Metabolite Profiling of Alangium salviifolium Bark Using Advanced LC/MS and GC/Q-TOFTechnology

There is an urge for traditional herbal remedies as an alternative to modern medicine in treating several ailments. Alangium salviifolium is one such plant, used traditionally to treat several diseases. In several reports, there are findings related to the use of this plant extract that demonstrate its therapeutic value. However, very few attempts have been made to identify the extensive metabolite composition of this plant. Here, we performed metabolite profiling and identification from the bark of A. salviifolium by extracting the sample in organic and aqueous solvents. The organic and aqueous extracts were fraction-collected using the Agilent 1260 Analytical Scale Fraction Collection System. Each of the fractions was analyzed on Liquid Chromatogaphy/Quadrupole Time-of-Flight LC/Q-TOF and Gas Chromatography/Quadrupole Time-of-Flight GC/instruments. The Liquid Chromatography/Mass Spectrometry (LC/MS) analyses were performed using Hydrophilic Ineraction Liquid Chromatography (HILIC), as well as reversed-phase chromatography using three separate, orthogonal reverse phase columns. Samples were analyzed using an Agilent Jet Stream (AJS) source in both positive and negative ionization modes. The compounds found were flavonoids, fatty acids, sugars, and terpenes. Eighty-one secondary metabolites were identified as having therapeutic potential. The data produced was against the METLIN database using accurate mass and/or MS/MS library matching. Compounds from Alangium that could not be identified by database or library matching were subsequently searched against the ChemSpider) database of over 30 million structures using MSMS data and Agilent MSC software.In order to identify compounds generated by GC/MS, the data were searched against the AgilentFiehn GCMS Metabolomics Library as well as the Wiley/NIST libraries

Discrete and combined effects of Ylang-Ylang (Cananga odorata) essential oil and gamma irradiation on growth and mycotoxins production by Fusarium graminearum in maize

In the present study, discrete and combined inhibitory effects of Cananga odorata essential oil (COEO) and irradiation were established on growth and production of deoxynivalenol (DON) and zearalenone (ZEA) by Fusarium graminearum in maize kernels. Chemical profile of COEO was characterized by GC-MS and a total of 35 chemical constituents were identified, and major compounds were linalool (29.15), germacrene-D (11.82), and thymol (8.45). The COEO and irradiation have inhibited the fungal growth and mycotoxins at 3.9 mg/g and 7.5 kGy, respectively. Distinct inhibitory activity of COEO and irradiation on fungal growth and mycotoxins was assessed by constructing linear regression curves. Regression models of COEO and irradiation have presented good coefficients of determination (R2) of 0.9886 and 0.9798 for fungal growth (log CFU), 0.97 and 0.9622 for DON, and 0.9811 and 0.9807 for ZEA, respectively. The linear regression models concluded that COEO and irradiation have dose-dependent inhibitory effect on fungal growth and mycotoxins. Further, combined inhibitory effect of COEO and irradiation on fungal growth and mycotoxins was assessed by checkerboard method. The combined treatment of COEO and irradiation was too shown decent coefficients of determination (R2) and found greatly effective. The combined treatments of COEO and irradiation have inhibited the fungal growth and mycotoxins in stored maize kernels much below than their discrete inhibitory levels, and it was noticed at 2.5 mg/g of COEO and 4 kGy of irradiation. The study concluded that combination of essential oil and irradiation could be highly efficient decontamination technique to diminish the fungal growth and mycotoxins in agricultural commodities

A novel igy-aptamer hybrid system for cost-effective detection of seb and its evaluation on food and clinical samples

In the present study, we introduce a novel hybrid sandwich-ALISA employing chicken IgY and ssDNA aptamers for the detection of staphylococcal enterotoxin B (SEB). Cloning, expression and purification of the full length recombinant SEB was carried out. Anti-SEB IgY antibodies generated by immunizing white leg-horn chickens with purified recombinant SEB protein and were purified from the immunized egg yolk. Simultaneously, ssDNA aptamers specific to the toxin were prepared by SELEX method on microtiter well plates. The sensitivity levels of both probe molecules i.e., IgY and ssDNA aptamers were evaluated. We observed that the aptamer at 250 ngmL(-1) concentration could detect the target antigen at 50 ngmL(-1) and the IgY antibodies at 250 ngmL(-1), could able to detect 100 ngmL(-1) antigen. We further combined both the probes to prepare a hybrid sandwich aptamer linked immune sorbent assay (ALISA) wherein the IgY as capturing molecule and biotinylated aptamer as revealing probe. Limit of detection (LOD) for the developed method was determined as 50 ngmL(-1). Further, developed method was evaluated with artificially SEB spiked milk and natural samples and obtained results were validated with PCR. In conclusion, developed ALISA method may provide cost-effective and robust detection of SEB from food and environmental samples

A novel igy-aptamer hybrid system for cost-effective detection of seb and its evaluation on food and clinical samples

In the present study, we introduce a novel hybrid sandwich-ALISA employing chicken IgY and ssDNA aptamers for the detection of staphylococcal enterotoxin B (SEB). Cloning, expression and purification of the full length recombinant SEB was carried out. Anti-SEB IgY antibodies generated by immunizing white leg-horn chickens with purified recombinant SEB protein and were purified from the immunized egg yolk. Simultaneously, ssDNA aptamers specific to the toxin were prepared by SELEX method on microtiter well plates. The sensitivity levels of both probe molecules i.e., IgY and ssDNA aptamers were evaluated. We observed that the aptamer at 250 ngmL(-1) concentration could detect the target antigen at 50 ngmL(-1) and the IgY antibodies at 250 ngmL(-1), could able to detect 100 ngmL(-1) antigen. We further combined both the probes to prepare a hybrid sandwich aptamer linked immune sorbent assay (ALISA) wherein the IgY as capturing molecule and biotinylated aptamer as revealing probe. Limit of detection (LOD) for the developed method was determined as 50 ngmL(-1). Further, developed method was evaluated with artificially SEB spiked milk and natural samples and obtained results were validated with PCR. In conclusion, developed ALISA method may provide cost-effective and robust detection of SEB from food and environmental samples

Phylogenetic tree of Macrolepiota bharadwajii

Phylogenetic tree of Macrolepiota bharadwajii constructed using MEGA v. X (Kumar et al. 2018) of the ITS-nrDNA sequence alignment by Neighbour-Joining Method (Saitou & Nei 1987). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1 000 replicates) are shown next to the branches (Felsenstein 1985). The evolutionary distances were computed using the Kimura 2-parameter method (Kimura, 1980) and are in the units of the number of base substitutions per site. This analysis involved 27 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were 811 positions in the final dataset. The phylogenetic position of Macrolepiota bharadwajii is indicated in bold.</p

Application of Activated Carbon Derived from Seed Shells of Jatropha curcas for Decontamination of Zearalenone Mycotoxin

In the present study, activated carbon (AC) was derived from seed shells of Jatropha curcas and applied to decontaminate the zearalenone (ZEA) mycotoxin. The AC of J. curcas (ACJC) was prepared by ZnCl2 chemical activation method and its crystalline structure was determined by X-ray diffraction analysis. The crystalline graphitic nature of ACJC was confirmed from the Raman spectroscopy. Scanning electron microscope showed the porous surface morphology of the ACJC surface with high pore density and presence of elemental carbon was identified from the energy dispersive X-ray analysis. From Brunauer–Emmett–Teller (BET) analysis, SBET, micropore area, and average pore diameter of ACJC were calculated as 822.78 (m2/g), 255.36 (m2/g), and 8.5980 (Å), respectively. The adsorption of ZEA by ACJC was accomplished with varying contact time, concentration of ZEA and ACJC, and pH of media. The ACJC has adsorbed the ZEA over a short period of time and adsorption of ZEA was dependent on the dose of ACJC. The effect of different pH on adsorption of ZEA by ACJC was not much effective. Desorption studies confirmed that adsorption of ZEA by ACJC was stable. The adsorption isotherm of ZEA by ACJC was well fitted with Langmuir model rather than Freundlich and concluded the homogeneous process of sorption. The maximum adsorption of ZEA by ACJC was detected as 23.14 μg/mg. Finally, adsorption property of ACJC was utilized to establish ACJC as an antidote against ZEA-induced toxicity under in vitro in neuro-2a cells. The percentage of live cells was high in cells treated together with a combination of ZEA and ACJC compared to ZEA treated cells. In a similar way, ΔΨM was not dropped in cells exposed to combination of ACJC and ZEA compared to ZEA treated cells. Furthermore, cells treated with a combination of ZEA and ACJC exhibited lower level of intracellular reactive oxygen species and caspase-3 compared to ZEA treated cells. These in vitro studies concluded that ACJC has successfully protected the cells from ZEA-induced toxicity by lowering the availability of ZEA in media as a result of adsorption of ZEA. The study concluded that ACJC was a potent decontaminating agent for ZEA and could be used as an antidote against ZEA-induced toxicity

Table_1_Combinational Inhibitory Action of Hedychium spicatum L. Essential Oil and γ-Radiation on Growth Rate and Mycotoxins Content of Fusarium graminearum in Maize: Response Surface Methodology.docx

<p>Nowadays, contamination of agricultural commodities with fungi and their mycotoxins is one of the most annoying with regard to food safety and pose serious health risk. Therefore, there is a requisite to propose suitable mitigation strategies to reduce the contamination of fungi and mycotoxins in agricultural commodities. In the present study, combinational inhibitory effect of Hedychium spicatum L. essential oil (HSEO) and radiation was established on growth rate, production of deoxynivalenol (DON) and zearalenone (ZEA) by Fusarium graminearum in maize grains. The HSEO was obtained from rhizomes by hydrodistillation technique and chemical composition was revealed by GC-MS analysis. A total of 48 compounds were identified and major compounds were 1,8-cineole (23.15%), linalool (12.82%), and β-pinene (10.06%). The discrete treatments of HSEO and radiation were effective in reducing the fungal growth rate and mycotoxins content, and the complete reduction was noticed at 3.15 mg/g of HSEO and 6 kGy of radiation. Response surface methodology (RSM) was applied to evaluate the combinational inhibitory effect of HSEO and radiation treatments on fungal growth rate and mycotoxins content. A total of 13 experiments were designed with distinct doses of HSEO and radiation by central composite design (CCD) of Stat-Ease Design-Expert software. In combinational approach, complete reductions of fungal growth, DON, and ZEA content were noticed at 1.89 mg/g of HSEO and 4.12 kGy of radiation treatments. The optimized design concluded that combinational treatments of HSEO and radiation were much more effective in reducing the fungal growth and mycotoxins content compared to their discrete treatments (p < 0.05). Responses of the design were assessed by second-order polynomial regression analysis and found that quadratic model was well fitted. The optimized design has larger F-value and adequate precision, smaller p-value, decent regression coefficients (R²) and found statistically significant (p < 0.05). In addition, correlation matrix, normal plot residuals, Box-Cox, and actual vs. predicted plots were endorsed that optimized design was accurate and appropriate. The proposed combinational decontamination technique could be highly applicable in agriculture and food industry to safeguard the food and feed products from fungi and mycotoxins.</p

Antagonistic activity of ocimum sanctum l. essential oil on growth and zearalenone production by fusarium graminearum in maize grains

The present study was aimed to establish the antagonistic effects of Ocimum sanctum L. essential oil (OSEO) on growth and zearalenone (ZEA) production of Fusarium graminearum. GC-MS chemical profiling of OSEO revealed the existence of 43 compounds and the major compound was found to be eugenol (34.7%). DPPH free radical scavenging activity (IC50) of OSEO was determined to be 8.5 mu g/mL. Minimum inhibitory concentration and minimum fungicidal concentration of OSEO on graminearum were recorded as 1250 and 1800 mu g/mL, respectively. Scanning electron microscope observations showed significant micro morphological damage in OSEO exposed mycelia and spores compared to untreated control culture. Quantitative UHPLC studies revealed that OSEO negatively effected the production of ZEA; the concentration of toxin production was observed to be insignificant at 1500 mu g/mL concentration of OSEO. On other hand ZEA concentration was quantified as 3.23 mu g/mL in OSEO untreated control culture. Reverse transcriptase qPCR analysis of ZEA metabolic pathway genes (PKS4 and PKS13) revealed that increase in OSEO concentration (250-1500 mu g/mL) significantly downregulated the expression of PKS4 and PKS13. These results were in agreement with the artificially contaminated maize grains as well. In conlusion, the antifungal and antimycotoxic effects of OSEO on F. graminearum in the present study reiterated that, the essential oil of O. sanctum could be a promising herbal fungicide in food processing industries as well as grain storage centers