Optimisation of a fluorimetric assay for the screening of potential alpha-glucosidase inhibitors in coloured plant extracts and foods

The frequently used method for the in vitro screening of inhibitors of yeast α-glucosidase is based on the hydrolysis of p-nitrophenol-α-D-glucopyranoside to yield p-nitrophenol and glucose. After adjusting to pH 8 to form the yellow nitrophenolate anion, the absorbance at 405 nm is measured [1,2]. During our investigations into potential α-glucosidase inhibitors from sugarcane products, we found a decrease in sensitivity when highly coloured extracts were assayed. Therefore, an optimised method of the α-glucosidase assay was developed, using the fluorogenic substrate 4-methylumbelliferyl-α-D-glucopyranoside (4-MUG), to ensure a more reproducible and reliable screening tool for highly coloured plant extracts and foods. The optimised conditions for the assay were determined: α-glucosidase (2mU/mL), 4-MUG (84µM), incubation time (20 minutes), incubation temperature (37°C) and assay buffer pH (5.5). A molasses extract was screened for α-glucosidase inhibition using the optimised conditions with 80% inhibition seen at 150µg/mL and no negative quenching effects at concentrations within the linear range of the instrument. The optimised assay was also used to determine IC50 values for acarbose and fucoidan. The results suggest that acarbose does inhibit yeast α-glucosidase, which contradicts earlier work [3]. Overall, this optimised assay will be a valuable tool for the screening of highly coloured plant extracts and foods for α-glucosidase activity

A comparative study of seed morphology in relation to desiccation tolerance and other physiological responses in 71 Eastern Australian Rainforest species

Seed characteristics were measured in 71 Eastern Australian rainforest species representing 30 families. Sensitivity to desiccation to low moisture contents (\u3c 10%) occurred in 42% of species. We estimate, based on findings from 100 species from this present study and previously published reports, that 49% of Eastern Australian rainforest species have non-orthodox seeds. Germination level and time to 50% germination were not significantly different between desiccation sensitive (DS) and desiccation tolerant (DT) seeds. The estimation of seed desiccation sensitivity based on predictors is an important tool underpinning ex situ conservation efforts. Seed characteristics differed significantly between DS and DT seeds; that is, DS seeds had: (i) larger fruits (19 949 mg vs 8322 mg); (ii) larger seeds (1663 mg vs 202 mg); (iii) higher seed moisture contents (49.7% vs 35.5% fresh weight); (iv) lower oil content (7.3% vs 24.8% yield); and (v) less investment in seed coats (0.19 vs 0.48 seed coat ratio). Only 25% of DS seeded species had oily seeds compared with 87% of DT seeded species. Most green embryos were DS. Seed coat ratio was the best predictor of seed DS (80% correctly predicted). Seed moisture content at maturity was also related to germination time. Mean seed size was correlated (−0.657, P = 0.01) with mean seed oil content in 46 species. Further research on seed storage physiology of possible oily and/or DS seeded species is crucial to ensure future long-term security of this biodiversity, particularly for species currently threatened in situ and/or of socioeconomic importance in Eastern Australian rainforests

Immunomodulatory Polysaccharide from Chlorophytum borivilianum Roots

Chlorophytum borivilianum Santapau & Fernandes (Liliaceae) is an ayurvedic Rasayana herb with immunostimulating properties. The polysaccharide fraction (CBP) derived from hot water extraction of C. borivilianum (CB), comprising of ~31% inulin-type fructans and ~25% acetylated mannans (of hot water-soluble extract), was evaluated for its effect on natural killer (NK) cell activity (in vitro). Human peripheral blood mononuclear cells (PBMCs), isolated from whole blood on a Ficoll-Hypaque density gradient, were tested in the presence or absence of varying concentrations of each C. borivilianum fraction for modulation of NK cell cytotoxic activity toward K562 cells. Preliminary cytotoxicity evaluation against P388 cells was performed to establish non-cytotoxic concentrations of the different fractions. Testing showed the observed significant stimulation of NK cell activity to be due to the CBP of C. borivilianum. Furthermore, in vivo evaluation carried out on Wistar strain albino rats for humoral response to sheep red blood cells (SRBCs) and immunoglobulin-level determination using enzyme-linked immunosorbent assay (ELISA), exhibited an effectiveness of C. borivilianum aqueous extract in improving immune function. Present results provide useful information for understanding the role of CBP in modulating immune function

Current Understanding of Acute Bovine Liver Disease in Australia

Acute bovine liver disease (ABLD) is a hepatotoxicity principally of cattle which occurs in southern regions of Australia. Severely affected animals undergo rapid clinical progression with mortalities often occurring prior to the recognition of clinical signs. Less severely affected animals develop photosensitization and a proportion can develop liver failure. The characteristic histopathological lesion in acute fatal cases is severe, with acute necrosis of periportal hepatocytes with hemorrhage into the necrotic areas. Currently there are a small number of toxins that are known to cause periportal necrosis in cattle, although none of these have so far been linked to ABLD. Furthermore, ABLD has frequently been associated with the presence of rough dog’s tail grass (Cynosurus echinatus) and Drechslera spp. fungi in the pasture system, but it is currently unknown if these are etiological factors. Much of the knowledge about ABLD is contained within case reports, with very little experimental research investigating the specific cause(s). This review provides an overview of the current and most recently published knowledge of ABLD. It also draws on wider research and unpublished reports to suggest possible fungi and mycotoxins that may give rise to ABLD