B. Holmbom ÅA 1
Knots in trees –An exceptionally rich source
of bioactive polyphenols
Bjarne Holmbom
Åbo Akademi
IAWS Annual Plenary MeetingSt. Petersburg, 16 June 2009
B. Holmbom ÅA 4
Analysis: The spruce knot contained 10% of lignans !
The start for extensive research on knots
B. Holmbom ÅA 5
1998
Analysis10% lignans !
More spruceknots
Knots from other treespecies
Technicalsep. of knots
Applications
Holmbom et al., 2007
0
30
60
90
120
6-7 5-6 4-5 3-4 2-3 1-2 0-1 0-1 3-4 5 10 20 cm
Tree 3, LK 9.5
0
30
60
90
120
6-7 5-6 4-5 3-4 2-3 1-2 0-1 0-1 3-4 5 cm
Tree 6, LK 4.5
Knot in stem Outer branch
Lignans, mg/g
Compr. w.
Upper w.
Willför et al., 2003 a
0.0 %0.1 %0.1 - 5 % 6 - 29%
Hydroxymatairesinol (HMR)
Lignans in Picea abies
70-85% of the lignans
O
OMeO
OH
OH
OMe
OH
Willför et al., 2003 a
B. Holmbom ÅA 9
1998
Analysis10% lignans !
More spruceknots
Knots from other treespecies
Technicalsep. of knots
Applications
60+ species
B. Holmbom ÅA 10
Picea hydrophilic knot extractives, mg/g
0
50
100
150
200
250
Other comp.Other lignansHMR
P. abies P. glauca P. mariana P. sitchensisLK DK LK LK LKDK DKDK
LK: Living knotDK: Dead knot
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Larix hydrophilic knot extractives, mg/g
0
50
100
150
200
250
Other comp.Other lignansSecoisolaricir.
L. decidua L. lariciana L. sibirica
LK LK LKDK DK DK
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Pinus hydrophilic knot extractives, mg/g
0
20
40
60
80
100
120
140
160
180
Other comp.Other lignansLariciresinol
P. banksiana P. contorta P. resinosa P. sibirica P. sylvestris
LK LKa LK LK LKLKbDK DK DK DK
<0.05 % 0.4 – 2.9%
(-)-Nortrachelogenin (NTG)
Lignans inPinus sylvestris
O
O
OH
OMe
MeO
OH
OH
80-95% of the lignans
Heartw. Sapw.
In pine knots also
Resin acids 9 – 30% Stilbenes 1 – 7%
33 knots 7 trees
Willför et al., 2003 b
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Hydrophilic extractives in hardwood knots
050
100150200250300350400
Bet
ula
pend
ula
Pop
ulus
gran
dide
ntat
a
Pop
ulus
trem
ula
Pop
ulus
trem
uloi
des
Euc
alyp
tus
glob
ulus
Aca
cia
cras
sica
rpa
Aca
cia
man
gium
Fagu
ssy
lvat
ica
mg/g d.w.
Pietarinen et al., 2005 b
Pietarinen et al., 2005 a
Flavonoids in Populus species
05
101520253035404550
Populusgrandidentata
Populus tremula Populustremuloides
mg/ g d.w.
OthersDihydrokaempferol + glycoside
Kaempferol + glycoside
Naringenin + glycoside
OH
OH
O
O
OH
OH
OH
O
O
OH
OH
Dihydrokaempferol
NaringeninPietarinen et al., 2005 a
B. Holmbom ÅA 16
Flavonoids in Acacia mangium and A. crassicarpa
0
5
10
15
20
25
30
35
40
Acacia mangium Acacia crassicarpa
mg/g d.w.
Teracacidin
Keto-teracacidin
Biflavonoids
Oligomeric flavonoids
Melacacidin
Iso-melacacidin
Pietarinen et al., 2005 b
B. Holmbom ÅA 17
1998
Analysis10% lignans !
More spruceknots
Knots from other treespecies
Technicalsep. of knots
Applications
”ChipSep” Productionof knots
B. Holmbom ÅA 18
Stemwood Chipping Screening PulpingTMPCTMP
Kraft pulpOver-thick chips
Knots “Normalwood ”
ChipSep
B. Holmbom ÅA 19
The ChipSep ProcessFI Pat Appl. Aug. 2000 by C. Eckerman and B. Holmbom
Over-thickchips
Rechipped
Knotwood
Normalwood
Warm air
ChipDryer
Sep. tank
Pulping
B. Holmbom ÅA 20
1998
Analysis10% lignans !
More spruceknots
Knots from other treespecies
Technicalsep. of knots
ApplicationsHMR as dietary supplementAntioxidants and biocides
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The spruce lignanHydroxymatairesinol (HMR)
• Strong antioxidant • Inhibits breast cancer growth (Univ. of Turku)
• Hormos Medical Corp., Turku, product development• Clinical test on humans: no adverse effects• New dietary ingredient clearance from US FDA 2004• License Hormos Linnea S.A. in 2005• Production of knots (Separation Research)
O
OMeO
OH
OH
OMe
OH
B. Holmbom ÅA 25
Recent research has shown that plant lignans have a positive influence on the development of breast, prostate and colon cancer which rely specifically on oestrogens in order to progress.
Lignans also help to maintain good cardiovascular health and to moderate other oestrogen-dependent health problems such as menopause symptoms and osteoporosis.
www.hmrlignan.com
On the market in 2006
B. Holmbom ÅA 26
MeO
OH
OH
OMe
OHOH
Secoisolariciresinol
MeO
OH
O
OH
OH
OMe
Lariciresinol
O
O
OH
OMe
MeO
OH
OH
Nortrachelogenin
O
OMeO
OH
OH
OMe
OH
HMR
O
OMeO
OH
OH
OMe
Matairesinol
In many species alsoflavonoids
and stilbenes
Knots: the richest source of polyphenols in nature
B. Holmbom ÅA 27
Next polyphenol products ?
HMR also in functional foodsNatural cosmeticsTechnical antioxidantsNatural biocides
B. Holmbom ÅA 28
1998
Analysis10% lignans !
More spruceknots
Knots from other treespecies
Technicalsep. of knots
Applications
6-29% lignans
60+ species5-15% polyphenols
”ChipSep” Productionof knots
HMR as dietary supplementAntioxidants and biocides
A new platform for production of bioactive compounds from trees
B. Holmbom ÅA 29
Key publications IPohjamo, S.P., Hemming, J.E., Willför, S.M., Reunanen, M.H.T. and Holmbom, B.R. (2003), Phenolicextractives in Salix caprea wood and knots, Phytochemistry 63:2, 165-169Willför, S., Hemming, J., Reunanen, M., Eckerman, C. and Holmbom, B. (2003 a), Lignans and lipophilicextractives in Norway spruce knots and stemwood, Holzforschung 57:1, 27-36Willför, S., Hemming, J., Reunanen, M. and Holmbom, B. (2003 b), Phenolic and lipophilic extractives in Scots pine knots and stemwood, Holzforschung 57:4, 359-372Willför, S.M., Ahotupa, M.O., Hemming, J.E., Reunanen, M.H.T., Eklund, P.C., Sjöholm, R.E., Eckerman, C.S.E., Pohjamo, S.P. and Holmbom, B.R. (2003), Antioxidant activity of knotwood extractives and phenoliccompounds of selected tree species, J. Agr. Food Chem. 51:26, 7600-7606Holmbom, B., Eckerman, C., Eklund, P., Hemming, J., Nisula, L., Reunanen, M., Sjöholm, R., Sundberg, A., Sundberg, K. and Willför S. (2004), Knots in Trees – A New Rich Source of Lignans, Phytochem. Rev.2(3):331-340. Pietarinen, S., Willför, S. and Holmbom, B. (2004) Wood resin in Acacia mangium and Acacia crassicarpawood and knots, Appita J. 57:2, 146-150.Lindberg, L.E., Willför, S.M. and Holmbom, B.R. (2004) Antibacterial effects of knotwood extracts on paper mill bacteria, J. Ind. Microbiol. Biotechnol. 31, 137-147.Eklund, P.C., Willför, S., Smeds, A., Holmbom, B.R. and Sjöholm, R. (2004) A new lariresinol-type butyrolactone lignan derived from hydroxymatairesinol and its identification in spruce wood, J. Nat. Prod. 67:6, 927-931.Willför, S., Reunanen, M., Eklund, P., Sjöholm, R., Kronberg, L., Fardim, P., Pietarinen, S. and Holmbom, B. (2004), Oligolignans in Norway spruce and Scots pine knots and Norway spruce stemwood, Holzforschung 58: 345-354.Willför, S., Nisula, L., Hemming, J., Reunanen, M. and Holmbom, B. (2004), Bioactive phenolic substances in industrially important tree species. Part 1: Knots and stemwood of spruce species, Holzforschung, 58: 335-344.Willför, S., Nisula, L., Hemming, J., Reunanen, M. and Holmbom, B. (2004), Bioactive phenolic substances in industrially important tree species. Part 2. Knots and stemwood of different fir species. Holzforschung 58: 650-659.
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Key publications IIPietarinen, S., Willför, S., Sjöholm, R. and Holmbom, B. (2005), Bioactive substances in important tree species. Part 3. Knots and stemwood of Acacia crassicarpa and Acacia mangium. Holzforschung, 59: 94-101.Willför, S., Elund, P., Sjöholm, R., Reunanen, M., Sillanpää, R., Von Schoultz, S., Hemming, J., Nisula, L. and Holmbom, B. (2005), Bioactive substances in important tree species. Part 4. Identification of two new 7-hydroxy divanillyl butyrolactol lignans in some spruce, fir and pine species. Holzforschung, 59: 413-417.Pietarinen, S., Hemming, J., Willför, S., Vikström, F. and Holmbom, B. (2005, Wood resin in Bigtooth and Quaking Aspen wood and knots. J. Wood Chem. Technol. 25(1-2): 27-39.Markus, H., Mäki-Arvela, P., Kumar, N., Kul´kova, N.V., Eklund, P., Sjöholm, R., Holmbom, B., Salmi, T. and Murzin, D.Yu. (2005), Hydrogenolysis of hydroxymatairesinol over carbon-supported palladium catalysts, Catal. Lett. 103(1-2): 125-131.Willför, S.M., Sundberg, A.C., Rehn, P.W., Holmbom, B.R and Saranpää, P.T. (2005), Distribution of lignans in knots and adjacent stemwood of Picea abies, Holz-Roh Werkst. 63: 353-357.Markus, H., Mäki-Arvela, P., Kumar, N., Heikkilä, T., Lehto, V.-P., Sjöholm, R., Holmbom, B., Salmi, T. and Murzin, D.Yu. (2006), Reactions of hydroxymatairesinol over supported palladium catalysts, J. Catalysis238(2): 301-308.Pietarinen, S.P., Willför, S.M., Ahotupa, M.O., Hemming, J. and Holmbom B.R. (2006), Knotwood and bark extracts : strong antioxidants from waste materials. J. Wood Sci. 52:436-444.Willför, S.M., Smeds, A.I. and Holmbom, B.R. (2006), Chromatographic analysis of lignans. J. Chromatogr. A1122: 64-77.Pietarinen, S.P, Willför, S.M., Vikström, F.A. and Holmbom, B.R. (2006): Aspen knots, a rich source of flavonoids. J. Wood Chem. Technol., 26(3): 245-258.Smeds, A.I., Eklund, P.C., Sjöholm, R.E., Willför, S.M., Nishibe, S., Deyama, T. and Holmbom, B. (2007)Quantification of a broad spectrum of lignans in cereals, oilseeds and nuts. J. Agric. Food Chem. 55: 1337-1346. Neacsu, M., Eklund, P.C., Sjöholm, R.E., Pietarinen, S.P., Ahotupa, M.O., Holmbom, B.R. and Willför, S.M. (2007): Antioxidant flavonoids from knotwood of Jack pine and European aspen. Holz-Roh Werkst 65: 1-6.Willför, S., Hafizoğlu, H., Tümen, I., Yazici, H., Arfan, M., Ali, M. and Holmbom, B. (2007): Extractives of Turkish and Pakistani tree species. Holz-Roh Werkst. 65:3, 215-221.
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Key publications IIINeacsu, M., Micol, V., Pérez-Fons, L., Mallavia, R., Holmbom, B. and Willför, S. (2007) A novel antioxidant phenyl disaccharide from Populus tremula knotwood. Molecules 12(2): 205-217.Välimaa, A-L., Honkalampi-Hämäläinen, U., Pietarinen, S., Willför, S., Holmbom, B. and von Wright, A. (2006): Antimicrobial and cytotoxic knotwood extracts and related pure compounds and their effects on food-associated micro-organisms. Int. J. Food Microbiol. 115(2): 235-243.Piispanen, R., Willför, S., Saranpää, P., Holmbom, B. (2008) Variations of lignans in Norway spruce (Picea abies [L.] Karst.) knotwood: within-stem variation and the effect of fertilisation at two experimental sites in Finland. Trees 22, 317-328.
Book chapterHolmbom, B., Willfoer, S., Hemming J., Pietarinen, S., Nisula, S., Eklund, P. and Sjoeholm, R. (2007) Knots in trees – a rich source of bioactive polyphenols. In: Materials, Chemicals and Energy from Forest Biomass (Ed. D.S. Argyropoulos), ACS Symposium Series 954, ACS, 2007, pp. 350-362.
PatentsC. Eckerman and B. Holmbom: Förfarande för återvinning av kvistnötsmaterial från överstor flis / Method for recovery of knotwood material from over-size chips. Finn. Pat No 112041 (31.10.2003), US Pat. No 6,739,533 (25.5.2004)B. Holmbom, C. Eckerman, J. Hemming, M. Reunanen, K. Sundberg and S. Willför: A method for isolating phenolic substances or juvabiones from wood comprising knotwood. PCT Int. Appl. 2002, WO 2002098830 A1, 12 December 2002, 31 pp.S. Willför, C. Eckerman, J. Hemming, B. Holmbom, S. Pietarinen and A. Sundberg: Novel use of knotwoodextracts, Finn. Pat. Appl. Nr 20031642, Nov. 12, 2003
B. Holmbom ÅA 32
Major financial support
Academy of Finland (Finnish Centre of ExcellenceProgramme)Tekes (Finnish Funding Agency for Technology and Innovations)Keksintösäätiö (Foundation for Finnish Inventions)Hormos MedicalUPM-Kymmene
B. Holmbom ÅA 33
Team WorkÅA, Wood and Paper Chemistry
Rainer Ekman (†), Markku Reunanen, Christer Eckerman, Jarl Hemming, Andrey Pranovich, Lari Vähäsalo, Stefan Willför, Kenneth Sundberg, Suvi Pietarinen, Linda Nisula, Thomas Holmbom, Reija Harlamow
ÅA, Organic ChemistryRainer Sjöholm, Patrik Eklund, Annika Smeds
University of Turku Risto Santti, Sari Mäkelä, Niina Saarinen, Markku Ahotupa
Personnel at Hormos Medical and UPM-Kymmene