Assessment of biological activity and UPLC–MS based chromatographic profiling of ethanolic extract of Ochradenus arabicus

Natural products from wild and medicinal plants, either in the form of crude extracts or pure compounds provide unlimited opportunities for new drug leads owing to the unmatched availability of chemical diversity. In the present study, the cytotoxic potential of crude ethanolic extract of Ochradenus arabicus was analyzed by MTT cell viability assay in MCF-7 adenocarcinoma breast cancer cells. We further investigated its effect against oxidative stress induced by anticancer drug doxorubicin. In addition, Ultra Performance Liquid Chromatography–Mass Spectrometry (UPLC–MS) based chromatographic profiling of crude extract of O. arabicus was performed. The MTT assay data showed that the extract is moderately toxic to the MCF-7 cells. However, its treatment alone does not induce oxidative stress while doxorubicin increases the level of oxidative stress in MCF-7 cells. Whereas, simultaneous treatment of plant extract and doxorubicin significantly (p < 0.05) decreased the level of intracellular reactive oxygen species (ROS) and lipid peroxidation while an increase in the reduced glutathione and superoxide dismutase activity was observed in time and dose dependent manner. Hence, our finding confirmed cytotoxic and antioxidant potential of crude extract of O. arabicus in MCF-7 cells. However, further investigations on O. arabicus as a potential chemotherapeutic agent are needed. The analysis of bioactive compounds present in the plant extracts involving the applications of common phytochemical screening assays such as chromatographic techniques is discussed.     

Embryo apoptosis identification: Oocyte grade or cleavage stage?

Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced.Abbreviations: ART, assisted reproductive technologies; BO, Brackett and Oliphant; BSA, bovine serum albumin; CaI, calcium ionophore; CC, cumulus cells; COC, cumulus–oocyte complex; CO₂, carbon dioxide; CR1aa, Charles Rosenkran’s 1 amino acid; DNA, deoxyribonucleic acid; DO, denuded oocyte; EA, early apoptosis; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; FSH, follicle stimulating hormone; GSH, glutathione; hpi, hours post insemination; IVC, in vitro culture; IVF, in vitro fertilization; IVM, in vitro maturation; IVP, in vitro produced; LA, late apoptosis; LH, luteinizing hormone; PBS, phosphate buffered saline; PI, propidium iodide; PS, phosphatidylserine; TUNEL, terminal deoxynucleotidyl transfer-mediated dUTP nick end-labeling.     

Thermotolerance and molecular chaperone function of an SGT1-like protein from the psychrophilic yeast, <Emphasis Type="Italic">Glaciozyma antarctica</Emphasis>

The ability of eukaryotes to adapt to an extreme range of temperatures is critically important for survival. Although adaptation to extreme high temperatures is well understood, reflecting the action of molecular chaperones, it is unclear whether these molecules play a role in survival at extremely low temperatures. The recent genome sequencing of the yeast Glaciozyma antarctica, isolated from Antarctic sea ice near Casey Station, provides an opportunity to investigate the role of molecular chaperones in adaptation to cold temperatures. We isolated a G. antarctica homologue of small heat shock protein 20 (HSP20), GaSGT1, and observed that the GaSGT1 mRNA expression in G. antarctica was markedly increased following culture exposure at low temperatures. Additionally, we demonstrated that GaSGT1 overexpression in Escherichia coli protected these bacteria from exposure to both high and low temperatures, which are lethal for growth. The recombinant GaSGT1 retained up to 60 % of its native luciferase activity after exposure to luciferase-denaturing temperatures. These results suggest that GaSGT1 promotes cell thermotolerance and employs molecular chaperone-like activity toward temperature assaults.     

Pre-harvest aflatoxins and <Emphasis Type="Italic">Aspergillus flavus</Emphasis> contamination in variable germplasms of red chillies from Kunri,Pakistan

Various cultivars of red chilli were collected from a small town named Kunri, located in the province Sindh, Pakistan. This town is a hub of red chilli production in Asia. A total of 69 samples belonging to 6 cultivars were obtained and analysed for the occurrence of aflatoxins and Aspergillus flavus, to explore the potential of resistant and susceptible germplasm. Aflatoxins were detected by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), while A. flavus was isolated and identified using agar plate, blotter paper, deep freezing and dilution techniques. Molecular characterization using internal transcribed spacer (ITS) 1/4 and A. flavus specific FL1-F/R primers confirmed the identity of A. flavus. The data revealed that 67 and 75% samples contaminated with aflatoxin B₁ (AFB₁) and with A. flavus, respectively. A highly susceptible chilli cultivar was ‘Nagina’, showing 78.8% frequency of total aflatoxins (1.2–600 μg/kg) and a mean of 87.7 μg/kg for AFB₁ and 121.9 μg/kg for total aflatoxins. A. flavus was detected with 93% frequency and 2.14 × 10⁴ colony forming units. In contrast, cultivars ‘Kunri’ and ‘Drooping Type’ were found to be resistant, with low levels of aflatoxins and fungal counts. The study was conducted for the first time to explore two potential cultivars that were less susceptible towards A. flavus and aflatoxin contamination. These cultivars could be preferably cultivated and thereby boost Pakistan’s chilli production.     

Extraction of microalgae derived lipids with supercritical carbon dioxide in an industrial relevant pilot plant

Microalgae are capable of producing up to 70% w/w triglycerides with respect to their dry cell weight. Since microalgae utilize the greenhouse gas CO₂, they can be cultivated on marginal lands and grow up to ten times faster than terrestrial plants, the generation of algae oils is a promising option for the development of sustainable bioprocesses, that are of interest for the chemical lubricant, cosmetic and food industry. For the first time we have carried out the optimization of supercritical carbon dioxide (SCCO₂) mediated lipid extraction from biomass of the microalgae Scenedesmus obliquus and Scenedesmus obtusiusculus under industrrially relevant conditions. All experiments were carried out in an industrial pilot plant setting, according to current ATEX directives, with batch sizes up to 1.3 kg. Different combinations of pressure (7–80 MPa), temperature (20–200 °C) and CO₂ to biomass ratio (20–200) have been tested on the dried biomass. The most efficient conditions were found to be 12 MPa pressure, a temperature of 20 °C and a CO₂ to biomass ratio of 100, resulting in a high extraction efficiency of up to 92%. Since the optimized CO₂ extraction still yields a crude triglyceride product that contains various algae derived contaminants, such as chlorophyll and carotenoids, a very effective and scalable purification procedure, based on cost efficient bentonite based adsorbers, was devised. In addition to the sequential extraction and purification procedure, we present a consolidated online-bleaching procedure for algae derived oils that is realized within the supercritical CO₂ extraction plant.