Induction of antimicrobial peptides from Bacillus thuringiensis challenged Spodoptera litura larvae and investigation of the antimicrobial properties of hemolymph samples

Antimicrobial peptides constitute key factors in insect humoral immune response against invading microorganisms. In this study, biochemical approach was identified antimicrobial peptides which appeared in larval hemolymph of Spodoptera litura after bacterial challenge. HPLC profile showed two major peaks in two samples, Brassica oleracea and Ricinus communis fed S. litura that were collected at 5 min interval. It was shown to be active against Gram-positive and Gram-negative bacteria. The highest zone of inhibition was observed in Staphylococcus aureus and Escherichia coli in B. oleracea fed S. litura hemolymph fraction II and R. communis fed S. litura hemolymph fraction I and it also contributes the increased antioxidant, lysozyme, and less hemolytic activity were increase in treated groups. TLC activity was tested with hemolymph extract samples, pink color pots was identified the protein present in the samples. An SDS-PAGE result shows that high expression of antimicrobial peptide present in the treated sample. The appearance of peptides with such different properties in insect hemolymph in response to immune challenge indicates the complexity of the insect immune system.

Polysaccharide Based Polymers Produced by Scabby Cankered Cactus Pear (Opuntia ficus-indica L.) Infected by Neofusicoccum batangarum: Composition, Structure, and Chemico-Physical Properties

Neofusiccocum batangarum is the causal agent of scabby canker of cactus pear (Opuntia ficus-indica L.). The symptoms of this disease are characterized by crusty, perennial cankers, with a leathery, brown halo. Characteristically, a viscous polysaccharide exudate, caking on contact with air, leaks from cankers and forms strips or cerebriform masses on the surface of cactus pear cladodes. When this polysaccharide mass was partial purified, surprisingly, generated a gel. The TLC analysis and the HPLC profile of methyl 2-(polyhydroxyalkyl)-3-(o-tolylthiocarbomoyl)-thiazolidine-4R-carboxylates obtained from the mixture of monosaccharides produced by acid hydrolysis of the three EPSs examined in this research work [the polysaccharide component of the exudate (EPSC) and the EPSs extracted from asymptomatic (EPSH) and symptomatic (EPSD) cladodes] showed the presence of d-galactose, l-rhamnose, and d-glucose in a 1:1:0.5 ratio in EPSC while d-galactose, l-rhamnose, d-glucose, and d-xylose at the same ratio were observed in EPSH and EPSD. The presence of uronic acid residues in EPSC was also showed by solid state NMR and IR investigation. Furthermore, this manuscript reports the chemical-physical characterization of the gel produced by the infected cactus pear.

Uncovering the potential of actinobacterium BLH 1-22 isolated from marine sediment as a producer of antibiotics

Actinobacteria have been known as producers of many bioactive compounds. The present study examines ten marine Actinobacterial isolates, aiming to investigate their potential as producers of antimicrobial compounds. The secondary metabolites were extracted from these Actinobacteria using ethyl acetate, and the crude extracts were tested for their bioactivity against Escherichia coli, Bacillus subtilis, and Micrococcus luteus. The antibacterial screening showed that the crude extracts of these Actinobacteria inhibit the growth of indicator strains. The extracts of isolate BLH 1-22 were further analysed using high-performance liquid chromatography (HPLC), which showed potential compounds with peak and retention time similar to the antibiotic standards (i.e., erythromycin, ampicillin, tetracycline, and penicillin). In addition to the HPLC profile, molecular identification showed that the isolate BLH 1-22 was similar to Micromonospora chalcea (99.6%). Further genome characterization of the strain, as well as purification and fractionation of the metabolite extracts, are important to obtain a comprehensive study on the potential of isolate BLH 1-22 as antibiotic compound producers. This study reported the potential of Micromonospora BLH 1-22 isolated from marine sediment. Hence, it also highlighted the potential of Actinobacteria isolated from Indonesian environments for bioprospecting studies.

Cloning, Expression and Structural Modeling of the MlrA Protein from Novosphingobium sp. KKU25s for Microcystin Degradation

MlrA is a gene involved in the degradation of toxic cyanobacterial microcystins. This gene encodes microcystinase, mlrA, the 1st enzyme in the pathway that breaks down toxic cyanobacterial microcystins. In this study, primers were designed, and polymerase chain reaction (PCR) was performed to amplify the mlrA gene in Novosphincgobium sp. KKU25s. A PCR product of 752 base pairs was obtained. The nucleotide sequence of the mlrA gene of Novosphingobium sp. KKU25s was similar to that of Sphingomonas sp. ACM-3962 (98 % similarity). The mlrA gene of Novosphingobium sp. KKU25s was further cloned into the pGEM T-Easy plasmid to obtain the nucleotide sequence of the mlrA gene. The gene was also ligated into the pET32a plasmid for gene expression. Expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and verified using SDS-PAGE. The expressed protein was approximately 22 kilodaltons. The cell-free extract (CE) containing the crude protein from confirmed recombinant cells showed high activity in the biodegradation of [Dha7] MC-LR. [Dha7] MC-LR at an initial concentration of 30 mg L-1 and was completely biodegraded within 30 h. A distinct product derived from [Dha7] MC-LR appeared with a decrease in the [Dha7] MC-LR peak in the HPLC profile. The product (m/z 999.51) showed a molecular weight of 18, which is higher than that of native [Dha7] MC-LR (m/z 981.50), and was determined to be a linearized peptide fragment of [Dha7] MC-LR using LC-MS analysis. The 3-dimensional structure of microcystinase was predicted from the amino acid sequence deduced from the mlrA gene by the Swiss Model and Phyre2 programs. The structure contained a predicted alpha helix. The predicted 3-dimensional structure was also similar to that of a protein in the CAAX protease group. HIGHLIGHTS Research focused on characterization of microcystinase (MlrA) protein First research worked on the degradation of [Dha7] MC-LR by MlrA This work is useful for the applications aimed at the removal of MCs in freshwater environments GRAPHICAL ABSTRACT

HPLC Profile of Phenolic Contents, Antioxidant and Antidiabetic Activities of Methanolic Extract of the Leaves of Sarcocephalus latifolius(Bruce, Smith) Grown in North Central Geopolitical Zone, Nigeria

Preliminary phytochemical screening of the n-butanol fraction of the methanol leaf extract of Sarcocephalus latifoliuswas carried out;its phenolic contents were quantified by HPLC. The in vitroantihyperglycemic and antioxidant activities of the fraction were evaluated.The results of the present study indicated that the leaves’extract contained phytochemicals,like phenols, flavonoids, alkaloids and glycosides.Phenolic compounds,such as garlic acid, catechin, ferrulic acid, p-coumaric acid, apigen-7-glucose, kaemferol, quercetin and amentoflavone,were quantified by HPLC. Moreover, the fraction of leaves’extract of S.latifoliusdemonstrated significant antioxidant and antihyperglyce-mic activities.The presence of these phytochemicals,especially phenols and flavonoids,in the leavesof S.latifoliustherefore justifiesitsmedicinal usefulness,as this plant has the potential to protect oxidative stress-induced ailments

The Influence of Solvent, Host, and Phenological Stage on the Yield, Chemical Composition, and Antidiabetic and Antioxidant Properties of Phragmanthera capitata (Sprengel) S. Balle

Phragmanthera capitata was reported to possess many biological properties making it a good candidate for the formulation of a phytomedicine with multiple effects. In this work, we studied some factors likely to modify these therapeutic properties with the aim to contribute to its standardization as an improved traditional medicine. P. capitata parasitizing Persea americana, Psidium guajava, and Podocarpus mannii were harvested at three phenological stages (vegetative, flowering, and fruiting stages). The extracts were prepared by maceration in n-hexane, ethyl acetate, ethanol, methanol, and distilled water. The total phenolic, flavonoid, flavonol, and tannin contents were measured using appropriate methods. The antioxidant potential of extracts was investigated using TAC, DPPH scavenging, and FRAP methods. The α-amylase and α-glucosidase inhibitory activities of extracts were determined using enzymatic methods. The ethyl acetate extracts with the best phenolic content were subjected to HPLC analysis. The extraction yields were higher with methanol. The ethyl acetate extract of P. capitata harvested from P. guajava showed a stable HPLC profile during the development of the plant, while extracts from the plant collected from P. americana and P. mannii showed both qualitative and quantitative variations according to phonological stages of the plant. The inhibition of α-amylase was more pronounced for P. capitata harvested from P. guajava, decreasing during flowering and fruiting, while inhibition of α-glucosidase was not influenced by the phenological stage and the host of the plant. The α-amylase inhibitors were better extracted by ethyl acetate and those of α-glucosidase by ethanol or methanol. The phenolic contents and antioxidant properties of the extracts were influenced by the phenological stage of P. capitata and its hosts. These results suggest that it is preferable to harvest P. capitata during flowering or during fruiting stages on any host. None of the used solvents permitted an optimal extraction of active principles form P. capitata, suggesting that the mixture of solvents must be considered in further studies.