Optimized extraction of Pinus taeda knotwood sawmill residues as source of antifungal compounds

Abstract

In Uruguay, Pinus taeda is the dominant conifer in industrial plantations, generating large volumes of knot-containing offcuts during processing. These residues are highly enriched in extractives, offering opportunities for valorization within a circular bioeconomy. This study examined the recovery of bioactive compounds from P. taeda knotwood through ethanol extraction, process optimization, Gas Chromatography coupled with Mass Spectrometry and Flame Ionization Detection (GC–MS/FID) analysis, and antifungal evaluation. The effects of extraction temperature, ethanol concentration, and liquid–solid (L/S) ratio on extraction yield, total phenolic content, and FRAP antioxidant activity were assessed, and response models were developed to identify optimal conditions. Maximum extraction yield (20.8%) occurred at 47 °C, 100% ethanol concentration, and L/S of 8.3. The highest phenolic content (5.7 g gallic acid equivalents/100 g) was predicted at 61 °C, 100% ethanol, and L/S 15, while the greatest antioxidant capacity (15.8 mmol ascorbic acid equivalents/100 g) was achieved at 75 °C and L/S 15, independently of ethanol concentration. Two extraction conditions, representing the best compromise among yield, phenolics, and antioxidant performance, were selected for further characterization and antifungal tests. GC–MS/FID analysis showed that stilbenes and terpenoids dominated the extracts. Antifungal assays against Trametes versicolor and Gloeophyllum trabeum revealed strong inhibition, reaching up to 65% and 97% after seven days. Overall, the results demonstrate that P. taeda knotwood residues can be efficiently valorised via ethanolic extraction to obtain bioactive fractions with high antioxidant and antifungal activity, supporting sustainable and circular approaches for wood protection.

Graphical abstract

Keywords

Pinus taeda / Knotwood / Phenolic compounds / Antioxidant activity / Antifungal / Circular bioeconomy / Wood preservation

References

[1] Abad MJ, Ansuategui M, Bermejo P (2007) Active antifungal substances from natural sources. Arkivoc 2007(7):116–145

[2] Alonso R (2021) Poblaciones fúngicas que afectan la producción de pino en Uruguay. PhD Thesis, Universidad de la República. Facultad de Ciencias - PEDECIBA

[3] Babbar N, Oberoi HS, Uppal DS, Patil RT. Total phenolic content and antioxidant capacity of extracts obtained from six important fruit residues. Food Res Int, 2011, 44: 391-396.

[4] Bakrim S, Machate H, Benali T. Natural sources and pharmacological properties of pinosylvin. Plants, 2022, 11(12): 1541.

[5] Balasundram N, Sundram K, Samman S. Phenolic compounds in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses. Food Chem, 2006, 99: 191-203.

[6] Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluating antimicrobial activity: A review. J Pharm Anal, 2016, 6: 71-79.

[7] Barbero-López A, Chibily S, Tomppo L, et al.. Pyrolysis distillates from tree bark and fibre hemp inhibit the growth of wood-decaying fungi. Ind Crops Prod, 2019, 129: 604-610.

[8] Belt T, Keplinger T, Hänninen T, Rautkari L. Cellular level distributions of Scots pine heartwood and knot heartwood extractives revealed by Raman spectroscopy imaging. Ind Crops Prod, 2017, 108: 327-335.

[9] Belt T, Awais M, Mäkelä M. Chemical Characterization and Visualization of Progressive Brown Rot Decay of Wood by Near Infrared Imaging and Multivariate Analysis. Front Plant Sci, 2022, 13: 940745.

[10] Berezjuk R, Nop P, Marík R, et al.. Towards more accurate grading: Non-destructive methods for assessing low-quality sawlogs. Holzforschung, 2025.

[11] Boragno L (2025) Resultados de la Cartografía Nacional Forestal 2024. Montevideo

[12] Box GEP, Behnken DW. Some New Three Level Designs for the Study of Quantitative Variables. Technometrics, 1960, 2: 455-475.

[13] Celimene CC, Micales JA, Ferge L, Young RA. Efficacy of pinosylvins against white-rot and brown-rot fungi. Holzforschung, 1999, 53: 491-497.

[14] Chen AYY, Olsen T. Chromated copper arsenate–treated wood: a potential source of arsenic exposure and toxicity in dermatology. Int J Womens Dermatol, 2016, 2: 28-30.

[15] Clausen TP, Reichardt PB, Bryant JP (1986) Pinosylvin and pinosylvin methyl ether as feeding deterrents in green alder. J Chem Ecol 12(12): 2117–2131

[16] Coniglio R, Gaschler W, Clavijo L. Water-based system to prevent the yellowing of opaque coatings on knotted pine wood. J Coat Technol Res, 2023, 20: 781-788.

[17] Coniglio R, Gaschler W, Dieste A. Knot extractives responsible for the yellowing of white-coated pine wood. Eur J Wood Wood Product, 2023, 81: 1109-1117.

[18] Daniel G (2016) Fungal degradation of wood cell walls. Secondary xylem biology: origins, functions, and applications. Elsevier Inc., 131–167

[19] Dieste A. Mitigación del impacto ambiental de madera tratada químicamente, 2014. Montevideo, Dirección Nacional de Industrias. Ministerio de Industria, Energía y Minería

[20] Dieste A, Baño V, Cabrera MN et al (2018) Forest-based bioeconomy areas. Strategic products from a technological point of view. Montevideo

[21] Felhofer M, Prats-Mateu B, Bock P, Gierlinger N. Antifungal stilbene impregnation: Transport and distribution on the micron-level. Tree Physiol, 2018, 38: 1526-1537.

[22] Gabaston J, Richard T, Cluzet S, et al.. Pinus pinaster Knot: A Source of Polyphenols against Plasmopara viticola. J Agric Food Chem, 2017, 65: 8884-8891.

[23] Gérardin P, Hentges D, Gérardin P et al (2023) Knotwood and branchwood polyphenolic extractives of silver fir, spruce and douglas fir and their antioxidant, antifungal and antibacterial properties. Molecules 28 https://doi.org/10.3390/molecules28176391

[24] Hames B, Ruiz R, Scarlata C, et al.. NREL 42620: Preparation of Samples for Compositional Analysis, 2008. Colorado, National Renewable Energy Laboratory

[25] Hillis WE (1987) Heartwood and tree exudates. Springer

[26] Hovelstad H, Leirset I, Oyaas K, Fiksdahl A. Screening Analyses of Pinosylvin Stilbenes, Resin Acids and Lignans in Norwegian Conifers. Molecules, 2006, 11: 103-114.

[27] Hu H, Tan W, Xi B. Lignin-phenol monomers govern the pyrolytic conversion of natural biomass from lignocellulose to products. Environ Sci Ecotechnology, 2021, 8: 100131.

[28] Intharuksa A, Kuljarusnont S, Sasaki Y, Tungmunnithum D (2024) Flavonoids and other polyphenols: bioactive molecules from traditional medicine recipes/medicinal plants and their potential for phytopharmaceutical and medical application. Molecules 29

[29] Jablonsky M, Nosalova J, Sladkova A, et al.. Valorisation of softwood bark through extraction of utilizable chemicals. A review. Biotechnol Adv, 2017, 35: 726-750.

[30] Kebbi-Benkeder Z, Colin F, Dumarçay S, Gérardin P. Quantification and characterization of knotwood extractives of 12 European softwood and hardwood species. Ann Sci, 2015, 72: 277-284.

[31] Kirker GT, Hassan B, Mankowski ME, Eller FJ (2024) Critical review on the use of extractives of naturally durable woods as natural wood protectants. Insects 15

[32] Le Man H, Behera SK, Park HS. Optimization of operational parameters for ethanol production from Korean food waste leachate. Int J Environ Sci Tech, 2010, 7: 157-164.

[33] Lee DG, Lee SJ, Rodriguez JP, et al.. Antifungal activity of pinosylvin from pinus densiflora on turfgrass fungal diseases. J Appl Biol Chem, 2017, 60: 213-218.

[34] Li X, Yao L, Xiong B, et al.. Inhibitory Mechanism of Pinosylvin Monomethyl Ether against Aspergillus flavus. J Agric Food Chem, 2022, 70: 15840-15847.

[35] Miranji EK, Kipkemboi PK, Kibet JK. A Review of Toxic Metals and Hazardous Organics in Wood Treatment Sites and Their Etiological Implications. J Chem Reviews, 2022, 4: 40-66

[36] Montgomery DC (2017) Design and analysis of experiments. Wiley

[37] Moore JR, Cown DJ (2015) Processing of wood for wood composites. Wood Composites. Elsevier Inc., pp 27–45

[38] Morais S, Fonseca HMAC, Oliveira SMR et al (2021) Environmental and health hazards of chromated copper arsenate-treated wood: a review. Int J Environ Res Public Health 18

[39] Nisula L (2018) Wood extractives in conifers: a study of stemwood and knots of industrially important species. Abo Akademi University

[40] Palombo V, Clavijo L, Cabrera MN (2024) Determining the pulping conditions and properties of unbleached pulp from uruguayan Pinus Taeda. Ingeniería 29 https://doi.org/10.14483/23448393.21172

[41] Parham RA, Gray RL. Formation and Structure of Wood. The Chemistry of Solid Wood, 1984. Washington DC, American Chemical Society: 3-56.

[42] Periferakis A, Periferakis AT, Troumpata L et al (2025) Pinosylvin: a multifunctional stilbenoid with antimicrobial, antioxidant, and anti-inflammatory potential. Curr Issues Mol Biol 47

[43] Prior RL, Wu X, Schaich K. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agric Food Chem, 2005, 53: 4290-4302.

[44] Qi J, Li F, Jia L et al (2023) Fungal Selectivity and Biodegradation Effects by White and Brown Rot Fungi for Wood Biomass Pretreatment. Polym 15. https://doi.org/10.3390/polym15081957

[45] Rasul A, Millimouno FM, Ali Eltayb W et al (2013) Pinocembrin: A novel natural compound with versatile pharmacological and biological activities. Biomed Res Int 2013. https://doi.org/10.1155/2013/379850

[46] Richard B, Bénard A, Dumarçay S, Colin F (2024) Wood, knots and bark extractives for oak, beech and Douglas fir: a dataset based on a review of the scientific literature. Ann Sci 81. https://doi.org/10.1186/s13595-024-01223-0

[47] Sandberg D, Fink G, Hasener J et al (2023) Process Control and Grading in Primary Wood Processing. Springer Handbook of Wood Science and Technology. Springer Science and Business Media Deutschland GmbH, pp 1019–1073

[48] Santos MB, Sillero L, Gatto DA, Labidi J (2022) Bioactive molecules in wood extractives: Methods of extraction and separation, a review. Ind Crops Prod 186

[49] Shmulsky R, Jones PD (2011) Forest Products and Wood Science An Introduction: Sixth Edition, Sixth. Wiley-Blackwell

[50] Singleton VL, Rossi JA. Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. Am J Enol Vitic, 1965, 16: 144-158.

[51] Sjöström E. Extractives. Wood Chemistry: Fundamentals and Applications, Second, 1993. San Diego, Academic: 90-108.

[52] Sluiter A, Ruiz R, Scarlata C et al (2005) Determination of Extractives in Biomass

[53] Sluiter A, Hames B, Ruiz R et al (2008) Determination of Ash in Biomass

[54] Sluiter A, Hames B, Ruiz R et al (2012) Determination of structural carbohydrates and lignin in Biomass

[55] Tascioglu C, Yalcin M, Sen S, Akcay C. Antifungal properties of some plant extracts used as wood preservatives. Int Biodeterior Biodegradation, 2013, 85: 23-28.

[56] Tran TM, Atanasova V, Tardif C, Richard-Forget F. Stilbenoids as Promising Natural Product-Based Solutions in a Race against Mycotoxigenic Fungi: A Comprehensive Review. J Agric Food Chem, 2023, 71: 5075-5092.

[57] Valette N, Perrot T, Sormani R, et al.. Antifungal activities of wood extractives. Fungal Biol Rev, 2017, 31: 113-123.

[58] Vek V, Poljanšek I, Humar M, et al.. In vitro inhibition of extractives from knotwood of Scots pine (Pinus sylvestris) and black pine (Pinus nigra) on growth of Schizophyllum commune, Trametes versicolor, Gloeophyllum trabeum and Fibroporia vaillantii. Wood Sci Technol, 2020, 54: 1645-1662.

[59] Venkatesan T, Choi YW, Kim YK. Impact of Different Extraction Solvents on Phenolic Content and Antioxidant Potential of Pinus densiflora Bark Extract. Biomed Res Int 2019, 2019.

[60] Venkatesan T, Choi YW, Kim YK. Comparative evaluation of the impact of extraction solvent and time on the yield and antioxidant potential of Pinus densiflora needle and bark extracts. Wood Sci Technol, 2020, 54: 587-598.

[61] Vovchuk CS, González Garello T, Careaga VP, Fazio AT (2024) Promising Antifungal Activity of Cedrela fissilis Wood Extractives as Natural Biocides against Xylophagous Fungi for Wood Artwork of Cultural Heritage. Coatings 14. https://doi.org/10.3390/coatings14020237

[62] Willför S, Hemming J, Reunanen M, Holmbom B. Phenolic and lipophilic extractives in Scots pine knots and stemwood. Holzforschung, 2003, 57: 359-372.

[63] Willför SM, Ahotupa MO, Hemming JE, et al.. Antioxidant Activity of Knotwood Extractives and Phenolic Compounds of Selected Tree Species. J Agric Food Chem, 2003, 51: 7600-7606.

[64] Xavier L, Barrenengoa M, Dieste A et al (2021) Valorization of Pinus taeda bark: source of phenolic compounds, tannins and fuel. Eur J Wood Wood Product 1–19. https://doi.org/10.1007/s00107-021-01703-4

[65] Xavier L, Coniglio R, Bermúdez F et al (2025) Harnessing polyphenols from pulp industry residues of juvenile eucalyptus wood: potential for adhesive applications. Bioresour Bioprocess 12. https://doi.org/10.1186/s40643-025-00914-4

[66] Zheng YZ, Deng G, Zhang YC (2022) Multiple free radical scavenging reactions of flavonoids. Dyes Pigm 198. https://doi.org/10.1016/j.dyepig.2021.109877

[67] Zink-Sharp A. The Mechanical Properties of Wood. Wood Quality and its Biological Basis, 2003. Oxford, CRC

Funding

Johann Heinrich von Thünen-Institut, Bundesforschungsinstitut für Ländliche Räume, Wald und Fischerei (4249)

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