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Knots in trees –An exceptionally rich source

of bioactive polyphenols

Bjarne Holmbom

Åbo Akademi

IAWS Annual Plenary MeetingSt. Petersburg, 16 June 2009

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Knot

As it happened in 1998 - - -

1998A crooked spruce tree was cut down

A knot was sawn out

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Analysis: The spruce knot contained 10% of lignans !

The start for extensive research on knots

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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

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Much higher concentrations of lignans in the north

Piispanen et al., 2008

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

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1998

Analysis10% lignans !

More spruceknots

Knots from other treespecies

Technicalsep. of knots

Applications

60+ species

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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

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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

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1998

Analysis10% lignans !

More spruceknots

Knots from other treespecies

Technicalsep. of knots

Applications

”ChipSep” Productionof knots

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Stemwood Chipping Screening PulpingTMPCTMP

Kraft pulpOver-thick chips

Knots “Normalwood ”

ChipSep

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The ChipSep ProcessFI Pat Appl. Aug. 2000 by C. Eckerman and B. Holmbom

Over-thickchips

Rechipped

Knotwood

Normalwood

Warm air

ChipDryer

Sep. tank

Pulping

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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

Farm production of knots 2005 -

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Continuous ChipSep separatorChips in

Knot productNormal wood out

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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

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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

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Next polyphenol products ?

HMR also in functional foodsNatural cosmeticsTechnical antioxidantsNatural biocides

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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

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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

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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

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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