Materials, Chemicals, and Energy from Forest Biomass - American

Chapter 22

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Knots in Trees: A Rich Source of Bioactive Polyphenols Bjarne Holmbom, Stefan Willfoer, Jarl Hemming, Suvi Pietarinen, Linda Nisula, Patrik Eklund, and Rainer Sjoeholm Process Chemistry Centre, Aabo Akademi University, Turku/Aabo, Finland, FI 20500

Knots in trees, i.e. the branch bases inside stems, contain extraordinary high amounts o f polyphenols, which are potent natural antioxidants and biocides. Studies of more than 50 tree species have shown that knots, in most of the studied species, contain remarkably higher amounts of polyphenols than the adjacent stemwood, for many species 20-100 times higher. Knots o f softwood species typically contain 5-15% (w/w) o f polyphenols, with lignans as the dominating group. Pine species contain a high percentage of stilbenes in their knots, while flavonoids are abundant in knots of certain hardwood species. Spruce knots from Northern Finland contain on average about 10% o f lignans. The dominating spruce lignan 7-hydroxymatairesinol (HMR) is a strong antioxidant and has moreover been found to inhibit growth of certain tumors. Production of H M R and marketing it as dietary supplement in the U S A has recently been started. In a large pulp mill using Norway spruce wood it is possible to sort out knots and extract up to 100 tons of H M R per year.

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© 2007 American Chemical Society In Materials, Chemicals, and Energy from Forest Biomass; Argyropoulos, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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Knots in Trees Knots are the branch bases inside the tree stems. Already in the 1930s, knots were reported to contain high contents of extractives (/). In studies by Wegelius (2) and Boutelje (3), the distribution of extractives in stemwood, branches and knots of spruce was determined. However, the knot extractives were not analyzed. The first studies that included analysis of knot extractives were on radiata pine (Pinus radiata) (4), and parana pine (Araucaria angustifolia) (5). Although the knots of parana pine were reported to contain much lignans, over 20% (w/w), the paper did not arouse any new interest in knot extractives. Ekman (6) investigated the distribution of lignans in Norway spruce trees and found high contents in the heartwood of branches (4-6%) and in roots (2-3%), but he did not analyze any knots. In 1998, we analyzed a spruce knot, and found that it contained as much as 10% (w/w) of lignans. This discovery gave the starting signal for extensive research on knots; first on knots in Norway spruce (Picea abies) and Scots pine (Pinus sylvestris), and later on knots in many more tree species. Up to now we have analyzed knots in more than 50 species. Knots in trees are of little value in manufacturing of pulp. In fact, the knots are detrimental to pulping and pulp quality, and should preferably be separated before pulping. A process for separation of clean knot material for further extraction of polyphenols has recently been developed (7). In this paper we summarize some of our research on polyphenols in knots. We have documented that knots are a very rich source of polyphenols, and especially of lignans, maybe even the richest in all nature. Many different polyphenols could be produced in industrial scale from knots. The main lignan in spruce knots, 7-hydroxymatairesinol (HMR), has been found to be a strong antioxidant, and furthermore to be anticarcinogenic. Recently, clearance was obtained from F D A to market H M R in dietary supplements in the U S A . The industrial production of H M R that now has started includes separation of spruce knot material, extraction with ethanol, and precipitation of H M R as adduct with potassium acetate.

Polyphenols in Knots We have during some years analyzed the polyphenols present in knots and compared the contents in knots with those in heartwood and sapwood samples from the same trees. We have analyzed fresh wood samples from at least 2-3 trees per species, including at least one living knot (having a living branch attached) and one dead knot from each tree. The wood samples have first been extracted with hexane to remove the lipophilic extractives, and thereafter the

In Materials, Chemicals, and Energy from Forest Biomass; Argyropoulos, D.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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polyphenols have been extracted with acetone or ethanol containing 5% water (8). The polyphenols have been analyzed primarily by G C and G C - M S as trimethylsilyl derivatives. Most of the polyphenols have also been isolated in pure form for structural studies, primarily by N M R spectroscopy, and for various biotests. A large number of lignans have been identified in softwood species. Structures of some common softwood lignans are presented in Fig. 1. In addition to ordinary lignans we have also found substantial amounts of lignans with three or more phenylpropane units, called oligolignans (9).

Matairesinol (MR)

Secoisolariciresinol (Seco)

Todolactol

Figure 1. Structures of common lignans in softwood tree knots.

Large Amounts of Lignans in Knots of Spruce Species

Norway Spruce (Picea abies) A study of more than 30 knots in seven Norway spruce trees (8) verified that knots contain much larger amounts of lignans than the ordinary stemwood, commonly over 100 times larger than in the adjacent stem heartwood. The studied knots contained 6-24% (w/w) of lignans. Later we have found even higher amounts, up to 29%, of lignans in spruce knots from Northern Finland. The lignan contents vary widely between trees, and even between knots in the


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same tree. Lignans are mainly found in the knots, i.e inside the stem, and their content drops to a level below 1% at 10-20 cm outwards in the branches. The typical distribution of lignans in Norway spruce trees is illustrated in Fig. 2.

Figure 2. Typical amounts of lignans (in % w/w) in Norway spruce trees, oligolignans excluded

The lignans in spruce knots are composed mainly of HMR, comprising 7085% of the lignans in the knots. HMR occurs in two stereoisomers forms, in a ratio of about 3:1. It was recently unambiguously proven that the major isomer has the 7S,8R,8'R configuration, while the minor isomer, eluting earlier in GC, has the 7R,8R,8 R configuration (10). The lignan contents were found to be at the same level from the middle of the tree stem out to the outer branch (8). However, the lignan contents were found to decrease in the radial directionfromthe center of the knot towards the outerwood (77). The contents were generally higher in the upper part of the knots (opposite wood) than in the lower parts, where dense compression wood is formed.


354 In addition to the ordinary dimeric lignans, Norway spruce knots also contain lignans with three and four phenylpropane units. Their amounts are 2 6% (w/w) of the spruce knots. The trimeric lignans, also called sesquilignans, are comprised of the major spruce lignans with an additional guaiacylglycerol unit bound to one of the phenolic hydroxyl units in the lignans (9).


Other Spruce Species We have analyzed the polyphenols in knots and stemwood of other spruce species (12). White spruce (Picea glauca) contains almost the same amounts of lignans as Norway spruce (Picea abies) (Fig. 3). The lignan composition is also similar, with H M R as the main lignan. Black spruce (Picea mariana) contains less lignans and the relative amount of H M R is also lower. Sitka spruce (Picea sitchensis) contains only a small amount of lignans. Korean spruce (Picea koraiensis) contains much H M R , but Serbian Spruce (Picea omorika) contains only a small amount of H M R . Red spruce (Picea pungens) contains only a small amount of lignans and has secoisolariciresinol as the main lignan component. The todolactol isomers in paper (12) were incorrectly reported as isomers of liovil.

Average amounts of lignans and oligolignans in knotwood (mg g" dry wood) 180 1

160 140

• Oligolignans H Other lignans • Todolactol M 7-Hydroxymatairesinol

120 100 80 60 40 20 0

P. abies

P. glauca P. mariana P. sitchensis P. omorika P. koraiensis P. pungen

Figure 3. Lignans and oligolignans in knots of spruce species (12).


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High Lignan Content in Knots of Fir Species Fir (Abies) species are also used for pulp and paper production, especially in Canada, Northern U S A , China and Central Europe. Like spruces, firs also have many knots since the branches usually remain on the tree stems even at mature age and in dense forest stands. We have studied several fir species (75) and average data are presented in Fig. 4. Fir knots contain typically 3-7% of lignans, and 3-5% of oligolignans. The oligolignans are composed mainly of guaiacylglycerol ethers and coumarates o f the dominating lignans. Alpine fir (Abies lasiocarpa), growing in western Canada, contained only small amounts of lignans. In all firs, secoisolariciresinol was the most abundant lignan. Balsam fir (Abies balsamea) contained a substantial amount also of lariciresinol. Typical for firs is that they also contain juvabiones, which are nonphenolic, cyclic sesquiterpenoid acids. Like lignans, also juvabiones are accumulated in the knots, with typical contents of 1-2%.

1

Average amounts of lignans, oligolignans, and juvabiones in knotwood (mg g*) dry wood 140 • Juvabiones • OligoBgnans D OtherGgnans Β Lariciresinol • Hydroxymatairesinol 100 Β Secoisolariciresinol I 80 60

A. sibihca A. lasiocarpa A. balsamea

A. alba

A. amabilis

A. veitchii A. A. concolor sachalinensis

Figure 4. Lignans, oligolignans andjuvabiones in knots of fir species (13).

Lignans Found in Knots of Pine and Other Softwood Species Considerable amounts of lignans have also been found in knots of several pine species. Scots pine (Pinus sylvestris), the predominant pine in Europe,



356 contained 0.4-2.9% of lignans in the knots (14) (Fig. 5). In addition to lignans, the knots contain 9-30% of resin acids and 1-7% of stilbenes. The predominant lignan, comprising 80-95% of the knot lignans, is (-)nortrachelogenin, also named (->wikstromol (/J). N o lignans could be detected in the ordinary stemwood. Pine knots also contain stilbenes of the pinosylvin type, as well as small amounts of oligolignans. However, the main extractives in pine knots are resin acids and other terpenoids making the recovery of pure lignans more difficult than for spruce and fir knots, which contain lignans as the dominating extractives.

Figure 5. Typical amounts of lignans in Scots pine (Pinus sylvestris) (14).

Several other pine species, as well as species of the families Larix, Pseudotsuga, Tsuga and Thuja have also been analyzed (16, 17). There are large differences between the pine species. Some pine species also contain flavonoids, in substantial amounts. Knots of Larix species (larches) contain mainly secoisolariciresinol and lariciresinol, similar to fir species. Knots of Tsuga species (hemlocks) are similar to spruce knots and contain H M R as the main lignan. Knots of Pseudotsuga menziesii (Douglas fir) contain both flavonoids and lignans.


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Polyphenols in Knots of Hardwoods A number of hardwood species have also been analyzed. Results have been published for a willow species (Salix caprea) (18), three aspen species (19) and two acacia species (20). Most hardwoods contain flavonoids in their knots. Salix caprea was found to contain the flavonoids dihydrokaempferol, catechin, gallocatechin, dihydromyrcetin and taxifolin. Knots contained more flavonoids than the stemwood of the same tree. Knots in aspen trees contained 3-5% of flavonoids (Fig. 6). The dominating components were dihydrokaempferol and naringenin (Fig. 7).

Average amounts of flavonoids in knotwood (mg g-1 dry wood) 80 70 60

• • • •

Other flavonoids Kaempferol + glycoside Dihydrokaempferol + glycoside! Naringenin + glycoside 1

50 40 30 20 10 0

P. grandidentata

P. tremula

P. tremuloides

Figure 6. Flavonoids in knots of aspen species (19).

"Keto-teracacidin"

Melacacidin

Isomelacacidin

Figure 7. Structures of major flavonoids in knots of aspen and acacia species.


358 The main polyphenols in Acacia mangium were teracacidin and ketoteracacidin, whereas Acacia crassicarpa contained mainly melacacidin and isomelacacidin (18). Also in these species, knots contained larger amounts of polyphenols than the stemwood.


Polyphenols are Bioactive Compounds The lignans, flavonoids and stilbenes found in large amounts in knots most probably have a defense function in the trees, although the defense mechanisms have not been elucidated. Common to all these polyphenols is their antioxidative and radical-scavenging properties. Hydrophilic extracts of knots were documented to have a high antioxidative potency and/or a radical scavenging capacity (16). Pure lignans and the flavonoid taxifolin were also strong antioxidants. However, when tested on bacteria isolated from paper mill waters, only extracts of knots containing the stilbenes pinosylvin and its monomethyl ether, (mainly from pine species) showed antibacterial effects (21). Pinosylvin is also know to be a natural fungicide. The large interest in the bioactivity of polyphenols is mainly due to their widespread occurrence in foods and their favorable effects on health. Flavonoids and stilbenes have been in the focus of health research for a long time, whereas lignans only more recently have attained broader interest (22). Lignans are of special interest because they are converted by bacteria in the large intestine to enterolactone and enterodiol. These lignan metabolites are considered to be associated with decreased risk for breast cancer and cardiovascular disease (23).

HMR in Spruce Lignan has Promising Bioactive Properties The bioactivity of H M R has been studied extensively during more than ten years by the research group of Risto Santti and Sari Mâkelâ at the University of Turku, Finland. The research has been carried out in cooperation with our and other groups. It has been shown that in both rats and humans H M R can be metabolized to the known mammalian lignan enterolactone, which has antitumorigenic effects for hormone-dependent cancers (23, 24, 25). Furthermore, H M R is a powerful antioxidant, similar to other lignans (16), and may be able to decrease the oxidation of LDL-particles and thus support cardiovascular health. H M R and other lignans can also inhibit overactivity of phagocytes and lymphocytes (26). H M R has also been shown to be non-toxic (27). Based on the research, H M R has been proposed as a chemopreventive agent against certain cancers, hormone-dependent diseases and cardiovascular diseases



359 (28). Recently, clearance was obtained from the F D A to market H M R in dietary supplements in the U S A and in 2006, products containing H M R came on the market. H M R is also of great interest because it can be used as a starting material for the production of other potentially valuable lignans. H M R is easily converted to matairesinol (Fig. 1) by catalytic hydrogenolysis (29, 30). H M R can also be reduced to secoisolariciresinol (29). Several other lignans can also be prepared from H M R , such as lariciresinol, cyclolariciresinol, enterolactone and enterodiol (10, 29). In alkaline conditions H M R is converted mainly to conidendric acid and in acidic conditions to conidendrin (31).

Industrial Production of HMR When tree stems are chipped industrially, most of the wood knots will form thick chips. In the following chip screening knots will be enriched in the overthick chip fraction. Today, this over-thick fraction is usually rechipped and used in the pulp production. However, knots are not desired in the wood furnish for mechanical pulping since knots are detrimental to the pulp quality (32). In chemical pulping the knots mostly remain uncooked and must be sorted out after the pulping (33). Therefore, knots should be taken out from the pulp wood furnish. A convenient method for separation and purification of knotwood material from wood chips, named ChipSep, has been developed (7). In the method, overthick chips are ground to splinters, the splinters are mildly dried and then separated by sedimentation in water. The knotwood material sediments while the normal stemwood material floats to the water surface. Industrial production of H M R was started in 2005. Over-thick spruce chips from the UPM-Kymmene pulp and paper mill in Kajaani in Northern Finland is used as raw material. The chips are taken out in the wood handling before the thermomechanical pulping (Fig. 8). Knot material is then separated by the ChipSep process operated in continuous mode. The obtained knot material, with over 90% knot material, contains about 10% of lignans and has a H M R content of 6-8% (w/w). The lignans are extracted from the knot material by ethanol, and the major isomer of H M R is precipitated from the solution by addition of potassium acetate, in principle according to the method described already in 1957 (34). The H M R potassium acetate adduct has a purity of 90-95%. In the UPM-Kymmene mill, processing about 1000 tons of spruce wood per day, it would be possible to separate knot material for an annual production of over 100 tons of H M R .



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Figure 8. Outline ofproduction of HMR lignan from Norway spruce.

Conclusions The conclusion is that knots of certain spruce and fir species constitute an exceptionally rich source of lignans. In certain spruce knots, the amount of lignans can exceed even 20% (w/w) and the knots can contain hundreds of times more lignans than the adjacent stemwood in the same tree. Some spruce species, especially Norway spruce and white spruce, contain on the average 5-10% lignans in the knots, with 7-hydroxymatairesinol (HMR) comprising 70-85% of the lignans. Knots in firs commonly contain 3-7% of lignans. The most common lignans in firs are secoisolariciresinol followed by lariciresinol. Pine species contain both lignans, stilbenes and flavonoids in their knots, but the large amounts of resin acids and other terpenoids complicate their extraction and purification. Certain hardwood species contain substantial amounts of flavonoids in their knots. The polyphenols occur in knots in free form and are easily extracted by ethanol, or other polar solvents. Not only H M R , but also other lignans, could be produced in large scale from knots at pulp and paper mills, either directly by extraction and purification, or by semisynthesis from H M R . Pure lignans, stilbenes and flavonoids can be extracted from knots and provide material for research on these potentially valuable bioactive compounds. Much research is presently being done in order to understand their biological and physiological properties, and consequently to create the base for applications in medicine and nutrition, or as natural antioxidants and antimicrobial agents in technical products.


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Acknowledgements Financial support has been received from the National Technology Agency (TEKES), the Academy of Finland, the Foundation for Research of Natural Resources in Finland, and the companies UPM-Kymmene, Raisio Chemicals (now Ciba Specialty Chemicals) and Hormos Nutraceutical. We are also grateful for the fruitful cooperation with Risto Santti, Sari Mâkelâ, Niina Saarinen and Markku Ahotupa at University of Turku. This work is part of the activities at the Abo Akademi Process Chemistry Centre within the Finnish Centre of Excellence Programme by the Academy of Finland.

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Materials, Chemicals, and Energy from Forest Biomass - American

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