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.

A PPARγ ligand present in Actinidia fruit (Actinidia chrysantha) is identified as dilinolenoyl galactosyl glycerol.

Activity-guided fractionation of Actinidia fruit species, including Kiwifruit, has identified DLGG (dilinolenoyl galactosyl glycerol) as a potent PPARγ (peroxisome-proliferator-activated receptor γ) ligand. DLGG is a type of MGDG (monogalactosyl diacylglycerol) and is present in all chloroplasts, and therefore all green fruits and vegetables. PPARγ is a ligand-activated transcription factor that regulates glucose metabolism and inflammation. An ethyl acetate extract of Actinidia chrysantha was fractionated by HPLC and the PPARγ-binding activity was detected by FP (fluorescence polarization). Linoleic and α-linolenic acids in A. chrysantha were readily detected as PPARγ ligands. Slow-binding PPARγ ligands were detected in several hydrophobic fractions. High-resolution MS identified DLGG as one of these ligands and confirmed that its binding is non-covalent. DLGG is a slow-binding PPARγ ligand with an IC50 of 1.64 µM, ±0.093 achieved after 45 min equilibration. DLGG is the first example of a form of DAG (diacylglycerol) that is a PPARγ ligand. In addition, DLGG is the first reported glycolipid ligand for PPARγ and also the first non-covalent, slow-binding PPARγ ligand.

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.

Apple peels, from seven cultivars, have lipase-inhibitory activity and contain numerous ursenoic acids as identified by LC-ESI-QTOF-HRMS.

Apple peel contains numerous phytochemicals, many of which show bioactivity. This study investigated the identity of triterpenoid compounds contained in ethanolic extracts of peel from seven apple cultivars. Using HPLC-ESI-QTOF-HRMS, accurate mass information was obtained for 43 compounds, and chemical identity was inferred from the calculated elemental composition, fragment masses, ms/ms, and a limited set of authentic standards. Compounds were identified as triterpene acids and tentatively identified as ursenoic (or oleanoic) acid derivatives containing hydroxyl, oxo, and coumaroyloxy groups. These apple skin extracts exhibited lipase-inhibitory activity, which may be linked to the ursenoic acid content. Furthermore, both triterpene content and lipase-inhibitory activity varied by cultivar.