Multipurpose exploitation of european forest resources: from wood and bark to bioactive compounds

The removal of knotwood from the chips prior to pulping and bleaching in pulp mills can provide considerable benefits in terms of pulping and bleaching costs (chemical consumption), pulp and paper quality, as well as water and energy consumption and effluent qualities. The recovery of bark from sawmills and pulp mills can also be considered. As TMP and CTMP mills are mostly concerned by the knot wood removal and bark recovery, an estimation of the benefit during mechanical pulping process has been made, based on a process data and quality of the raw materials and end-products, from a pulp mill. The economy and reliability of the exploitation of wood wastes have also been studied. Thanks to CTP and AAU own research know-how, it has been emphasised that one of the main benefits of removing knotwood from chips in TMP or CTMP mills are better pulp strength and brighter pulp with less dark specks. The separated knots is a rich raw material for extraction of lignans and other polyphenols., depending on the tree species. The knotwood separation process was tested in large laboratory scale on over-sized chips of Abies alba obtained from the pulp and paper mill. The knotwood was enriched to a high degree in the fractions, giving knotwood material with a lignan content of 5-9% (50-90 kg/ton). Theoretically and considering that the studied pulp mill burns 1250 m3/year (1 100 T/year) of Abies alba knots, 6 tons of lignans could be extracted per year. These results are promising and production of lignans from Abies alba should be explored further. Bark wastes and especially from poplar, common oak, fir and pines are materials rich in flavanoids and other phenolic compounds. A large-scale study was carried out with industrial barks of populus deltoides supplied by a French pulp mill. It was shown that populus deltoides bark contained 1% (10 kg/ton) of two strong anti-oxidants. In addition, specific phenolic compounds responsible for anti-adhesive activities were also quantified in poplar barks. Theoretically and considering that the studied pulp mill releases 7 000 tons/year of bark wastes, 70 tons of flavanoids could be extracted per year. Barks are therefore interesting wastes for their high contact in phenolic compounds, which could reasonably well exploited in the future, without sorting.

The antioxidative effect of wood extractives was first established by the evaluation of the capacity of extractive compounds for inhibition of lipid peroxidation (PEROX-test) and peroxyl radical scavenging (TRAP-test) in vitro. In both tests, the detection of free radicals is based on sensitive cheluminescent techniques. Validity of the assays has been tested by various structurally in related antioxidant and peroxyl radical scavenging molecules. An evaluation of the inhibition of lipid peroxidation has been carried out on the first ethanol crude extracts.32 samples of wood extractives have been tested and the different test samples showed the antioxidative effect, even though at different levels based on the nature of the samples.During a first series of test, Pinus pinaster knotwood and Pinus nigra knotwood were demonstrated as the most potent samples. However, in a second series of tests, Larix decidua and Abies alba knotwood both showed strong lipid peroxidation preventing and peroxyl radical scavenging potentialSix properties of the extracts were determined (which will be detailed in other results' description):- inhibition of lipid peroxidation- scavenging of peroxyl radicals- Inhibition of oxidation of low density lipoprotein (LDL) lipids- Inhibition of myeloperoxidase activity- inhibition of oxidative burst- effects on DNA oxidabilityBiological effects of wood extractives were studied by a carefully selected repertoire of biotests. In some cases the potency of wood extractives even exceeded that of the reference compounds. Biological activity was found in samples from most wood species, different parts of trees (knots, sapwood, bark) and in their water and ethanol extracts. In particular, various extracts of Larix decidua, Abies alba and Pinus nigra showed strong potential in several test systems.

There is an increasing need for the introduction of novel bioactive compounds to fight problems of biocide resistance by microbial strains. Large crude extracts and fractions of barks and knots from pulp wastes (pulp and paper industries) have been studied to detect the most effective anti-microbial extracts against micro-organisms and the conditions of their implementation to promote wood extractives as anti-microbial agents (bactericide, bacteriostatic, fungicide and fungistatic), slimicide and dispersing agent. The anti-microbial and anti-adhesive effects of crude extracts were two of the main expected results foreseen. It is based on an original protocol based on broth micro-dilution in order to screen the anti-microbial activity of crude extracts towards five non-pathogenic bacterial strains, which have been identified. In a first phase, 21 extracts have been tested against microorganisms to conclude that 5 extracts were shown to be the most potent ones: ethanol extracts of knots from Pinus nigra (black pine), Pinus pinaster (maritime pine), Populus deltoids (poplar), Eucalyptus globulus (eucalypt) and Qercus robur (oak). Finally, the most effective anti-microbial extracts or fraction as well as the most effective concentrations were determined for each bacteria. The most potent extracts were studied for anti-adhesive effect on 3 materials (stainless steel, PTFE and glass). Two samples of hexane and water extracts of barks (Eucalyptus globulus and Pinus pinaster) have demonstrated interesting effect toward bacterial adhesion.

The aim of these tests was to study how extractive compounds affect the ability of neutrophils and other cells of the host s defence to produce reactive oxygen species in vitro. Extractives have been tested for their ability to depress overactivity of the cellular defence functions. It is today well known that the microbicidical action of human neutrophils is due to the production of reactive oxygen species, hypochlorus acid and tyrosyl radical. Reactive species produced by the oxidative burst and myeloperoxidase activity are the ultimate damaging agents. It seems therefore logical that a direct way for attenuation of the damage due to overactivity would be effective inhibition of oxidative burst and myeloperoxydase activity. Three potent extracts inhibit ROS under the specified experimental conditions: - Populus deltoides knots and bark extracts, Altogether 36 samples of wood extracts were tested. The effects of wood extractives were compared to appropriate reference compounds, hydroxymatairesinol and nitecapone (a synthetic drug with strong myeloperoxidase inhibiting activity). Strongest of the wood extractives showed potency that was comparable to that of nitecapone and stronger than effect of hydroxymatairesinol.

Anti-tumorigenic properties have been screened with the in vitro test of compounds to regulate spontaneous apoptosis and proliferation in tumour cell battery and to modulate the sensitivity on physiologically induced apoptosis. Cell proliferation has been determined as cell population growth in the presence of varying concentrations of the test compound. The compounds that directly induce cell death and inhibit cancer cell proliferation would be useful for cancer therapeutics. Based on previous testing, three samples of wood extracts with strong antioxidant potential were selected for testing of the effects on DNA oxidability. Effects on oxidation of DNA were studied by ability of wood extractives either to cause oxidation or prevent UV-induced oxidation of calf thymus DNA in vitro. The dose and spectrum of UV-light was comparable to what European populations are exposed during a normal sunny summer day. DNA was isolated by conventional extraction procedures, and DNA oxidation measured by the relative amount of 8-oxo-deoxyguanosine. This test is regarded as an indication of the ability of test substances either to cause or prevent oxidation of DNA. Since the test is qualitative or, at best, only semi-quantitative in nature, comparison to potency of reference compounds is not scientifically justified. None of the studied samples caused DNA oxidation, and all of them showed distinct ability to prevent UV-induced DNA damage in vitro: Dead knotwood of Abies alba, living knotwood of Larix decidua and Pinus nigra knotwood.

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