Rama Addepalli

In vitro Anti-Thrombotic Activity of Extracts from Blacklip Abalone (Haliotis rubra) Processing Waste

Waste generated from the processing of marine organisms for food represents an underutilized resource that has the potential to provide bioactive molecules with pharmaceutical applications. Some of these molecules have known anti-thrombotic and anti-coagulant activities and are being investigated as alternatives to common anti-thrombotic drugs, like heparin and warfarin that have serious side effects. In the current study, extracts prepared from blacklip abalone (Haliotis rubra) processing waste, using food grade enzymes papain and bromelain, were found to contain sulphated polysaccharide with anti-thrombotic activity. Extracts were found to be enriched with sulphated polysaccharides and assessed for anti-thrombotic activity in vitro through heparin cofactor-II (HCII)-mediated inhibition of thrombin. More than 60% thrombin inhibition was observed in response to 100 μg/mL sulphated polysaccharides. Anti-thrombotic potential was further assessed as anti-coagulant activity in plasma and blood, using prothrombin time (PT), activated partial thromboplastin time (aPTT), and thromboelastography (TEG). All abalone extracts had significant activity compared with saline control. Anion exchange chromatography was used to separate extracts into fractions with enhanced anti-thrombotic activity, improving HCII-mediated thrombin inhibition, PT and aPTT almost 2-fold. Overall this study identifies an alternative source of anti-thrombotic molecules that can be easily processed offering alternatives to current anti-thrombotic agents like heparin.

In vitro anti-inflammatory activities of blacklip abalone (Haliotis rubra) in RAW 264.7 macrophages

ABSTRACT Abalone (Haliotis sp) has been used as a traditional functional food for many years by different cultures believing that consumption provides health benefits. We investigated the anti-inflammatory activity from blacklip abalone visceral waste. An extract was prepared from the viscera using digestion with the food grade proteases papain and bromelain followed by anion exchange chromatography (AEC). The anti-inflammatory potential of the extract and AEC fractions were investigated in comparison to a plant-derived positive control (quercetin) through the inhibition of lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 monocytes. Significant anti-inflammatory activity was observed in response to the abalone extract and the unbound AEC material over a concentration (based on collagen) range of 12–96 µg/mL. Overall, results indicated that blacklip abalone extract has anti-inflammatory activity in vitro, warranting further investigation into the bioactive constituents that may be potential anti-inflammatory therapeutics.

Biological fate of food nanoemulsions and the nutrients they carry – internalisation, transport and cytotoxicity of edible nanoemulsions in Caco-2 intestinal cells

Edible nanoemulsions are promising delivery systems with the potential to enhance nutrient/drug solubilisation, digestibility, bioavailability and potentially facilitate direct cellular uptake. However, the high potential of edible nanoparticles has also led to concerns about their biological fate and whether these nanoparticles or the active ingredients they carry pose (new) toxicological risks. Here we outline the development of new sub 50 nm edible nanoemulsions that allow us to probe the duality of enhanced nutrient solubilisation and bioavailability with potential toxicological side effects. The toxicity and biological fate of the edible nanoemulsions was investigated using Caco-2 cells to facilitate cell viability assays, transport of nanoemulsions across an in vitro intestinal model and internalisation visualised by confocal microscopy. These experiments demonstrate that edible nanoemulsion toxicity is not just a function of surfactant composition, but more critically a synergistic effect between surfactants and their physical location. Critically the presence of reactive ingredients (β-carotene) leads to a dramatic increase in nanoemulsion toxicity that may counteract the benefits associated with enhanced solubilisation/cellular uptake. Such research into the biological fate of edible food nanoemulsions and the nutrients they carry is important not only because nanotechnology in food is an emotive topic, but also because these insights may inform public policy decisions.

Anti-coagulant and anti-thrombotic properties of blacklip abalone (Haliotis rubra): in vitro and animal studies

Sulphated polysaccharides with anti-thrombotic and anti-coagulant activities have been found in various marine biota. In this study, a previously characterised anti-thrombotic and anti-coagulant extract from blacklip abalone was fractionated by anion exchange chromatography (AEC), pooled (on a sulphated polysaccharide basis) and administered to Wistar rats via oral gavage (N = 8) for assessment as an oral therapeutic. To ensure that the preparation had anti-coagulant activity prior to oral administration, it was assessed in rat blood by thromboelastography (TEG) significantly increasing reaction (R) time (or time until clot formation). Following in vitro confirmation of anti-coagulant activity, 40 mg of the preparation was orally administered to rats with blood samples collected at 2, 4, and 6 h post-gavage. Assessment of all blood samples by TEG showed some prolongation of R time from 355 to 380 s after 4 h. Dosing of the post-gavage blood samples with the abalone preparation to confirm anti-thrombotic activity in vitro revealed residual anti-coagulant activity, further suggesting that oral administration did increase anti-coagulant potential in the collected blood but that bioavailability was low. Assessment of tissues and haematological parameters showed no obvious harmful effects of the abalone preparation in animals. In summary, even though oral administration of fractionated and pooled blacklip abalone extract to rats delayed clotting after 4 h, bioavailability of the preparation appeared to be low and may be more appropriate for intravenous administration as an anti-thrombotic or anti-coagulant therapeutic.

Indospicine cytotoxicity and transport in human cell lines

Indospicine, a non-proteinogenic analogue of arginine, occurs only in Indigofera plant species and accumulates in the tissues of animals grazing on Indigofera. Canine deaths have resulted from the consumption of indospicine-contaminated meat but only limited information is available regarding indospicine toxicity in humans. In this study three human cell lines, Caco-2 (colorectal adenocarcinoma), HT29-MTX-E12 (colorectal adenocarcinoma) and HepG2 (hepatocellular carcinoma), were used to investigate the cytotoxicity of indospicine and its metabolite 2-aminopimelic acid in comparison to arginine. Indospicine and 2-aminopimelic acid were more cytotoxic than arginine, displaying the highest toxicity in HepG2 liver cells. Intestinal transport in vitro also revealed a 2-fold higher transport rate of indospicine compared to arginine. The sensitivity of HepG2 cells to indospicine is consistent with observed canine hepatotoxicity, and considering the higher in vitro transport of indospicine across an intestinal barrier, it is possible that similar ill effects could be seen in humans consuming contaminated meat.