The second Newsletter for the research project called “Condensed tannins from Norwegian pine and spruce bark - antiparasitic effects and potential commercial exploitation

Aasen, Inga Marie

Athanasiadou, Spiridoula

Blomstrand, Berit Marie

Enemark, Heidi

Mahnert, Karl-Christian

Ptochos, Sokratis

Steinshamn, Håvard

Sørheim, Kristin

Woolsey, Ian

English

Organic Eprints

Dear readerWe are now one and a half years into the BarkCure project and we are on the track. All the bark material that we are to use in the project is now collected from the sawmills and in storage. We have six mas-ter batches all together, two from pine and four from spruce. Condensed tannins have been extracted from all master batches using three different types of solvents. The extracts are currently being used in different types of laboratory tests to evaluate their efficacy against gastro-intestinal parasites. Preliminary results indicate that there are promising effects, and our Newsletter no.2PhD student Berit Marie Blomstrand presen-ted some of these results at an international conference in Spain this autumn. Based on results from the ongoing laboratory tests, we will scale up the extraction of the most promising extracts this coming spring. These extracts will be tested in sheep infected with gastro-intestinal nematodes. This animal experiment will be run at Scotland’s Rural College, one of our partners in Edinburgh next summer. We also communicate our research activities and results on our project web page www.barkcure.noBest regardsHåvard SteinshamnProject leaderDecember 2018     www.barkcure.no   Project facts:  BarkCure is funded by the Norwegian Research Council through the BIONÆR programme.NOK 11 mill. fundingProject period: June 2017-October 2020.Project owner: Norwegian Centre of  Organic Agriculture (NORSØK)Project leader: Håvard Steinshamn, NIBIOPage 1In front: Kristin Sørheim, NORSØK, Sokratis Ptochos, SRUC, Berit  Marie Blomstrand, NORSØK, Spiridoula Athanasiadou, SRUC. Back: Håvard Steinshamn, NIBIO, Ian Woolsey, Veterinærins-tituttet, Karl-Christian Mahnert, Norsk Treteknisk Institutt, Stig Milan Thamsborg, UCPH, Heidi Enemark, Veterinærinstituttet, Inga Marie Aasen, SINTEF. Photo: Anita Land  www.barkcure.no                                                                                                                                                                                                       December 2018The Norwegian Institute of Wood Technolo-gy performs the debarking process creating the value chain. One important question is what would be the minimum price per ton of bark that the industry partners have to realize for production of condensed tannins (CT) in bark to be competitive in comparison to the utilization of bark today (gardening products, energetic recovery). This will be investigated by interviewing key personnel and industrial partners.Value chain bark/ selection of bark materialKarl-Christian Mahnert karl.mahnert@treteknisk.noPage 2Figure 1. Moisture content and wood percentageDrum debarking (Photo: logprollc.com) Rotary debarking (Photo: http://www.directindustry.com)In 2018, the task has been to estimate the wood percentage and moisture content in the bark material produced during the previous summer and winter. Preliminary results indicate a lower moisture content in the bark collected last winter (no water spray) and a lower wood percentage in the summer bark (exception Master Batch II: intermittent production) (figure 1).Mixing of master batch. Photo: Karl-Christian Mahnert  www.barkcure.no                                                                                                                                                                                                       December 2018For analyses and characterisation of CTs, the bark was dried and milled before extraction with water and water-miscible solvents. As expected, higher CT-yields were obtai-ned when solvents containing methanol or acetone were used, compared to water only. However, the yield in water is of interest with respect to potential industrial produc-tion, as water extraction will be cheaper than using solvents.Quantitative analyses were performed using spectrophotometric assays and chroma-tographic methods, after degradation of the complex polymers to monomers. The overall CT-yields were highest from spruce bark, however, the CT-content of the dried extracts was highest from pine, which is in accordance with preliminary results from in vitro assays (see below). For spruce, the bark from rotary-debarking had higher CT-content than the bark from drum-debar-king. Extracts from the summer batch (9 extracts) for in vitro evaluation were delivered in January 2018, while extracts from the winter batch were ready in June 2018. Further characterisation of the CT-extracts by chromatographic methods and mass spectrometry (LC-MS) is on-going and indi-cates different ratios between the two main classes of monomers in spruce and pine.Chemical characterization, isolation and fractionation of bark CTExtracts of CT have been prepared from bark ‘Master batches’ collected by The Norwegian Institute of Wood Technology. The summer-batches were received in August 2017, and the winter-batches in April 2018. Inga.M.Aasen@sintef.noCharacterisation of CT-extracts, Janne Beate Øiaas Page 3Inga Marie AasenPhoto: Berit Marie BlomstrandIsolation and fractionation of bark CT’s Spruce, drum debarkingPhotos: Janne Beate ØiaasPine, rotary debarking Spruce, rotray debraking  www.barkcure.no                                                                                                                                                                                                       December 2018                            So far, nine bark extracts from the summer batch (sampled during autumn 2017) were tested (spring 2018). The nine bark extracts from the winter batch (sampled in March-April 2018) are now being tested. Efficacy against Teladorsagia circumcincta has been assessed with an egg hatch test and a larval motility test with a real time cell analyser. The same methods will be applied to test the extracts against Trichostrongylus colubri-formis (planned February 2019).Preliminary results show that pine extracts have high efficacy independent of extraction method, with the acetone extract giving the highest egg hatch inhibition (EH% <10). Water and methanol extracts of spruce had little or no effect (EH% >90) (figure 2).Preliminary results from the larval motility test with real time cell analysis show that methanol and acetone pine extracts de-monstrated a reduction in larval motility in all concentrations. Water extracts and methanol pine 1 extract showed motility reducing efficacy in the two highest concen-trations.Preliminary conclusion: CT extracted from Pine bark, by means of methanol and acetone, demonstrated the highest anthelmintic efficacy in both methods tested. There seems to be a positive correlation between anthelmintic efficacy and the CT content of the extracts.Biological activity of bark CT extracts against sheep nematodesIn 2018 we started to test the biological efficacy of bark CT extracts on selected ovine nematodes in vitro. Teladorsagia cicumcincta larva. Photo: Berit M. Blomstrandberit.blomstrand@norsok.noPage 4Sokratis Ptochossokratis.ptochos@sruc.ac.ukBerit Marie BlomstrandSpiridoula Athanasiadouspiridoula.athanasia-dou@sruc.ac.ukBerit M. Blomstrand: - Lab work at SRUC. Photo: Sokratis PtochosFigure 2. Two extracts influence on nematode egg hatch (EH%)The extracts from both the summer and the winter batch will be tested on different life cycles  stages of cattle nematodes, and we will use different methods for the different stages. The life cycle of gastro-intestinal ne-matode is illustrated in figure 3, where the different stages, L1-L5, are indicated.These methods used will be Larval feeding inhibition assay (L1), Larval migration assay (L3) and Egg hatch assay (egg-L1). These tests are due ultimo January 2019. This autumn we have assessed the in vitro anti-protozoal efficacy of all the 18 bark extracts on Cryptosporidium parvum, a protozon causing diarrhea in both humans and animals.  We have tested the ability of the extract to inhibit the intracellular growth of C. parvum (figure 4). Thus far the results indicate that the acetone and methanol pine extracts show a promising efficacy, while the water extracts show no growth inhibition.Page 5  www.barkcure.no                                                                                                                                                                                                       December 2018Ian Woolsey ian.woolsey@vetinst.noBiological activity of bark CT extracts against cattle nematodes and sheep protozoaThe plan for next summer is to test in vitro anthelmintic efficacy of selected bark extract against cattle nematodes and antiprotozoal efficacy against sheep Eimeria spp field isola-tes.  Furthermore, we will collect Eimeria spp field isolates and purify them. Thereafter, the cell cultures will be infected, and diffe-rent extracts will be tested for their ability to inhibit sporozoite invasion into, and growth inhibition of Eimeria in the cells. The results from the in vitro tests of the bark extracts will determine the experimental design for assessment in sheep, which will take place in Scotland in July 2019.Figure 3. Life cycle of gastro intestinal nematodes. Ill: Anne de BoerFigure 4. Life cycle of Cryptosporidium parvum (www.pixsharp.com)Heidi Enemarkheidi.enemark@vetinst.noberit.blomstrand@norsok.noBerit Marie BlomstrandNovember 2017Project owner contact:Kristin Sørheim, NORSØKGunnars veg 66630 Tingvollkristin.sorheim@norsok.noResearch partners: Industry Partners: Project coordination:Håvard Steinshamn, NIBIOGunnars veg 66630 Tingvollhavard.steinshamn@nibio.no     www.barkcure.no                                                                                                                                                                                                       Dec mber 2018

Newsletter Barkcure No. 2

https://orgprints.org/34240/2/Barkcure%20Des%202018%20%20trykk%2013%20des%202018.pdf

Newsletter Barkcure No. 2

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