The maximum growth of E. cloacae was obtained at 30°C, pH 7, with the addition of maltose and KI to the media. The free radical scavenging activity exhibited good antioxidant activity (72.19%, IC 50 = 1.266 mg/ml) on using 2.0 mg/ml of the crude extract. The extract showed a high antiviral activity towards Newcastle disease virus and avian influenza virus A(H5N1).

Characterization of natural bioactive compounds produced by isolated bacteria from compost of aromatic plants

This study aimed to highlight the importance of compost from aromatic plants as a stunning source for several bio active compounds generated from their inhabited thermophilic bacteria. Some of the isolated compounds could have a potential role in the treatment of microbial infections.

Balanites aegyptiaca ameliorates insulin secretion and decreases pancreatic apoptosis in diabetic rats: Role of SAPK/JNK pathway

SAPK-JNK pathway performs a significant role in the pathogenesis of type 2 diabetes. Balanites aegyptiaca (BA) is used as an anti-diabetic agent in folk medicine however its hypoglycemic mechanism is not fully elucidated. The current study aimed to evaluate the effect of crude extract, butanol, and dichloromethane fractions from BA on the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK-JNK) pathway in experimental diabetic rats. Six groups of male Wistar rats were included: normal control, diabetic, diabetic rats treated with crude, butanol or dichloromethane fraction from BA (50 mg/kg BW) and diabetic rats treated with gliclazide as a reference drug for one month. Our results suggested a protective role of treatment of diabetic rats with BA against oxidative stress-induced SAPK-JNK pathway. Moreover, BA treatment produced a reduction in plasma glucose, HbA1c, lactic acid, lipid profile, malondialdehyde levels and produced an increase in insulin, reduced glutathione levels, catalase and superoxide dismutase activities compared with untreated diabetic rats. Moreover, it decreased apoptosis signal-regulating kinase 1, c-Jun N-terminal kinase 1, protein 53 and increased insulin receptor substrate 1 in rat pancreas while it increased glucose transporter 4 in rat muscle. Analysis of BA extracts by LC-HRMS revealed the presence of different saponins with reported hypoglycemic effect. In conclusion, BA exerted hypoglycemic, hypolipidemic, insulinotropic and antioxidant effects. Additionally, it reduced apoptosis in pancreatic β-cells and increased glucose uptake in muscle. These results suggest that the hypoglycemic effect of BA is due to the inhibition of the SAPK-JNK pathway.

Acylated Iridoids and Rhamnopyranoses from Premna odorata (Lamiaceae) as Novel Mesenchymal–Epithelial Transition Factor Receptor Inhibitors for the Control of Breast Cancer

Phytochemical investigation of Premna odorata Blanco, Lamiaceae, leaves afforded three new acylated iridoid glycosides 1–3 and two new acylated rhamnopyranoses 9 and 10, in addition to ten known compounds. The structures of the new compounds were confirmed using extensive 1D and 2D NMR analysis. Molecular modeling study suggested the potential of the acylated rhamnopyranoses to bind at the c‐Met kinase domain. Cell‐free Z′‐LYTE™ assay testing revealed the good c‐Met phosphorylation inhibitory activity of 9, followed by 8, and 10, with IC50 values of 2.5, 6.9, and 12.7 μM, respectively. The (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) cell proliferation assay testing against the human c‐Met expressing highly invasive MDA‐MB‐231 suggested compound 9 as the most active with IC50 value of 13.3 μM. Testing of compound 9 against multiple phenotypic breast cancer cell lines including MCF‐7, BT‐474 cells, and MDA‐MB‐468 proved enhanced activity against the highly c‐Met expressing triple‐negative breast cancer cell lines. Acylated rhamnopyranoses are potential novel c‐Met inhibitors appropriate for future optimizations to control c‐Met‐dependent breast malignancies. Copyright

Premna odorata Volatile Oil as a New Mycobacterium tuberculosis Growth Inhibitor for the Control of Tuberculosis Disease

Aims: This study aimed to identify and compare Premna odorata Blanco volatile oil (VO) for the first time; isolated from different plant organs (leaves, young stems, and flowers) with evaluating the oil antituberculosis (anti-TB) activity. Study Design: Experimental design was carried out by using hydrodistillation method, GC/MS analysis and MeDipro Mycobacterium tuberculosis (MTB) Antigen ELISA Technique (MMA-ELISA) accompanied by polymerase chain reaction (PCR) analysis. Place and Duration of Study: This study was carried out at Faculty of Pharmacy, Beni-Suef University, between May to July 2017. Methodology: P. odorata VO was identified using GC/MS analysis, the oil anti-TB activity was evaluated using in vitro and in vivo MMA-ELISA accompanied by PCR analysis. Original Research Article Elmaidomy et al.; EJMP, 21(4): 1-11, 2017; Article no.EJMP.38375 2 Results: GC/MS analysis revealed that P. odorata VO consisted of monoterpenes, sesquiterpenes, diterpenes and higher alkanes; where monoterpenes and sesquiterpenes were represented the major oils fractions. Trans-caryophyllene (29.403% & 14.638%) and β-phellandrene (22.390% & 11.701%) were the major compounds in the leaves and young stems oils, respectively. While αpinene (38.160%) was a characteristic component of the flowers oil. MMA-ELISA showed that a dose of 100 μl/ml in vitro and 300 μl/ml in vivo; the leaves, young stems, and the flowers oils separately had significant anti-TB activities with measured values > 1.5 μg/ml MTB antigen; while the three organs oils in combination 1:1:1 increased the potency of the oils against MTB with measured values < 1.5 μg/ml MTB antigen with PCR negative analysis. Conclusion: P. odorata VO exhibited Anti-TB activity which Anti-TB could be related to the presence of cyclic terpenes (major) and acyclic oxygenated terpenes (minor) compounds.