Actinidin diversity: discovery of common and selective substrates for actinidin isoforms and Actinidia cultivars.

The actinidin proteinase family has a striking sequence diversity; isoelectric points range from 3.9 to 9.3. The biological drive for this variation is thought to be actinidin's role as a defense-related protein. In this study we map mutations in the primary sequence onto the 3D structure of the protein and show that the region with the highest diversity is close to the substrate binding groove. Non-conservative substitutions in the active site determine substrate preference and therefore create problems for quantification of actinidin activity. Here we use a peptide substrate library to compare two actinidin isoforms, one from the kiwiberry cultivar 'Hortgem Tahi' (Actinidia arguta), and the other from the familiar kiwifruit cultivar 'Hayward' (Actinidia chinensis var. deliciosa). Among 360 octamer substrates we find one substrate (RVAAGSPI) with the useful property of being readily cleaved by all the functionally active actinidins in a set of A. arguta and A. chinensis var. deliciosa isoforms. In addition, we find that two substrates (LPPKSQPP & ILRDKDNT) have the ability to differentiate different isoforms from a single fruit. We compare actinidins from 'Hayward' and A. arguta for their ability to digest the allergenic gluten peptide (PFPQPQLPY) but find the peptide to be indigestible by all sources of actinidin. The ability to inactivate salivary amylase is shown to be a common trait in Actinidia cultivars due to proteolysis by actinidin and is particularly strong in 'Hortgem Tahi'. A mixture of 10% 'Hortgem Tahi' extract with 90% saliva inactivates 100% of amylase activity within 5 minutes. Conceivably, 'Hortgem Tahi' might lower the glycaemic response in a meal rich in cooked starch.

JAK2 and AMP-kinase inhibition in vitro by food extracts, fractions and purified phytochemicals.

We have identified a range of food phytochemicals that inhibit Janus Kinase 2 (JAK2) and Adenosine Monophosphate Kinase (AMPK). A mutated and dysregulated form of JAK2, a tyrosine kinase, is associated with several diseases including Crohn's disease. Using an in vitro, time-resolved fluorescence (TR-FRET) assay, we tested 49 different types of food extracts, plus 10 concentrated fractions of increasing hydrophobicity from each extract, to find foods containing JAK2 inhibitors. The food extracts tested included grains, meat, fish, shellfish, dairy products, herbs, mushrooms, hops, fruits and vegetables. Several fruits were potent inhibitors of JAK2: blackberry, boysenberry, feijoa, pomegranate, rosehip and strawberry, which all contain ellagitannins, known inhibitors of kinases. These fruits are in the Rosales and Myrtales plant orders. No other foods gave >1% of the maximal JAK2 inhibitory activities of these fruits. AMPK, a sensor and regulator of energy metabolism in cells, is a serine-threonine kinase which is reported to be activated by various flavonoid phytochemicals. Using a TR-FRET assay, we tested various fruit extracts for AMPK activation and inhibition. Ellagitannin containing foods scored highly as AMPK inhibitors. Despite several reports of AMPK activation in whole cells by phytochemicals, no extracts or pure compounds activated AMPK in our assay.

The combination of analytical-scale HPLC separation with a TR-FRET assay to investigate JAK2 inhibitory compounds in a Boysenberry drink.

We report the detection of JAK2 inhibitory activity in a Boysenberry (Rubus loganbaccus x R. baileyanus Britt.) drink using a combination of analytical-scale high performance liquid chromatography (HPLC) with a high-sensitivity time-resolved fluorescence coupled with fluorescence resonance energy transfer (TR-FRET) method. Phytochemical components of a Boysenberry drink were separated by reversed phase HPLC , and 84 separate fractions were collected. HPLC fractions corresponding to the ellagitannin and ellagic acid peaks observed in the chromatogram inhibited JAK2 activity. Anthocyanins, while they were the major phytochemical components of the Boysenberry drink, had no JAK2 inhibitory activity even though anthocyanins have previously been shown to be anti-inflammatory. This study demonstrates the usefulness of combining rapid analytical-scale HPLC separation with a highly sensitive fluorescence bioassay for characterising bioactivity in complex plant extracts. Ellagic acid was found to have an IC(50) of 92 nM against JAK2 and complete inhibition of JAK2 activity was observed in HPLC fractions of Boysenberry extract which had been diluted several hundred fold. To the best of our knowledge, this is the first demonstration that ellagitannins and other natural ellagic acid analogues are potent inhibitors of JAK2. Thus a drink containing Boysenberry juice concentrate may have anti-inflammatory properties.

Changes in markers of inflammation, antioxidant capacity and oxidative stress in smokers following consumption of milk, and milk supplemented with fruit and vegetable extracts and vitamin C.

Two milk-based beverages delivering twice the average daily antioxidant intake were formulated, based on synergistic combinations of fruit and vegetable extracts, and containing vitamin C (1.00 mg/ml) for shelf stability. Smokers (n = 42) consumed prototype milk A, B or non-supplemented milk (no extracts or vitamin C; 200 ml) twice daily for 6 weeks. Fasting and post-prandial (2 h after milk consumption) blood samples were collected at baseline and the end of each treatment. Non-supplemented milk significantly reduced fasting inflammatory cytokines (interleukin (IL) 6, IL-1ß, tumour necrosis factor-α) compared to baseline. Both supplemented milk-based beverages significantly increased fasting plasma vitamin C concentrations and antioxidant potential and decreased serum uric acid, compared to non-supplemented milk. The beverages did not induce post-prandial oxidative stress or inflammation. Therefore, regular consumption of the supplemented milks may confer health benefits because of increased antioxidant potential or through mechanisms resulting from increased vitamin C or decreased uric acid concentrations.

Demonstrating the safety of manuka honey UMF 20+in a human clinical trial with healthy individuals.

Honey is an established traditional medicine with a variety of putative nutritional and health effects, including antibacterial, antioxidant, anti-inflammatory and prebiotic. The aim of the present study was to investigate the safety of consuming manuka honey, UMF 20+, on healthy individuals by establishing whether UMF 20+caused an allergic response (as measured by IgE levels), changed major commensal and beneficial microbial groups in the gut and/or affected levels of one of the most common advanced glycation endpoints, N-(carboxymethyl)-lysine (CML). The study had a randomised, double-blind cross-over design. A total of twenty healthy individuals aged 42-64 years were recruited. We tested two different honeys- a multiflora honey and UMF 20+, both produced by Comvita New Zealand Ltd (Te Puke, New Zealand). Multiflora honey or UMF 20+(20 g) was consumed daily for 4 weeks, with a 2-week 'washout' period in between. Blood samples were collected every week for each intervention period and used to measure total IgE levels in serum and advanced glycation endproducts - a consequence of methyglyoxal accumulation. Faecal samples were collected at the beginning and end of each 4-week period. DNA was extracted from faecal samples and the levels of a number of microbial groups in the gut, both beneficial and commensal, were analysed. Neither product changed the levels of IgE or CML or altered gut microbial profiles during the trial, confirming that UMF 20+is safe for healthy individuals to consume. Despite anecdotal evidence suggesting that manuka honey is good for digestive health, we observed no beneficial effects on lower gut bacterial levels with either honey in this healthy population.

A fluorescence polarization assay to quantify biotin and biotin-binding proteins in whole plant extracts using Alexa-Fluor 594 biocytin.

A high-throughput fluorescence polarization assay has been developed for the detection of biotin and biotin-binding proteins in whole leaf extracts. Various groups are investigating the insecticidal properties of avidin and other biotin-binding proteins expressed in leaves of transgenic plants. The methods commonly used to quantify biotin and avidin in leaf extracts are enzyme-linked immunosorbent assay (ELISA) and Western blotting. Here we describe a homogeneous fluorescence polarization (FP) method that quantifies transgenic avidin in whole leaf extract by the simple addition of the fluorescent avidin ligand Alexa-Fluor 594 biocytin (AFB). The FP assay exploits the fact that AFB excites and emits in regions of the spectrum that are relatively free of background fluorescence in leaf extract. Transgenic leaf avidin can be quantified within 1-2 h by the FP method, in comparison with 1-2 days for ELISA and Western blotting. The FP method can also measure the amount of biotin in control leaves, not expressing avidin. Functional avidin levels of 1.54 microM (26.1 microg/g leaf tissue) were detected in tobacco leaves expressing vacuole-targeted avidin. Control leaves had biotin levels of around 0.74 microM (approximately 0.18 microg/g leaf tissue). Reagent costs are minimal: typically AFB is used at concentrations of 1-10 nM, avidin is used at 1-100 nM, and sample volumes are 20 microL in 384-well microplates.