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Detoxification of thujaplicins in living western redcedar (Thuja plicata Donn.) trees by microorganisms Jin, Lehong 1987

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DETOXIFICATION OF THUJAPLICINS IN LIVING WESTERN REDCEDAR (Thuja piicat a Donn.) TREES BY MICROORGANISMS by LEHONG JIN B. Eng. Nanjing Forestry University 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the FACULTY OF GRADUATE STUDIES (Department of Forestry) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February, 1987 ©Lehong J i n , 1987 In p resen t i ng this thesis in part ial fu l f i lment o f the requ i remen ts for an a d v a n c e d d e g r e e at t he Univers i ty o f Br i t ish C o l u m b i a , I agree that the Library shal l m a k e it f reely avai lable for re fe rence and s tudy . I fur ther agree that pe rm iss i on for ex tens i ve c o p y i n g o f this thesis for scho la r l y p u r p o s e s may be g ran ted by the h e a d o f m y depa r tmen t o r b y his o r he r represen ta t i ves . It is u n d e r s t o o d that c o p y i n g o r pub l i ca t i on of this thesis fo r f inanc ia l ga in shal l no t b e a l l o w e d w i t h o u t m y wr i t t en p e r m i s s i o n . D e p a r t m e n t T h e Un ivers i t y o f Bri t ish C o l u m b i a 1956 M a i n M a l l V a n c o u v e r , C a n a d a V 6 T 1Y3 DE-6(3/81) ABSTRACT Thujaplicins are the major components in the steam-v o l a t i l e fraction of western redcedar {Thuja pi i cat a Donn.) (WRC) heartwood extractives. They are consided to be highly toxic to fungi and are c h i e f l y responsible for WRC heartwood decay resistance. This study proves that this t r a d i t i o n a l concept of t o x i c i t y i s not completely correct. Thujaplicins are toxic to common decay fungi isolated from decayed WRC wood in l i v i n g trees or wood in service, such as Poria al bipel I uci da Baxter. On the other hand, when a fungus such as Sporothrix sp. invades sound heartwood of l i v i n g WRC, thu j a p l i c i n s do not provide resistance but instead are altered by that fungus, so that their t o x i c i t y to decay fungi i s destroyed. Evidence obtained in th i s study indicates that the mechanism of t h u j a p l i c i n t o x i c i t y to common decay fungi involves the reactive keto-enolic group. As example, t h u j a p l i c i n t o x i c i t y disappears i f thi s reactive group i s blocked by laboratory methylation. In l i v i n g trees d e t o x i f i c a t i o n by Sporot hri x sp. i s demonstrated to involve a process of oxidative dimerization and isomerization of the thu j a p l i c i n s to a new lactone compound. This compound i s i i proven to have no t o x i c i t y to decay fungi, such as Porta al bipel I uci da Baxter. The dimerization and isomerization destroy the r e a c t i v i t y of the keto-enolic group and thus t h u j a p l i c i n t o x i c i t y . I s o l a t i o n , p u r i f i c a t i o n , and determination of the chemical structure of the new lactone compound produced from th u j a p l i c i n s during Sporothrix sp. infect i o n was carried out by chemical, chromatographic and spectroscopic methods. This naturally occurring compound has not been isolated previously and there are no previous reports in the l i t e r a t u r e about a compound with t h i s structure. Following IUPAC rules, the compound i s named as 3,3,4,7,7,8-hexamethyl-2,6-dioxa-1,5-anthracene-dione, and given the t r i v i a l name 'Thujin'. B i o l o g i c a l experiments ca r r i e d out in th i s study c l e a r l y show that in l i v i n g WRC trees, fungal attack involves a succession of microorganisms. Three early stage attacking fungi were consistantly isolated from discolored WRC heartwood. They are i d e n t i f i e d as Sporothrix sp. Ki rschst ei ni eI I a thuji na (Peck) Pomerleau & Etheridge and Phialophora sp. B i o l o g i c a l roles of these fungi are demonstrated based upon the results of wood block bioassays and chemical analysis of wood blocks treated with the three f u n g a l i s o l a t e s . TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS V LIST OF TABLES ix LIST OF FIGURES xi ACKNOWLEDGEMENTS xiv INTRODUCTION 1 LITERATURE REVIEW 7 2.1. Importance of western redcedar to the B r i t i s h Columbia forest industry 7 2.2. The chemical composition of WRC wood 11 2.3. Fungi and decay in WRC 21 2.4. Tropolones, tropolone derivatives and their properties 27 2.5. B i o l o g i c a l a c t i v i t i e s of wood extractives 45 2.6. Possible mechanisms of extractive t o x i c i t y 58 MATERIALS AND METHODS 69 3.1. Sample selection 69 3.2. Isolation cf microorganisms 74 3.3. Determination of extractives 77 3.4. I d e n t i f i c a t i o n of WRC Heartwood extractive fractions 83 3.5. Wood block bioassay 85 v 3.6. Isolation and i d e n t i f i c a t i o n of an unknown compound from discolored heartwood extractives- 92 a) Solvent fractionation 92 b) Column chromatography 93 c) Thin layer chromatography 95 d) C r y s t a l i z a t i o n and elemental analysis of the unknown compound 96 e) Mass spectrometry 97 f) Nuclear magnetic resonance spectrometry (proton and carbon-13 NMR) 97 g) V i s i b l e and ultraviolma-thujaplicin h) Infrared Spectrometry 98 3.7. Toxicity test bioassays 100 a) Toxicity of t h u j a p l i c i n 100 b) Toxicity of the unknown compound 102 c) Toxicity of methylated t h u j a p l i c i n 104 3.8. Examining o r i g i n of the new unknown compound 107 3.9. Detection of metal chelates from discolored WRC heartwood toluene extractives 110 RESULTS AND DISCUSSION 112 4.1. Microorganisms isolated and their d i s t r i b u t i o n in heartwood of WRC 112 4.2. Extractive differences between sound and discolored WRC heartwood 119 4.3. B i o l o g i c a l and chemical roles of Sporothrix sp. K. thujina and Phialophora sp. in l i v i n g WRC trees 131 4.4. Elucidation of the unknown compound structure 1 43 4.5. Mechanisms of thu j a p l ic in ' s t o x i c i t y 177 4.6. Future perspectives 183 CONCLUSIONS 185 LITERATURE CITED 188 v i APPENDIXES 201 Appendix 1 . l i s t of Synonymi for the Fungi Used in the Text. 201 Appendix 2. Thujaplicin Content from Old Growth WRC by Colorimetric Method 202 Appendix 3. Thujaplicin Content from Old Growth WRC by Gas Chromatography Method 203 Appendix 4. Thujaplicin Content from Second Growth WRC by Gas Chromatography Method 204 Appendix 5. Check L i s t of Fungi Collected on Western Redcedar in B r i t i s h Columbia from 1943 to 1945 205 Appendix 6. Results of Analysis of Variance for the WRC Heartwood Weight Loss 208 Appendix 7. Results of Multiple Range Tests for Wood Block Bioassay 212 Appendix 8. Chemical S h i f t s of Some Protons 213 Appendix 9. Chemical S h i f t s of Some Carbon-13 Resonances 214 Appendix 10. IR Data for Selected Carbon-Hydrogen Absorption Bands 215 Appendix 11. IR Data for Selected X-H Absorption Bands 216 v i i Appendix 12. IR Data for Selected Carbon-Carbon Absorption Bands 217 Appendix 13. IR Data for Selected Carbon-Oxygen Absorption Bands 218 v i i i LIST OF TABLES Table 1. The Significance of WRC in B.C. as a Forest Resource and Timber Cut in 1980. 8 Table 2. The Volume of Different Grades of WRC Log Export from B.C. in 1980. 8 Table 3. Chemical Composition of Western Redcedar, Western Hemlock and Douglas-fir Wood. 12 Table 4. Composition of Steam V o l a t i l e O i l from WRC Butt Heartwood. 16 Table 5. L i s t of Some Trees Containing Terpenoid Tropolones— 29 Table 6. Quantitative Analyses of Tropolones in Cupr es s aceae . 30 Table 7. Carbon-13 Chemical Shif t s of Several Derivatives of 2-methoxytropone in CDClj. 43 Table 8. Results of Tox i c i t y Tests for a Number of Heartwood Extractives (Decay as a percentage of decay in control) . 47 Table 9. Concentration Series for Standard Gamma-thujaplicin Solutions. 81 Table 10. Frequency of Microorganism Isolation on Malt Agar from WRC Heartwood v s . Sample Age. 1 1 3 ix Table 11. BE Extractives and Thujaplicin Content of the WRC Heartwood. 120 Table 12. TLC Rf Values for Four WRC Heartwood Extracts Using BE (9:1) Solvent. 127 Table 13. TLC Rf Values of Known WRC Extractives Using BE (9:1) Solvent. 129 Table 14. Results of Weight Loss and Extractive Analysis of WRC Sound Heartwood Blocks Treated with Sporothrix sp. , K. t huj i na and Phialophora sp. 133 Table 15. Data of the Low Resolution Mass Spectrum of the Unknown Compound. 148 Table 16. Broad Band and Attached Proton Test Carbon-13 NMR Spectra Data of the Unknown Compound. 157 Table 17. Atomic Weight of Isotopes Used in High Resolution Mass Spectroscopy. 162 Table 18. Fourier Transform IR Spectral Data of the Unknown Compound. 171 x LIST OF FIGURES Figure 1. The V o l a t i l e Components of WRC Heartwood Extractives. 14 Figure 2. The Non-volatile Components of WRC Heartwood Extractives. 18 Figure 3. Structures of Methyl Ether, Conjugated Acid Cations and Amine Salts of Tropolone. 35 Figure 4. Oxidation of H i n o k i t i o l with Hydrogen Peroxide. 38 Figure 5. Anionic Substitution and Rearrangement of Tropolone. 39 Figure 6. The Complex Structure between Tropolone, Magnesium and S-Adenosyl Methionine (COMT) . 62 Figure 7. The Chemical Structures of the WRC Extractive F e r r i c Chelates. 67 Figure 8. Tree from Which the Wood Samples Were Taken. 70 Figure 9. Four 0.9 Meter Bolts Cut from the Selected Tree. 71 Figure 10. Cross Sections of the Selected Tree Showing Color Variations. . 73 Figure 11. Radial Pieces S p l i t from a Quadrant and Used in the Isolation of Microorganisms (A) and Extractives (B and C). 75 xi Figure 12. Calibra t i o n Curve: Absorbance vs Gamma-thujaplicin F e r r i c Chelate Concentrations. 82 Figure 13. Growing WR1, WR2 and WR3 Cultures Isolated from Discolored WRC Heartwood (Piece A). 86 Figure 14. WR1, WR2 and WR3 Cultures Used in the Wood Block Bioassay. 88 Figure 15. Pet r i Dish Prepared for Toxic i t y Tests. 101 Figure 16. Inhibition of Porta al bipel I uci da Baxter by Beta-thujaplicin at Sixteen Different Concent ract ions. 103 Figure 17. Inhibition of Porta albi pel Iuci da Baxter by the Unknown Compound, Methylated Thujaplicins and Thujaplicins. 105 Figure 18. Histogram (No.1) of Percentage Frequency D i s t r i b u t i o n for S t e r i l e Wood and WR1, WR2 and WR3 Fungi Infections vs. Sample Age. 114 Figure 19. Histogram (No.2) of Percentage Frequency Distributions for S t e r i l e Wood and WR1, WR2 and WR3 Fungi Infections v s . Sample Age. 115 Figure 20. TLC Plates of BE Extractives from WRC Discolored and Light-straw Colored Heartwoods. 126 Figure 21. Low Resolution Mass Spectram of the Unknown Compound. 1 47 Figure 22. Proton NMR Spectrum of the Unknown Compound. 152 x i i Figure 23. Broad Band and Attached Proton Test Carbon-13 Spectra of the Unknown Compound. 156 Figure 24. High Resolution Mass Spectrm of the Unknown Compound. 1 63 Figure 25. Chemical Structure of "Thujin". 167 Figure 26. UV Spectrum of the Unknown Compound. 168 Figure 27. V i s i b l e Spectrum of the Unknown Compound. 169 Figure 28. Fourier Transform IR Spectrum of the Unknown Compound. 170 x i i i ACKNOWLEDGEMENTS I wish to express my sincere gratitude and deep appreciation to my research supervisor, Dr. E. P. Swan, Adjunct Professor, U.B.C., Research S c i e n t i s t , Forintek Canada Corp. and my academic supervisor Dr. J . W. Wilson, Professor, Director of Forestry Graduate Studies, U.B.C. for thei r firm guidance, kind advice, understanding and constant encouragement throughout the course of this study. I also express my special acknowledgement to Dr. B. van der Kamp, Associate Professor, (Forest Pathology) U. B. C. for his guidance, rewarding discussions, and assistance in the b i o l o g i c a l phase of the research. Many thanks are due to Dr. S. Z. Chow, Director of Research, Canadian Forest Products Ltd. and Dr. F. G. Herring, Professor, Department of Chemistry, U.B.C., members of the supervisory committee, for their interest and support given to me. I am also in indebted to J. Nault, B. Daniel and J. Clark, technologists, Forintek Canada Corp. for their technical assistance during the experimental phase of the study. Special thanks are extended to the Department of Chemistry, U.B.C. for providing spectroscopic services and elemental analyses; to Forintek Canada Corp. for providing xiv working space and research f a c i l i t i e s ; and to Dr. M. P. C o r l e t t , I d e n t i f i c a t i o n Service, Biosystematic Research In s t i t u t e , Agriculture Canada, Ottawa, for i d e n t i f y i n g three fungi isolated during the study. I am very grateful to the Ministry of Education, People's Republic of China and The University of B r i t i s h Columbia, Canada for their generous f i n a n c i a l support in the form of a National Overseas Graduate Scholarship (for the f i r s t year) and a University Graduate Fellowship (for three years), respectively. F i n a l l y , I would l i k e to thank my parents for their love, understanding and encouragement which have been a main source of strength. xv INTRODUCTION Western redcedar (WRC)(Thuja pii cat a Donn.) is one of the most important coniferous woods of B r i t i s h Columbia (B.C.) and i s recognized as one of the world's most durable woods. Its natural-resistance to decay i s indicated by the fact that f a l l e n WRC trees may sometimes remain on the forest floor for centuries with l i t t l e apparent decay except in the thin sapwood layer. On the other hand, in certain areas of B. C , there i s s t i l l a serious problem of WRC heartwood decay, especially in mature and overmature trees. "On the B. C. coast extensive columns of butt and trunk rot, often associated with basal scars, are common. In some areas in the i n t e r i o r of the province, standing WRC trees consist of l i t t l e more than a s h e l l of sapwood, the heartwood being completely destroyed by decay. In B. C. as a whole, the amount of decay expressed as a percentage of the t o t a l gross volume of wood is greatest for WRC (32%). This i s more than with any other major conifer (average 12%)" (van der Kamp, 1975). This discrepancy needs to be explained by further research. It i s long understood from WRC chemical studies that the water-soluble materials contained in the heartwood give the wood much of i t s d u r a b i l i t y . These water soluble extractives contain two main groups with fungicidal properties, the steam-volatile tropolone derivatives and a 1 mixture of non-volatile phenolic derivatives (Barton and Gardner, 1954). B i o l o g i c a l tests have shown so far that one group of tropolones, the t h u j a p l i c i n s , are c h i e f l y responsible for WRC heartwood decay resistance. It i s worthwhile to point out that these results are based on choosing well known decay fungi as test material (Roff, 1961). Wood decay i s the f i n a l result of a sequence of events. Studying chemical responses of the tree to the f i n a l organisms in t h i s sequence may misrepresent the decay process. It i s possible that before these fungi attack l i v i n g trees the t o x i c i t y factor of th u j a p l i c i n s and other extractives i s already altered by early attacking fungi. Therefore, the t o x i c i t y of t h u j a p l i c i n s to various fungi under certain conditions remains an important problem. Investigations of WRC decay in B. C. have shown that t h i s involves a succession of organisms (van der Kamp, 1975). The major heart-rotting organisms are * Basidiomycet es . The heart-rotting fungi of l i v i n g WRC on the coast, in decreasing order of detection frequency, *: The fungal names used through out the text are those appeared in the o r i g i n a l publications. Their current names are l i s t e d in Appendix 1 according to Compendium of Plant Disease and Decay Fungi in Canada 1960-1980 (Ginns, 1986). 2 are Poria al bipel I uci da Baxter. (white r ing r o t ) , Poria. asiatica ( P i l a t ) Overh. (brown cubical pocket and butt rot) and Forties pi ni Lloyd. (white p i t t e d trunk r o t ) . In the i n t e r i o r of the province the most important organisms are Poria asiatica (Pilat) Overh., Poria weirii Murr. (yellow ring rot) and Fomes pi ni (Thore) Lloyd. (Buckland, 1946). However, an interesting question i s which fungus is the f i r s t to interact with s t e r i l e heartwood tissues. The work done by van der Kamp (1975) indicated that fungi which attack at early stages caused wood discolo r a t i o n and may a l t e r the extractive properties. He found three d i f f e r e n t fungi in several coastal samples. They were not f i n a l l y i d e n t i f i e d at that time. He gave the code names as: WR1 (possibly Cyl i ndrocephal um sp.) WR2 (possibly Ki rschstei ni el I a thuji na (Peck) Pomerleau & Etheridge.) and WR3 (unknown). Questions ari s e on the role of the WRC heartwood extractives, e s p e c i a l l y t h u j a p l i c i n s , and how fungi detoxify the t h u j a p l i c i n s and destroy the natural toxins, rendering the heartwood subject to decay. Mechanisms of t h u j a p l i c i n t o x i c i t y to certain groups of fungi and some fungi's detoxifying effects on t h u j a p l i c i n s are unknown. Nothing has been reported on these matters. 3 A c c o r d i n g to the c h e m i c a l s t r u c t u r e s of t h u j a p l i c i n s , i t i s r e a s o n a b l e to assume that the r e a c t i v e k e t o - e n o l i c group i s the key f u n c t i o n a l group which may be r e s p o n s i b l e f o r these a c t i o n s . T h i s a s sumpt ion , however, has not been proven e x p e r i m e n t a l l y . T e s t i n g the t o x i c i t y of t h u j a p l i c i n d e r i v a t i v e s whose k e t o - e n o l i c group a c t i v i t y has been b l o c k e d and a n a l y s i s of c h e m i c a l s t r u c t u r e s of d e t o x i f i e d or b iodegraded t h u j a p l i c i n s c o u l d p r o v i d e v a l u a b l e i n f o r m a t i o n . These a s p e c t s r e q u i r e c l a r i f i c a t i o n . To i n v e s t i g a t e the e f f e c t s of t h u j a p l i c i n t o x i c i t y on v a r i o u s fung i and to examine the mechanisms of i t s s e l e c t i v e t o x i c i t y c o u l d serve to s t i m u l a t e r e s e a r c h on new f u n g i c i d e s and i n s e c t i c i d e s of t e c h n i c a l i m p o r t a n c e . A l s o , i t c o u l d s t i m u l a t e f u r t h e r u t i l i z a t i o n of WRC heartwood e x t r a c t i v e s which now have l i t t l e commerc ia l use . Our knowledge i n t h i s f i e l d i s s t i l l not f u l l y d e v e l o p e d . A f t e r almost t h i r t y y e a r s of c h e m i c a l s t u d i e s on WRC e x t r a c t i v e s c a r r i e d out i n the Western F o r e s t r y Research L a b o r a t o r y ( F o r i n t e k Canada C o r p . ) , the t e c h n i q u e s are a v a i l a b l e to separate these e x t r a c t i v e s . In t u r n , t h i s p r o v i d e s m a t e r i a l s f or examining decay r e s i s t a n c e mechani sms. C u r r e n t u n d e r s t a n d i n g of the r o l e of WRC heartwood 4 e x t r a c t i v e s i n d e c a y r e s i s t a n c e i s i n a d e q u a t e . T o i m p r o v e t h i s u n d e r s t a n d i n g , s e v e r a l q u e s t i o n s m u s t b e a n s w e r e d . W i t h s u c h q u e s t i o n s i n m i n d , t h e t h r e e o b j e c t i v e s o f t h e r e s e a r c h a r e t o : 1) E x a m i n e t h e i n t e r a c t i o n b e t w e e n t h u j a p l i c i n s a n d l e s s e r k n o w n f u n g i w h i c h a t t a c k WRC h e a r t w o o d i n l i v i n g t r e e s a t e a r l y s t a g e s , a n d t o r e v e a l t h e r o l e o f t h u j a p l i c i n s i n l i v i n g t r e e s i n o p e r a t i o n a g a i n s t f u n g a l a t t a c k ; 2) S e p a r a t e , a n a l y s e , a n d i d e n t i f y t h e m a j o r p r o d u c t s w h i c h f o r m f r o m t h u j a p l i c i n s a f t e r t r e a t m e n t w i t h i s o l a t e d f u n g i ; a n d 3) E x a m i n e t h e c h e m i c a l m e c h a n i s m o f t h e t o x i c s e l e c t i v i t i e s o f t h u j a p l i c i n s t o v a r i o u s f u n g i . T w o m a j o r h y p o t h e s e s w i l l b e t e s t e d i n t h i s s t u d y i n o r d e r t o a c h i e v e t h e s e o b j e c t i v e s . 1) T h e t o x i c i t y o f t h u j a p l i c i n s t o f u n g i v a r i e s b e t w e e n f u n g i . T h e r e a r e t w o c o n s e q u e n t t e s t h y p o t h e s e s f o r t h i s m a j o r h y p o t h e s i s a s : A ) S t e r i l e WRC h e a r t w o o d i s i n v a d e d f i r s t b y m i c r o o r g a n i s m s w i t h a h i g h t o l e r a n c e t o t h u j a p l i c i n s a n d s u c h o r g a n i s m s d e t o x i f y t h u j a p l i c i n s a n d d e s t r o y t h e s e 5 n a t u r a l t o x i n s of WRC heartwood, r e n d e r i n g the wood s u b j e c t t o f u r t h e r decay by the same or new organisms; and B) T h u j a p l i c i n s a r e h i g h l y t o x i c t o the w e l l known f u n g i such as Poria al bi pel I uci da B a x t e r . and Poria w e i r i i Murr. and the decay caused by th e s e f u n g i i s r e s t r i c t e d t o thos e a r e a s i n which t h u j a p l i c i n s have d i s a p p e a r e d or been d e a c t i v a t e d . 2 ) The s e l e c t i v i t y of t h u j a p l i c i n t o x i c i t y t o v a r i o u s f u n g i i s based on a c t i v i t y of the k e t o - e n o l i c group i n d i f f e r e n t f u n g a l e n v i r o n m e n t s . There a r e a l s o two consequent t e s t hypotheses f o r t h i s major h y p o t h e s i s a s : A) D e a c t i v a t i o n of t h i s group by e a r l y a t t a c k i n g f u n g i might a r i s e from o x i d a t i o n , p o l y m e r i z a t i o n or h y d r o l y s i s of t h u j a p l i c i n s ; and B) A c t i v a t e d t o x i c i t y of t h u j a p l i c i n s t o decay f u n g i might be due t o the f o r m a t i o n of a metal c h e l a t e w i t h m e t a l i o n s i n s i d e the wood, or w i t h m e t a l i o n s a s s o c i a t e d w i t h f u n g a l enzymes, or f o r m a t i o n of amine t h u j a p l i c i n s a l t s w i t h a m i n o - a c i d s of the f u n g i , and f o r m a t i o n of hydrogen bonding between t h u j a p l i c i n and f u n g a l p r o t e i n , w i t h any or a l l of th e s e mechanisms d e a c t i v a t i n g f u n g a l growth. 6 LITERATURE REVIEW 2.1. Importance of western redcedar to the B r i t i s h Columbia forest industry Western redcedar (WRC) {Thuja piicat a Donn.), a major tree species in the forests of northwestern North America, p a r t i c u l a r l y in the coastal areas cf B r i t i s h Columbia (B.C.) is one of the largest trees in the P a c i f i c Region. It frequently reaches heights of 45 to 60 metres and diameters of 2.5 metres .or more (Barton,1962). B.C. accounts for 86.7% of WRC in a l l of Canada, as 876 m i l l i o n m3 out of 1010 m i l l i o n m (Canadian Forestry S t a t i s t i c s , 1980). It constitutes 11.3% (913.5 m i l l i o n m ) of the B. C. forest resource (Ministry of Forestry, 1980). The t o t a l annual WRC 3 harvested in B.C. in 1980 amounted to 2.03 m i l l i o n m in the 3 i n t e r i o r and 6.42 m i l l i o n m in the coast respectively (Table 1) (Ministry of Forestry, 1980). WRC also provided valuable foreign exports as Grade I, II, and III lumber (Table 2). It constituted 10.4% (3.891 3 3 m i l l i o n m out of 37.418 m i l l i o n m ) of the t o t a l lumber exported from B.C. in 1982 ( S t a t i s t i c s Canada, 1983). The WRC trees rarely occur in extensive pure stands but 7 Table 1. The Significance of WRC in B. C. as a Forest Resource and Timber Cut in 1980. Forest d i s t r i c t Prince George Prince Rupert Vancouver Cariboo Kamloops Nelson Forest resource Timber cut coast i n t e r i o r 23.5 329.8 432.0 20.5 56.0 51 .7 (million m ) 1 .08 5.34 0.08 0.33 0.06 0.18 0.52 0.36 Tatal 913.5 Source: Ministry of Forestry, Table 2. The Volume of Different B. C. in 1980. Grade 1 2 3 Total Source: Ministry of Forestry, 6.42 2.03 (1980). Grades of WRC Log Exports from 3 Volume (m ) 27,288.9 42,144.8 95,123.2 164,556.9 (1980). are commonly associated with several other tree species. In the P a c i f i c coast forests, i t grows with Douglas-fir, western hemlock, Sitka spruce, P a c i f i c s i l v e r and noble f i r s . In the Interior of B.C., i t i s usually found with western white pine, western larch, western hemlock, Douglas-f i r , and Engelman spruce (Bolsinger, 1979). WRC wood i s used in a variety of products, because of i t s well recognized reputation of higher d u r a b i l i t y under service conditions. The main products include shakes and shingles, siding, poles and posts, fence material, casket stock, outdoor furniture, paneling and many special items. It i s also used as a p a r t i a l (15 to 30%) wood furnish for pulping with other species (Swan and Jiang, 1970). WRC pulp is known to have excellent paper-making properites. Paper-making tests with WRC, hemlock, Douglas-fir and southern pine bleached Kraft pulps (Murray and Thomas, 1961) indicated that WRC ranked f i r s t in burst, fold and t e n s i l e properties. Due to the fineness of i t s f i b e r s , WRC also ranked f i r s t in opacity and smoothness. It also contributed higher tear strength. Although WRC can produce a very good quality pulp, the demand for WRC as pulping material in comparison with other western Canadian species, such as Douglas-fir and 9 western hemlock i s r e l a t i v e l y lower. The main reason which discourages use of this wood as pulping material i s i t s higher extractives content, together with i t s low density. As a consequence, a r e l a t i v e l y higher chemical requirement and a longer pulping time are needed. The o v e r a l l pulp y i e l d per^ unit volume of raw material is only 60% of that obtained from Douglas-fir (Barton and MacDonald,1971). Accelerated corrosion in mild-steel digesters and evaporators i s another processing problem encountered in Kraft pulping of WRC due to the formation of extractive metal chelates (Gardner, 1963). If the commercial use of these extractives in WRC can be found, i t w i l l lead to a better u t i l i z a t i o n by pre-extraction of WRC heartwood before pulping. The cost of pre-extraction could be compensated with the p r o f i t generated by extractive byproducts. 10 2.2. The chemical composition of WRC wood The structural constituents of WRC wood, namely c e l l u l o s e , hemicelluloses and l i g n i n , occur in roughly the same proportion as in other coniferous woods. The non-structural components of wood, which can be normally extracted by water, or organic solvents, are defined as wood extractives. An unusual c h a r a c t e r i s t i c of WRC wood is i t s high heartwood extractives content. Lewis' (1950) work (Table 3) showed that t o t a l hot water extractives in WRC was almost twice as much as in western hemlock and Douglas-fir. The WRC heartwood extractives a f f e c t u t i l i z a t i o n far out of proportion to the amount present. They are responsible for the odor, taste, and color of WRC heartwood. In addition, they af f e c t the d u r a b i l i t y , permeability, ease of pulping and other properties (Gardner et al, 1966). In essence, WRC extractives d i s t i n g u i s h the heartwood in appearance and properties from a l l other woods. The WRC heartwood extractives obtained by hot water extraction may be separated readily into v o l a t i l e and non-v o l a t i l e fractions by steam d i s t i l l a t i o n . The v o l a t i l e fraction consists mainly of a class of organic compounds c a l l e d tropolone derivatives. In WRC heartwood, from old 1 1 Table.3. Chemical Composition of WRC, Western Hemlock and Douglas-fir Wood. Alpha Hemi- Total water Species c e l l u l o s e c e l l u l o s e Lignin extractives Ash WRC 47.5 13.2 29.3 10.2 0.24 Western hemlock 48.8 14.7 28.8 5.3 0.47 Douglas-fir 53.8 1.3.3 26.7 5.92 0.28 Source: Lewis, (1950). 12 growth, the content of this group of compounds ranged from 0% at the p i t h to 1.2% in the outer straw colored heartwood (MacLean and Gardner, 1956b). Their results are shown in Appendix 2. Recent research by Nault (1986) indicated that by using more sensitive a n a l y t i c a l methods, higher amounts of tropolone derivatives can be detected. His results showed that in old growth trees, t h i s group of components ranged from 0.014% to 1.774% in heartwood, while in fast growth trees, the amounts varied from 0.011% to 0.525% (Appendixes 3, 4). There are at least nine components in the steam v o l a t i l e f r a c t i o n of WRC extractives, alpha-,beta- and gamma-thujaplicins (Gardner, 1958); beta-dolabrin (Gardner and Barton, 1958); and b e t a - t h u j a p l i c i n o l , thujic acid, and methyl thujate (MacLean and Gardner, 1956b). Other substances present have been i d e n t i f i e d as nezukone and carvacrol methyl ether (MacLean, 1970). The chemical structures of these are shown in Figure 1. The t h u j a p l i c i n s are highly toxic to wood destroying fungi, their t o x i c i t y being of the order as that of sodium pentachlorophenate (Barton and MacDonald, 1971). Beta-t h u j a p l i c i n o l i s a t h u j a p l i c i n containing an additional v i c i n a l hydroxyl group which considerably modifies i t s 1 3 F i g u r e 1. T h e V o l a t i l e C o m p o n e n t s o f WRC H e a r t w o o d E x t r a c t i v e s . S o u r c e : B a r t o n a n d M a c D o n a l d , ( 1 9 7 1 ) . 14 r e a c t i v i t y and t o x i c i t y (Roff, 1961). The non-tropolone components of the steam v o l a t i l e o i l , thujic acid and methyl thujate are also seven-membered ring compounds. Thujic acid i s a c o l o r l e s s , odourless c r y s t a l l i n e organic acid with low fungal t o x i c i t y . Methyl thujate is responsible for the c h a r a c t e r i s t i c odour of WRC and has some t o x i c i t y to the carpet beetle and case-making clothes moth (Barton, 1962). Gardner and Barton (1958) measured the r e l a t i v e concentrations of these compounds. Beta-thujaplicin has a s l i g h t l y higher concentration than gamma-thu'japlicin. Alpha-t h u j a p l i c i n i s present only in minor amounts. The beta-t h u j a p l i c i n o l i s approximately one-tenth of the t o t a l t h u j a p l i c i n content. Beta-dolabrin, nezukone and carvacrol methyl ether are present in only trace amounts (Table 4). The non-volatile WRC heartwood extractives are mainly polyphenolic fractions. They account for 5 to 15% of the heartwood. U n t i l now, eleven lignans have been i d e n t i f i e d (Barton and MacDonald, 1971). They are: p l i c a t i c acid (Gardner et al, 1959; 1960; 1966); p l i c a t i n (Gardner et al, 1959; 1960; 1966); t h u j a p l i c a t i n (Gardner et al , 1966); t h u j a p l i c a t i n methyl ether (MacLean and Murakami, 1966a); dihydroxythujaplicatin (MacLean and MacDonald, 1967); 15 Table 4. Composition of Steam V o l a t i l e O i l from WRC Butt Heartwood. components o i l ( % ) wood(%) methyl thujate, other neutrals 21.1 0.17 thujic acid 10.4 0.08 tropolones 68.5 0.56 beta-thujaplicinol 8 0.07 gamma-thujaplicin 24 0.20 beta-thujaplicin 35 0.30 alpha-thujaplicin 1 0.01 beta-dolabrin 0.04 0.0003 Source: Gardner and Barton, (1958). 16 hydroxythujaplicatin methyl ether (MacLean and Murakami, 1966b); dihydroxythujaplicatin methyl ether (MacLean and Murakami, 1967); plicatinaphthalene (MacLean and MacDonald, 1969b); plicatinaphthol (MacLean and MacDonald, 1969a); gamma-thujaplicatene (MacDonald and Barton, 1970); and beta-ap o p l i c a t i t o x i n (MacDonald and Barton, 1973). The chemical structures of these are shown in Figure 2. The chemistry of these interesting lignans has been discussed (Gardner el al, 1971). Of these, the main component (30 to 40% of the mixture or 1 to 5% O.D. wood basis) i s a very strong organic acid which was heat and l i g h t s e n s i t i v e . It i s c a l l e d p l i c a t i c acid. The water soluble (organic solvent insoluble) portions of WRC extractives are non-phenolic carbohydrates, consisting of simple sugars, L-arabinose, pectic compounds and hemicelluloses. Small amounts of protein are also present (Barton and MacDonald, 1971). The formation and r e l a t i v e amounts of WRC heartwood extractives and th e i r varying concentration from outer heartwood to p i t h has been explained by changes in metabolism (more hydroxylation and less O-methylation) with age (Swan et at, 1969). The hydroxylation of the tropone ring (nezukone to t h u j a p l i c i n s to beta-thujaplicinol) 1 7 Figure 2. The Non-volatile Components of WRC Heartwood Ext r a c t i v e s . m e t h y l ether H O -O H XIV D i h ^ d r o i y l h u j O p N c o t i n C H 3 O ' Y 0 C H 3 O H XV H y d ' o x y t n u j o p l i c a t i n m e t h y l e t h e r O H XVI D i h y d r o x y l h u j o p l t c o t i n m e t h y l ether - C H , > *o H O ' ^ " O C H 3 O H XVII P l i c o t l n o p h t h s l e n e O H XVIII Plicotinophthol XIX Y-Thujcplieofene x x B-apoplicatitoxin Source: Barton and MacDonald, (1971). 18 appeared to occur much faster, and wholly at the sapwood-heartwood boundary. They also discussed the p o s s i b i l i t y of t h u j a p l i c i n s ' i n h i b i t i n g a f f e c t on the O-methylation enzyme system. Lignans of WRC were thought to be formed via a main hydroxylation sequence from the f i r s t member of the family, t h u j a p l i c a t i n to hydroxy-thujaplicatin to dihydroxy-t h u j a p l i c a t i n to p l i c a t i n to p l i c a t i c acid and plicatinaphthol (Swan and Jiang, 1970). On the other hand, methylation formed t h u j a p l i c a t i n methyl ether, hydroxy-t h u j a p l i c a t i n methyl ether and dihydroxy-thujaplicatin methyl ether (Swan and Jiang, 1970). During the past three decades, the large scale investigation of WRC extractives has provided much useful information on both i t s chemistry and methodology. With the aid of modern types of chromatography, such as paper, thin layer, column, vapour-phase and liquid-phase, and combinations of these techniques, separations of very complex mixtures l i k e lignans have become possible. The use of these methods of separation together with powerful new instruments for s t r u c t u r a l determinations, as for example v i s i b l e , UV, and IR spectroscopy, o p t i c a l rotary dispersion, nuclear magnetic resonance (proton and carbon-13 NMR), mass spectra and X-ray crystallography has lead to 19 better understanding of these extractive structures (Swan et al, 1969). 20 2.3. Fungi and decay in WRC Because i t i s an organic material, wood i s subject to several forces of degradation, including fungi, bacteria insects and marine borers. Decay in wood is caused mainly by fungi. Fungi contain no chlorophyll and are heterotrophic organisms, so they must obtain their nourishment from energy supplying, tissue building, organic substances previously produced by green, chlorophyll bearing plants. They must consume preformed organic matter. They may l i v e as saprophytes which digest and consume dead plants or animals, their parts, or their wastes. Alternately, fungi may l i v e as parasites and assimilate tissues or parts of l i v i n g plants and animals. Fungi usually enter and ramify within plants in the form of minute threads or filaments, c a l l e d hyphae , which consist of c h i t i n tubes with occasional cross walls (septa). Digestive enzymes are liberated from the fungal c e l l into the immediate environment where food molecules are solublized and then absorbed into the fungal c e l l . Wood, such as trees, logs and manufactured products is subject to fungal decay which discolors and destroys i t , and causing s i g n i f i c a n t economic loss and various 21 u t i l i z a t i o n problems. Basham and Morawski (1964) found that approximately 22 m i l l i o n cubic feet of timber, which was roughly 6.3% of the annual harvest, was wasted annually in Ontario because of heart rot or s t a i n . In the province of B r i t i s h Columbia, the amount of decay expressed as percentage of the t o t a l gross volume of wood was 12% on average for major conifers, while for WRC i t was 32% ( B r i t i s h Columbia Forest Service, 1957). The decay fungi belong to the most advanced class of fungi in the evolutionary scale, the Basi di omycetes. This class name and that of a subdivision containing the decay producing species, the Hymenomycetes are increasingly seen in l i t e r a t u r e in the subject of wood decay. Two p r i n c i p a l types of wood decay fungi are recognized: Brown-rot and Whi te-rot. Brown-rot fungi preferably attack softwoods. They secrete enzymes which decompose the polysaccharides of the c e l l wall, leaving the l i g n i n matrix nearly undigested and result in decayed wood, brown in color. White-rot fungi degrade l i g n i n , as well as polysaccharides. Preferably they start to metabolize l i g n i n and hemicelluloses, the c e l l u l o s e fraction i s degraded only at a l a t e r stage (Liese, 1970). The degraded wood i s white and soft. 22 Brown and white rots are commercially and e c o l o g i c a l l y extremely important as they are the types found in standing l i v i n g trees and cause serious s t r u c t u r a l f a i l u r e . Soft-rot fungi mostly belong to the Ascomycetes or Fungi imperfecti which are able to degrade polysaccharides and l i g n i n . Because of their p r e f e r e n t i a l growth within the c e l l wall in contract with the brown and white rot fungi whose hyphae develop inside the c e l l lumen, soft rot has been considered as a wood destroyer (Liese, 1970). Although soft-rot destroys wood, i t is o r d i n a r i l y much less serious than brown and white rots in standing trees because i t progresses slowly into wood from the surface, and commonly does not go as deeply. However, th i s type of rot is most important in wood in service and i s found in both softwoods and hardwoods. In a large scale investigation of decay and decay fungi in WRC i n i t i a t e d in 1943, a t o t a l of 615 trees were examined in 11 l o c a l i t i e s on the coast and 110 trees in eight l o c a l i t i e s in the i n t e r i o r . Seventy-seven species of Basi di omycet es were c o l l e c t e d on l i v i n g and dead WRC in B. C. from 1943 to 1945. A check l i s t of these fungi i s shown as Appendix 5 (Buckland, 1946). From the study, six fungi have been reported associated with brown cubical rots 23 of l i v i n g WRC trees. Two of these, Poria asiatica Overh. and Polyporus balsameus Peck. were of considerable importance. Another four which occurred infrequently were Fomes pi ni col a (sw) Cke., Polyporus schweinitzii Fr., Coni ophor a cer ebelI a Perso. and Meruit us spp. During the course of investigation, four fungi capable of producing white rots of importance and four of possible significance in l o c a l i z e d areas have been found in WRC. Poria al bi pel I uci da Baxter., Poria weirii Murr., Fomes pini (Thore) Lloyd. and Poria subacida (Peck) Sacc. were i d e n t i f i e d as major wood destroying fungi. The four of secondary importance were i d e n t i f i e d as Fomes annosus (Fr.) Cke., Armillaria mellea (Fr.) Quel., Omphali a campanella (Fr.) Quel, and Fomes ni grol imi tat us (Romell) Egel. From th i s investigation, Buckland concluded that the p r i n c i p a l fungi responsible for decay in WRC wood were somehow associated with the geographical location of the tree. Poria al bipel I uci da Baxter. and Poria weirii Murr. occured on the coast and i n t e r i o r but as heart-rotting organisms, the former was only s i g n i f i c a n t l y important on the coast and the l a t t e r in the i n t e r i o r . The reason for t h i s i s unknown. Basi di omycet es causing decay in WRC wood in service 24 appeared to be t o t a l l y d i f f e r e n t from those in standing trees. Southam and E h r l i c h (1950), Duncan and Lombard (1965), Eslyn (1970), Clark and Smith (1979), Schefter, et al (1983) and S e t l i f f and Cserjesi (1985) reported about 30 species of fungi found in service poles and shakes. A rather interesting concept was established in the research area of forest pathology in the 1970's. More and more evidence suggested that decay fungi never occured alone either in standing trees, in slash or in wood in use. Wood decay was the f i n a l result of a sequence of events ( M e r r i l l and Shigo, 1979). On the other hand, va r i a t i o n in the natural decay resistance of heartwood was possibly an important factor governing the rate., d i r e c t i o n and pattern of microbial attacking processes (Wilkes, 1982 a, b). Studying the t o x i c i t y of natural protective components to those organisms at the end of t h i s sequence w i l l not lead to a f u l l understanding of the t o t a l process. The roles of non-hymenomycetous pioneer organisms began to draw s c i e n t i f i c c u r i o s i t y because they were isolated consistently from the discolored margins of decay columns, while wood decay fungi were consistently recovered only from advanced decayed wood. Manion and Zabel (1979) postulated that these 25 "pioneers" had an i n i t i a l detoxifying role in standing trees, and then after a preconditioning period, new infections occurred as other decay fungi became ac t i v e . The study done by Shortle (1979) pointed out that successful invasion by both "pioneers" and decay fungi appeared to be affected by factors associated with the defense levels of the tree. In research done by van der Kamp (1975), three non-decay fungi were isolated from the straw-colored, red and brown stained sound wood areas of WRC. They were not f i n a l l y i d e n t i f i e d , but were given code numbers WR1, WR2 and WR3. Poria al bi pel Iuci da Baxter, was isolated from a narrow zone at the edge of the sound wood. These results supported the hypothesis that decay of WRC involved a succession of organisms. 26 2 . 4 . T r o p o l o n e s , t r o p o l o n e d e r i v a t i v e s a n d t h e i r p r o p e r t i e s Tropolone and tropolone derivatives are generic terms applied to unsaturated seven-membered carbon ring compounds, 2-hydroxy-2,4,6-cycloheptatrien-1-one and i t s derivatives. In 1945, Dewar (1945a, b, c) proposed a seven-membered enolone structure which was a novel aromatic system and he gave the name of tropolone to the parent structure of such a system. This was the incentive that led to l a t e r discovery of some dozens of natural tropolones derivatives. The simplest of these and the f i r s t to be conclusively characterized as tropolone derivatives were the isomeric t h u j a p l i c i n s . They were alpha-, beta-, and gamma-th u j a p l i c i n s . In- 1933, Anderson and Sherrard (1933) discovered a toxic "phenolic" substance, c i o H 1 2 ° 2 ' m*P* in WRC and l a t e r an isomer (m.p. 52-52.5°) that c r y s t a l l i z e d from the mother liquor on standing for a long period of time (Anderson and Gripenberg, 1948). In 1948, Erdtman and Gripenberg (1948) obtained from heartwood of the same tree cu l t i v a t e d in Sweden, besides the substance of m.p. 82°, a t h i r d isomer of m.p. 34°. They were c a l l e d as alpha-, beta-and gamma-thujaplicins in the increasing order of their melting points ( H i l l i s , 1962). 27 Beta-thujaplicin, also known as h i n o k i t i o l because of i t s presence in the essential o i l of "Taiwan hinoki" (Ch a m a e c y p a r i s taiwanensis Masamune et Suzuki.) was the subject of much independent study in Japan. Nozoe (1956) obtained h i n o k i t i o l from alkaline hydrolysis of i t s red iron complex, h i n o k i t i n . As a result of extensive research on h i n o k i t i o l in the period 1940-1947, Nozoe, with a number of associates, independently proposed an unsaturated carbon ring structure and l a t e r i t s identity with beta-thujaplicin was proven by di r e c t comparison (Nozoe, 1950). Tropolones are di s t r i b u t e d widely in nature. They can be found in terpenoids (Nozoe, 1950), fungal metabolites (Just and Williams, 1962; Blank et al, 1966; Ng et al, 1968; Blank et al , 1969), alkaloids (Santavy, 1950), glucosides and pigments (Nozoe, 1956). WRC and several other species of the family Cupr es s aceae are r i c h in terpenoid tropolones. In Table 5, several kinds of important terpenoid tropolones from the heartwoods and essential o i l s of some tree species are l i s t e d (Nozoe, 1956). The quantitative analysis of these tropolones corresponding to species of Cupressaceae was provided by Zavarin et al (1959) using paper chromatography (Table 6). The tropolone contents in these heartwoods varied greatly between trees, with age of tree, 28 Table 5. List of Some Trees Containing Terpenoid Tropolones. Botanical name Common name Geographical location Type oi tropolone" References Chamaccyparis taiwa- Taiwan-hinoki• Formosa a. P (133. 134) nensis M A S A M U N E et (Taiwan) S U Z U K I C. formosensis M A T S U M . Benihi* Formosa (trace) (133) C. nootkatensis S P A C H . Alaskan U . S . A . Nk (37) Yellow cedar Thuja plicata D . D O N Western U . S . A . «• P- Y {14, 15, 80, Red cedar 246) Thuja plicata D . D O N Sweden a, y (80. 92) T. occidentalis L . Northern Sweden a. y (93) White cedar T. occidentalis L . Nioi-hib?.* Japan a. p (126) T.Slandishii C A R R . Nezuko* Japan P ("7) Thujopsis dolabrata Hiba* Japan <*. P (796) Z I K B . et Zucc. Libocedrus decurrens Incense cedar U . S . A . P- Y (245- 247) T O R R E Y Cupressus macrocarpa Monterey New Zealand p. Nk (57. 246) H A R T W . cypress • Juniperus chinensis L . Byakushin* Japan P. Nk (128) * Japanese common name. *• a, p\ y: Thujaplicins; Nk: Nootkatin. Source: Nozoe, (1956). 29 Table 6. Quantitative Analyses of Tropolones in Cupr es s aceae. 0- T* 6-Thuja- Thuja- Thuja- .Noot- Doln- Tliuja-Totai Method of Species plicin plicin pliein kntin brin mucin plicinol T-U T-10 T-4.5 T-O.l Analysis Cuprcssus pypmaea (Lcinm.) Sarg. — 0.1 — 0.1 0.001 0.4 — 0.4 0.3 0.1 — 1.4 Clirom. C. targenlii, Jeps. — 0.03 — 0.1 0.01 — 0.03 — — — — 0.17 Chrom. C. abramsiana, C. B. Wolf — 0.04 — 0.02 0.001 — — 0.01» — — 0.07 Chrom. C. govenxana, Gord. — 0.008 — 0.0008 0.0004 0.005 — 0.05» — — • 0.02 Chrom. C. ariionica, Greene — 0.0003 0.002 0.008 — — 0.0003 — — — — 0.01 Chrom. C. gempervirent, L.' — 0.2 — 2.Q — — — — — — — 2.2 Chrom. & Prep. C. macrocarpa, Uartw.1'*1' + — 0.2 * — — — — — — — 0.2 Chrom. & Prep. C. toruhsa1-' — — — + — — — — — — — • Unkn. Unkn. C. macnabiana1* + +• + — — ' - — — — — — Unkn. Unkn. Chamaeeyparis lawtoniana* 0.0002 — — — • — — — — — — 0.0002 Chrom. . (A. Murr) Pari Ch. noolkalcnsis,' (D. Don) Spach,'( — — — 0.1 — — — — — — — 0.1 Chrom. & Prep. ! Ch.tyoidei*(BS.P.) + + + — — — — — — — — Unkn, Chrom. ! Ch.formosensia* Mateum.'-' — — — — — — — — — — Unkn. Chrom. & Prep. Ch. taiwanensis,' Masam. ct Su*. , , M~ l , + + — — — — — — — — — Unkn. Chrom. & Prep. ; Ch. obluta,' Sieb. et Zucc.»M» - - — — — — - — — — — — Prep. o • Numbers denote the Approximate amount of ft tropolone in %, based on dry weight of wood. ' In this case it has not been determined which of the two compounds is present, or whether both o r e . ' These analyses hove been taken from the literature. TABLE II" or- 0- V 0-Thuja- Thuja- Thuja- Noot- Thuja- Method of Species plicin plicin plicin kntin Dolabrin Pygmacin plicinol T - l l T-10 T-4.5 T-O.l Total Analysis Papuaecdrui torriccllentii, Li Junipcrut occidcnlalit,1 Hook — 0.07 — _ — — — — — _ 0.05 0.12 Chrom. — — — — — — — • — — — — — Chrom. / . communis, L. — 0.001 — 0.003 — 0.0003 — — — 0.0000 — 0.005 Chrom. /. monospcrma, (Englm) Sarg — 0.0002 — 0.008 0.0001 0.008 — — — — — 0.1G Chrom. oslcospermn, (Torr) Littlo — 0.0001 — Traco — — — — — — — 0.0001 Chrom. /. deppcana, Stcud. — 0.003 — 0.04 0.0002 — — — — — — 0.043 Chrom. J. mexieana, Sprong. — — — — — — — — . — — — — Chrom. • /. virginiana,1 L.* /. chinenais," L.* — — — — — — — — — — — — Chrom. — 0.0004 — 0.008 — — — — — — — 0.0084 Prep. • Numbers denote the approximate amount of tropolone in %, based on dry weight of wood. * These analyses have been taken from the literature. Source: Zavarin el al , ( 1959). tree growth s i t e and point of sampling within the stem. They may be isolated from wood in the laboratory either by steam d i s t i l l a t i o n or by extraction with organic solvents. The hydroxyl group of th u j a p l i c i n s is enolic in nature and possesses properties c h a r a c t e r i s t i c of enols and phenols. The a c i d i t y , enhanced by the adjacent carbonyl group, i s midway between that of carboxylic acids and phenols. The actual a c i d i t y of each individual compound varies considerably depending on the presence or absence of the substituents and the character and the position of the l a t t e r . The a c i d i t y increases s l i g h t l y from alpha- to gamma- to beta-thujaplicins with pKa values of 7.8, 7.3, and 7.1 respectively (Raa and Goksoyr, 1965). The keto-enolic structure of t h u j a p l i c i n s gives great p o s s i b i l i t i e s for chelation. Color reactions of th u j a p l i c i n s with solutions of f e r r i c and cupric s a l t s are highly c h a r a c t e r i s t i c . Treatment with f e r r i c s a l t solutions gives an immediate b r i l l i a n t red coordination complex, which i s insoluble in water but readily soluble in benzene and chloroform. An excess of f e r r i c ion converts the red complex to a green derivative which is very soluble in water (MacLean and Gardner, 1956a). With cupric s a l t s , a green complex i s formed, which i s insoluble in water but soluble 31 in benzene, chloroform and alcohols. The copper complexes are readily c r y s t a l l i z e d from organic solvents and are useful in i s o l a t i o n and characterizations of natural tropolones. A simple but expensive method of preparing large samples of t h u j a p l i c i n s used copper screens in commercial drying kilns to c o l l e c t the green copper t h u j a p l i c i n chelate deposit (Gardner et al, 1957). Decomposition of the copper chelates with hydrogen sulphide y i e l d s an o i l y mixture of the t h u j a p l i c i n s . This procedure has proven to be a convenient method of obtaining research samples of t h u j a p l i c i n s . In addition to t h u j a p l i c i n s , several other important terpenoid tropolone derivatives have been characterized such as b e t a - t h u j a p l i c i n o l (Gardner, et al, 1957), beta-dolabrin (Nozoe et al , 1953, Gardner and Barton, 1958), and nootkatin (Barton, 1976). Among them, both beta-thujaplicinol and beta-dolabrin were isolated from WRC, while nootkatin was isolated from yellow cedar. Nootkatin has the formula C15 H18°2' m*P* 95°. Its c h a r a c t e r i s t i c color complex with iron and copper solutions and i t s u l t r a v i o l e t and infrared absorption spectra indicated that i t was a tropolone. Like the t h u j a p l i c i n s found in WRC, nootkatin was considered to be very toxic to decay fungi together with chamic and 32 chaminic acids, which were assumed to be responsible for the d u r a b i l i t y of yellow cedar (Barton, 1976). Tropolone derivatives were found not only in trees, but also isolated from the metabolite of microorganisms, such as from Sepedonium chrysospermum (Bull.) Fr. (Divekar, et al, 1965). There were two tropolone derivatives i d e n t i f i e d from the organism. One of them, named sepedonin, ^11 H12^5' 3,6,9-trihydroxy-methyl-1,3,4,7-tetrahydrocyclo-heptapyran-7-one), predominated in growing cultures and was believed to be the d i r e c t product of metabolism. The other, an anhydro derivative of sepedonin, was probably formed spontaneously in the culture f i l t r a t e during the i s o l a t i o n process by chemical dehydration (Divekar et al, 1965). Tropolone derivatives exist in nature mostly as monomers. Only one ditropolonoid has been reported as a natural compound so far (Baggaley and Norin, 1968). Its t r i v i a l name i s utahin. Utahin was isolated from the heartwood of Juniper us utahensis Lemm. by Runeberg ( 1 960). It i s a yellow, o p t i c a l l y inactive, high melting compound, m.p. 313°. The formation of utahin probably involved the symmetrically oxidative coupling of two simple tropolone molecules (Baggaley and Norin, 1968). Chemically, tropolone and i t s derivatives undergo 33 j f a c i l e e l e c t r o p h i l i c substitution and tropenoid compounds in general submit more eas i l y to nucleophilic substitutions as well as to rearrangement to benzenoid compounds (Nozoe, 1956). The enolic hydroxyl in the tropolone ring i s the source of i t s a c i d i t y , and hence the a c i d i t y disappears when the tropolone is e s t e r i f i e d or e t h e r i f i e d . The methyl ether (Figure 3A) could be obtained by treating a tropolone with diazomethane, or i t s sodium or s i l v e r salt with methyl iodide (Doering, 1951; Raa and Goksoyr, 1965). Tropolones are resistant to hot concentrated acids, since they are s t a b i l i z e d in the form of their conjugated acid cations (Figures 3B and 3C). However, tropolones are highly l a b i l e in the presence of a l k a l i or a non-aromatic primary amine (Nozoe, 1956; Poh, 1974). Poh (1974) reported that tropolones reacted s p e c i f i c a l l y and rapidly with a l l non-aromatic primary amines in non-polar solvents, such as carbon tetrachloride and benzene, to y i e l d the corresponding yellow s o l i d amine tropolone s a l t s (Figure 3D). The reaction between tropolones and non-aromatic primary amines is an acid-base reaction. Since secondary and t e r t i a r y a l i p h a t i c amines are as basic as primary a l i p h a t i c amines (pKa about 10), they 34 F i g u r e 3. S t r u c t u r e s o f M e t h y l E t h e r , C o n j u g a t e d A c i d C a t i o n s , a n d A m i n e S a l t s o f T r o p o l o n e . 35 are expected to also react with tropolones. Poh demonstrated that non-aromatic secondary and t e r t i a r y tropolone s a l t s were soluble in carbon tetrachloride and benzene, whereas non-aromatic primary amine tropolone s a l t s were insoluble in these solvents. Because aromatic amines are weak bases (pKa less than 5), they do not abstract the a c i d i c proton of a tropolone to a s u f f i c i e n t extent. These amine tropolone sa l t s react rapidly with aqueous bases to give back the o r i g i n a l amines and tropolones (Poh, 1974). Oxidation with a l k a l i n e hydrogen peroxide can degrade the tropolone ring. It was reported (Nozoe, 1956) that hydrogen peroxide oxidation of beta-thujaplicin ( h i n o k i t i o l ) gave f i r s t isopropyl-3-hydroxytropolone which was then rapidly cleaved to c i s , cis-muconic acid. Because of formation of a dihydroxy intermediate, beta-isopropyl-l e v u l i n i c acid was also produced during beta-thujaplicin oxidation. It was also found that persulfate oxidation of tropolone or beta-thujaplicin resulted in hydroxylation of 3- and 5- positions (Nozoe et al, 1953). In the case of gamma-thujaplicin i t yielded beta-isopropyl-levulinic acid and beta-isopropyl-muconic acid (probably in i t s trans form). For alpha-thujaplicin, only the compound beta-isopropyl-muconic acid was formed under the influence of 36 hydrogen peroxide. The reaction process of hydrogen peroxide oxidation i s shown as Figure 4. Tropolones were found to e a s i l y undergo e l e c t r o p h i l i c substitution. The positions e a s i l y substituted were as expected 3, 5, and 7 in the tropolone ring (Pauson, 1955). The mechanisms of e l e c t r o p h i l i c substitution of tropolones seemed to be similar to those of benzenoid compounds, espe c i a l l y phenols. E l e c t r o p h i l i c substitutions taken place e a s i l y in tropolones were: azo coupling n i t r o s a t i o n ; sulfonation (by sulfamic acid); hydroxymethylation; and halogenation (Nozoe, 1956). The e l e c t r o p h i l i c substitution processes were markedly influenced by the s t e r i c effect of the neighboring groups in the tropolone series. It was observed (Nozoe, 1956) that troponoids containing substituents such as halogens and methoxyl groups, could e a s i l y eliminate these substituents in the form of an anion and then either substitute with another substituent or rearrange to benzenoid structures (Figure 5). By means of modern physical measurements, such as u l t r a v i o l e t (UV), infrared (IR), nuclear magnetic resonance (NMR) spectra, and x-ray and electron d i f f r a c t i o n , a great deal of the structural information about t h i s group of natural compounds was available. 37 Figure 4. Oxidation of H i n o k i t i o l with Hydrogen Peroxide. CC, Hinokitiol I ICXXXI1.) (CXXXMl.) • • 1 voprof>y!-ru.cumuconu: »cid. OH HO 1 O I / O C O O H C H a r \ f ' /""V'' / — C O O H . -< O H -< ° -< fCXXXV.) ICXXXVI.J ICXXXVU.) -< COOH (CXXXVII1.) Source: Nozoe, (1956). 38 Figure 5. Anionic S u b s t i t u t i o n and Rearrangement of Tropolones. Source: Nozoe, (1956). 39 The UV spectra of troponoids were si m i l a r , to some extent, to those benzenoid compounds with the same wavelength range but d i f f e r e n t i n t e n s i t y . As noted by H i l l i s (1962), the UV absorption exhibited a maximum of very intense absorption (log e, 4.25-4.7) in the 228 to 270 urn region (Region 1) and two bands of somewhat lesser intensity (loge. , 3.5-4.0) in the 300 to 340 and 350 to 380 urn regions (Region 2). The band in Region 1 was common to tropones and tropolones. It was hardly affected by the presence of a l k y l groups but sh i f t e d s l i g h t l y to longer wavelength when halogen, hydroxyl or ester groups were present. IR spectra of tropolones have been reported in several studies (Nozoe, 1956; Gardner et al, 1957). The bands in the spectrum at 1265, 1440, 1468, 1558, 1618, and 3165 cm - 1 have been considered to be c h a r a c t e r i s t i c of the tropolone nucleus (Gardner et al, 1957). Besides those bands, the C-H stretching vibration of the tropolone ring was similar to that of benzenoid compounds and appeared as a band of weak intensity in the region 3060 to 3010 cm 1 . The C=0 stretching vibr a t i o n of tropones appeared at 1645 cm 1 in carbon tetrachloride and with higher intensity at 1651 cm 1 in the gaseous state. For tropolones, the carbonyl band was located at 1613, 1620 and 1628 cm 1 ( s o l i d , solution, gas, 40 respectively) indicating a displacement of 20 to 25 cm toward the lower frequency region as compared with tropones. This observation, as well as the fact that the OH band of tropolone was displaced from the normal hydroxyl frequency (3600 cm 1) to a lower frequency region (3165 cm indicated the presence of intramolecular hydrogen bonding (Nozoe, 1956). NMR spectroscopy is a very useful method for structural assignment and isomer characterization. No systematic report has been found giving information on proton (1H) NMR spectra of tropone and tropolone derivatives. This might be due to d i f f i c u l t i e s of the small chemical s h i f t range in proton NMR spectra for the various strongly coupled non-equivalent ring protons. These protons often exhibited second-order multiplets, which were tedious to analyse completely (Bagli and st-Jacques, 1978). On the other hand, because of larger chemical s h i f t s and greater spectral s i m p l i c i t y , some proton decoupled carbon-13 NMR data of tropone and tropolone derivatives have been found in several studies (Weiler, 1972; Bagli and st-Jacques, 1978; Bagli et al, 1979). The chemical s h i f t s in ppm from TMS using CDClg as solvent for the tropone nucleus seemed very simple and 41 distinguishable. They were C-1: 187.7, C-2 and C-7: 141.7, C-3 and C-6: 135.8, C-4 and C-5: 134.4 (Bagli and s t -Jacques, 1978). The carbon-13 chemical s h i f t s for the ring carbons of various substituted tropones are l i s t e d in Table 7 (Bagli et al, 1979). There was a rapid equilibrium between the two tautomers (or resonance structures) of tropolone, as follows: The proton decoupled carbon-13 NMR spectra showed that t h i s compound behaved as a symmetric structure. There were only four s i n g l e t l i n e s in the spectrum ( 1H NMR spectrum of over 20 l i n e s ) . The chemical s h i f t s for C-1 and C-2, C-3 and C-7, C-4 and C-6 had the same values respectively (Weiler, 1972). Neither proton NMR nor carbon-13 NMR spectroscopic data of tropolones derivatives iso l a t e d from WRC heartwood have been reported so far. There has been much speculation on the biogenesis of the natural tropolones. With regard to wood tropolones, Erdtman (1952) discussed the good p o s s i b i l i t y that 42 Table 7. Carbon-13 Chemical S h i f t s of Several Derivatives of 2-methoxytropone in CDC1-, Solution. Compound C-1 C-2 C-3 C-4 C=5 C-6 C-7 c-8 H 180. 1 165. 0 112. 2 132. 4 1 27. 6 136. 3 1 36. 3 55. 9 3-Br 179. 2 162. 9 1 27. 9 1 37. 9 1 28. 1 1 3 4 . 7 1 38. 0 59. 1 7-Br 1 73. 6 162. 6 112. 2 1 32. 8 1 25. 1 1 39. 6 1 37. 5 56. 5 5-Br 179. 2 164. 6 111. 2 1 34. 0 1 22. 2 1 39. 6 1 35. 7 56. 2 3,7-Br 2 173. 1 159. 8 127. 6 137. 7 125. 2 1 38. 1 1 39. 1 59. 5 5,7-Br 3 173. 2 161 . 8 111. 5 1 34. 5 1 19. 3 1 42. 6 1 36. 9 56. 8 5-Cl 178. 8 164. 0 110. 6 130. 4 133. 2 137. 2 1 35. 6 56. 0 3-CH 2C0 2CH 3 181 . 4 164. 4 1 32. 0 1 35. 5 1 29. 4 136. 8 1 38. 6 58. 7 7-CH2C02CH3 1 78. 7 164. 1 112. 2 1 32. 2 1 26. 6 137. 5 1 42. 2 56. 1 3 - C H 3 181. 1 163. 7 1 36. 2 1 34. 8 1 29. 0 1 37. 5 1 36. 2 58. 0 7 - C H 3 179. 1 162. 6 111. 7 1 30. 3 1 26. 4 135. 5 1 45. 6 55. 6 3-OCH3 180. 7 154. 5 1 58. 6 1 27. 8 1 29. 7 1 33. 2 140. 5 58. 6 7-OCH3 1 73. 7 161 . 7 114. 1 1 25. 7 1 25. 7 114. 1 161. 7 56. 2 6-OCH3 177. 8 165. 4 108. 2 1 29. 9 1 24. 0 163. 2 113. 2 55. 0 5-OCH3 178. 6 159. 0 112. 9 107. 1 1 58. 5 132. 1 1 36. 2 54. 9 Source: Bagli et al, (1979). 43 tropolones were derived d i r e c t l y from terpenes or had a common precursor. Although t h u j a p l i c i n s did not possess the usual isoprenoid structure, they might be considered as of terpenoid o r i g i n because they were often found together with terpenoid phenols, such as carvacrol, thymoquinone, and thymohydroquinone and with terpenoid carboxylic acids, such as rhodinic acid and alpha-, beta-chamic acid (Nozoe, 1956). Nootkatin, one of the tropolone derivatives found in yellow cedar, has been referred to as a sesquiterpene tropolone (Barton, 1976), which might be derived from a sesquiterpene d i r e c t l y or by secondary introduction of the isopentenyl side chain to beta-thujaplicin ( H i l l i s , 1962). In the case of microorganism metabolite tropolones, biosynthesis experiments have suggested that they were formed via the acetate mechanism. Carbon-13 biosynthetic studies conducted by Wright and his co-workers (1969) indicated that sepodonin, a tropolone metabolite of Sepedonium chr ys os permum (Bull.) Fr., was formed by methylation of a polyketide intermediate derived from acetate and malonate, followed by a stereospecific rearrangement and c y c l i z a t i o n . 44 2 . 5 . B i o l o g i c a l a c t i v i t i e s o f wood e x t r a c t i v e s The heartwood of many trees, in contrast to the accompanying sapwood, may be remarkably resistant to attack of fungi, insects and sometimes even marine borers (Wise, 1944). It i s now generally accepted that high heartwood d u r a b i l i t y p a r a l l e l s the presence of extraneous non-structural components, such as tropolones (Barton and MacDonald, 1971), resins (Richardson, 1978), tannins (Hart and H i l l i s , 1972), lignans (MacRae and Towers, 1984), and other phenolics. It had been established for a long time that two major groups of extractives in WRC, tropolone derivatives (especially thujaplicins) and water soluble phenols (lignans) have fungicidal properties. Rennerfelt (1948) tested the t o x i c i t y of t h u j a p l i c i n s . He found that decay fungi were inh i b i t e d with concentration between 0.001 and 0.002 percent of gamma-thujaplicin (10-20 ppm). Phenol, tested in the same way, inhibited the decay fungi at 0.1 to 0.2 percent concentration. He concluded that t h u j a p l i c i n s were about 10 times more e f f e c t i v e than pinosylvin, the most potent fungicide in hard pine heartwood (Rennerfelt, 1945). After comparison of i n h i b i t i n g e f f e c t s 45 between t h u j a p l i c i n and sodium pentachlorophenol, a synthetic product which has become widely used as an eff e c t i v e fungicide against decay and blueing fungi on timber, he indicated that their a c t i v i t i e s were of the same order. In the case of blueing fungi, Rennerfelt (1945) also showed alpha-, beta-, and gamma-thujaplicins to be toxic in low concentrations with beta being the most toxic, followed by gamma, then alpha using the agar d i f f u s i o n method. Rudman conducted research on causes of natural d u r a b i l i t y in timber (Rudman, 1962; 1963). A number of heartwood extractives and related compounds were examined by a semi-micro technique which used a wood substrate in the form of sawdust. Results of his research are shown in Table 8 (Rudman, 1963). He found that beta and gamma th u j a p l i c i n s , and beta-thujaplicinol were the most toxic to a l l the species of decay fungi tested by comparison with other extractive chemicals at 1 percent w/w concentration in wood sawdust. He also indicated t h u j a p l i c i n s to be as toxic as the naphthopyran lapachonon (Rudman, 1963). The antifungal a c t i v i t y of hinoki {Chamaecyparis obtusa Endl.) extractives which i s r i c h in th u j a p l i c i n s was examined and proved to be highly toxic to a c u l t i v a t i o n culture media of Basi di omycet es (Kinjo and Yaga, 1986). 46 Table 11 Results of To x i c i t y Tests for a Number of Heartwood Extractives (Decay as a percentage of decay in c o n t r o l ) . Fungi C. elivacea 1779 L. ttpldtut 7516 L. trabta 7520 P. monlicola 7522 Pfrcfntapf Concentration w/w ot Extiatt lvt In Sawdutt 0.11 0.33 1.00 0.11 0.33 1.00 0.11 0.33 1.00 0.11 0.33 1.00 B e n t t n c D e r i v a t i v e * 104 92 28 106 69 39 78 72 1 76 70 87 109 110 69 64 62 76 76 45 30 65 103 81 B5 77 75 66 68 61 71 75 53 78 76 84 eo 109 102 114 120 105 62 102 101 60 76 85 100 60 66 104 115 107 68 63 65 100 65 62 105 103 110 68 112 88 62 65 60 62 85 60 107 126 104 85 66 70 65 64 11 62 63 79 06 103 120 86 65 79 68 68 60 104 100 69 115 115 110 78 78 88 64 66 80 78 88 106 62 69 64 60 60 6 69 89 78 85 63 66 104 104 65 67 64 71 102 88 102 113 64 100 63 76 69 61 84 64 61 87 59 103 103 68 N a p h t h o p y r a n 108 48 0 65 44 13 93 0 I 61 61 0 Q u l n o n o 101 87 101 69 58 54 69 67 47 100 79 45 102 103 107 105 78 104 67 66 63 78 69 78 Anlhraq-jinont-2-carboxyllc acid . . 09 64 87 62 66 65 67 64 7 44 25 6 C o u m a r l n s 69 82 83 63 103 67 66 89 92 B7 84 63 106 107 102 63 62 46 117 110 121 70 67 66 90 67 69 65 67 0 62 81 12 61 100 34 106 60 109 — — 71 — — 109 118 72 70 n o 75 — 106 64 — 100 95 105 65 77 24 88 73 0 63 71 0 es 62 100 60 60 2 60 BS 0 75 88 70 104 66 107 30 4 0 78 B9 23 85 71 68 S t U b e n e s 64 86 83 74 71 88 87 65 89 86 60 100 91 69 63 101 62 69 107 104 0 65 91 62 65 64 BB 70 68 39 87 66 86 69 64 42 66 62 61 68 80 43 64 65 66 82 107 101 T t o p o l o n e a 112 69 62 63 64 29 — — 75 60 — 113 113 30 53 7 0 0 0 1 • 35 0 0 102 60 8 84 31 6 81 6 10 63 11 0 F l a v o n o l d * d-CBtfchin 60 63 60 63 80 86 71 84 75 68 62 69 89 83 63 79 79 82 72 65 72 Ta*lfolin 66 66 88 70 55 40 66 63 62 87 77 81 62 69 65 104 84 61 83 61 86 71 77 83 92 61 — 66 79 78 61 89 87 Rcbiiiftln 78 66 68 49 36 14 62 104 62 83 74 86 61 88 87 81 65 64 61 78 66 62 85 84 66 85 62 64 Arorr.aflf ndrln 83 eo 66 66 114 69 86 86 Aromadrndrin-7-mtthylether . . . 62 62 89 68 61 76 89 61 64 65 66 60 9B 69 76 69 75 69 64 67 61 ' 65 106 BB 104 67 62 111 107 67 -103 63 B6 77 82 67 62 87 81 102 83 66 64 86 79 106 103 69 76 87 59 100 105 63 87 80 B7 66 68 103 -— — 102 107 84 101 68 84 67 86 73 64 — 62 67 63 100 104 69 80 76 88 __ 69 61 64 78 102 too 75 62 87 66 97 95 82 80 63 107 I OB 106 60 102 101 63 90 71 89 80 81 es 69 100 84 63 68 62 81 15 64 61 86 2-HydTexy-4 l6-dimethoxychalkone . 63 83 79 62 63 69 66 88 69 99 79 64 65 101 86 B2 69 64 95 106 98 87 98 102 L l g n a n t • P8 63 66 78 63 85 66 101 93 96 101 109 62 69 86 BO 64 6 109 107 n o 80 100 65 — — — 81 96 82 97 86 88 13 63 90 C o m m e r c i a l F u n g i c i d e s B3 52 0 . 16 11 2 92 66 0 65 36 0 61 0 0 74 17 II 73 B2 0 90 74 — 88 44 0 0 2 0 0 0 0 0 0 0 0 0 0 28 2 6 0 0 0 *> Ether-methanol extracted mountain ash ( £ . rifnans) heartwood was uted a i the tubttrate. Source: Pudman, (1963). 4 7 Toxicity tests of the water soluble phenolic fracti o n of WRC heartwood extractives conducted by Roff and Atkinson (1954) showed that the water soluble phenols in WRC were also toxic to decay fungi, though they were much less toxic than t h u j a p l i c i n s . The t o x i c i t y to fungi of this fr a c t i o n was of the same order as that of zinc chloride (Roff and Atkinson, 1964), which was roughly one two-hundredth of that of the t h u j a p l i c i n s . However, since the concentration of the phenolic fracti o n in the inner heartwood of WRC was frequently 20 to 100 times that of the thu j a p l i c i n s (Barton, 1962), i t was apparent that t h i s fraction of the extractives also had a certain role as a natural preservative. A l l previous reports on the higher antifungal a c t i v i t y of t h u j a p l i c i n s were based on the results obtained by using well known decay fungi, Basidi omycet es , as testing organisms. These results were useful for considering t h u j a p l i c i n s as a potential wood preservative in lumber treatment in service. However, these results f a i l to explain the real role of t h u j a p l i c i n s in standing trees, simply because the results of interaction between th u j a p l i c i n s and non-hymenomycetous "pioneer" organisms could affect the whole process of microorganism progression in standing WRC trees. 48 The a n t i b a c t e r i a l a c t i v i t y of beta-thujaplicins was demonstrated by Turst and Coombs (1973). They showed that beta-thujaplicin was a broad-spectrum a n t i b a c t e r i a l compound. It was bacterostatic for gram-positive and gram-negative species. At concentrations greater than 100 ug/ml, i t was b a c t e r i c i d a l for a number of gram-negative species. Coombs and Turst (1973) found that the a n t i b a c t e r i a l a c t i v i t y of beta-thujaplicin was lost following exposure to laboratory l i g h t . Photochemical decomposition of beta-t h u j a p l i c i n was detected during their study by u l t r a v i o l e t spectral analysis of the compound. The e f f e c t s of beta-t h u j a p l i c i n on the metabolism of yeast had been investigated by Raa and Goksoyr (1965). The i n h i b i t i o n in respiration caused by t h u j a p l i c i n and i t s cupric chelate seemed to act very much in the same manner, but the l a t t e r compound was 70 to 100 times more to x i c . Previous work (Barton and MacDonald, 1971) indicated that there was a strong relationship among wood color, the amount of wood extractives and the decay resistance of WRC heartwood. The v a r i a t i o n in the pattern of decay resistance p a r a l l e l e d v a r i a t i o n in content of the extractives (mainly t h u j a p l i c i n s ) . The color of WRC heartwood ranged from light-straw through pinkish and tan-brown to a dark 49 chocolate brown. According to the results of MacLean and Gardner, (1956b; 1958), wherever a color t r a n s i t i o n from l i g h t to darker color was present in a sample, the t r a n s i t i o n was accompanied by a sharp drop in the content of t h u j a p l i c i n s , the dominant natural preservative in the wood, together with the water soluble phenols. The change in t h u j a p l i c i n content with color was much more pronounced than i t was for water soluble phenols. These phenomena were further observed by Roff (1961). Work carr i e d out recently (Doyle, 1985) on testing the d u r a b i l i t y of second growth and fast-grown WRC wood against a number of decay fungi showed a similar pattern to the old growth wood. It was apparent in his study that decay resistance increased with r a d i a l distance from the p i t h . A rather interesting piece of research done by Wilcox and P i i r t o (1976) provided evidence concerning the relationship between color variatio n across a section and the natural decay resistance of redwood (Sequoia sempervirens (D.Don.) Endl.). Redwood has a wide degree of natural v a r i a b i l i t y in both the shade and intensity of heartwood color. In their study, water and ethanol were used as extracting solvents. Their results showed that dark colored wood had the highest extractives content. When the 50 darkness of wood color increased, the content of ethanol-soluble extractives increased. They demonstrated that as content of ethanol soluble extractives increased, that i s , the color of the wood darkened, weight loss of the wood against an exceptionally aggressive wood destroyer, P. moniicola, tended to decrease. Thus, they generalized that in redwood, darkness of heartwood color appeared to be a r e l a t i v e l y good index of decay resistance, with the darkest boards tending to have the highest resistance. The patterns of coloration of wood v s . decay resistance in WRC and redwood were opposite. This kind of contradiction may be due to difference in methods; species of wood; or the microorganisms used, and indicated a need for further detailed research on the relationship of chemical structural changes and dis c o l o r a t i o n , and the mechanisms of extractive t o x i c i t y in natural d u r a b i l i t y . Heartwood extractives of other trees also showed varied b i o l o g i c a l a c t i v i t y . The hexane, chloroform, and methanol extractives of the heartwood of Madura pomifera (Raf.) Schneid were reported (Wang and Hart, 1983) to be toxic to some wood decay fungi, such as Coriolus versicolor (L.) Quel. and Gloeophyllum trabeum (Pers. ex.Fr.) Mull. The major fr a c t i o n of the extractives responsible for the decay 51 resistance, according to their r e s u l t s , appeared to be a mixture of t e t r a - and penta-hydroxystilbenes with tetrahydroxystilbenes predominating. The t o x i c i t y of extractable lignans from sound and Fomes annosus (Fr.) Karst. affected heartwood of Norway spruce were examined by Shain and H i l l i s (1971). Hydroxymatairesinol, matairesinol, and conidendrin were i d e n t i f i e d from the extractives. Bioassay tests using F. annosus (Fr.) Karst. in their study indicated that conidendrin was not i n h i b i t o r y at any of the concentrations tested, whereas matairesinol and hydroxymatairesinol s i g n i f i c a n t l y i n h i b i t e d mycelial growth. The effect of concentration was not s i g n i f i c a n t for matairesinol, but was highly s i g n i f i c a n t for hydroxymatairesinol, which was more inh i b i t o r y than the former at a l l concentrations tested. They suggested a r e l a t i o n s h i p between s o l u b i l i t y of the fungal i n h i b i t o r s and decay resistance of these i n h i b i t o r s . They discovered that a compound with r e l a t i v e l y low aqueous s o l u b i l i t y (e.g. pinosylvin) would have to be active at lower concentrations than one with r e l a t i v e l y high aqueous s o l u b i l i t y . Doi hi stroma pi ni Hulbary i s a s i g n i f i c a n t leaf pathogen of young Pi nus radiata D.Don. plantations in New Zealand. 52 Bioassays of selected fatty and resin acids using Dot hi stroma pi ni Hulbary. as test organism (Franich et al , 1983) have shown that a long chain fatty acid ( s t e a r i c ) , omega-hydroxy fatty acids and oxidized resin acids (7-keto and 7-hydroxydehydroabietic acids; 13-hydroxypodocarpa-8,11 ,13-trien-18-oic acid) were highly f u n g i s t a t i c . The compounds inhi b i t e d both Dot hi stroma pi ni Hulbary. spore germination and mycelial growth in v i t r o . The s i g n i f i c a n t antifungal e f f e c t s of selected bark extractives of Carya ovata M i l l . , Quercus rubra (red oak), and P. strobus (white pine) were observed on the growth medium of Lenzites trabea (Harun and J r , 1985). The b i o l o g i c a l a c t i v i t i e s of Japanese red pine (Pinus densiflora S. et Z.) and Japanese white birch (Betula pi at yphylI a Sukatcev ver. japonica Hara) extractives in kraft black liquors and kraft bleach effluents were reported by Sameshima and his coworkers (Sameshima et al 1978; 1980 and 1986). They found that the extractives obtained by d i e t h y l ether extraction and steam d i s t i l l a t i o n had r e l a t i v e l y higher termite k i l l i n g a c t i v i t y (Sameshima et al , 1980). Six phenols and four fatty and resin acids from the extractives were responsible for the t o x i c i t y . Their t o x i c i t y l e v e l s were determined by gas-liquid chromatography 53 (Sameshima et al, 1986). Many works have pointed out that the d u r a b i l i t y of wood in service and the decay resistance of heartwood in l i v i n g trees were dependent on the t o x i c i t y of i t s extractives, their concentration and their s p e c i f i c i t y to certain species of fungi. As reported by a number of researchers, some phenolic extractives had no toxic effect to certain species of fungi. On the other hand, extractives might have been modified or u t i l i z e d by the fungi as an energy source in v i t r o . Nootkatin, one of natural tropolones isolated from heartwood of yellow cedar (Chamaecyparis noolkatensis (D. Don) Spach.) had been tested e a r l i e r by Rennerfelt and Nacht (1955) and had been found to be fungistatic at concentrations of about 0.001 percent and fungicidal at 0.005 percent against a number of wood-destroying fungi. Later in 1970, several unidentified fungi which cause black stain in yellow cedar were isolated (Smith, 1970) and the tolerance of these fungi to nootkatin was examined (Smith and C s e r j e s i , 1970). They found a considerable decrease of nootkatin content without s i g n i f i c a n t weight loss in clear yellow cedar blocks af t e r six unidentified blackstain fungi grew on them. They suspected that the lower concentration 54 of nootkatin after the treatment was due to the degradation of nootkatin by these fungi, and this effect resulted in a great decrease in the decay resistance of the wood to other unidentified wood-destroying fungi. No attempts of i s o l a t i o n and i d e n t i f i c a t i o n of degraded compounds were made in the study. In 1975, van der Kamp (1975) isolated three fungi, whose code numbers were WR1, WR2 and WR3, from old growth WRC on the B. C. coast from light-straw, red and brown stained sound wood. The bioassays for testing the t o x i c i t y of t h u j a p l i c i n s to these three fungi were performed (van der Kamp, 1986). He found that both WR1 and WR2 invaded s t e r i l e heartwood and quickly reduced even high t h u j a p l i c i n concentrations to trace amounts and then caused a considerable reduction in the natural decay resistance of the wood in test blocks. He supported the hypothesis that decay of WRC involved a succession of organisms and the early invaders (possibly WR1) somehow destroyed the toxic natural extractives present in the heartwood. Work done by Edmonds (1976) was similar to the work by Shain and H i l l i s (1971). In addition to the phenolic compounds catechin and leucocyanidin, the lignans: matairesinol and hydroxymatairesinol, which were tested in 55 Shain and H i l l i s ' (1971) work, were also used by Edmonds. The test organism was also the same: F. annosus (Fr.) Karst. The only difference was that these compounds were isolated from the d i f f e r e n t tree species, one was from Norway spruce and the other from western hemlock. Edmonds' result (1976) indicated that extractives from western hemlock heartwood were not capable of i n h i b i t i n g the growth of F. annosus (Fr.) Karst. Though leucocyanidin i n h i b i t e d growth of the fungus at higher concentrations in culture media, i t was present in only small amounts in wood extractives, and apparently did not show any ef f e c t at naturally occurring l e v e l s . On the other hand, i t was proved by him that the F. annosus (Fr.) Karst. was capable of modifying and u t i l i z i n g these phenolic extractives. Edmonds' result contradicted that of Shain and H i l l i s (1971). The contradiction may ari s e from the test organism F. annosus (Fr.) Karst. used in both experiments. According to Korhonen (1978) F. annosus consisted of at least two c o l s e l y related i n t e r s t e r i l i t y groups which on average d i f f e r from each other in many properties. One group was found mostly on Norway spruce (S group), which was the most important cause of butt rot of Norway spruce. The other one was found only on pine (P group). If the F. annosus used in 56 both experiments were not the same, the d i f f e r e n t results may by the consequence. Two of early stage attacking fungi, Phi al ophora parasitica A j e l l o et al. and PaeciI omyces spp. were used in the study of the t o x i c i t y tests of tallowwood (Eucalyptus microcorys F. Muell.) heartwood extractives (Wilkes and Heather, 1983). They found that these two microfungi were apparently quite tolerant to naturally occurring extractives from clear wood and the modified extractives of discolored tissues. This tolerance enhanced the fungus' a b i l i t y to act as pioneer invaders. The a b i l i t y of fungi to u t i l i z e naturally occurring heartwood extractives was examined by their growth in a nutrient solution containing water-soluble extractive of tallowwood heartwood as the only source of carbon (Wilkes and Heather, 1983). Their results suggested that these organisms remove or modify natural preservatives to a form less i n h i b i t o r y to the Basi di omycetes or even to a form which can be used as source of energy. The PaeciIomyces spp. was more capable than P. parasitica A j e l l o et al. of using extractives in nutrient solution as the sole source of energy. 57 2.6. P o s s i b l e mechanisms of e x t r a c t i v e t o x i c i t y I t has been supposed by some workers t h a t compounds in heartwood e x t r a c t i v e s prevent the i n v a s i o n of fung i or i n s e c t s e i t h e r m e c h a n i c a l l y , c h e m i c a l l y , b i o c h e m i c a l l y or by combinat ions of these p r o c e s s e s . I t has been demonstrated by Hart and H i l l i s (1972) that i n h i b i t i o n of the w o o d - r o t t i n g fung i P. monticola M u r r . in heartwood of white oak {Quercus alba L . ) was due to the presence of water s o l u b l e e l l a g i t a n n i n s . The a d d i t i o n of PVP ( p o l y v i n y l p y r r o l i d o n e ) or Tween 80 ( p o l y o x y e t h y l e n e s o r b i t a n monooleate) to the growth medium t o t a l l y overcame the f u n g i s t a t i c e f f e c t s of the e l l a g i t a n n i n s . PVP and Tween 80 are c a p a b l e of s p l i t t i n g t a n n i n - p r o t e i n complexes and thus regenera te enzyme a c t i v i t y . O r d i n a r i l y , the t a n n i n -p r o t e i n complex have been e x t r a o r d i n a r i l y s t r o n g . They suggested tha t the e l l a g i t a n n i n e f f e c t was p r o b a b l y due to i n t e r a c t i o n between t a n n i n molecu le s and f u n g a l p r o t e i n s , and f o r m a t i o n of a hydrogen-bonding t a n n i n - p r o t e i n complex which a f f e c t e d the f u n g a l membrane p e r m e a b i l i t y . The t o x i c e f f e c t s of t a n n i n on fung i had been r e p o r t e d e a r l i e r in white oak heartwood ( Z a b e l , 1948). The t o x i c i t y of p o d o c a r p i c a c i d (PCA) , a k i n d of 58 l i g n a n , d e p o s i t e d i n h e a r t s h a k e s of s e v e r a l Dacrydium spp. was i n v e s t i g a t e d (Bauch et al, 1977). The f u n g i s t a t i c PCA c o n c e n t r a t i o n was 0.05 p e r c e n t of heartwood w i t h Chaetomium globosum (Kunze. ex. F r . ) s t r . and 0.25 p e r c e n t w i t h Coniophora puteana (Schum. ex. F r . ) K a r s t . s t r . Those workers s u g g e s t e d t h a t PCA i n the t i s s u e might a c t as the m e c h a n i c a l b l o c k a g e f o r b a c t e r i a , f u n g i and i n s e c t s . The p h e n o l i c s t i l b e n e compound p i n o s y l v i n , formed a f t e r sapwood was i n f e c t e d w i t h the Si rex spp. fungus, c o u l d p l a y an i m p o r t a n t r o l e i n r e s t r i c t i n g the spread of t h e fungus ( H i l l i s and Inoue, 1968). P i n o s y l v i n was found t o be r a t h e r more t o x i c i n most c a s e s and a more u n i v e r s a l p o i s o n than i t s monomethyl e t h e r , which was an i n h i b i t o r i n 0.01 t o 0.02 p e r c e n t c o n c e n t r a t i o n . - I t was proposed t h a t p i n o s y l v i n 1 s i n h i b i t i o n e f f e c t was due t o both a f u n g i t o x i n and i n a c t i v a t i o n of the e x t r a - c e l l u l a r enzymes. Study on the e f f e c t s of WRC e x t r a c t i v e b e t a - t h u j a p l i c i n on m e t a b o l i s m of y e a s t was r e p o r t e d by Raa and Goksoyr (1965). They showed t h a t b e t a - t h u j a p l i c i n i n c o n c e n t r a t i o n s -4 -6 above 10 M and c u p r i c t h u j a p l i c i n c h e l a t e above 10 M i n h i b i t e d t h e r e s p i r a t i o n of b a k e r ' s y e a s t , the endogenous r e s p i r a t i o n as w e l l as the r e s p i r a t i o n a f t e r a d d i t i o n of g l u c o s e , e t h a n o l or sodium a c e t a t e . T h e i r experiment 59 d e m o n s t r a t e d t h a t b e t a - t h u j a p l i c i n c o u l d f o r m m e t a l c h e l a t i n g c o m p o u n d s w i t h c o p p e r , i r o n ( I I ) a n d o t h e r s , w h i c h w e r e f a r more t o x i c t h a n t h e f r e e b e t a - t h u j a p l i c i n . T h e y d i s c u s s e d t h e p o s s i b i l i t i e s t h a t i n h i b i t i o n o f b e t a -t h u j a p l i c i n a n d i t s c h e l a t i n g c o m p o u n d s mus t h a v e a c t e d on t h e g l y c o l y t i c p a r t o f t h e r e s p i r a t o r y p a t h w a y a n d on t h e e l e c t r o n t r a n s f e r s y s t e m i n t h e c i t r i c a c i d c y c l e . The i n h i b i t i o n o f s u c c i n a t e a n d a c e t a t e r e s p i r a t i o n s h o w e d t h a t t h e r e s p i r a t o r y p a t h w a y f o r t h e s e s u b s t r a t e s was b l o c k e d . T h e y c o n c l u d e d t h a t one p o s s i b l e t a r g e t o f t h u j a p l i c i n a n d c u p r i c c h e l a t e may h a v e b e e n s u c c i n i c d e h y d r o g e n a s e . H o w e v e r , t h e y s p e c u l a t e d t h a t t h e s e two c o m p o u n d s i n a c t i v a t e d t h e enzyme s y s t e m s , b u t d i d n o t a c t e n t i r e l y on t h e same enzyme s y s t e m . I n t h e c a s e o f b a c t e r i a , Aeromonas spp. a n d o t h e r g r a m -n e g a t i v e s p e c i e s , b e t a - t h u j a p l i c i n i n h i b i t i o n a p p e a r e d t o i n t e r f e r e w i t h c e l l w a l l i n t e g r i t y , a n d c e l l d e a t h r e s u l t e d f r o m l y s i s by o s m o t i c p r e s s u r e ( T u r s t a n d C o o m b s , 1 9 7 3 ) . A n o t h e r p o s s i b l e mode o f a c t i o n o f b e t a - t h u j a p l i c i n t o c e r t a i n g r o u p s o f b a c t e r i a m i g h t h a v e b e e n t o d i s r u p t membrane f u n c t i o n o f t h e b a c t e r i a (Hugo a n d B l o o m f i e l d , 1 9 7 1 ) . I n h i b i t i o n o f t h e enzyme c a t e c h o l O - m e t h y l t r a n s f e r a s e 60 (COMT) by tropolones has been reported (Belleau and Burba, 1963). In the study, the enzyme was p u r i f i e d and a catechol methylation reaction was car r i e d out with COMT in the presence of tropolone derivative and without the tropolone derivative. The inh i b i t o r y a c t i v i t y of tropolone derivatives toward the COMT was observed. It was confirmed that tropolones acted as s p e c i f i c i n h i b i t o r s of the (COMT) enzyme. A structure similar to that for COMT was proposed for the complex between tropolones and s-adenosyl methionine (Figure 6). The mechanism by which WRC th u j a p l i c i n s i n h i b i t decay fungi was unknown. No experiments have been reported in this matter, so far. The most common hypothesis was that the presence of the reactive keto-enolic group was necessary for toxic action. Concentrations of minerals have been shown to be greater in discolored and decayed wood in l i v i n g trees than in healthy clear wood of the same trees on wt/wt basis (Shigo and Sharon, 1970). Rennerfelt (1962) compared sound heartwood and decayed heartwood of spruce stems as to metal ion contents. He found a. remarkable increase in the potassium and calcium content in decayed heartwood. He suggested that active accumulation of metal ions i s caused 61 Figure 6. The Complex Structure between Tropolone, Magnesium and S-Adenosyl Methionine. *: Dot l i n e s represent hydrogen bonds between proteins and the complex. Source: Belleau and Burba, (1963). 62 by l i v i n g hyphae from the transpiration stream. Safford and Shigo (1974) found that the concentration of potassium, manganese, calcium and magnesium were a l l higher in decayed heartwood. It was not clear so far i f metal ion accumulation in decayed heartwood was due to the metabolic a c t i v i t y of the fungus or a defence mechanism of the tree, or both. Metal elements, especially iron, were very important for microorganisms to grow. Iron, because of i t s a b i l i t y either to accept or donate electrons, participated in a variety of oxidation reduction reactions, making i t indispensable for a variety of enzymatic reactions in almost a l l organisms. Microorganisms produced siderophores as secondary metabolites (Moody, 1986). These siderophores, such as catechol and hydroxamate type compounds, contained available oxygen atoms. These oxygen atoms can bind f e r r i c iron to form a stable complex (Neiland, 1981). Once siderophores capture iron, to be of any use, i t must be transported into the c e l l . This was accomplished either by the uptake of the intact iron-siderophore complex or by removal of the iron at the c e l l surface by membrane receptors, which had the special function to recognize iron-siderophore complex (Moody, 1986). Currently, two possible 63 mechanisms for the removal of iron from a siderophore were considered. Hydrolysis of the siderophore followed by iron removal or, in situ reduction and release of the iron (Raymond et al ,1984). If the process of translocating iron was disrupted by other forces, the microorganisms would not survive. Cowling and Brown (1969) stated that the wood c e l l wall c a p i l l a r y structure was too small to allow the migration of decay enzymes into wood. Therefore, the a b i l i t y of brown-rot fungi to i n i t i a t e decay in the secondary c e l l wall and the rapid, widespread depolymerization of the c e l l u l o s e did not appear to be attributable solely to enzymatic mechanism. From the results of a series of studies (1972a; 1972b; 1973; 1974a; 1974b), Koenigs proposed for the f i r s t time that fungi may have attacked c e l l u l o s e and partly decay wood via an H20 2 _iron non-enzymatic system. The rate of c e l l u l o s e oxidation by the iron-H2C>2 system was dependent on the rate of decomposition of H2^2 t o hydroxyl and hydroperoxy r a d i c a l s , which were the active oxidizing agents. It has been established (Buchanan et al , 1976) that e x t r a c e l l u l a r H2^2 P r°duced by brown-rot fungi decomposed much more rapidly in the presence of Fe(II) as compared to F e ( l I I ) . Schmidt et al (1981) demonstrated that oxalic 64 acid, which was a fungal secretion product, reduced naturally occurring f e r r i c iron (Fe III) in wood to the ferrous (Fe II) state. Experiments conducted by Nicholas (1981) found that oxalic acid was also one of the degradation products in the H 20 2 -Fe (II) reaction with c e l l u l o s e . This endogenous oxalic acid participated in the reduction of more f e r r i c irons to ferrous irons in the system, which accelerated the whole degradation process. Thus, the system of H2^2 n o n _ e n z y i n a t i - c decay was well established. Such a system could have functioned in a preparatory way by acting on the wood pore structure, thus increasing the wood's s u s c e p t i b i l i t y to subsequent enzymatic attack. If tropolone derivatives in WRC can capture either Fe(III) in the wood or Fe(II) in the process to form chelates, the non-enzymatic H 2 0 2 i r o n system w i l l be blocked. Among WRC heartwood extractives, a c h a r a c t e r i s t i c of tropolone derivatives was their a b i l i t y to combine rapidly with metals, such as copper, f e r r i c ion, etc. to form a stable chelate. The s t a b i l i t y constants of CuT 2 and FeT 2 were calculated to be 14 X 10 1 4 and 7 X 10 1 1. The data for formation constants of the tropolone metal complexes and derivatives were also reported e a r l i e r by Bayant and his co-workers (1953; 1954a; 1954b; 1954c). Similar chelates can 65 be formed w i t h c a t e c h o l . Some s t r u c t u r e s of th e s e c h e l a t e complexes a r e shown i n F i g u r e 7. One v e r y l i k e l y a c t i o n of t h u j a p l i c i n s ' t o x i c i t y t o decay f u n g i i s the f o r m a t i o n of me t a l c h e l a t e s w i t h those i r o n s , which a r e e s s e n t i a l f o r f u n g a l s u r v i v a l or f o r m i c r o o r g a n i s m s u c c e s s i o n . On the o t h e r hand, m e t a l i o n s show t o x i c e f f e c t s a t h i g h e r c o n c e n t r a t i o n s t o f u n g a l growth. T h i s i s one of the b a s i c mechanisms of the m e t a l complex wood p r e s e r v a t i v e s . T h e i r p r e v e n t i v e b e h a v i o r p r o b a b l y i s due t o the c a t i o n ' s d i r e c t t o x i c a c t i o n , or t h e i r a p p a r e n t , r e p e l l e n t a c t i o n , or t h e i r c h e m i c a l m o d i f i c a t i o n t o the s u b s t r a t e so t h a t i t has become r e s i s t a n t t o the f u n g a l enzyme system. The most common such p r e s e r v a t i v e i n use i s copper-chromium-arsenic s a l t s (CCA) ( R i c h a r d s o n , 1978). T h e r e f o r e , t h u j a p l i c i n m e t a l c h e l a t e s may be more t o x i c t o decay f u n g i than t h u j a p l i c i n s t h e m s e l v e s . In a d d i t i o n t o the c a p a b i l i t y of t r o p o l o n e d e r i v a t i v e s t o e a s i l y form m e t a l c h e l a t e complexes, o t h e r a c t i v i t i e s of the k e t o - e n o l i c group s h o u l d be ta k e n i n t o account when the mechanism of t o x i c i t y i s c o n s i d e r e d . The a b i l i t y t o u t i l i z e l a r g e m o l e c u l e s i n the wood c e l l w a l l depends on the a b i l i t y of the fungus t o d i g e s t the m o l e c u l e s , which i n t u r n depends on the enzymes w i t h which the fungus i s equ i p p e d . Enzymes 66 Figure 7. The Chemical Structures of the WRC Extractive F e r r i c Chelates. I Fe + 3 beta-thujaplicin f e r r i c chelate Fe + 3 J 3 catechol f e r r i c chelate Source: Barton and MacDonald, (1971). 67 are b u i l t up of proteins which in turn consist of the combination of various amino-acids. The a b i l i t y of tropolone derivatives to form amine-tropolone salts (Poh, 1974) and also to form hydrogen bonds with proteins provide possible mechanisms for toxic action. 68 MATERIALS and METHODS 3.1. Sample selection The WRC wood sample for this study was c o l l e c t e d at the U.B.C. Research Forest, Maple Ridge, B. C. in the Gwendoline Lake area, which has WRC trees over 350 years old. Several trees were examined using a 30 cm power borer to obtain samples which were observed for color v a r i a t i o n (general pattern) and decay l e v e l . Several desirable trees were found, but only one of these was f e l l e d . The tree sampled was approximately 30 m in height -(Figure 8) and 84 cm in diameter at breast height. The tree age was determined by counting growth zones throughout a cross-section with assistance of a hand lens. The age of the tree was 420 years. Starting at breast height, four 0.9 m length bolts were cut (Figure 9) and removed, and one 0.9 m bolt was cut and removed from 20 m above the stump. Each bolt was s p l i t into four lengthwise sections in order to transport them to the U.B.C. campus laboratory. Sections were la b e l l e d and then stored in a cold room (2°C) at the Faculty of Forestry, U. B. C. immediately after a r r i v a l . In order to maintain the wood freshness, two of 69 Figure 8. Tree from Which the Wood Samples Were Taken 70 F i g u r e 9. Four 0.9 Meter B o l t s Cut from the S e l e c t e d Tree. the most representative sections taken at breast height were kept in a p l a s t i c bag in a freezer at about -5°C. The color pattern of the tree cross-section i s shown in Figure 10. The wood color variation followed t y p i c a l patterns for this species. It ranged from white in sapwood, light-straw in outer heartwood, through pinkish, brown and tan-brown in inner heartwood, to dark-brown in the central area. In addition to t h i s normal color pattern, a pocket of pink-brown wood was observed in one quadrant. This can be seen c l e a r l y (Figure 10B) in the outer heartwood to inner heartwood region. This discolored pocket was surrounded by straw-colored heartwood and can be traced to an injury scar at breast height (Figure 10A). The quadrant with the discolored pocket provided very good samples for comparison of chemical composition of discolored and sound WRC heartwoods. 72 F i g u r e 10. Cross S e c t i o n s of the S e l e c t e d Tree Showing C o l o r V a r i a t i o n s . 10A: Cross s e c t i o n at 10B: Cross s e c t i o n at 0.9 ra breast h e i g h t . above breast h e i g h t . 73 3.2. Isolation of microorganisms Microorganisms were isolated from one quadrant of the bolt taken at breast height. The quadrant involved a large amount of light-straw colored heartwood on one side and a large amount of discolored heartwood on the other side of the same growth zones. The quadrant was further s p l i t r a d i a l l y lengthwise into two sections and one of the sections, including the discolored pocket, was further s p l i t into Pieces A, B and C (Figure 11). The exposed r a d i a l surface of Piece A consisted of five color zones: White colored sapwood (7 growth zones); Light-straw colored outer heartwood (16 growth zones); Reddish colored light-brown colored inner heartwood (53 growth zones); and Brown to dark-brown colored inner heartwood (134 growth zones). Piece A was used i s o l a t i n g microorganisms, while Pieces B and C were used in extractive studies. Fungi grow normally as saprophytes or parasites on naturally occurring animal or plant products. These products can be extremely complex materials which provide a number of nutrients, including some unknown, for the growth of the fungus. A primary requisite for laboratory c u l t i v a t i o n of fungi i s selection of a suitable substratum. 74 F i g u r e 1 1 . R a d i a l P i e c e s S p l i t from a Quadrant and Used in the I s o l a t i o n of M ic roorgan isms (A) and E x t r a c t i v e s (B and C ) . A B C 7 5 There are two types of media used in laboratory work, natural media and synthetic media. Natural media, such as malt extract and yeast extract, are the materials on which fungi would o r d i n a r i l y grow in nature. These natural media are autoclaved and used d i r e c t l y for fungal growth in laboratory experiments. A disadvantage of natural media is that they can never be p r e c i s e l y duplicated for studies as they are of unknown composition. A synthetic medium consists of chemicals (such as D-glucose, asparagine, magnesium sulphate, or biotin) which are of known composition and concentration. This is the only type of medium which may be precisely duplicated and i s suitable for physiological studies. Unfortunately, some fungi may grow poorly or not at a l l on synthetic media. In the present study, i s o l a t i n g and developing fungi from heartwood of the sampled tree as "normally" as possible was of most importance, therefore, the natural medium was preferred. A fresh uncontaminated r a d i a l wood surface from Sample A was exposed under s t e r i l e conditions on a laminar a i r flow bench. Small chips of wood removed from the r a d i a l surface were placed in Petri dishes on a culture medium consisting of 30 ml of 2% agar, 3% malt extract and 0.5% yeast extract. There were four chips in each dish. A minimum of four chips 76 were removed from every f i v e annual rings. The Petri dishes were maintained at room temperature in a dark incubator for about two months with regular examination. 3.3. Determination of extractives Wood specimens used for extractives determination were obtained from Pieces B and C (Figure 11). Pieces were cut into small wood s l i c e s and then divided into four groups according to growth zone and color v a r i a t i o n . The five groups were: 1. White colored sapwood from 413 to 420 years old (S-P); 2. Discolored outer heartwood from 303 to 412 years old (D-O); 3. Light-straw colored outer heartwood from 303 to 412 years old (L-O); 4. Discolored inner heartwood from 198 to 302 years old (D-I); and 5. Light-straw colored inner heartwood from 198 to 302 years old (L-I). The samples were a i r - d r i e d , and ground in a Willey m i l l to pass a fifty-mesh screen. Moisture content of these samples were determined by oven-drying. They were 57.5%, 77 31.36%, 27.45%, 28.22% and 23.56% for S-P, D-0, L-0, D-I and L-I, respectively. Nearly ten grams oven-dry (O.-D.) basis of wood meal for each sample was extracted (using four thimbles with size of 33 X 88 mm in Soxhlet extraction apparatuses) for twenty-four hours with reagent grade benzene and ethanol (BE) at r a t i o of 2:1 as solvent. The r a t i o of solvent to wood was 300 ml solvent to ten grams of wood (O.-D.). The solvent solubles were transferred to a pre-weighed flask and concentrated by means of a rotary vacuum evaporator at 35° to 40°C. The concentrates were then dried under vacuum in a desiccator u n t i l constant weight was obtained. The yiel d s of dried extracts were calculated, as: % dried = wt. of (flask + soluble) - wt. of flask extract X 100 wt. of wood sample (O.-D.) For determining tropolone derivative content in the extractives, each concentrated extractive mixture was dilu t e d to 50 ml with the same BE mixture. The methods of Gardner and Barton (1958), and MacLean and Gardner (1956a) were used, with the modification as suggested by Nault (1984). Five ml of each extractive mixture was transferred to a clean beaker and further d i l u t e d to 10 ml with BE (2:1) solvent. The diluted solutions were extracted with 10 ml 1 78 N LiOH to remove a c i d i c compounds. A small separatory funnel was used with shaking for about one minute for each extraction. The aqueous layer was a c i d i f i e d with 3 ml 4N H 2S0 4 and then extracted twice with 10 ml of chloroform:hexane (3:2) solvent. Thujaplicins were dissolved in the chloroform:hexane solution which was then mixed with 5 ml of f e r r i c acetate (1% w/w Fe) solution. The f e r r i c acetate solution was made from equal volumes of fresh f e r r i c chloride (0.179 M) and sodium acetate (0.537 M) solutions. The mixture of f e r r i c acetate and tropolone solution was well shaken and the organic layer (chloroform:hexane) was removed and saved. The f e r r i c chelates of th u j a p l i c i n s were dissolved in t h i s organic layer. The aqueous layer was washed with an additional 10 ml of chloroform:hexane and the washings were added to the organic layer. The f e r r i c chelate solution was made up to a f i n a l volume of 100 ml. Nault (1984) found from c a l i b r a t i o n curves he generated that the absorbance of both beta- and gamma-thujaplicins at the same concentrations were the same. Therefore, only gamma-thujaplicin was selected as a standard solution in t h i s study due to only a small amount of beta-thujaplicin being av a i l a b l e . 79 A pure gamma-thujaplicin sample was supplied by Forintek Canada Corp. Standard solutions of the gamma-th u j a p l i c i n f e r r i c chelate were prepared for absorbance measurements at the same time by using exactly the same method as for the extractives. The standard solutions consisted of a series of concentrations of gamma-thujaplicin (Table 9). According to Nault's results (1984), the chelated t h u j a p l i c i n was at the maximum after one day. On the basis of his r e s u l t s , a l l absorbances in t h i s study were measured 24 hours aft e r the tropolone solutions were reacted with f e r r i c acetate. Due to higher sample solution concentration, 2 ml of solution for each sample was taken from the 100 ml f e r r i c chelate solution and then di l u t e d to 10 ml for spectroscopic measurement. The absorbances of both standard solutions and sample solutions were measured in the 425 nm region on a Beckman Model 24 spectrophotometer. The chloroform:hexane (3:2) solvent was used as a blank. A c a l i b r a t i o n curve was prepared after recording the UV absorbance for the standard gamma-thujaplicin f e r r i c chelate solutions (Figure 12). The BE (2:1) extractives and t h u j a p l i c i n contents in the fiv e samples were measured. 80 Table 9 . Concentration Series for Standard Gamma-thujaplicin Solutions. Concentrations of Absorbance standard gamma-thujaplicin chelate solution (g/IOOml) 5 . 1 0 X 1 0 " 3 1 . 1 9 0 3 . 4 0 X 1 0 " 3 0 . 7 8 9 2 . 5 5 X 1 0 - 3 0 . 6 4 0 1 . 2 5 X 1 0 - 3 0 . 3 0 1 5 . 1 0 X 1 0 ' 4 0 . 1 2 1 0 . 0 0 0 . 0 0 0 81 Figure 12. C a l i b r a t i o n Curve: Absorbance vs. Gamma-thujaplicin F e r r i c Chelate Concentrations. Concentration (g/l00mlX10-3) 82 3.4. I d e n t i f i c a t i o n of WRC heartwood extractive fractions Thin layer chromatography (TLC) has been found to be a convenient method for separating and ide n t i f y i n g the various components of WRC heartwood extractives. It allowed a reasonable choice of absorbents, such as s i l i c a gel or c e l l u l o s e . Whatman K6F s i l i c a gel (40A) plates, 20 X 20 cm, 250 urn thickness and 20 X 5 cm, 250 urn thickness, were used in t h i s study. The WRC heartwood extractives obtained from D-O, D-I, L-O, and L-I were spotted on a 20 X 20 cm K6F plate a short distance (1 cm) above the edge and were eluted by the flow of mobile phase benzene:ethanol (9:1) which was pre-selected according to e a r l i e r experiments. The mobile phase slowly covered the rest of the plate by c a p i l l a r y flow, which carr i e d sample components upward through the absorbent bed. Development of the chromatogram was stopped when the leading edge of the mobile phase neared the top of the plate. TLC experiments were repeated many times in order to obtain the best r e s u l t s . Detection of chromatographic zones was performed by various methods. Some strongly colored compounds were marked d i r e c t l y on the plate. Some other compounds that were naturally fluorescent were seen as bright zones when the plates were examined under u l t r a v i o l e t 83 l i g h t . For compounds which were non-colored and non-fluorescent, two special detection techniques were applied. One detection method was to expose the TLC plate to iodine vapor for about two minutes. The iodine vapor does not react with the organic compounds, but is p r e f e r e n t i a l l y condensed in the solute zones, apparently being bound to the organic molecules purely by van der Waals forces. Thus, the zones appear as yellow spots against a white background. The advantages of th i s detection method are that the process is e n t i r e l y reversible and universal. Once the plate i s removed from the iodine tank, the spot disappears in a few minutes. This technique is p a r t i c u l a r l y useful when solute zones must be recovered for further analysis. Colored spots corresponding • to the components in t h i s study could be detected quite well after the treatment. A second detection method was to spray an equal mixture of concentrated s u l f u r i c and n i t r i c acids on the plate, followed by heating the plate for two to three minutes to retain the color bands. Individual chromatographic spots corresponding to extractive components were located and characterized by means of Rc values. 84 3.5. Wood block bioassay This experiment was based on the results of e a r l i e r experiments (see Section 3.2). The i s o l a t i o n of microorganism experiments showed that there were three early stage attacking fungi. The Code Nos. WR1, WR2 and WR3 were assigned (Figure 13) . One of the purposes of the wood block test was to examine decay resistance of light-straw colored (L-0) heartwood to these three fungi. From chemical composition analysis experiments (see section 3.4) with discolored and sound WRC heartwood, i t was found that there was a new compound present in discolored heartwood which was not present in sound heartwood. Therefore, another objective of the wood block bioassy experiment was to study the chemical changes of sound (light-straw colored) heartwood after in f e c t i o n by the above three fungi. The wood sample was obtained from the same piece used for determination of heartwood extractives. The wood piece was cut lengthwise into a 6.5 X 6.5 cm long s t i c k . The s t i c k was cut into 0.5 cm blocks with cross-section on the major surface. A l l blocks were in the same growth zone range with a very small v a r i a t i o n in height within stem 85 Figure 13. (maximum 30 cm). The blocks were numbered sequentially. A l l the blocks were dried in a humidity chamber at a constant temperature (25°C) and r e l a t i v e humidity (70%) u n t i l equilibrium was reached, and then weighed to the nearest 0.001 g. After weighing the blocks, ten were taken to O.-D. to determine the moisture content of the blocks at equilibrium. Then, the O.-D. weights of the test blocks were calculated. The fungi used in t h i s experiment were WR1, WR2 and WR3 which were isolated in the e a r l i e r experiment (Section 3.2.) as described. Cultures were grown on media with 2% agar, 3% malt extract and 0.5% yeast extract in 25 mm deep glass Petri dishes. In order to keep the fungi growing evenly on the surface of the culture media, a well growing WR1 fungal colony was transferred into a s t e r i l e container containing s t e r i l i z e d d i s t i l l e d water. The mycelium of WR1 was dispensed in the solution and then transferred to the culture media surface by a metal wire loop. The metal wire loop was moved across the entire surface several times to spread WR1 mycelium evenly. After about three weeks, the WR1 Petri dishes were ready for use (Figure 14A). For WR2 and WR3, small chips of the fungal culture were transferred to the glass P e t r i dishes and spread as evenly as possible. 87 Figure 14. After about three weeks, WR2 and WR3 Petri dishes were also ready for use (Figures 14B and 14C). The sound heartwood blocks (L-0) were divided into nine groups with four blocks in each group. A l l the blocks of each group were surface s t e r i l i z e d by flaming. S t e r i l e d i s t i l l e d water was pipetted on the main surface of the blocks to give a moisture content of 70%. Seven of the nine groups were placed on top of the growing fungi in the assigned P e t r i dishes without contacting the culture media. The Petri dishes were placed in an incubator in the dark at 25° C. The other two groups of wood blocks served as two sets of control samples. Four blocks of one set were placed on the top of the culture media in the Petri dishes lacking growing fungi. Another four blocks were placed in the empty Petri dishes. These two set of control samples were stored in the same incubator with other groups during the treatment period. The d e t a i l s of each treatment were: Group 1 (1-1, 1-2, 1-3, 1-4), Group 2 (2-1, 2-2, 2-3, 2-4) and Group 3 (3-1, 3-2, 3-3, 3-4) were treated with WR1, WR2 and WR3, respectively, for an eight week period; Group 4 (4-1, 4-2, 4-3, 4-4) was treated f i r s t with WR1 for four weeks and then treated with WR2 for four weeks; Group 5 (5-1, 5-2, 5-3, 5-4) was treated with WR1 for 89 four weeks and then treated with WR3 for another four weeks; Group 6 (6-1, 6-2, 6-3, 6-4) was treated with WR2 for four weeks and then treated with WR3 for another four weeks; Group 7 (7-1, 7-2, 7-3, 7-4) was treated with WR1, WR2 and WR3 one after another at four week inte r v a l s ; Group 8 (8-1, 8-2, 8-3, 8-4) was placed on the top of culture media for eight weeks (as control l e v e l 2); and Group 9 (9-1, 9-2, 9-3, 9-4) was placed in empty Petri dishes for eight weeks (as control l e v e l 1). After treatments, the blocks were cleaned by a brush and dried in the humidity chamber at constant temperature (25°C) and r e l a t i v e humidity (70%) u n t i l equilibrium was reached. After weighing the blocks to the nearest 0.001 g, nine blocks which were taken from the nine respective groups were oven-dried and the O.-D. weights of a l l treated blocks were then obtained. The weight loss of each group after treatment was calculated. S t a t i s t i c a l analysis of a l l the data was then carried out. Due to inhomogenous variance for the nine groups O.-D. percentage weight loss, the square root data transformation was used. Analysis of variance and multiple range tests were then performed. Extractive analyses for each group of wood block 90 samples were performed using the methods as described e a r l i e r . The results were compared with those from control samples. 91 3.6. I s o l a t i o n and i d e n t i f i c a t i o n o f an unknown compound f r o m d i s c o l o r e d h e a r t w o o d e x t r a c t i v e s According to results of previous experiments (Section 3.4. and 3.5.), i t was demonstrated p o s i t i v e l y that a new unknown compound was present in discolored heartwood (D-0, D-l) extractives and in the WR1 treated heartwood extractives, but not in WR2 or WR3 treated heartwood extractives. Isolation, p u r i f i c a t i o n and determination of the chemical structure for th i s compound was attempted. a) S o l v e n t f r a c t i o n a t i o n A 400 gram sample of a mixture of outer (D-0) and inner (D-l) discolored heartwood was extracted with BE (2:1) solvent. After evaporation of the solvent, the extractives were dried in a desiccator. To fractionate the extractives, the dried BE extractives were dissolved in ether and f i l t e r e d to remove insoluble materials. The ether insoluble materials were dissolved in ethyl acetate. The ethyl acetate insolubles were f i l t e r e d and then dissolved in ethanol. The ether, ethyl acetate and ethanol extractives were examined by TLC with BE (9:1) as developing solvent. 92 It was found that the new compound was present mainly in the ethyl acetate soluble f r a c t i o n . Further separation was accomplished by using column chromatography. b) Column chromatography Elution column chromatography was used with absorbent based on types of compounds to be separated. Among absorbents, such as c e l l u l o s e , starch, sugars, magnesium s i l i c a t e , s i l i c i c acid, f l u o r i s i l and alumina, only s i l i c i c acid ( s i l i c a gel) is useful for the separation of esters and acids. About 150 grams of s i l i c a gel ( B i o - s i l A, 200-400 mesh) was oven-dried at 130°C for four hours and kept in a desiccator u n t i l i t was cool. D i s t i l l e d water was added in the r a t i o of 50 ml water to 100 gram s i l i c a g e l . A column 50 mm in diameter was used in thi s study. A plug of glass wool was placed in the bottom of the column, and a layer of sand was added on top of the plug to provide an even base for the absorbent column. The eluting solvents were chosen by t r i a l experiments. Because the least polar solvent used in thi s experiment was benzene, the column was packed by adding the absorbent slurry in benzene u n t i l the desired amount of s i l i c a gel had been placed. After the 93 column had been prepared, the solvent l e v e l was lowered to the top of the column by draining i t from the bottom of the column. The dried ethyl acetate soluble f r a c t i o n of discolored heartwood extractives were dissolved in BE (9:1) solvent, which was the least polar solvent that would dissolve the extractive mixture. The extractive solution was c a r e f u l l y added to the top of the column. The absolute amount of extractives added depended on the amount of s i l i c a gel in the column with a maximum of one gram of dried extractives for every 100 grams of s i l i c i c a c i d . The column was eluted with solvent mixtures of increasing p o l a r i t y , s t a r t i n g with pure benzene and gradually increasing the percentage of ethanol to give an equal mixture of BE (50:50). This gradient elution was used to remove each fractio n i n d i v i d u a l l y . Four fractions were f i n a l l y c o l l e c t e d . The four fractions were run on K6F s i l i c a gel TLC plates of 250 urn thickness, and the results showed that the unknown compound was mainly present in the f r a c t i o n obtained by elution with a BE (85:15) solvent mixture. 94 c) Thin layer chromatography Preparative thin layer chromatography was used in th i s experiment to further separate the unknown compound from others in the fractions c o l l e c t e d by column chromatography described in the Section above. Whatman PLK5F s i l i c a gel plates, 20 X 20 cm, 1000 urn thickness were used. The fraction obtained by column chromatography which contained the unknown compound in the discolored heartwood (D-0 and D-I) extractives was applied on the plate as a band across the plate instead of spots. A BE ( 9 : 1 ) solvent system was used for developing the plate. Following development, u l t r a v i o l e t l i g h t (both short and long wavelength) was then employed to locate the band of unknown compound. The band was scraped from the plate with care. The material scraped from the plate was c a r e f u l l y ground to very fine p a r t i c l e s and then the unknown compound was leached from the absorbent ( s i l i c a powder) using ethanol. This procedure was repeated many times. A l l the unknown compound c o l l e c t e d in th i s way was applied to thick TLC plates again using the same solvent system, in order to gain higher sample purity. The eventual t o t a l amount of unknown compound obtained in th i s way was about ten 95 milligrams. Quantitative measurement of th i s unknown compound was performed also by preparative TLC. An ethanol solution with 0.06745 g vacuum dried BE extractives was applied on the plate. The plate was developed by BE ( 9 : 1 ) solvent. The recovered new compound was vacuum dried and the y i e l d was then caculated. d) C r y s t a l l i z a t i o n and elemental analysis of the unknown compoud C r y s t a l l i z a t i o n of the unknown compound p u r i f i e d by the preparative TLC method was attempted. The sample was dissolved in 5 ml of ethyl acetate and then the solvent was evaporated with nitrogen gas to about 2 ml. Heptane was added to the solution. The r a t i o of ethyl acetate to heptane was about 1:3. The f i n a l solution was evaporated to 5 ml. The solution was kept in a freezer for about 5 weeks u n t i l c r y s t a l l i z a t i o n was achieved. A c r y s t a l l i z e d sample was submitted for micro-elemental analysis which was carr i e d out by the Micro-analysis Lab., Dept. of Chemistry, U. B.C.. The result of t h i s analysis w i l l be discussed l a t e r . 96 e) Mass spectrometry Mass spectra of th i s unknown compound from the discolored heartwood (D-0 and D-I) extractives were obtained from the Mass Spectrometry Center, Department of Chemistry, U. B. C. A t o t a l of four spectra were obtained. The four samples used for these mass spectra were p u r i f i e d from 1000 urn TLC plates at d i f f e r e n t times. Two preparations were used to obtain low resolution spectra, and these two spectra were i d e n t i c a l . The ionization temperature for low resolution mass spectra was 100°C. The other two preparations were used to obtain higher resolution spectra. The mass range scanned for these higher resolution spectra was from 30.9984 to 692.9569 mass units. The ionization temperature was 150°C. f) Nuclear magnetic resonance spectrometry (proton and carbon-13 NMR) Both proton and carbon-13 nuclear magnetic resonance (NMR) spectra were taken by NMR Services, Department of Chemistry, U. B. C. The machine used was a Varian XL-300. Because of the small amount of sample available, Fourier 9 7 transform NMR was employed in a l l cases in order to enhance s e n s i t i v i t y . The samples were prepared in deuterochloroform solution with tetramethylsilane (TMS) as an internal standard. Because only about 5 mg of sample was available, the carbon-13 spectra were run overnight. g) V i s i b l e and u l t r a v i o l e t spectrometry A Beckman Model 24 spectrometer located at Forintek Canada Corp. was used for q u a l i t a t i v e analysis of the unknown compound. The sample was dissolved in ethanol solvent. The sample prepared for the v i s i b l e spectrum was at a concentration of 250 ug/ml, while the u l t r a v i o l e t spectrum sample was at 25 ug/ml. The reference beam contained the solvent as appropriate blank. h) Infrared spectrometry Preliminary IR spectrometry of the sample was performed using a Perkin-Elmer 680 series IR spectrometer. Because of the low machine s e n s i t i v i t y , unsatisfactory spectra were obtained even though an 80 minute scan time was used. A more sati s f a c t o r y IR spectrum was run on a Fourier transform 98 I R s p e c t r o m e t e r i n t h e D e p a r t m e n t o f C h e m i s t r y , U . B . C . V e r y s m a l l a m o u n t s ( a b o u t 20 ug) o f u n k n o w n c o m p o u n d i n e t h a n o l s o l u t i o n w e r e p i p e t t e d o n t o t h e s u r f a c e o f a s i l v e r c h l o r i d e c r y s t a l p l a t e . When t h e e t h a n o l was e v a p o r a t e d , t h e c r y s t a l p l a t e was c a r e f u l l y s e c u r e d b e t w e e n t h e m e t a l r e t a i n e r s o f t h e c e l l . 9 9 3.7. T o x i c i t y test bioassays Poria al bipe11 uci da Baxter. was selected as the microorganism to be used in t o x i c i t y test bioassays because i t i s the most common decay organism isolated from the WRC heartwood. Because of the great d i f f i c u l t y for obtaining large amount of the new compound, following p a r t i c u l a r technique was used instead of using standard t o x i c i t y test methods. The bioassays were performed in Pe t r i dishes containing 30 ml of 2% agar, 1% malt extract and 0.5% yeast extract culture media. After the culture media solutions s o l i d i f i e d in the Petri dishes, four uniform 5 mm diameter holes were made and the culture media inside these holes were c a r e f u l l y removed as shown in Figure 15. a) T o x i c i t y of th u j a p l i c i n s A series of beta-thujaplicin solutions at sixteen d i f f e r e n t concentrations was prepared in 10% ethanol. They were at: 5.00; 4.00; 3.75; 3.33; 2.50; 2.00; 1.89; 1.67; 1.25; 1.00; 0.940; 0.830; 0.625; 0.500; 0.469; and 0.417 mg/ml. Then, 0.1 ml of each solution was transferred by syringe into individual holes as shown in Figure 15. Four 100 Figure 15. Petri Dish Prepared for Toxicity Tests. 101 Petri dishes were used for each t r i a l . The Petri dishes were lying on a laminar flow a i r bench without a cover for two hours to allow evaporation of ethanol and d i f f u s i o n of solution to the media. A plug from a Petri dish containing evenly growing Poria al bipel I uci da Baxter, fungus was placed in the center of the dish. The plates were examined regularly. After 96 hours, d i f f e r e n t patterns of fungal growth i n h i b i t i o n for the sixteen d i f f e r e n t solution concentrations were observed (Figure 16). There were three r e p l i c a t i o n s of th i s experiment. The same experiments were ca r r i e d out by using gamma-th u j a p l i c i n and a mixture of beta- and gamma-thujaplicin solutions. b) T o x i c i t y of the unknown compound The t o x i c i t y test for the unknown compound was based on comparison between e f f e c t s with beta-thujaplicin and the unknown compound on fungal growth i n h i b i t i o n . A solution of 0.125 mg b e t a - t h u j a p l i c i n / 0.1 ml 10% ethanol was used. This was extracted from about 20 mg O.-D. of WRC heartwood. This assumes an average t h u j a p l i c i n content of 0.6% in O.-D. wood. 1 02 Figure 16. Inhibition of Poria albipel I ucida Baxter. by Beta-thujaplicin at Sixteen Different Concentrations. Concentrations are (mg beta-thujaplicin/ml ethanol): 1:5.00; 2:4.00; 3:3.75; 4:3.33; 5:2.50; 6:2.00; 7:1.89; 8:1.67; 9:1.25; 10:1.00; 11:0.940; 12:0.830; 13:0.625; 14:0.500; 15:0.469; 16:0.417. 103 The amount of unknown compound in 20 mg O.-D. WRC heartwood was isolated as follows. On average, the BE extractives comprised 15% of the t o t a l O.-D. weight of WRC heartwood. Therefore, for 20 mg O.-D. heartwood, about 3.15 mg of BE extractives would be present. A 3.15 mg sample of discolored (D-0 and D-I) heartwood BE extractives was run on a 1000 urn TLC plate and the unknown compound band was recovered by scraping. The s i l i c a powder was ground and the compound was leached by ethanol. The dried compound was dissolved in 0.1 ml 10% ethanol solvent and then transferred by syringe into one hole in the Petri dish. In each plate, two holes ( l e f t and right) were f i l l e d with 0.1 ml of 1.25 mg/ml beta-thujaplicin and other two holes (up and bottom) were f i l l e d with 0.1 ml of unknown compound solution. After 48 hours, the r e l a t i v e i n h i b i t i n g e f f e cts were compared (Figure 17A). At the same time, four solutions of the unknown compound were prepared at higher concentrations of 5, 2.5, 1.67, and 1.25 mg/ml and tested by the same method (Figure 17B) . c) T o x i c i t y of methylated t h u j a p l i c i n Ethanol solutions of 5 mg/ml beta- and gamma-104 Figure 17. Inhibitio n of Poria al bi peI I ucida Baxter, by the Unknown Compound, Methylated Thujaplicins and Thujaplicins. 1 7A 17B 1 7A: Left and right holes f i l l e d with 0.1 ml of 1.25 mg/ml beta-t h u j a p l i c i n ; top and bottom f i l l e d with unknown compound. 1 7B: Unknown compound at four higher concentractions: 5.00,2.50,1.67 and 1.25 mg/ml. 1 7C: Left and right holes f i l l e d with 0.1 ml of 1.25 mg/ml beta-t h u j a p l i c i n ; top and bottom f i l l e d with the same amount of methylated beta-thujaplicin. 1 7 C 105 t h u j a p l i c i n mixtures were prepared. The solution was methylated by addition of a double volume of etheral diazomethane. After shaking the solution for two minutes, the solvent was evaporated by heating at about 60°-70°C. The methylated t h u j a p l i c i n s were dissolved in 20% ethanol solvent at a concentration of 5 mg/ml. A 0.1 ml methylated sample of the solution was transferred to each hole in the culture media in a prepared Petri dish. A t o t a l of four holes were made on each dish in which two of the holes were f i l l e d with methylated t h u j a p l i c i n s and the other two with 0.1 ml of 1.25 mg/ml beta- and gamma-thujaplicin mixture. After 48 hours, the r e l a t i v e i n h i b i t i n g e f f e c t s were compared (Figure 17C). 106 3.8. Examining o r i g i n of the new unknown compound E a r l i e r experiments (Section 3.4.) showed that the new unknown compound appeared in heartwood extractives only following WR1 treatment. The wood block bioassay (Section 3.5.), however, did not t e l l the o r i g i n of t h i s unknown compound. The present experiment was so designed that major components of WRC heartwood extractives were examined i n d i v i d u a l l y under treatment with WR1 to find their r elationship with this new compound. Sapwood was used in t h i s experiment. White colored sapwood (S-P) was cut into small pieces with the dimensions 40 X 5 X 2 mm. The transverse surface was the largest face on each piece. A l l the sapwood pieces were extracted by BE (2:1) solvent in a shaker for four hours. The extracted sapwood pieces were divided into six sets with four sapwood pieces within the set. The f i r s t set of sapwood pieces was used as a control sample for extracted sapwood and these were l e f t in storage. The second set of sapwood pieces was a i r - d r i e d and d i s t i l l e d water was pipetted onto the surfaces to keep the moisture content high enough for fungal growth. These 1 07 sapwood pieces were placed on top of evenly growing WR1 in Petri dishes without dir e c t contact with the culture media. The dishes were examined regularly and after 15 days, the WR1 could be observed on the top surfaces of the pieces. The sapwood pieces were taken from the dishes and cleaned with a brush. The t h i r d set of sapwood samples was treated the same way as the second set except that there was an addition of t h u j a p l i c i n solution before being placed on top of the WR1. The sapwood pieces were soaked in concentrated beta-t h u j a p l i c i n ethanol solution u n t i l the ethanol had evaporated. The amount of t h u j a p l i c i n in the solution was about 1% of O.-D. weight o.f the t h i r d set of sapwood samples. The sapwood pieces were then a i r - d r i e d and d i s t i l l e d water was pipetted onto their surfaces to keep the moisture content high enough for fungal growth. After 15 days, the WR1 was observed on the top surfaces of the pieces. The same experiments were performed for the solutions of gamma-thujaplicin (Set 4), beta- and gamma-thujaplicin mixture (Set 5), and p l i c a t i c acid (Set 6). A l l six sets of samples were ground to wood meal fine enough to pass a 40-mesh screen. They were extracted 108 separately with BE (2:1) solvent for 24 hours. The extractive solutions were examined by TLC. A multiple internal r e f l e c t i o n infrared (MIRIR) spectral study of the reaction of micro-sections of WRC sapwood with fungus WR1 and t h u j a p l i c i n s was also performed (by a Perkin-Elmer 680 series IR spectrometer). Some technical d i f f i c u l t i e s were encountered. This measurement f a i l e d to produce meaningful r e s u l t s . 109 3.9 Detection of metal chelates from discolored WRC heartwood toluene extractives About 350 g of discolored WRC heartwood (mixture of D-0 and D-I) was extracted with toluene solvent for 24 hr. After evaporating most of the solvent, one-third of the toluene extractives was diluted to 50 ml with toluene. This toluene solution was extracted with 15 ml of 0.1% NaOH (twice) and then was washed with 15 ml of d i s t i l l e d water (twice). Theoretically, the materials remaining in the toluene f r a c t i o n should be thujone, the new unknown compound and other non-phenolic components soluble in toluene, which are a l l weakly colored. A very unusual dark red solution color was observed, which i s a c h a r a c t e r i s t i c of Fe(III)-t h u j a p l i c i n chelate. F i f t y ml of the toluene f r a c t i o n was reacted with 2 ml of 6 N NaOH and 2g/25 ml (H 20) of Na2S for 3 hr. while s t i r r i n g . Dark black-green pre c i p i t a t e s resulted. The prec i p i t a t e s were vacuum f i l t e r e d through a layer of C e l i t e A n a l y t i c a l F i l t e r - A i d , and then oven dried. A solution of 0.06 N H2SO^ was added to the toluene extractives obtained after f i l t r a t i o n . The reaction was detected with a change of solution color, from dark red to 1 10 brown to a f i n a l yellow-white. The addition of ^SC^ was stopped, as soon as a white color appeared. The entire solution was extracted by ethyl ether and then the ethyl ether extractives were washed with d i s t i l l e d water. After evaporating the ethyl ether from the extractives, t h u j a p l i c i n s were detected as the reaction products in the ethyl ether solutions on a TLC plate. 1 1 1 RESULTS AND DISCUSSION 4.1. Microorganism i s o l a t i o n and d i s t r i b u t i o n in WRC heartwood Microorganisms isolated in th i s study supported e a r l i e r findings reported by van der Kamp (1975). There were three fungi consistently isolated from wood samples examined in thi s study. They were l a b e l l e d as WR1, WR2 and WR3 respectively (Figure 13). A t o t a l of 328 attempts were made to i s o l a t e these fungi from WRC wood chips. Among 328 chips, only those which yielded pure cultures of WR1, WR2 or WR3 were counted. The i s o l a t i o n frequency for the three fungi from various colored zones of the WRC heartwood i s given in Table 10. The percentage frequency histograms of the occurence for s t e r i l e wood, WR1, WR2 and WR3 fungal infections versus sample age in the tree are shown in Figures 18 and 19. There were 26 sapwood chips used. Various organisms appeared from them, but no attention was paid to these organisms. WR1, WR2 and WR3, however, were never isolated from the sapwood. Among 26 chips, 12 were s t e r i l e . Among WR1, WR2 and WR3, WR1 was the slowest growing fungus iso l a t e d . After about two months incubation, a 1 1 2 Table 10. Frequency of Microorganism Isolations on Malt Agar from WRC Heartwood Samples v s . Sample Age. age 412-396 395-382 381-362 361-342 341-324 color L-0 R-0 R-B-0 R-B-0 R-B-0 attempts 36(%) 24(%) 32(%) 38(%) 36(%) s t e r i l e 28(77.8) 8(33) 8(25) 6(15.8) 5(13.9) WR1 4(11.1) 16(67) 14(43.8) 13(34.2) 12(33.3) WR2 0(0) 0(0) 10(31.2) 13(34.2) 8(22.2) WR3 0(0) 0(0) 0(0) 0(0) 7(19.4) age 323-294 293-264 263-234 233-198 color B-I B-I B-I B-I attempts 32(%) 38(%) 34(%) 32(%) s t e r i l e 3(9) 2(5.3) 3(8.8) 1 (3) WR1 7(22) 10(27.8) 2(5.6) 0(0) WR2 8(25) 6(16.7) 4(11.1) 4(12.5) WR3 14(44) 14(38.9) 23(63.9) 27(84.5) "attempts" represents the number of chips. The numbers in brackets represent the percentage of s t e r i l e , WR1, WR2, or WR3 chips present. L-O: Light-straw colored outer heartwood. R-O: Red-colored outer heartwood. R-B-O: Red-brown colored outer heartwood. B-I: Brown-darkbrown inner heartwood. 1 1 3 Figure 18. Histogram (No.l) of Percentage Frequency D i s t r i b u t i o n for S t e r i l e Wood and WR1,WR2 and WR3 Fungi Infections vs. Sample Age. >> O c Q) D cr (D 0) cn D -t— c o d) Q L . 100 90 80 70 60 50 40 30-20-10 0 / / I Legend EZ3 STERILE m Sporothrix sp. E l thujina | | />/w a/ ophora sp, l lHI Age of the Growth Zone in Year from the Pith F i g u r e 19. H i s t o g r a m (NO.2) of Percentage Frequency D i s t r i b u t i o n s f o r S t e r i l e Wood and WR1, WR2 and WR3 Fung i I n f e c t i o n s vs. Sample Age . 100 80 60 40 20 H 100-0- 40H <D 1 - 20 H U_ (D 100 H Ui CO 8 0 C 6 0 -2 40H Q_ 20 100-1 80 60 40 20 H 21 STERILE Sporot hri x sp . XZZZ2-K. t huj i na Phialophora s p . , 6 V ,6V,6V;6> o> l<vfc l<vfc A g e of the Growth Zone in Year from the Pith 115 l i v i n g WR1 culture from some of the wood chips developed a very c h a r a c t e r i s t i c appearance. It was white-greyish in color, with a maximum radius of 30 mm for a developed colony on the malt extract medium. After transfer to a fresh medium, the cultures developed into a convoluted mass of conidia with few hyphal segments (Figure 13A). WR2 (Figure 13B) was consistently dark black in color. There were no spores produced in the culture. Van der Kamp ( 1975) tentatively i d e n t i f i e d WR2 as Ki rschstei ni el I a t huji na (Peck) Pomerleau & Etheridge because of the resemblence of WR2 to the one isolated and t e n t a t i v e l y i d e n t i f i e d by Smith (1970) as the above. WR3 (Figure 13C) had irregular shapes and i t s color was a mixture of black and grey. Cultures of WR1, WR2 and WR3 were sent to the I d e n t i f i c a t i o n Service, Biosystematic Research Institute, Agriculture Canada for f i n a l i d e n t i f i c a t i o n . The report comfirmed van der Kamp's (1975) tentative i d e n t i f i c a t i o n on WR2 as Ki rschst ei ni el I a thuji na (Peck) Pomerleau & Etheridge. On the basis of described colony and hypha features including pointed incrustations on surface of the hyphae. WR1 was t e n t a t i v e l y i d e n t i f i e d as Sporothrix sp. which was quite d i f f e r e n t from van der Kamp's report as 116 Cyl i ndr oce phal urn. WR3 i s suggested to be Phialophora sp.,a hyphomycete l i k e culture. In order to avoid confusion, in the following text, Sporothrix sp. , K. t huj i na and Phialophora sp. w i l l be used instead of WR1, WR2 and WR3 respectively. The o r i g i n a l cultures of WR2 and WR3 are available at I d e n t i f i c a t i o n Service, Biosystematic Research Inst i t u t e , Agriculture Canada, Ottawa, with system number: DAOM 196473 and DAOM 196486 respectively. An obvious pattern of microorganism d i s t r i b u t i o n in WRC heartwood was observed in th i s study. It can be seen from Figures 18 and 19 that in WRC heartwood, s t e r i l e wood occurred mainly in growth zones from 396 to 412, which was a straw-colored heartwood area adjacent to the sapwood. Sporothrix sp. appeared mostly in growth zones 382 to 395, which corresponded to the reddish colored heartwood, also c a l l e d the edge area of the discoloration zone. K. t huji na occurred mostly in growth zones 342 to 361 and was found in both reddish-brown and brown colored zones of the heartwood. Phialophora sp. was found from growth zones 198 to 324 and with greatest frequency at growth zones of 198 to 233. It is clear that the occurence of these microorganisms in WRC discolored heartwood followed the order that Sporothrix sp. f i r s t moved outwards from the pith , then K. t huji na and 11 7 then Phialophora sp. An interpretation of t h i s microorganism d i s t r i b u t i o n pattern was given by van der Kamp (1975) and i s a further subject of the present study. Sporothrix sp. is a fungus which has the capacity to destroy or a l t e r natural toxic compounds, such as t h u j a p l i c i n s and perhaps others. This action would f a c i l i t a t e the invasion by K. thujina and then Phialophora sp. K. thujina and Phialophora sp. in turn appears to affect the wood in such a manner that i t becomes more susceptible to decay fungi, such as Poria al bipel I uci da Baxter., which o r d i n a r i l y would be inhibited by naturally occurring toxic compounds. 1 18 4.2. E x t r a c t i v e d i f f e r e n c e s between sound and d i s c o l o r e d WRC heartwood The amount of wood e x t r a c t i v e s i n t r e e s i s a f f e c t e d by s e v e r a l f a c t o r s i n c l u d i n g age, p o s i t i o n of the sample w i t h i n the t r e e , g e o g r a p h i c l o c a t i o n , s e a s o n a l v a r i a t i o n s and g e n e t i c d i f f e r e n c e s ( H i l l i s , 1962). In t h i s s t u d y , a comparison of d i f f e r e n c e s i n e x t r a c t i v e c o m p o s i t i o n between sound and d i s c o l o r e d heartwood was made w i t h i n the same WRC t r e e and a t the same l e v e l w i t h i n the t r e e , t h e r e b y e l i m i n a t i n g some s o u r c e s of v a r i a t i o n . D i f f e r e n c e a l o n g the r a d i u s a t one l e v e l were examined. The y i e l d s of BE (2:1) e x t r a c t i o n s f o r f i v e groups of samples: D i s c o l o r e d o u t e r heartwood ( D - 0 ) ; D i s c o l o r e d i n n e r heartwood ( D - l ) ; L i g h t - s t r a w c o l o r e d o u t e r heratwood (L-O); L i g h t - s t r a w c o l o r e d i n n e r heartwood ( L - I ) and Sapwood (S-P) a r e g i v e n i n the T a b l e 11. The c o n c e n t r a t i o n of e x t r a c t i v e s was d e t e r m i n e d based on the O.-D. weight of wood. In summary, t h e r e were t h r e e r e s u l t s from t h i s s t u d y : 1. Heartwood has a much h i g h e r BE (2:1) e x t r a c t i v e s c o n t e n t than sapwood; 2. W i t h i n the same growth zones, d i s c o l o r e d heartwood has a h i g h e r c o n c e n t r a t i o n of BE e x t r a c t i v e s than l i g h t -119 Table 11. Benzene ethanol (BE) (2:1) Extractives and Thujaplicin (TH) Content of the WRC Heartwood. Age Code BE extract ives content (O.D. %) TH Unknown content compound (O.D. %) (O.D. %) 413-303 303-198 413-303 303-198 420-413 D-0 D-I L-0 L-I S-P 15.45 12.04 12.55 9. 1 1 3.20 0.230 0.120 0.730 0.552 0.012 0.291 0. 187 None None None D-O: discolored outer heartwood. D-I: discolored inner heartwood. L-O: light-straw colored outer heartwood. L-I: light-straw colored inner heartwood. S-P: sapwood. 120 straw colored heartwood; and 3. Across a radius, BE extractives increase from inner heartwood to outer heartwood. There i s evidence that amounts of polyphenolics and other extractives in the c e l l wall increase when sapwood i s transformed to heartwood. Barton and Gardner (1954) reported a higher holocellulose (66.5%) and c e l l u l o s e content (54.2%) and a much lower extractive content (2.6%) in the sapwood of WRC r e l a t i v e to the heartwood. Similar results in other species, such as Norway spruce (Picea abies (L.) Karst.) have been reported (Johansson and Theander, 1974). They found that starch was common in sapwood rays but not in the heartwood where i t seemed to be replaced by phenols. The formation of phenolics in heartwoods i s part of the tree defence mechanism. In l i v i n g sapwoods, however, the l i v i n g parenchyma may react p h y s i o l o g i c a l l y when disturbed by fungal infection (Shain, 1967). The lower extractive content in sapwoods might be due to naturally higher resistance to microorganism attack on l i v i n g sapwood. In heartwoods, the accumulated phenolic extractives can prevent such attacks. Results obtained in th i s study agree with these concepts. The color of WRC heartwood i s subject to marked 121 v a r i a t i o n . Because color of the wood i s generally due to extractives contained therein, i t is usually true that a darker color denotes a higher extractive content. This i s also true in WRC according to results obtained in this study. There have been no previous reports comparing WRC extractive contents in discolored and sound heartwood taken from the same growth zones. This study found that outer discolored (D-0) heartwood had a BE (2:1) extractive content 2.90% greater than the outer sound (L-O) heartwood, while inner discolored (D-I) heartwood had an extractive content 2.93% greater than sound inner (L-l) heartwood (Table 11). There is evidence of factors a f f e c t i n g the amount of extractives in heartwoods. The amount appears to be partly inherited and partly due to environmental factors, and i t i s d i f f i c u l t to separate these variables. In t h i s study, the results showed that the amount of BE (2:1) extractives increased with distance from the p i t h to the most recently formed heartwood which follows known patterns (Barton and MacDonald, 1971; Nault, 1984). The t h u j a p l i c i n contents of f i v e groups of samples were measured by a colorimetric method as described (Section 3.3.). The results are l i s t e d in Table 11. Similar to BE (2:1) extractives within the heartwood, t h u j a p l i c i n content 122 increased from p i t h to outer heartwood. Sapwood only possessed trace amounts of th u j a p l i c i n s in comparison to the heartwood. It was observed, however, that the pattern of t h u j a p l i c i n concentration vs. heartwood coloration was di f f e r e n t from that of BE (2:1) extractives. Although discolored heartwood (D-0 and D-I) had a higher t o t a l BE (2:1) extractive content, light-straw colored heartwood had a much higher t h u j a p l i c i n content than discolored heartwood in the same growth zones. The outer light-straw colored heartwood contained 3.2 times as much th u j a p l i c i n s as the outer discolored heartwood, while the inner l i g h t straw-colored heartwood had 4.0 times the content of the inner discolored heartwood. It i s now generally accepted that the high decay resistance and d u r a b i l i t y of WRC heartwoods p a r a l l e l the presence of extractives. However, i t i s worthwhile to point out that the extractives mentioned in this sense only mean those components or fractions in the extractives which are highly toxic to certa i n groups of fungi. In WRC the dominant natural preservative i s the t h u j a p l i c i n f r a c t i o n in the extractives. Hence, a higher BE extractive content in discolored heartwood (D-0 and D-I) does not necessarily 123 r e p r e s e n t a h i g h e r t o x i c i t y t o c e r t a i n g r o u p s o f f u n g i . T h e l i g h t - s t r a w c o l o r e d h e a r t w o o d ( L - 0 a n d L - I ) , t h o u g h i t h a s a l o w e r c o n c e n t r a t i o n o f B E e x t r a c t i v e s t h a n d i s c o l o r e d h e a r t w o o d , p o s s e s s e s h i g h e r d e c a y r e s i s t a n c e t h a n d i s c o l o r e d h e a r t w o o d d u e t o i t s h i g h e r t h u j a p l i c i n c o n t e n t . D i s c o l o r a t i o n o f t h e w o o d c h a n g e s t h e l i g h t - s t r a w c o l o r e d h e a r t w o o d i n t o b r o w n c o l o r e d h e a r t w o o d p r i o r t o a d v a n c e d d e c a y . I n t h e s a m e g r o w t h z o n e s , w h e n w o o d c o l o r c h a n g e d f r o m l i g h t - s t r a w t o b r o w n , t h e t h u j a p l i c i n c o n c e n t r a t i o n d r o p p e d r e m a r k a b l y . T h i s o b s e r v a t i o n s t r o n g l y i n d i c a t e s t h a t d u r i n g t h e d i s c o l o r a t i o n p r o c e s s ( i n t h i s c a s e , i t w a s a m i c r o o r g a n i s m a t t a c k p r o c e s s ) , t h e e x t r a c t i v e c o n t e n t o f t h e h e a r t w o o d s o m e h o w c h a n g e d , p a r t i c u l a r l y f o r t h i s g r o u p o f c o m p o u n d s . A s a r e s u l t o f t h i s c h a n g e , t h e t r o p o l o n e c o n t e n t d e c r e a s e d , w h i c h s u g g e s t s t h a t t h e t h u j a p l i c i n s m i g h t h a v e b e e n s t r u c t u r a l l y a l t e r e d o r d e g r a d e d b i o c h e m i c a l l y t o n e w c o m p o u n d s b y t h o s e m i c r o o r g a n i s m s c a u s i n g d i s c o l o r a t i o n . I t i s e x p e c t e d t h a t t h i s c h a n g e o f t h e h e a r t w o o d t h u j a p l i c i n c o n t e n t c e r t a i n l y a f f e c t e d t h e p r o p e r t i e s o f t h e h e a r t w o o d i n t e r m s o f n a t u r a l r e s i s t a n c e o f t h e h e a r t w o o d t o f u r t h e r f u n g a l a t t a c k . T h e d e c r e a s e o f t h e t h u j a p l i c i n c o n c e n t r a t i o n i n d i s c o l o r e d h e a r t w o o d e x t r a c t i v e s w a s n o t t h e o n l y m a j o r 1 24 phenomenon observed in this study. A more interesting finding i s that in addition to the normal chemical composition present in light-straw colored heartwood, there was a new, previously unknown compound c l e a r l y present in discolored heartwood extractives. This new compound was detected by TLC (Figure 20). The same observation was confirmed by repeated experiments. Four samples used for these experiments were obtained from Nault (1986). According to Nault (1986) these samples had a lower t h u j a p l i c i n content and certain degree of d i s c o l o r a t i o n . The TLC analysis of BE (2:1) extractives of these four samples indicated that three out of the four samples contained the unknown compound chromatographic spot. Thin layer chromatography was the most important method used in t h i s study to separate and i d e n t i f y the various WRC heartwood extractive components. The positions of the components on the TLC plates were marked under UV l i g h t (both short and long wavelengths) and then the plates were exposed to iodine vapor. The R^  values for a l l spots on the experimental plates were calculated and are l i s t e d in Table 12. The R^  values of known lignans in WRC heartwood extractives using BE (9:1) eluting solvent were published by MacDonald and Swan (1970). The known components in the 125 Figure 20. TLC Plates of BE Extractives from WRC Discolored and Light-straw Colored Heartwoods. «o m> 3s**. 3fn~ MS? m D - 0 L - 0 D-I L-I 126 Table 12. TLC Rf Values for Four WRC Heartwood Extracts Using BE (9:1) Solvent. Spot # D-0 D-I L-0 L-I 1 0.00 0.00 0.00 0.00 2 0.05 0.05 0.05 0.05 3 0.09 0.09 0.09 0.09 4 0.14 0.14 0.14 0.14 5 None None 0.17 0.17 6 0.19 0.19 0.19 0.19 7 0.25 0.25 0.25 0.25 8 0.30 0.30 0.30 0.30 9 0.33 0.33 0.33 0.33 10 0.35 0.35 0.35 0.35 1 1 0.40 0.40 0.40 0.40 1 2 0.45 0.45 0.45 0.45 1 3 0.48 0.48 0.48 0.48 1 4 0.79 0.79 None None 15 0.86 0.86 0.86 0.86 D-O: Discolored outer heartwood. D-I: Discolored inner heartwood. L-O: Light-straw colored outer heartwood. L-I: Light-straw colored inner heartwood. 127 v o l a t i l e f r a c t i o n of WRC heartwood extractives were i d e n t i f i e d by running against pure compounds with BE (9:1) solvent by TLC. R^  values of known WRC heartwood extractives are l i s t e d in Table 13. A comparison of the R^  values of the spots on the plates of D-O, D-I, L-0, and L-I to the known R^  values was made. The differences in extractive composition between sound and discolored heartwood extractives are obvious. The known components were i d e n t i f i e d in both sound and discolored heartwood extractives by R^  values. In addition to these known component spots on the plates, there were several unknown spots which appeared on both discolored and sound samples. Since only the major components were isolated and i d e n t i f i e d by previous work, i t i s reasonable to expect the presence of some unknown spots. As long as the unknown spots occurred in both sound and discolored wood extractive samples (Table 12, Spots # 4,6, and 9), they did not represent a s i g n i f i c a n t difference between the two kinds of extractives. Special attention was paid to one of the new, unknown spots appearing on the plates of discolored heartwood extractive samples (D-0, D-I) which was not found in sound heartwood extractives (L-0, L-I). This new spot has an Rf 128 Table 13. TLC Rf Values of Known WRC Extractives Using BE (9:1) Solvent. Name of the compound R^  value Lignans: P l i c a t i c acid 0.00 P l i c a t i n 0.05 Plicatinaphthol 0.10 Dihydroxythujaplicatin 0.15 Plicatinaphthalene 0.16 Dihydroxythujaplicatin 0.17 methyl ether Thujaplicatin 0.21 Gamma-thujaplicatene 0.23 Hydroxythujaplicatin 0.25 methyl ether Thujaplicatin 0.30 methyl ether * V o l a t i l e Fraction : Beta-thujaplicinol 0.35 Beta-thujaplicin 0.40 Gamma-thujaplicin 0.45 Thujic acid 0.47 Methyl thujate 0.85 Source: MacDonald and Swan, (1970). *The data obtained by using pure samples in t h i s study. 129 value of 0.79 which i s c l e a r l y d i f f e r e n t from other known compounds reported (Table 13). Quantitative measurement of this new compound by the preparative TLC method (Section 3.6c) showed that the concentration of i t in outer discolored heartwood (D-0) was 0.291% and for inner discolored heartwood (D-I), i t was 0.187%. Further investigation of t h i s compound was carried out. A detailed elucidation of i t s structure, an evaluation of i t s t o x i c i t y to certain fungi and a possible mechanism of formation of thi s compound in discolored heartwood are discussed l a t e r . 130 4 . 3 . B i o l o g i c a l a n d c h e m i c a l r o l e s o f Sporothrix s p . , K. thujina a n d Phialophora s p . i n l i v i n g WRC t r e e s T h u j a p l i c i n s a n d o t h e r h e a r t w o o d e x t r a c t i v e s w h i c h a r e r e s p o n s i b l e f o r n a t u r a l d e c a y r e s i s t a n c e i n WRC h e a r t w o o d s , may i n t e r a c t w i t h v a r i o u s f u n g i e x i s t i n g w i t h i n t h e l i v i n g t r e e . I n t h i s s t u d y , t h r e e k i n d s o f e a r l y s t a g e a t t a c k i n g f u n g i (Sporothrix s p . , K. thujina a n d Phialophora s p . ) w e r e i s o l a t e d f r o m d i s c o l o r e d WRC h e a r t w o o d . The f r e q u e n c y d i s t r i b u t i o n o f t h e f u n g i ( T a b l e 1 0 , F i g u r e s 18 a n d 19) i n d i c a t e d t h a t Sporothrix s p . may be t h e f i r s t f u n g u s a t t a c k i n g t h e s t e r i l e h e a r t w o o d , w i t h a t t a c k by K. thujina a n d Phialophora s p . f o l l o w i n g . The wood b l o c k b i o a s s a y was d e s i g n e d t o e x a m i n e t h e e f f e c t s o f t h e s e t h r e e f u n g i on p e r c e n t a g e w e i g h t l o s s e s o f s o u n d h e a r t w o o d b l o c k s , t h u j a p l i c i n , BE ( 2 : 1 ) e x t r a c t i v e s c o n t e n t a n d n a t u r a l d e c a y r e s i s t a n c e i n d i v i d u a l l y , a n d s e q u e n t i a l l y w i t h d i f f e r e n t a t t a c k i n g o r d e r s . I n o r d e r t o k e e p t h e e x p e r i m e n t a s c l o s e a s p o s s i b l e t o r e a l c o n d i t i o n s i n t h e l i v i n g t r e e , f r e s h c u t l i g h t - s t r a w c o l o r e d h e a r t w o o d , w h i c h c o n t a i n e d a h i g h c o n c e n t r a t i o n o f t h u j a p l i c i n s a n d t o t a l e x t r a c t i v e s , was u s e d . The r e s u l t s o f w e i g h t l o s s e s , BE ( 2 : 1 ) e x t r a c t i v e s a n d t h u j a p l i c i n c o n t e n t s o f WRC s o u n d 131 heartwood blocks treated with Sporothrix sp. , K. thujina and Phialophora sp. are shown in Table 14. The wood block bioassay experiment was a completely randomized design experiment with nine d i f f e r e n t treatments There were four repeated measurements within each treatment. Two lev e l s of control treatments were used in the experiment. It i s noted that there is a 2.85% reduction of BE (2:1) extractives and 0.026% reduction for t h u j a p l i c i n content from the wood blocks in the empty Petri dishes (Group 9) to the wood, blocks placed on the top of the culture media without fungi growing on i t (Group 8). Similar to chemical loss, there is also a s i g n i f i c a n t weight loss between the two control lev e l s (difference:0.635%-0.021%=0.614%). This observation t e l l s one that there i s a chemical loss, as well as a weight loss due to dir e c t contact between the wood blocks and water culture media. In this experiment, treatment blocks did not d i r e c t l y contact the culture media, but were placed on the surface of the growing fungal cultures. Mass or chemical exchange, however, between testing blocks and culture media are s t i l l possible through translocation by fungi or due to very close proximity between the blocks and culture media. Therefore, taking a conservative approach, a l l conclusions drawn from 132 Table 14. Results of Weight Loss and Extractive Analyses of WRC Sound Heartwood Blocks Treated with Sporothrix sp., K. thujina and Phialophora sp. Group No. (n=4) Weight Loss Mean (*) SD. BE EXT (O.-D.)% TH Thujin (O.-D.)% (8 weeks with WR1) 1.401 (d) 0.172 (8 weeks with WR2) 1.552 (d) 0.110 (8 weeks with WR3) 0.896 (c) 0.107 10.84 8.75 10.51 (4 weeks with WR1, then 4 weeks with WR2) 5.476 (f) 0.256 9.36 (4 weeks with WR1, then 4 weeks with WR3) 3.629 (e) 0.659 10.97 (4 weeks with WR2, then 4 weeks with WR3) 1.250 (d) 0.143 9.21 (4 weeks with WR1, with WR3) 5.561 (f) then 4 weeks with WR2 0.616 9.01 0.081 + 0.412 0.509 0.090 + 0.10.1 + 0.426 then 4 weeks 0.079 + 8: (control l e v e l 2: 8 weeks with culture medium) 0.635 (b) 0.260 10.93 0.587 9: (control l e v e l 1: 8 weeks in empty Petri dishes) 0.021 (a) 0.016 13.78 0.613 *: a,b,c,d,e and f represent six homogeneous subsets among nine O.-D. weight loss means according to Duncan's Multiple Range Test with 95% confidence l e v e l . BE EXT: Benzene ethanol extractives. TH: Thujaplicins 133 t h i s experiment w i l l be based on comparing treatment results with that of the second control l e v e l (Group 8). The chemical analysis of BE (2:1) extractives and t h u j a p l i c i n provide strong evidence for b i o l o g i c a l roles of the three early attacking fungi. Since Sporothrix sp. was the only major organism isolated near the edge of the discolored WRC heartwood, i t is reasonable to believe that t h i s fungus interacts with heartwood chemicals e a r l i e r than other isolated microorganisms. It is suggested by observations in t h i s experiment that Sporothrix sp. plays a major role in reducing natural decay resistance of the WRC heartwood. Every time Sporothrix sp. was involved (Groups 1, 4, 5, and 7) t h u j a p l i c i n was reduced to approximately 1/7 of the i n i t i a l amount. The fact that a high proportion of the t h u j a p l i c i n content could not be detected suggests that attack of the light-straw colored heartwood by Sporothrix sp. somehow changed the chemical form of the t h u j a p l i c i n s The wood block bioassay showed that the blocks exposed to Sporothrix sp. had a considerable loss of t h u j a p l i c i n content, but, at this stage the weight loss of the blocks was very small (Table 14). This demonstrates that Sporothrix sp. functions uniquely in d e t o x i f i c a t i o n of the 1 34 compounds toxic to other fungi, in th i s case t h u j a p l i c i n s . It i s also suggested by th i s result that the energy needed for Sporothrix sp. growth may not originate from wood c e l l wall components. There are p o s s i b i l i t i e s that the enzymes in Sporothrix sp. have the capacity to degrade or a l t e r t h u j a p l i c i n to a form with much lower t o x i c i t y . At the same time, t h i s reaction i t s e l f might provide the energy source for Sporothrix sp. growth. During the chemical comparison of sound and discolored heartwood extractives, i t was p o s i t i v e l y shown that there was a new previously unknown compound present only in extractives from discolored heartwood (Figure 20). The sound and discolored heartwood samples were taken d i r e c t l y from the freshly cut natural wood. The chemical composition of BE (2:1) extractives obtained from each treated wood group were examined by TLC. This same new unknown compound was found again in Groups 1, 4, 5 and 7 of treated wood block extractives (Table 14). These wood blocks were light-straw colored and did not contain t h i s new compound before the treatments. These four groups of samples underwent d i f f e r e n t sequences of treatment, but the common points are that a l l four treatments started with Sporothrix sp., and a l l four showed 135 major losses of t h u j a p l i c i n . The appearance of t h i s new unknown compound only in those heartwood extractives obtained from the treatments involving Sporothrix spp, but not in extractives obtained from treatments with K. thujina, or Phialophora sp. , or K. thujina and then Phialophora sp. , indicated that t h i s compound is a special product yielded by interaction between sound heartwood and Sporothrix sp. Although Sporothrix sp. i s an organism proven to be responsible for reduction of t h u j a p l i c i n content and the occurrence of the new unknown compound, there i s no direct evidence r e l a t i n g t h u j a p l i c i n s to t h i s new compound. It i s possible that the new compound may originate from other extractive components, such as lignans, under infection by Sporothrix sp. The relationship between t h u j a p l i c i n s and p l i c a t i c acid to the unknown compound was proven by the sapwood bioassay (see Section 3.8). Beta-, gamma-thujaplicins and p l i c a t i c acid were introduced to pre-extracted WRC sapwood pieces, and the pieces were then treated with Sporothrix sp. for 15 days. TLC results with the extractives showed that the unknown compound was found neither in pre-extracted sapwood control samples, nor in pre-extracted sapwood samples treated with Sporothrix sp. but lacking addition of t h u j a p l i c i n s or 136 p l i c a t i c acid. This result c l e a r l y demonstrated that the pre-extracted sapwood does not contain t h i s unknown compound. It also eliminated the p o s s i b i l i t y that the unknown compound was produced by Sporothrix sp. interacting with the pre-extracted wood complex or was produced by Sporothrix sp. i t s e l f . The p o s s i b i l i t y that the unknown compound originated from lignans was excluded, since the new compound was not found in extractives obtained from sapwood containing p l i c a t i c acid and exposed to Sporothrix sp. The unknown compound was only found in sapwood pieces treated with Sporothrix sp. and containing t h u j a p l i c i n s . It becomes obvious that the presence of both the microorganism Sporothrix sp. and t h u j a p l i c i n s i s a precondition for the formation of th i s unknown compound. The quantity of t h u j a p l i c i n s reduced by K. thuji na i n d i v i d u a l l y was smaller when compared to the quantity reduced by Sporothrix sp. In the l i v i n g tree, K. t huji na was isolated in the reddish and light-brown colored heartwood, in which the t h u j a p l i c i n content was very low, so i t seems that the p r o b a b i l i t y that K. t huji na functions as a destroyer of t h u j a p l i c i n s i s very low. It i s interesting to find that though K. t huji na i s not a t h u j a p l i c i n destroyer in l i v i n g trees, i t has a higher 1 37 capacity to decrease the BE (2:1) extractives amounts than Sporothrix sp. and Phialophora sp. The l i g h t straw colored WRC heartwood BE (2:1) extractives were almost the same by the end of the treatments for Groups 1, 3 and 5 samples. Samples treated with K. t huji na (Groups 2, 4, 6 and 7) had an obvious reduction of BE (2:1) extractives compared to other samples (Table 14). It i s also true that a f t e r the Sporothrix sp. treated blocks were exposed to K. thuji na, the weight loss was higher than the weight loss caused by treatment with Sporothrix sp. and then followed with Phialophora sp. These observations suggest that the K. thuji na might be active against components other than the th u j a p l i c i n s in WRC heartwood extractives. These components may also possess some natural t o x i c i t y to decay fungi. Changes in the chemical structure of extractive components could change their s o l u b i l i t y in a BE (2:1) solvent system, as well. The samples treated with Phialophora sp. alone showed a minor loss of t h u j a p l i c i n content from 0.587% to 0.509%. No doubt Phialophora sp. would not interact with t h u j a p l i c i n s d i r e c t l y in the light-straw colored heartwood so i t would not function as a pioneer. The results of Analysis of Variance of weight loss 1 38 (Appendix 6) indicate that there is s i g n i f i c a n t variation between treatment means. In other words, the means of the percentage O.-D. weight losses for nine wood block groups are s i g n i f i c a n t l y d i f f e r e n t at a 95% confidence l e v e l . Multiple Range Tests (Appendix 7) further i l l u s t r a t e these differences. Results show that there are six homogeneous subsets among the nine O.-D. weight loss means. Weight loss means caused by Treatments 4 and 7 are s t a t i s t i c a l l y equal to each other, and are s i g n i f i c a n t l y greater than a l l other weight loss means. Treatment 5 also resulted in a s i g n i f i c a n t weight loss, which was s i g n i f i c a n t l y greater than Treatments 1, 2, 3, and 6. It i s interesting to find from the results (Table 14) that wood blocks treated with Sporothrix sp. , K. thujina and Phialophora sp. i n d i v i d u a l l y (Groups 1, 2, and 3) did not show a very severe weight loss. Those blocks treated with K. thujina f i r s t and then with Phialophora sp. (Group 6) also did not show severe weight los s . The higher weight losses occurred only in three treatments (Groups 4,5 and 7) a l l started with Sporothrix sp. and then followed with K. thujina or with Phialophora sp. This observation strongly demonstrated that Sporothrix sp. can not cause serious weight loss of the wood block d i r e c t l y , but that interaction 1 39 between Sporothrix sp. and sound heartwood is a precondition for s i g n i f i c a n t weight loss caused by later attacking fungi. In other words, natural decay resistance in the sound heartwood which prevents advanced decay is somehow lessened or removed by the action of Sporothrix sp. Thereby, a strong synergistic e f f e c t i s shown between Sporothrix sp. and K. t huj i na or Sporothrix sp. and Phialophora sp. Results with Groups 4, 5, and 7 (Table 14) indicate that the treatments s t a r t i n g with Sporothrix spp then following with K. t huji na caused a much more severe percentage weight loss than followed by Phialophora sp. (Group 5). Thus, i t i s obvious that the synergistic e f f e c t between Sporothrix sp. and K. t huji na i s much higher than that of Sporothrix sp. and Phialophora sp. The order of treatment with Sporothrix sp. followed by K. t huji na i s the same as occurs naturally in l i v i n g WRC. Thus, sequential attacks by Sporothrix sp. and then K. t huji na in l i v i n g WRC trees appear to be one of essential steps for destroying the natural toxin of WRC sound heartwood and allowing further advanced decay. The role of Phialophora sp. i s not well demonstrated so far. van der Kamp (1975) proved that there is no antagonistic e f f e c t between K. t huji na and Phialophora sp. 140 The wood blocks treated with K. thujina followed with Phialophora sp. (Group 6) had a weight loss of 1.2500% about half that by K. thujina ( 1 .552%) and Phialophora sp. (0.896%) i n d i v i d u a l l y . This suggests that there i s neither an antagonistic nor synergistic e f f e c t between K. thujina and Phialophora sp. Based on the facts observed in these experiments, b i o l o g i c a l and chemical roles of Sporothrix sp. , K. thujina and Phialophora sp. in l i v i n g WRC trees can be summarized as follows. Sporothrix sp. acts as a pioneer attacking sound heartwood and destroying the natural decay resistance factor provided by t h u j a p l i c i n s . This attack then prepares wood for further fungal attacks. The phenomenon of s i g n i f i c a n t reduction of t h u j a p l i c i n content accompanying appearance of an unknown new compound during Sporothrix sp. attack of light-straw colored WRC heartwood is of interest. It may be assumed that the missing t h u j a p l i c i n s were degraded or converted to other compounds l i k e t h i s new unknown compound, which may not be toxic to decay fungi. The determination of the chemical structure and t o x i c i t y of t h i s unknown compound i s a key to solving the mystery of t h u j a p l i c i n d e t o x i f i c a t i o n mechanisms during Sporothrix sp. invasion. 141 K. thujina attacks the WRC heartwood after Sporothrix spp. has provided favorable conditions. The K. thujina i n f e c t i o n may interact with other toxic components in WRC heartwood extractives and further reduce the natural decay resistance of WRC heartwood with d i f f e r e n t mechanisms. Phialophora spp., present in the dark-brown colored heartwood, may act as an intermediate between these pioneers and the invasion of decay fungi. The whole process involves a succession of microorganisms which attack s t e r i l e (light-straw colored) heartwood (by pioneers), gradually turning i t into discolored wood and f i n a l l y decayed wood (by decay fungi). 1 42 4.4 Elucidation of the chemical structure for the new unknown compound Separation of components in complex mixtures i s often aided by preliminary separations based on chemical properties of the components. Most of components in WRC heartwood extractives are phenolic compounds having a certain range of p o l a r i t y . The solvents used in preliminary separation were ethyl ether, ethyl acetate and ethanol. According to a previous study (Barton and MacDonald, 1971), a very strong organic acid, p l i c a t i c acid, comprises approximately 40% of the WRC heartwood extractives. It i s very soluble in water compared with other components in extractives, and i s p a r t i c u l a r l y insoluble in ethyl acetate (Gardner et al, 1959). Removal of the ethyl acetate insoluble material from the extractives separates the more strongly polar components, such as p l i c a t i c acid, from other compounds. Ethyl ether i s s l i g h t l y less polar than ethyl acetate. Separation of ethyl ether solubles from ethyl acetate solubles was performed for further fractionation of the extractives. The unknown compound was found by TLC to occur mainly in the ethyl acetate solubles. After the ethyl acetate 1 43 solubles went through a gradient elution chromatographic column, the unknown compound was found to be present in the fractio n eluted by the the BE (85:15) solvent mixture. This observation indicates that t h i s new compound does not possess a very strong polar functional group. The extent of absorption of a single component on a TLC plate depends on the p o l a r i t y of the molecule, the a c t i v i t y of the absorbent, and the p o l a r i t y of the mobile l i q u i d phase. The actual separation of components in a mixture i s dependent on the r e l a t i v e values of the absorption-desorption equilibrium constants for each of the components in the mixture. In general, when the absorbent and the developing solvent are fixed, the more polar functional groups in the compound w i l l be more strongly absorbed on the surface of the s o l i d phase of TLC (Peters et al, 1974). In other words, a less polar compound flows better with the solvent and has a higher R^  value. On the TLC plates, developed by BE (9:1), this new compound has an R^  value at 0.79 which i s considerably higher than most known R^  values for tropolones and lignans in WRC heartwood extractives. It can be deduced that the chemical structure of t h i s new compound i s less polar than most of the known compounds in WRC heartwood extractives as shown by i t s higher R^  value. 144 Since the R^  values for a l l the known lignans in WRC heartwood extractives are from 0.00 to 0.30 (MacDonald and Swan, 1970) in BE (9:1) solvent, i t is unlikely that t h i s compound has a lignan l i k e structure. When the TLC plate was examined under UV l i g h t , the unknown compound was strongly fluorescent. This observation indicates that the chemical structure of th i s compound may possess some chromophores or auxochromes, such as conjugated double bonds and carbonyl groups, or -OH and -OR groups, respectively. These unsaturated groups can undergo pi to * * pi and n to pi tr a n s i t i o n s in UV l i g h t energy range. After obtaining the p u r i f i e d compound, u l t r a v i o l e t (UV), infrared (IR), proton and carbon-13 nuclear magnetic resonance (NMR), low and high resolution mass spectra (MS) were taken for the compound. These four spectroscopic methods provide an immense amount of information about the structure of an organic molecule, often leading to a unique structure. There i s no universal way of considering each piece of information. In t h i s case, the best place to begin i s with the molecular ion in the mass spectrum, from which a molecular formula can be deduced. During recent years, extensive application of mass spectrometry as an a n a l y t i c a l tool in organic chemistry has 1 45 continued to p r o l i f e r a t e . This i s because the mass spectrometer has the capacity to produce a molecular ion and many fragment ions from the molecules under investigation. It then separates these ions according to their mass-to-charge r a t i o , and measures the r e l a t i v e abundances of each ion. If a molecular ion has s u f f i c i e n t s t a b i l i t y , a -5 li f e t i m e of approximately 10 seconds, i t w i l l be f u l l y accelerated and recorded at i t s corresponding m/e value in i t s mass spectrum. Thus, for the majority of compounds, mass spectrometry provides an exact and unambiguous method for determining molecular weight of a molecule (Howe et al, 1981). In t h i s study, two low resolution mass spectra of the unknown compound were recorded. These two samples, obtained by two separate sample p u r i f i c a t i o n s , gave i d e n t i c a l spectra (Figure 21). The corresponding mass-to-charge rati o s of the important peaks in the spectrum and their abundance data are l i s t e d in Table 15. The highest measured mass in the two i d e n t i c a l spectra was 301 (Peak 1) next to a more intense peak with mass 300 (Peak 2). The molecular ion and related isotopic species correspond to the peaks of the highest mass in the mass spectrum (Pasto and Johnson, 1969). In a number of cases, compounds give molecular ions of very 146 100 90 80 70 60 60 40 30 20 10 F i g u r e 21. Low Re s o l u t i o n Mass Spectrum of the Unknown Compound, 206 223 13 H . . . in .» 286 L - J H ' ' •• '"I ' ITI ' I ' I ' ITI ' I i 200 220 236 i i i I ' I ' I Y I ' I I'l i'i rl T j i i i i ' I T i i I |'i 240 260 280 ,269 300 I I'l I I'l | I I I 1 I I I I I | I I'l I I I I I I | I I I I I 300 320 340 100 _ 90 80 70 60 60 40 30 20 10 0 77 • III,., ...I, I I l ' l ' l ' | I I'l' 60 69 4 ?6 105 80 119 j 11 • 111111 j 1 111 1 j 111 1 |.l I [ 1 j 111 149 •5 170 1|63 181 100 120 140 160 180 200 Table 15. Data of the Low Resolution Mass Spectrum of the Unknown Compound. M E A S U R E D M A S S 301 300 266 ZB5 21B 269 257 255 251 23? 236 235 225 224 223 222 213 211 206 205 204 193 192 1S1 162 181 180 179 171 170 169 166 165 164 163 162 150 149 148 146 143 142 141 121 120 11S 118 108 107 106 105 104 103 NO. P O I N T S 25 35 25 43 14 29 25 25 29 25 35 21 35 35 43 17 35 21 35 43 35 35 29 35 29 51 35 43 43 51 El 35 43 51 51 43 51 59 43 43 S I 43 51 51 43 51 43 43 43 43 43 43 43 A B S O L U T E I N T E N S I T Y 71 1 . 3695. 2275. 8134. 596. 2219. 2162. 1881 . 161 1 . 1705. 4129. 1064. Z676 . 3311 . 17811 . 833. 4628. 955. 4681 . 15466. 3195. 4394 . 1998. 3239. 1971 . 6665. 2492. 4825. 6770. 52476. 10134. 7 307. 12466. 45579. 14700. 7952. 33188. 314064 . 11365. 19927. 10177. 6409. 51652. 45747. 10047. 56109. 8417. 9547. 19969. 14456. 37611. 20947. 11759. X I N T . B A S E 0 .5 1.2 B.l 2.6 0 .2 B.l B.l 0 .6 0 .6 0 .5 1 .3 0 .3 0 . 9 1 , 5, 0. 1 . 0 . 1 . 4 . 1 .0 1 .4 0 .6 1 .0 0 .6 2.1 0 .6 1 . 2. 16. 3. 2. 4 .0 14 .6 4 .7 2 .5 10.6 100.0 3.6 6 .3 3.2 2 .0 16.4 14.6 3 .2 17.9 2 .7 3 . 0 6.4 4 .6 12.0 6 .7 3 .7 X INT. NREF 0 . 5 1.2 0 .7 2 .6 0 .2 0 .7 0.7 0 .6 0 .6 0 .5 1.3 0 .3 0 .9 1 . 1 5.7 0 . 3 1.5 0 .3 1.5 4.9 1 .0 1 .4 0. 1 . 0. 2. 0 .8 1.5 2, 16. 3, 2. 4 .0 14.5 4.7 2 .5 10.6 100.0 3.6 C.3 3.2 2 . 0 16.4 14. 3. 17. 2. 3. 6 4 , 12.0 6.7 3 .7 X TOT. 1 ON 0 .0 0.2 0.1 0 .4 0 . 0 0.1 0 . 1 0.1 0.1 0.1 0 .2 0.1 0.1 0 .2 0 .9 0 . 0 0 .2 0 . 0 0 .2 0 .6 0 .2 0 .2 0.1 0 .2 0.1 0 .3 0.1 0.2 0 .3 2.6 0 .5 0.4 0.6 2.3 0 .7 0.4 1.6 15.6 0 .6 1.0 0 .5 0 .3 2.6 2 .3 0 .5 2.8 0.4 0 .5 1.0 0 .7 1.9 1 .0 0 .6 14B Table 15. (continued) MEASURED NO. MASS POINTS 97 43 96 35 95 43 94 43 93 43 92 35 91 43 85 71 84 51 83 35 83 35 82 43 81 43 60 43 79 43 76 43 77 43 71 43 70 35 69 51 68 35 67 43 66 21 65 35 59 35 58 29 57 43 56 35 55 43 ABSOLUTE X INT. INTENSITY BASE 15345. 4 .9 8963. 2 .9 5100S. 16.2 13940. 4.4 49294. 15.7 10166. 3.2 39061. 12.4 14686. 4.7 6155. 2 .0 12709. 4 .0 10708. 3.4 21964. 7.0 64014. 20.4 9485. 3 .0 22000. 7.0 12036. 3.8 50115. 16.0 10028. 3.2 63*4 . 2 .0 35524. 11.3 4659. 1.5 18514. 5 . 9 954. 0 .3 9325. 3 .0 4573. 1.5 1992. 0 .6 8099. 2 .6 5544. 1.8 9906. 3.2 X INT. X TOT NREF ION 4.9 0 .8 2 .9 0 .4 16.2 2 .5 4.4 0 .7 15.7 2.4 3.2 0 .5 12.4 1.9 4 .7 0 .7 2 . 0 0 . 3 4 .0 0 .6 3.4 0 . 5 7 . 0 1.1 20.4 3.2 3 .0 0 .5 7.0 1.1 3.8 0 .6 16.0 2 .5 3.2 0 .5 2 .0 0 .3 11.3 1.8 1.5 0.2 5.9 0 .9 0 .3 0 .0 3 .0 0 .5 1.5 0 .2 0 . 6 0.1 2.6 0 .4 1. B 0 .3 3.2 0 .5 149 low in t e n s i t y . The intensity of Peak 1 was 19.3% of Peak 2 (711/3565). The peak at 300 would appear to be a parent peak. From the natural isotopic abundance of carbon-13, th i s indicates approximately 18 carbons are present. The next set of r e l a t i v e l y intense peaks were at 286 and 285, with 285 being l i k e l y generated from the parent ion by loss of a CH 3 group. The highest intensity peak in the entire spectrum was at mass 149. This peak is rather interesting because i t s mass i s exactly half that of the parent ion, with the additional loss of two hydrogens. The study of the fragmentation pattern of t h i s spectrum indicated that t h i s unknown compound appeared to contain two fragments of the same mass. The presence of the d i s t i n c t fragments such as 300 (parent ion, M), M-15, M-2X15, M-44, M-2X44, M-2X14-2X44, and M/2 support this observation. The peak with mass of M-2X15 may be assigned to the loss of two CH 3 groups, while the peak with the mass of M-2X44 may be assigned to the loss of two -CO-O- groups in the molecule. In summary, information provided by a low resolution mass spectrum of t h i s compound showed that the molecular weight of this unknown compound i s 300 with approximately 18 carbons present. The chemical structure of t h i s compound i s possibly a symmetric dimer. 150 The information gained so far i s not adequate to f i n a l i z e the molecular formula. Analysis of a nuclear magnetic resonance (NMR) spectrum provided more valuable information. Pulse Fourier Transform proton and proton decoupling carbon-13 spectra were obtained in th i s study on the unknown compound. The proton NMR spectrum of the unknown compound of the present study i s shown in Figure 22. In a proton NMR spectrum, the number of signals in the spectrum indicates how many di f f e r e n t types of protons are present in the molecule. In this case the spectrum i s rather simple with only four singlet peaks. Their chemical s h i f t s are 0.0, 1.28, 2.3, and 7.3 ppm. The sharp signal at 0.0 ppm i s due to the 12 equivalent protons of the internal standard solution TMS. Therefore, there are only three type protons with d i f f e r e n t environments present in the molecule. The chemical s h i f t s of the signals show the st r u c t u r a l environment of the protons present, and the coupling of the signals indicates the relationships between d i f f e r e n t types of protons in the molecule. The chemical s h i f t s of standard methyl, methylene, and methine protons are l i s t e d in Appendix 2 (Nakanishi, 1962). In d i l u t e solution, the factors which influence chemical s h i f t s of protons are 151 Figure 22. Proton NMR Spectrum of the Unknown Compound, 1% 11 SO 10 • 0 IS 10 s IS 10 $ 10 i e io it SO IS predominantly intramolecular. Inductive e f f e c t s and anisotropic effects of chemical bonds are the two dominant factors. If the electron density around an atom i s reduced due to the inductive e f f e c t of an attached electronegative atom, resonance occurs at a lower value of the applied f i e l d and the nucleus experiences a greater deshielding. The chemical s h i f t s of the methyl hydrogens of CH^-X compounds are dependent on the electronegativity of the substituent X. From Appendix 8, i t is clear that when X changes from C to -C-C=C- and to C-0, the chemical s h i f t s of the methyl proton change from 0.9 to 1.1 to 1.3 ppm. For methylene and methine protons, the smallest chemical s h i f t s w i l l be 1.4 and 1.5 ppm respectively. In the spectrum of the unknown compound, the signal at 1.28 ppm can be assigned to the protons from methyl groups which are possibly attached to a substituent l i k e -C-0-. The peak at 2.3 ppm suggests the presence of a CH^-Ar or a -C-CH2-C=C- group. Since t h i s peak i s a si n g l e t , t h i s means there are not any neighbouring protons attached to the adjacent carbon atoms. Thus, the singlet at 2.3 ppm can only be from a methyl group in CH 3~Ar. Approximate chemical s h i f t s of protons attached to aromatic linkages are in the 6.0 to 9.0 ppm range (Appendix 1 53 8). The fourth peak in the unknown compound i s at 7.3 ppm and can be assigned to the aromatic protons in the molecule, or more precisely i t may come from an aromatic system involving a carbonyl group l i k e -CH=C-CO (6.5 to 8.0 ppm). There is a small shoulder in the fourth peak due to the residual protons in the deuterated chloroform solvent (7.25 ppm). Other very small peaks appearing in the spectrum are possibly due to sample impurities. In a proton NMR spectrum, the area of the signals t e l l the r e l a t i v e numbers of these d i f f e r e n t types of protons. Integration of the peak areas shows that the r e l a t i v e numbers of the three types of protons present in the compound are 1:3:6 for the signals at 7.3, 2.3 and 1.25 ppm respectively. In summary, the information obtained from the proton NMR spectrum of thi s compound indicates that i t possesses only three types of di f f e r e n t protons. None of these protons has any neighbouring protons. The three peaks at 7.3, 2.3 and 1.25 ppm can be assigned as protons from environments of -CH=C-CO (within an aromatic system), CH^-Ar and CH^-C-O-, with the number ra t i o s of 1:3:6 respectively. The proton NMR spectrum gives only information about the protons present in the molecule. Information about the 1 54 carbons present in the molecule can be recorded in a carbon-13 NMR spectrum. The chemical s h i f t range for carbon-13 in organic compounds i s 0-230 ppm ( r e l a t i v e to internal TMS), roughly 20 times the range observed for proton chemical s h i f t s , so the d i f f e r e n t type of carbons in the molecule can be e a s i l y recognized. Complexity in a carbon-13 spectrum can aris e due to carbon-proton coupling. To simplify carbon-13 spectra, a technique c a l l e d heteronuclear decoupling (or proton noise decoupling) is normally used. Carbon-13 spectra obtained in this way contain several sharp l i n e s , each of which represents a unique type of carbon atom in the molecule. Carbon-13 Broad Band (BB) proton decoupled and Attached Proton Test (APT) spectra are shown in Figure 23. The computer analysis of the spectral data is shown in Table 16. The carbon-13 BB proton decoupled spectrum c l e a r l y shows that there are at least eight carbon atoms in the molecule or that there are only eight d i f f e r e n t types of carbon atoms 3 present in the molecule. In general, sp carbon atoms resonate in the 0-60 ppm range ( r e l a t i v e to TMS) in the absence of electronegative atoms such as oxygen or halogens. In alkenes and benzenoid aromatics, these carbon atoms normally resonate in the 100-160 ppm region. Extreme 1 55 Figure 23. Broad Band and Attached Proton Test Carbon-13 Spectra of the Unknown Compound. C 1 3 B B A N D A P 11 E X P E R I M E N T S ' I ' ' 160 T 1 80 ' I ' 40 I I I I I I I I I I I 20 PPM 100 60 i i | i 0 A P T Table 16. Broad Band and Attached Proton Test Carbon-13 Spectra Data of the Unknown Compound. # CURSOR FREQ PPM INTEGRAL INTENS 1 5072 16897.696 167 .9457 .362 5.717 2 701 7 13930.131 1 38 .451 1 2.640 15.700 3 7203 13646.385 1 35 .631 0 2.528 16.112 4 7998 12432.486 123 .5661 2.338 22.892 5 8040 12368.493 1 22 .9301 2.364 16.903 6 1 1 046 7782.546 77 .3505 2.222 16.903 7 1 1066 7750.746 77 .0344 2.806 18.086 8 1 1087 7718.990 76 .7188 2.530 18.068 9 1 271 6 5233.955 62 .0201 1 .373 16.886 10 1 3829 3535.788 35 .1421 1 .322 16.893 1 1 1 441 7 2638.261 26 .221 6 7.829 81.081 12 1 61 36 15.389 . 1 530 .677 6.716 13 1 61 46 .206 .0021 10.324 78.630 APT SPECTRUM ANALYSIS: INDEX FREQ 1 D 10476.4 2 Q 3937.6 3 Q 1981.5 4 Q 12.7 PPM INTENSITY 138.89 260.055 35.20 17.174 26.27 65.493 .17 44.315 NO OF PROTONATED CARBONS: 4 CH 1 CH„ 0 CH, 3 157 p o l a r i z a t i o n of the pi-system of a double bond may cause resonance outside these l i m i t s . Carbonyl carbon resonances appear at a very low f i e l d , i . e . 155-230 ppm (Breitmaier and Voelter, 1974). The chemical s h i f t s of some t y p i c a l carbon-13 resonances are shown in Appendix 9 (Williams and Fleming, 1980). The eight types of carbon in the unknown compound f a l l into the three ranges mentioned above. Three of them 3 belong to sp carbon atoms. Four types of carbon are in the alkene and benzenoid aromatic region. Only one type of carbon i s present in the carbonyl carbon atom region of the spectrum. Carbonyl carbon atom resonances are extremely valuable in structure elucidation since they allow the nature of carbonyl function to be deduced. Acids, esters, and amines (ppm less than 180) can be d i f f e r e n t i a t e d from aldehydes and ketones (ppm greater than 180) (Williams and Fleming, 1980). In t h i s case, the single signal for a carbonyl carbon atom in the molecule (at 169.945 ppm) could be in an ester, lactone or acid but not in a ketone or an aldehyde. APT carbon-13 spectra are superior to normal carbon-13 and proton decoupled carbon-13 spectra for determining the numbers of protons bonded to a carbon in a molecule. In a 158 carbon-13 APT spectrum, the signals for quarternary and methylene carbons appear normal while the methine and methyl carbons are inverted. In the APT spectrum of the unknown compound (Figure 23), the signals at 76.72, 77.03 and 77.35 ppm are due to the deuterated chloroform solvent. There are fi v e other normal signals in the spectrum. According to computer analysis of the data (Table 16), no carbon in the molecule is s p l i t into a t r i p l e t form. Thus, there are not any methylene carbons present in the molecule. A l l fi v e normal signals in the spectrum are due to the presence of quarternary carbons. According to chemical s h i f t s , the 3 signal at 62.02 can be assigned as a sp quarternary carbon atom. Three types of carbons with ppm at 122.93, 123.57, 135.63 are the quarternary carbons in the alkene and benzenoid aromatic region. The quarternary carbon at 167.95 ppm i s a carbonyl type carbon. The computer analysis indicates one carbon i s s p l i t into a doublet and i s found at 138.89 ppm. This signal i s inverted, so i t i s assigned as the only methine carbon in the molecule. This methine carbon i s an alkene or 3 benzenoid aromatic carbon. Three types of methyl sp carbons are assigned according to the quartet s p l i t t i n g in 159 the spectral data. They are the inverted signals at 35.20, 26.27 and 0.17 ppm. The signal at 0.17 ppm i s caused by the TMS internal standard. One of the most s t r i k i n g features of carbon-13 spectra is the intensity variat ion between resonances. Low i n t e n s i t i e s are quite c h a r a c t e r i s t i c of carbons without d i r e c t l y bonded hydrogens. In summary, the information provided by the carbon-13 NMR spectra makes i t clear that in the unknown compound there are eight d i f f e r e n t types of carbons present, but only three types have protons bonded to them. The other fiv e types of carbons are a l l quarternary carbons. Gathering the evidence obtained from proton and carbon-13 NMR spectra, i t i s not d i f f i c u l t to f i n d that the r a t i o of the methine carbon to two types of methyl carbons i s 1:1:2 because their proton r a t i o i s 1:3:6. Therefore i t can be said that there are at least nine carbons and at least ten protons present in the molecule. Evidence from the low resolution mass spectral fragments shows that t h i s unknown compound i s symmetric, and could therefore be a dimer of a compound present during i t s formation, such as t h u j a p l i c i n s . It i s reasonable to assume the molecule should at least involve 2X9 carbons and 2X10 protons. The carbon at 160 167.9457 ppm in the carbon-13 NMR spectrum i s assigned to an acid, ester, or lactone carbonyl carbon but not an aldehyde or ketone carbonyl carbon, which means that t h i s carbonyl carbon bonds d i r e c t l y to two oxygens in the molecule. Since the compound has a symmetric dimer structure, i t w i l l have at least four oxygen atoms. The combination of a l l the above facts give a molecular formula for the unknown compound of ^i8 H20 O4 w^ f c^ a molecular weight of 300.1362 (a.m.u.). This molecular weight i s calculated based on a table of atomic weights for isotopes used in high-resolution mass spectrometry of organic compounds (Table 17) from Williams and Fleming, 1980. The mass of the molecular ion obtained in the low resolution mass spectrum was 300, which i s the same as the mass deduced from the above structural data. Two high-resolution mass spectra were c o l l e c t e d to accurately determine the molecular weight of the compound. One of these spectra i s shown in Figure 24. The spectrum has exactly the same pattern as those done at low resolution. The molecular weights given by the two high resolution spectra are s l i g h t l y d i f f e r e n t . One molecular wieght i s 300.1368 and the other is 300.2075, which i s 0.02% higher than the expected molecular weight. The composition 161 Table 17. Atomic Weight of Isotopes Used in High Resolution Mass Spectroscopy. Isotope Atomic Weight Natural Abundance (C12=12.000000) (percent) 1H 1.007825 99.985 2H 2.011402 0.015 1 2C 12.000000 98.9 1 3C 13.003354 1.1 1 4N 14.003074 99.64 1 5N 15.000108 0.36 1 6 0 15.994915 99.8 1 7 0 16.999133 0.04 1 8 0 17.999160 0.2 Source: Willams and'Fleming, (1980). 1 62 100 9B BB 7B SB 50 40 30 20 10 0 Figure 24. High Resolution Mass Spectrum of the Unknown Compound. I I I I I | M ' l 260 TW rrr . i f " . . . n f 111111 m 280 300 II I I I I J II IT 320 T T I | I II 340 II I I | I M I I I I I I | I I I I II I I I | I I I I I I I I I | I I M I I I I I [ I I I 360 380 400 420 440 TT from exact isotopic weights for mass 300.1368 is ^18 H20°4" The fragments appearing in the two spectra at 149.0594 and 149.0609 best match a mass of 149.0602 which has the composition CgHgO,,. This c l e a r l y demonstrates the symmetric, dimeric structure of the unknown compound. The same fragments previously discussed with respect to the low resolution mass spectra were also observed in the high resolution spectra. If the information obtained from both mass spectra and NMR spectra i s correct, the molecular weight of this compound i s 300.1368 and the molecular formula i s ^ 13H20^4" According to the molecular formula, the ra t i o of the numbers of carbons to hydrogens in the molecule i s 18:20. The amount of carbon and hydrogen contained in the molecule is expected to be 71.89% and 6.71% of the molecular weight. Results of micro-elemental analysis on a c r y s t a l i z e d form of the unknown compound showed that the percentages of carbon and hydrogen in the molecule are 72.14% and 6.78%, respectively. The difference beteween found and expected values are +0.16% for carbon and +0.07% for hydrogen, which are both acceptable according to the precision of the analysis (maximum 2%). This result strongly confirmed the previous conclusions. 164 The information outlined so far proves that t h i s unknown compound possesses a molecular weight of 300.1362 mass units with a molecular formula as C18 H20°4" T ^ e structure of this compound appears as a symmetric dimer. From sapwood bioassay res u l t s , i t has been demonstrated that t h u j a p l i c i n i s a precursor of t h i s unknown compound. It i s reasonable to assume that the symmetric structure of this unknown compound possibly contains the dimer of modified thujaplic ins. The number of double bonds and rings in the molecule i s given by the equation: C^^O double bond equivalents (DBE) (2a + 2) - b . DBE = (Williams and Fleming, 1980) 2 The (2a + 2) term is the number of hydrogens in a saturated hydrocarbon having 'a' carbon atoms. Every ring or double bond means two fewer hydrogen atoms, while 'b', the actual number of hydrogen atoms present from (2a + 2), i s subtracted. Dividing by two gives the t o t a l number of double bonds and rings in the molecule. In t h i s unknown compound, DBE = 9 , so the t o t a l number of double bonds plus rings is nine. There are dozens of st r u c t u r a l isomers of compounds 165 with the molecular formula Cig H20°4* Based on a l l the information obtained, the most reasonable structure so far i s shown in Figure 25. UV, v i s i b l e and IR spectra were obtained to further examine accuracy of the proposed structure. The UV and v i s i b l e spectra of the compound are shown in Figure 26 and 27. The position (285 nm) of the strong UV absorption peak indicates an extended system of conjugation including a carbonyl group. This i s in agreement with the structure * proposed from other spectroscopic evidence. The n to pi t r a n s i t i o n of diketones or quinones w i l l give a second peak which usually occurs in the v i s i b l e 340-440 nm region and gives r i s e to the l i g h t yellow color of some of these compounds (Williams and Fleming, 1980). The peak which appears at 415 nm in the unknown compound spectrum is probably due to t h i s t r a n s i t i o n . The FTIR spectrum of t h i s unknown compound is shown in Figure 28. The spectral data are l i s t e d in Table 18. The evidence provided by the IR spectrum i s quite conclusive. Selected standard carbon-hydrogen absorption band and X-H absorption band data are given in Appendix 10 and 11 (Pasto and Johnson, 1969). The oxygen-hydrogen stretching absorption of alcohols, 166 Figure 25. Chemical Structure of "Thujin". molecular foumula: c 1 s H 2 0 ° 4 molecular weight: 300.1362 (a.m.u.) 3,3,4,7,7,8-hexamethy1-2,6,-dioxa-1,5-anthracenedione 167 168 Figure 28. Fourier Transform IR Spectrum of the Unknown Compound. 3 8 0 0 . Q 3 2 0 0 . O 2 6 Q O . O 2 Q O O . D 1 7 0 0 . O 1 4 0 Q . O 1 I O O . Q B O O . O O W A V E N U M Q E R S < C M - l > S O O . O O 2 0 0 . O O Table 18. Fourier Transform IR Spectral Data of the Unknown Compound. Peak no. Wavenumbers (cm ) 1 3016 2 2962 3 2878 4 1733 5 1599 6 1574 7 1500 8 1459 9 1381 10 1261 11 1192 12 1126 13 1076 1 4 745 15 721 171 phenols, and in t e r - or intra-molecular hydrogen bonding appears as a broad band with a maximum absorption in the 3650 to 3200 cm 1 range. Only in the case of oxygen-hydrogen stretching of carboxylic acids w i l l the absorption be out of t h i s region, appearing as a very broad band with maximum absorption at approximately 2940 cm 1, extending down to nearly 2500 cm 1 owing to the very strong intermolecular hydrogen bonding between carbonyl groups. The lack of any absorption peaks in the IR spectrum of the unknown compound in thi s region emphasizes the absence of an OH group in the structure. Carbon-hydrogen absorption occurs in two regions, the C-H stretching region from 3300 to 2500 cm 1 and the bonding region from 1550 to 650 cm 1. The methyl group gives r i s e to two stretching bands which generally occur at 2960 cm 1 and 2870 cm 1. These two peaks are observed in the FTIR spectrum of the unknown compound at a wavelength of 2962 cm 1 and 2878 cm 1. These two bands are assigned to the asymmetric and symmetric stretching deformation involving the entire methyl group. Methyl groups also give r i s e to two peaks in the asymmetric and symmetric bending deformation region near 1460 cm 1 and 1380 cm 1. The 1380 cm 1 region i s extremely valuable for detecting presence of 1 72 methyl groups because t h i s region i s almost devoid of other types of absorption bands (Pasto and Johnson, 1969). Two peaks are observed in the spectrum of the unknown compound at 1459 cm 1 and 1381 cm 1 corresponding to these deformations. The t y p i c a l absorption bands corresponding to a l k y l methylene ( C r ^ ) and a l k y l methine (CH) were not found in the spectrum. Carbon-carbon single bond stretching bands are extremely variable in position and are usually quite weak in intensity. They usually appear in the wavelength range less than 1400 cm 1, which i s also c a l l e d the fingerprint region (Fessenden, 1982). In the fingerprint region, the spectrum is often quite complex and c o r r e l a t i o n of an individual band with a s p e c i f i c functional group usually cannot be made with accuracy. Therefore, these absorption bands are of l i t t l e p r a c t i c a l use in st r u c t u r a l determinations. Carbon-carbon double bond stretching absorption occurs in the 2000 to 1430 cm 1 region (Appendix 12) (Pasto and Johnson, 1969). In the spectrum obtained, there are four peaks with wavelengths of 1599, 1574, 1500, and 1459 cm 1 in this region. It i s well known that the s k e l e t a l C=C vibrations of aromatics give r i s e to a series of four bands in t h i s region. These bands occur very close to 1600, 1580, 173 1500 and 1450 cm (Appendix 13). These bands vary greatly in i n t e n s i t y . The observed IR data and the absorption pattern of the unknown compound in this region very c l o s e l y match aromatic -C=C- absorption data. These peaks prove the presence of a conjugated structure in the molecule. One of the most d i s t i n c t i v e bands in an IR spectrum i s the one a r i s i n g from the carbonyl group. This usually strong peak i s observed somewhere between 1640 and 1820 cm 1 . Appendix 13. l i s t s the band positions for a number of d i f f e r e n t types of compounds that contain the OO group. The carbonyl peak appears in the unknown compound IR spectrum at 1733 cm 1, which i s a c h a r a c t e r i s t i c carbonyl peak for an a c y c l i c ester or a six-membered ring lactone (Appendix 13).- Other nearby peaks in t h i s area are 1725 cm 1 for aldehydes and 1710 cm 1 for carboxylic acids (Appendix 13). Since there are no OH absorptions in the spectrum, the p o s s i b i l i t y of the carbonyl peak being due to a carboxylic acid i s eliminated. The carbon-13 NMR spectrum of the unknown compound has c l e a r l y demonstrated the lack of aldehyde and ketone carbonyl groups in the structure. Above a l l , the 1733 cm 1 absorption band i s closest to that for a lactone carbonyl, so t h i s band can be assigned to a lactone carbonyl in the molecule. 174 In summary, evidence obtained from UV and FTIR spectra of the unknown compound f u l l y supports the proposed chemical structure. Elucidating chemical structure of t h i s compound by j o i n t application MS, NMR (proton and carbon-13), FTIR, and UV spectroscopy provides conclusive evidence leading to the f i n a l i d e n t i f i c a t i o n of the unknown compound as the proposed structure. A l i t e r a t u r e search for any previous report on th i s compound was ca r r i e d out by searching Chemical Abstracts •through the period from 1920 to the end of 1985. No previous information about th i s compound has been found. Two of the closest s t r u c t u r a l l y related compounds found were: (Tamura et al , 1974) According to the International Union of Pure and Applied Chemistry (IUPAC) d e f i n i t i v e rules for nomenclature of organic compounds (Weast, 1978) t h i s compound can be named 3,3,4,7,7,8-hexamethyl-2,6-dioxa-1,5-anthracenedione. 175 A t r i v i a l name of 'Thujin' was given to t h i s compound (as suggested by Drs. Swan and Wilson) based on i t s source (from Thuja plicata Donn. and th u j a p l i c i n ) (Figure 25). 176 4.5. Mechanisms of Thujaplicin's T o x i c i t y It i s well recognized that t h u j a p l i c i n s in WRC extractives exhibit a strong t o x i c i t y to f i n a l decay fungi which attack l i v i n g trees and timber in service (Rennerfelt, 1948). Poria al bi pel I uci da Baxter. is considered the most common decay fungus isolated from l i v i n g WRC trees in the coastal area (Buckland, 1946). The t o x i c i t y of t h u j a p l i c i n to Poria al bi pel I uci da Baxter. was tested in t h i s study. There were sixteen d i f f e r e n t concentrations of t h u j a p l i c i n used. The fungal i n h i b i t i o n of t h u j a p l i c i n at these sixteen concentrations to the fungus i s shown in Figure 16. Obviously, t h u j a p l i c i n i s toxic to t h i s fungus. It i s believed that the reactive keto-enolic group i s the key functional group in t h u j a p l i c i n s responsible for fungal t o x i c i t y . A c t i v i t y of the keto-enolic group in beta-thujaplicin has only been tested once before, in an experiment conducted for the t o x i c i t y of t h u j a p l i c i n to yeast (Raa and Goksoyr, 1965). This assumption has never been tested for wood decay fungi. In order to test this hypothesis, the t h u j a p l i c i n solution with the highest concentration (5 mg/ml) used in the t h u j a p l i c i n t o x i c i t y test was methylated by etheral diazomethane. At 5 mg/ml, 1 77 t h u j a p l i c i n ' s i n h i b i t i o n effect to Poria al bipel I uci da Baxter. was very strong. The i n h i b i t i n g effect of methylated t h u j a p l i c i n to the same fungus i s shown in Figure 17C. The results of this test indicate c l e a r l y that methylated t h u j a p l i c i n has no t o x i c i t y to t h i s fungus, thus, i t can be p o s i t i v e l y concluded that the t o x i c i t y of t h u j a p l i c i n to certain groups of fungi is due to the presence of the reactive keto-enolic group. The t o x i c i t y w i l l disappear i f the a c t i v i t y of t h i s reactive keto-enolic group is blocked, as i s done by methylation. As discussed e a r l i e r , in l i v i n g trees, the infection of straw-colored heartwood by the f i r s t fungus Sporothrix sp. would convert the t h u j a p l i c i n s to a new compound (thujin) which has structure as demonstrated. The t o x i c i t y of thujin to Poria al bipel I uci da Baxter. was tested. The experiment was based on a comparison between the i n h i b i t i n g effects of beta-thujaplicin and thujin, which was obtained from the same amount of wood sample (Figure 17A). At the same time, four solutions of thujin with much higher concentrations were tested by the same method (Figure 17B). From Figure 17A, i t i s found that in the same amount of wood, the t h u j a p l i c i n present s i g n i f i c a n t l y inhibited fungal growth, but thujin present did not show any i n h i b i t i o n . It might be 178 argued, based on the result of Figure 17A alone, that thujin i s not toxic at t h i s concentration but might be toxic to the fungus at higher concentrations. The results given in Figure 17B eliminated t h i s assumption. The concentrations of thujin used in Figure 17B were much higher than the concentration of t h u j a p l i c i n used in the experiment shown in Figure 17A. It can be concluded from this set of experiments that the t o x i c i t y of t h u j a p l i c i n to Poria al bipel I uci da Baxter, i s lost when the t h u j a p l i c i n i s converted to thujin during the Sporothrix sp. in f e c t i o n . S t r u c t u r a l l y , i t i s clear that the mechanism of this d e t o x i f i c a t i o n involves the deactivation of the reactive keto-enolic group in the t h u j a p l i c i n by oxidative dimerization and isomerization of t h u j a p l i c i n to a lactone type compound. No attempts were made in t h i s study to is o l a t e and identi f y the intermediates in t h i s conversion process, so the actual biochemical pathway for the formation of thujin from t h u j a p l i c i n during the Sporothrix sp. infection is unknown. Based on structures of the s t a r t i n g and ending products, the properties of the compounds and the enzymatic nature of these processes, however, one of the possible biochemical pathway can be suggested. 179 When the s t a r t i n g compound i s b e t a - t h u j a p l i c i n , the f o l l o w i n g may occur. When the s t a r t i n g compound i s gamma-thujaplicin, the f o l l o w i n g may occur. 180 C l , The postulated formation of thujin is by enzymatic biochemical processes, unlike synthetic organic reactions under controlled conditions in a laboratory. An outstanding feature of enzyme a c t i v i t y in b i o l o g i c a l processes i s substrate s p e c i f i c i t y , which determines i t s b i o l o g i c a l function. Another c r i t i c a l b i o l o g i c a l feature of enzymatic reactions i s that their high degree of substrate and c a t a l y t i c s p e c i f i c i t y ensure synthesis of only s p e c i f i c biomolecular products without the concomitant production of byproducts. Therefore, the i s o l a t i o n and i d e n t i f i c a t i o n of the enzymes and intermediates involved in the above processes would be necessary steps before a greater knowledge of the natural processes can be gained. Also, further research i s the only way to test the proposed pathway. Although the exact pathway of t h i s process i s unknown, during the dimerization of t h u j a p l i c i n to thujin, the t h u j a p l i c i n s change from stressed seven carbon ring structures to the more stable six member ring form. It i s required by stoichiometry that in t h i s process, beside energy, two atoms of carbon are released. Carbon i s one of the most important essential elements for the growth of fungi, and i s required in greater quantities than any other 181 essential element by fungi. Thus, this release of carbon i s rather important, since i t implies a possible motivation behind the Sporothrix sp. infection process i s to generate carbon to support i t growth. 182 4.6. Future perspectives This study demonstrated the b i o l o g i c a l functions of three early stage attacking fungi on WRC heartwood in l i v i n g trees, p a r t i c u l a r l y the role of Sporothrix sp. The mechanism of Sporothrix sp. d e t o x i f i c a t i o n of t h u j a p l i c i n s by converting t h u j a p l i c i n s to a dimerized lactone compound, thujin, i s supported by evidence. The quantitative measurement of thujin showed, however, that concentration was 0.291% in discolored outer heartwood (D-O) and 0.187% in discolored inner heartwood (D-I). This accounted for about 60% and 45% of the t h u j a p l i c i n s which had disappeared during disco l o r a t i o n (from L-0 to D-0 and L-I to D-I r e s p e c t i v e l y ) . Where are the remaining t h u j a p l i c i n portions? One of the explanation could be that remaining t h u j a p l i c i n portions are the intermediates in biodegradation processes. On the other hand, an important unexpected finding resulted from toluene extraction of discolored WRC heartwood. There is a c e r t a i n amount of t h u j a p l i c i n chelate compounds present in discolored heartwood which were not noticable with BE solvent. These metal chelates were isolat e d for the f i r s t time from WRC heartwood extractives by t h i s work. The v e r i f i c a t i o n of the metal chelating was 183 achieved by carrying out a series of reactions and f i n a l l y i d e n t i f y i n g FeS (dark black-green colored p r e c i p i t a t e ) , and thu j a p l i c i n s by TLC as products. Another alternative explaination for t h u j a p l i c i n loss is that during heartwood disc o l o r a t i o n , accompanying the active accumulation of metal ions (Safford and Shigo, 1974), a portion of th u j a p l i c i n s in WRC heartwood form chelates with metal ions. The source of the metal ions and the influence of these metal chelates on the entire microorganism attacking process and consequently decay resistance would make an interesting research topic. The mechanism of t h u j a p l i c i n t o x i c i t y to decay fungi, such as Poria al bipel I uci da Baxter., has been proven to be due to a c t i v i t y of the keto-enolic group in t h u j a p l i c i n s . Detailed modes of action, however, remain unknown. Based on th u j a p l i c i n properties, several modes can be proposed. They involve p o s s i b i l i t i e s for formation of metal chelates with metal ions inside the wood, or with metal ions associated with fungal enzymes, or formation of amine t h u j a p l i c i n s a l t s with amino-acids of the fungi, or formation of hydrogen bonding between thujap l i c i n s and fungal protein. Any or a l l of these actions would i n h i b i t fungal growth. 184 CONCLUSIONS Thujaplicins which are responsible for natural decay resistance of WRC, interact with various fungi existing within l i v i n g WRC trees. In th i s study, three early stage attacking fungi (Sporothrix sp. , Ki r s chest er ni el I a t huj i na (Peck) Pomerleau & Etheridge and Phialophora sp.) in WRC discolored heartwood were isolated and i d e n t i f i e d . The frequency d i s t r i b u t i o n of the fungi indicated a clear fungal attacking pattern. Sporothrix sp. was the f i r s t fungus attacking sound heartwood. Sporothrix sp. alone did not cause serious weight loss of wood blocks d i r e c t l y , but did remove t o x i c i t y of th u j a p l i c i n s to decay fungi. This study demonstrated that interaction between Sporothrix sp. and sound WRC heartwood resulted in an oxidative dimerization and isomerization of t h u j a p l i c i n s to a new lactone compound, which was proven to be no longer toxic to decay fungi, such as Poria al bipel I uci da Baxter. Therefore, antifungal a c t i v i t y of t h u j a p l i c i n s are shown to be highly s e l e c t i v e . On one hand, they are very toxic to wood destrying fungi, on the other hand, they are not very toxic to some "pioneer" fungi, l i k e Sporothrix sp., which can even detoxicify t h u j a p l i c i n s . 185 It has been demonstrated that the chemical mechanism of t h u j a p l i c i n t o x i c i t y to decay fungi i s due to the presence of the reactive keto-enolic group. Toxicity disappears i f the a c t i v i t y of t h i s reactive group i s blocked, as was done in t h i s study by methylation of the group. This group is also altered by Sporothrix sp., thereby, removing th u j a p l i c i n ' s antifungal a c t i v i t y . The mechanism of Sporothrix sp. detoxifying t h u j a p l i c i n s involves, s t r u c t u r a l l y , deactivation of the reactive keto-enolic group of t h u j a p l i c i n s by oxidative dimerization and isomerization of i t to a lactone compound. According to i t s chemical structure, this lactone compound i s named 3,3,4,7,7,8-hexamethyl-2,6-dioxa-1,5-anthracene-dione. A t r i v i a l name of thujin i s also given. The i s o l a t i o n and i d e n t i f i c a t i o n of thujin i s achieved by the j o i n t application of chemical, chromatographic and spectroscopic methods. Relationship between the three early attacking fungi is established in t h i s study. 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P u b l . N o . 6 8 : 5 3 . Z a v a r i n , E . , S m i t h , R . M . a n d A . B . A n d e r s o n . 1 9 5 9 . P a p e r c h r o m a t o g r a p h y o f t h e t r o p o l o n e s o f Cupressaceae I I . J . O r g . C h e m . 2 4 : 1 3 1 8 - 1 3 2 1 . 200 Appendix 1. L i s t of Synonyrni for the Fungi Used in the Text. Fomes annosus (Fr.) Karst. Heterobasi di on annosum (Fr.) Bref. Fomes nigrol imi t at us (Romell) Egel. Phellinus ni gr ol i mi t at us (Rom.) B. & C . Fomes pi ni (Thore) Lloyd. Phellinus pini (Fr.) Karst. Fomes pi ni col a (sw) Cke. Fomitopsis pi ni col a (Fr.) Karst. Polyporus balsameus Peck. Tyromyces balsameus (Peck) Murr. Polyporus s c h w e i n i t z i i Fr. Phaeolus s c h w e i n i t z i i (Fr.) Pat. Poria al bipe11 uci da Baxter. Poria rivulosa (B. & C.) Cooke. Poria a s i a t i c a Overh. Tyromyces seri ceomolIis Rom. Poria monticola Murr. Tyromyces placenta (Fr.) Ryv. Poria w e i r i i Murr. PhelI i nus w e i r i i Murr. 201 Appendix 2. Thujaplicin Content from Old Growth WRC by Colorimetric Method. Sample Radius Ave. age, Thujaplicin from pith,mm yr. (o-d wood) Tree 1 38 26.0 0.000 butt 76 77.0 0.002 height 1 1 4 128.0 0.003 1 52 180.0 0. 1 30 190 231 .0 0.440 228 282.0 0.620 267 334.0 0.830 305 385.0 1 . 1 30 343 436.0 1.150 381 488.0 1 .220 Tree 2 1 52 197.0 0.009 butt 1 90 253.0 0.013 height 228 309.0 0.014 267 366.0 0.019 305 422.0 0. 120 343 478.0 0.580 381 534.0 0.610 . 419 591 .0 0.610 457 647.0 0.670 Tree 3 91 .0 0.110 breast 111.0 0. 150 height 135.0 0.510 161.0 0.410 191.0 0.500 218.0 0.580 255.0 0.600 . 284.0 0.630 Tree 3 7.0 0.001 33% 19.0 0.007 height 40.0 0.011 60.0 0.036 101.0 0.036 139.0 0.360 202.0 0.520 Source: MacLean and Gardner, (1956b). 202 Appendix 3. Thujaplicin Content from Chromatography Method. Sample #1 #2 #3 #4 Average Total rings thuja from pi ic i pith % 20.0 0.034 70.0 0.337 1 30.0 0.460 1 90.0 0.543 235.0 0.564 262.0 0.655 280.0 0. 100 62.0 0.248 184.0 0.602 289.0 0.867 379.0 0.852 469.0 0.970 544.0 0.543 587.0 0.974 615.0 0.057 40.0 0.068 1 25.0 0.377 210.0 0.352 310.0 0.763 410.0 0.865 475.0 0.747 558.0 0.919 610.0 0.051 50.0 0.320 1 25.0 0.706 295.0 0.690 385.0 0.570 475.0 0.622 565.0 0.377 640.0 0.575 683.0 0.621 703.0 0.015 Source: Nault, (1986). Old Growth WRC by Gas Sample Average Total #5 #6 #7 rings thuja from pi ic i p i t h % 67.0 0.024 1 79.0 0.238 254.0 0.352 314.0 0.392 389.0 0.651 464.0 1 .774 509.0 1 .742 539.0 0.707 566.0 0.072 20.0 0.014 70.0 0.333 1 30.0 0.607 175.0 0.840 205.0 1 .400 238.0 1.315 250.0 0. 125 15.0 0.040 90.0 0.044 195.0 0.093 270.0 0.020 330.0 0.004 375.0 0.121 383.0 0.278 412.0 0.032 203 Appendix 4. Thujaplicin Content from Second Growth WRC by Gas Chromatography Method. Sample Average Total rings thuja-from p l i c i n , p i t h % #1-1 2.5 0.014 7.5 0.011 12.5 0.058 17.5 0.145 25.0 0.280 35.0 0.323 45.0 0.091 #1-2 2.5 0.086 7.5 0.086 12.5 0.108 17.5 0.081 25.0 0.034 35.0 0.058 #1-3 2.5 0.021 7.5 0.058 12.5 0.060 17.5 0.111 25.0 0.190 35.5 0.366 45.5 0.224 #1-4 2.5 0.035 7.5 0.070 12.5 0.077 17.5 0.060 25.0 0.264 35.0 0.525 45.0 0.156 #1-5 10.0 0.022 22.5 0.058 32.5 0.169 37.5 0.376 45.0 0.348 55.0 0.426 Sample Average Total rings thuja-from p l i c i n , p i t h % #2-1 12.5 0.043 27.5 0.097 32.5 0.161 37.5 0.244 42.0 0.467 50.0 0.406 60.0 0.436 #2-2 5.0 0.099 12.5 0.134 17.5 0.186 25.0 0.249 35.0 0.415 45.0 0.315 #2-3 12.5 0.033 27.5 0.046 32.5 0.080 37.5 0.205 42.5 0.188 50.0 0.186 60.0 0.327 #2-4 2.5 0.058 7.5 0.007 12.5 0.030 17.5 0.130 25.0 0.154 35.0 0.169 #2-5 30.0 0.099 62.5 0.213 67.5 0.157 72.5 0.250 Source: Nault, (1986). 204 Appendix 5. C H E C K LIST OF F U N G I C O L L E C T E D O N W E S T E R N R E D CEDAR IN BRITISH C O L U M B I A F R O M 1943 TO 1945 Species Loca l i t y of collections Armillaria mellea (Fr . ) Quel . 12, 13, 18* Auricularia auricularis (Gray) M a r t i n 13 Collybia sp. 12 Coniophora betulae (Schum.) K a r s t . Coniophora puteana (Schum. ex Fr . ) K a r s t . Coniophora suffocala (Peck) Massee 13 16 16 Corlicium sp. Corlicium bicolor Peck Corlicium cebennense Bou rd . Corlicium coronilla H o h n . Corlicium livido-cacruleum Karst . . Corlicium racemosum Bur t . Corlicium radiosum F r . Corlicium sidphureum (Pers. ex F r . ) F r . 1 , 3 , 7, 11 3 3 3 1 ,3 12 12 1 Crcpidolus herbarum Peck 17 Flammula decorala M u r r . Flammula liqiiiritiac (Weinm.) Quel . 17 11 Fomes applanatus (Pers.) W a l l r . Fomes annosus (Fr.) C k e . Fomes nigrolimitatus (Romell) Ege l . Fomes Pini (Thore) L l o y d and var . abielis K a r s t . 3 1 ,3 , 7 , 9 7, 11 5, 9, 11, 12, 13, 15 Fomes pinicola (Swartz) C k e . 1 ,2 , 10 Gloeocyslidium ochroleucum Bres. 1 Helvetia sp. 3 Hypholoma fasiculare (Fr.) Quel . Hypholoma capnoides (Fr . ) Que l . 3 1 IJyynenochaele fidiginosa (Pers.) Bres. llymenochaete tabacina (Sow.) L e v . 13 1, 3, 5, 12, 13, 18 Lenziles saepiaria (Wulf . ) Fr. 16 * The numbers refer lo localities on the map 205 Appendix 5 . (continued) Species Locality of collections Mnnixmius scorodonius Fr. 3 Mcmlius Jugnx Fr. 1 Mycena griseiconica KaufTm. 5 Odonlia sp. Odontic alutacea (Fr.) B. & G. Odonlia alutacea subsp. flocctisa B. & G.? Odonlia aspera (Fr.) Bourd. Odonlia lactea Karst. 1.3, 11 1 16 9 12, 13 Omphalia campanella (Fr.) Quel. 4, 7, 8, 12, 15, 18 Peniophora sp. Peniophora crassa Burt. Peninphora Jlavo-ferruginea Karst. Peniophora san guinea (Fr.) Hres. Peniophora vcluiina (DC) Ckc? j Phlchia mellea Overh. 13 Polyporus ahietinus (Dicks.) Fr. Polyporus bnlsameus Peck Polyporus cacsius (Sclirail.) Fr. Polyporus cuneaius (Murr.) Overh. Polyporus dichrous Fr. Polyporus elcgans (Bull.) Fr. Polyporus hirsulns (Wulf.) Fr. Polyporus immitis Peck Polyporus perennis (L.) Fr. Polyporus Schweinitzii Fr. Polyporus sewipileatns Peck Polyporus undosus Peck Polyporus versicolor (L.) Fr. 13, 15, 18 12, 15, 17 19 5, 7, 11, 12, 13, 14, 15, 17, 18 13, 16, 18 3, 12 16, 18 10 5 5 13, 15, 18 1 5 Poria sp. Poria alhipellucida Baxter Poria asiatica (Pilat) Overh. Poria candidissima (Schw.) Cke. Poria isabellina (Fr.) Overh. Poria lenis Karst. Poria nigrcscens Bres. Poria sericeo-tnollis (Komcll) Baxter Poria sinuosa (Fr.) Sacc. Poria subacida (Peck) Sacc. Poria Weirii Murr. 1, 13 1, 2, 3, 4, 5, 6. 7, 8, 11, 13 1, 4, 5, 6, 7, 8, 12, 14, 15, 18, 19 1 1, S, 6 1 11 1 11 1 , 2 , 3 , 5, 7, 11, 12, 13, 18 1 ,3 .4 ,8 , 9, 10. 12, 13. 15, 17, 18 Psathyrella sp. 11 Schizophyllum commune Fr. 1 Stereum Chailletii Pers. Stereum rugosiusculum B. & C. Stereum sanguinolenlum Alb. & Schw. 3 3 18 Tomenlella sp. 1 Trameles carbo'naria (B. & C.) Overh. Trametes mollis (Sommerf.) Fr. Trameles sepium Berk.? 1, 16, 19 13 19 2 0 6 Appendix 5. (continued) L O W E R B R I T I S H C O L U M B I A Scale — 1 inch = 140 miles 132* 130* 128* 126* 124* 122" 120* S T U D Y O F D E C A Y IN WE5TERN R E D C E D A R L O C A L I T I E S I N V E S T I G A T E D 1 - Cowichan Lake 1 1 - Sk idegate Lake 2 - N i xon C r e e k 12 - Clearwater R iver 3 - M e a d e C r e e k 13 - Blue R iver 4 - Copper Canyon, Chemainus 14 - Upper Thompson River 5 - M u i r C r e e k , Sooke 1 5 - LaForme Creek 6 - Bonel l Creek, Nanoose Bay 16 - Illecillewaet R iver 7 - Elsie L a k e , A lbern i 1 7 - Begb ie C reek 8 - Tsable River, Fanny Bay 18- Mabel Lake 9 - C o u r t e n a y D is t r i c t 1 9 - Shuswap R ive r 10 - Port Moody Cedar Sawmill Localities investigated in the study of decay in western red ceda Source: Buckland, ( 1 9 4 6 ) . 207 Appendix 6, Results of Analysis of Variance for the WRC Heartwood Weight Loss. UNIVARIATE 1-WAY ANOVA ANALYSIS OF VARIANCE OF 4 . WL% N= 36 OUT OF 36 SOURCE DF SUM OF SORS MEAN SOR F-STATISTIC SIGNIF BETWEEN 8 139 .53 17.441 153.75 .0000 WITHIN 27 3.0629 .11344 TOTAL 35 142.59 (RANDOM EFFECTS STATISTICS) ETA= .9892 ETA -SQR= .! 9785 (VAR COMP= 4.3319 '/.VAR AMONG= 97.45) EQUALITY OF VARIANCES: DF= 8. 656.10 F= 4.2082 .0001 TREAT N MEAN VARIANCE STD DEV (1) 4 1.4005 .29684 -1 . 17229 (2) 4 1 .5521 .12223 - 1 .11056 (3) 4 .89627 .11459 -1 .10705 (4) 4 5.4758 .65591 -1 .25611 (5) 4 3.6285 .43461 .65925 (6) 4 1.2500 .20537 -1 .14331 (7) A 5.5609 .37924 .61582 (8) 4 .63486 .67357 -1 .25953 (9) 4 .21286 -1 .26849 -3 .16386 - 1 GRAND 36 2.2689 4.0741 2.0184 Conclusion: The variances of nine groups are not homogenous and data transformation i s needed. UNIVARIATE 1-WAY ANOVA ANALYSIS OF VARIANCE OF 7.SOWL% N= 36 OUT OF 36 SOURCE DF SUM OF SORS MEAN SOR F-STATISTIC SIGNIF BETWEEN WITHIN TOTAL 8 17.471 2.1838 205.25 .0000 27 .28727 .10640 -1 35 17.758 (RANDOM EFFECTS STATISTICS) ETA= .9919 ETA-SQR= .9838 (VAR COMP = .54330 '/.VAR AMONG* 98.OS) EQUALITY OF VARIANCES: DF= 8, 656.10 F«= 1.2880 .2466 Conclusion: After square root data transformation, the variance of nine groups are homogenous. 208 Appendix 6. (continued) ANALYSIS OF VARIANCE/COVARIANCE-REPORT PHASE NO. OF VARIATES 1 NO. OF COVARIATES 1 NO. OF OBSERVATIONS 36 INDEX A INDEX RANGE 9 REGRESSION COVARIATE COEFFICIENT STANDARD ERROR F-VALUE PROB WT1 0 .05778 0 .10998 TEST OF ALL COVARIATES TOGETHER 0 .2760 0 .6096 0 .2760 0 .6096 Conclusion: The covariate ( o r i g i n a l weight) signi f icant. ANALYSIS OF VARIANCE/COVARIANCE-REPORT PHASE IS not NO. OF VARIATES NO. OF COVARIATES NO. OF OBSERVATIONS INDEX RANGE A INDEX 9 1 0 36 ANALYSIS OF VARIANCE/COVARIANCE OVERALL MEANS SRWL 1.3325 OVERALL STANDARD DEVIATIONS SRWL 0 .7123 ANALYSIS OF VARIANCE/COVARIANCE FOR VARIABLE SRWLX SRC. SUM OF MEAN TESTED N 0 - SOURCE D . F . SQUARES SQUARE F VALUE F PROB AGAINST 1 TREATMENT 8 17.470663 2 .183832 205 .2525 0 .0000 2 2 ERROR 27 2 .872729E-01 1 .063973E-02 3 TOTAL 35 17.757936 Conclusion: There i s a s i g n i f i c a n t difference between the averages of the percentage O.-D. weight losses of the nine group. 209 Appendix 6. (continued). Or i g i n a l Data: (Weight Losses of WRC Heartwood Blocks Containing High Thujaplicin Content After Infection With Three 'Early Stage' Fungi (WR1, WR2 and WR3).) Code Origi n a l O.D. Weight Weight no O.D. weight after loss % (g) treatment (O.D.) (g) Group 1 : (8 weeks with WR1 ) 1-1 6. 1 02 6. 001 1 .6552 1-2 5.473 5. 403 1 .2790 1-3 5.997 5. 918 1 .3173 1-4 6.072 5. 990 1 .3505 Ave: 1 .4005 Group 2 : (8 weeks with WR2) 2-1 5.702 5. 614 1 .5433 2-2 6.010 5. 918 1 .5308 2-3 6. 1 74 6. 069 1 .7007 2-4 6.069 5. 982 1 .4335 Ave: 1 .5521 Group 3 : (8 weeks with WR3) 3-1 6. 1 06 6. 060 0 .7534 3-2 5.929 5. 872 0 .961 4 3-3 6. 1 43 6. 082 0 .9930 3-4 6.041 5. 988 0 .8773 Ave: 0 .8963 Group 4 : (4 weeks with WR1 , then 4 weeks with 4-1 5.928 5. 581 5 .8536 4-2 5.978 5. 662 5 .2860 4-3 5.738 5. 430 5 .3677 4-4 6.023 5. 698 5 .3960 Ave: 5 .4758 210 Appendix 6. (continued) Code Original O.D. Weight Weight no O.D. weight after loss % (g) treatment (O.D.) (g) Group 5: ( 4 weeks with WR1, then 4 weeks with WR3) 5-1 6. 1 29 5.919 3.4263 5-2 6.076 5.796 4.6083 5-3 5.743 5.556 3.2561 5-4 6.050 5.855 3.2231 Ave: 3.6285 Group 6: (4 weeks with WR2, then 4 weeks with 6-1 6.012 5.928 1.3972 6-2 6. 104 6.025 1.2942 6-3 5.876 5.814 1.0551 6-4 5.903 5.829 1.2536 Ave: 1.2500 Group 7: (4 weeks with WR1, then 4 weeks with 4 weeks with WR3) 7-1 7-2 7-3 7-4 5.651 6.135 5.764 6.031 5.323 5.828 5.469 5.650 Ave 5.8043 5.0040 5.1180 6.3174 5.5609 Group 8: (control l e v e l 2: 8 weeks with culture medium) 8-1 8-2 8-3 8-4 6. 137 5.820 6.048 6. 193 Ave: 6.115 5.786 6.001 6.1 32 0.3583 0.5842 0.6118 0.9850 0.6349 Group. 9: (control l e v e l 1: 8 weeks in empty Petri dishes) 9-1 9-2 9-3 9-4 5.871 6.213 5.954 5.834 5.869 6.213 5.953 5.832 Ave: 0.0341 0.0000 0.0168 0.0343 0.0213 21 1 Appendix 7. Results of Multiple Range Tests for Wood Bioassay. Block MEANS FOR SOURCE* FACTOR LEVELS A 1 2 3 4 5 6 7 B 9 TREATMENT DIVISOR 4 4 4 4 4 4 4 4 4 FREQ 4 4 4 4 4 4 4 4 4 MEAN 1 . 182 245 945 340 899 1 17 355 784 0 . 125 SRWL7O STD DEV 0.071 0 .044 0 .057 0 .054 0. 167 0 .065 0 . 130 0 . 161 0 .087 RANGE TESTS FOR SRWL DUNCAN'S MULTIPLE RANGE TEST , RANGES FOR ALPHA=0.05 2 .8995 3.0469 3.1481 3.2112 3.2624 3.3029 3 3351 3 3611 THERE ARE 6 HOMOGENEOUS SUBSETS(SUBSETS OF ELEMENTS, NO PAIR OF WHICH DIFFER BY MORE THAN THE SHORTEST SIGNIFICANT RANGE FOR A SUBSET OF THAT S IZE) WHICH ARE LISTED AS FOLLOWS ( 9 ) ( 8) ( 3) ( 6 , 1. 2) ( 5) ( 4 , 7) STUDENT I ZED RANGES FOR NEWMAN-KEUL'S TEST , ALPHA=0.05 2 .902 3 .507 3 .870 4.131 4 .334 4 .498 4 .639 4 .759 THERE ARE 6 HOMOGENEOUS SUBSETS(SUBSETS OF ELEMENTS, NO PAIR OF WHICH DIFFER BY MORE THAN THE SHORTEST SIGNIFICANT RANGE FOR A SUBSET OF THAT SIZE) WHICH ARE LISTED AS FOLLOWS ( 9) ( 8) ( 3) ( 6. 1. 2) ( 5 ) ( 4 , 7) STUDENTIZED RANGE FOR TUKEY'S TEST. ALPHA= 0 . 0 5 4 .759 THERE ARE 6 HOMOGENEOUS SUBSETS(SUBSETS OF ELEMENTS, NO PAIR OF WHICH DIFFER BY MORE THAN THE SHORTEST SIGNIFICANT RANGE FOR A SUBSET OF THAT SIZE) WHICH ARE LISTED AS FOLLOWS ( 9) ( 8. 3) ( 3, 6 . 1) ( 6. 1. 2) ( 5) ( 4 . 7) 2 1 2 Appendix 8. Chemical S h i f t s of Some Protons. Ap?roxiroart Chemical Shifts of Protons Arucbed to L'nsarumed Linkages Proton 6 t Proton i T R - C H O 9-4-100 oo-o* — C = C H - 45-60 40-5-5 A r - C H O 97-105 -O5-03 —C=CH—CO 5-8-67 3-3-4-2 H - C O — 0 8 0-8 2 1 8-20 —CH=C—CO 6 5-8-0 2-0-35 H—CO—N &-0-8: I 8-20 —CH==C—0 40-50 5-0-6-0 -Cs=C—H 16-3 1 6-9-s: —C=CH—O 6-0-8 1 1-9-40 Aiomatic — C H = C — N 37-50 50-63 proiont 6-0-9-0 1-0-40 — C = C H — N 57-80 2-0-43 Chcmictl Shifti of Methyl, Methylene and Methine Protons Methyl Protons Methylene Protons Methine Protons Proton 6 t C H j - C 0 9 9 1 C H 3 - - C - C = C 11 6-9 C H . - C - - O 13 87 C H j — 0 = C 1-6 8-4 C H j - A t 2 3 7 7 C H j - C O — R 22 78 C H , C O - A r 2-6 7-4 C H y—CO—O—R 2-0 80 C H i — C O — O — A r 24 76 C H j — C O - N — R 2-0 8-0 C H 3 — O - R 33 67 C H 3 - 0 — C = C 3-8 62 C H j — O - A r 38 62 C K j — O — C O — R 37 63 C H j - N 23 77 C H j - N 3-3 6-7 C H j - N — A r 3-0 7-0 C H 3 - S 2 1 7 9 C H j - C - N O , 16 8 4 C H j — C = C — C O 2-0 8-0 C - = C t C H j ) - C O l k 82 C H , — N — C O — R 2-9 71 Proton Proton C — C H — C — C — C H 2 — C O — N — R — C — C H j — O — R — C — C H , — O — H T H , — O — A r I j — O — C O — R — C — C H . - N —C—C\i\—NO, — C — C H j — C — N O , — C — C H j — C = C — C C = C t C H j ) — C O C — C H j — C l C — C H . . — B r C — C H j — 1 C — C H - — Q s N 22 3-4 3-6 43 4 1 2-5 2-4 4-4 2 1 2-4 2- 4 36 35 3- 2 23 7-8 6-6 64 57 5-9 75 7-6 56 7-9 7-6 7-6 64 65 68 7-7 5-9 41 :~o — C H — A r — C — C H — C O — R — C — C H — O H — C — C H - O — C O -— C — C H — N C — C H — B r C — C H — I C—CH—C==N C — C H - N — C O — R 1- 5 2- 0 3- 0 27 3-3 37 3-9 48 28 3 2 4 7 43 43 27 41 85 8-0 7-0 73 67 63 6 1 52 72 68 5 3 57 57 73 5-9 Source: Nakanishi, (1962). 2 1 3 Appendix 9. Chemical S h i f t of Some Carbon-13 Resonances. 220 200 ISO Details or substituent effects on " C chemical MO UO 120 K>0 80 shifts are given in Tables 3-19. 3-20 and 3-21. Source: Willam and Fleming, (1980). 214 Appendix 10. IR Data for Selected Carbon-Hydrogen Absorption Bands, Functional Group Frequency cm'1 Wavelength M Assignment Remarks t Alkyl - C H , 2960 3.38 » u s, lu 2870 3.48 *• m, lu 1460 6.85 s. lu 1380 7.25 m.gu - C H , - 2925 3.42 s. lu 2850 3.51 ». m, Iu 1470 6.83 scissoring f. lu 1250 -8.00 twisting and s, nu wagging I — C — H 1 2890 3.46 r w, nu 1 1340 7.45 c w, nu — O C O C H , 1380-1365 7.25-7.33 «t s, gu — C O C H , -1360 -7.35 s, gu — C O O C H , -1440 -6.95 s, gu -1360 -7.35 t. gu Vinyl - C H , 3080 3.24 » u m. lu 2975 3.36 m, lu - 1420 -7.0-7.1 a (in-plane) m, lu -900 -11 e (out-of-plane) s, gu c - < 3020 3.31 V m, lu mono-substituted 990 10.1 e (out-of-plane) s, gu 900 11.0 a (out-of-plane) s. gu ru-disubstituted 730-675 13.7-14.7 c (out-of-plane) s. gu //•a/u-disubstituted 965 10.4 a (out-of-plane) s, gu trisubstiiuied 840-800 11.9-12.4 a (out-of-plane) m-s, gu Acetylene *=C— H 3300 3.0 » m-s. gu Aldehyde 2820 3.55 9 m, lu •° 2720 -3 .7 overtone or m.gu combination H band Source: Pasto and Johnson, (1969). 215 Appendix 11. IR Data for Selected X-H Absorption Bands. Wavelength Functional group Frequency cm'1 Remarks Alcohols (nonbonded) primary secondary tertiary phenols intermolecularly H-bondcd intramolecularly H-bonded 3640 3630 3620 3610 3600-3500 3400-3200 3600-3500 2.72 2.73 2.74 2.75 2.78-2.86 2.94-3.1 2.78-2.86 m. gu. usually determined in di-lute solution in nonpolar solv-ents. m. dimeric. rather sharp s, polymeric, usually quite broad m-s. much sharper than inter-molecular hydrogen bonded O H : is not concentration dependent. Amines R N H , R 2 N H A r N H R -3:500 -2.86 m. gu. -3400 - 2.94 m . g u . » , 1640-1560 6.1-6.4 m-s. gu. corresponds to scissoring deformation. 3500-3450 2.86-2.90 w-m. gu. r 3450 2.90 w-m,gu Pvrroles. indoles 3490 2.86 w-m. gu Ammonium salts N H 4 * - N - H j ^ N * H , 3300-3030 3.0-3.3 s. gu 1430-1390 7.0-7.2 s.gu 3000 — 3.0 s. gu. usually quite broad 1600-1575 6.25-6.35 S. gu. au 1490 - 6.7 s. gu. B, 2700-2250 3.7-4.4 s. gu. vu and »„ usually broad or a group of bands ; N * H 1600-1575 6.25-6.35 m. gu. a 2700-2250 3.7-4.4 s. gu. », P N H - band is weak and of no practical utility. Mercaptans — S H 2600-2550 3.85-3.92 s, gu band is often very weak and can be missed if care is not exercised. Source: Pasto and Johnson, (1969). 216 Appendix 12. IR Data f o r Se l e c t e d Carbon-Carbon Absorption Bands. Functional group Frequency em'1 Wavelenglh u Remarks Alicyclic C—C(»c— c> monosubstituted 1645 l.l-disubstiluted 1655 ci*-1.2,-disubst. 1660 fra/u-l.2-disubst. 1675 trisubstituted 1670 tetrasubstiluied 1670 6.08 6.04 6.02 5.97 5.99 5.99 m. these bands are of little m, utility in assigning mo-rn, stitution and stereo-w, chemistry; the C — H w, out-of-plane bending w, bands in the fingerprint region are recommended for this purpose. Conjugated C—C(»c—e) 1650 and 1600 6.06 and 6.25 s. gu, 1625 6.16 s. gu, 1600 6.25 l . gu. with aromatic with C—O Cyclic C—C(»c-c ) 6-membered ring 1660-1650 6.03-6.06 m. of limited utility due to and larger closeness to the alicyclic region. monosubstituted 1680-1665 5.95-6.00 m. 5-m. unsubstituted 1615 6.19 m. can be of great utility monosubstituted 1660 6.02 m. although the ring size disubstituted 1690 5.92 w. must be assigned first 4-m. unsubstituted 1660 6.02 m. for those compounds 3-m unsubstituted 1640 6.10 m. whose absorption falls monosubstituted 1770 5.65 m. into regions consistent disubstituted 1880 5.32 m. with other types of absorption bands (nu-clear magnetic reson-ance spectra can be very useful in this respect). Aromatic C — C 1600 6.24 » - s . in-plane skeletal vibra-1580 6.34 s. tions. the intensities of 1500 6.67 w-s. the 1600 and 1500 cm* 1 1450 6.9 s, may be rather weak. Source: Pasto and Johnson, (1969). 217 A p p e n d i x 13. IR D a t a f o r S e l e c t e d C a r b o n - O x y g e n A b s o r p t i o n Ban Frequency Wavelength Functional group cm'1 M Remarks C—0 Single Bonds C Primary C — O — H 1050 9.52 s. gu. »c—o (See discussion for sub-stituent effects.) Secondary C — O — H 1100 9.0S s. gu. » . c _ 0 Tertiary C — O — H 1150 8.68 S, gu, r c _ 0 Aromatic C — 0 — H 1200 8.33 S, gu. » c _ o Ethers-acyclic 1150-1070 8.7-9.35 s. gu. an'.isymmetric VQ—O—C C — C — o — C 1270-1200 and 7.9-8.3 and s. gu. antisymmetric »c—o—C 1070-1020 9.3-9.8 s, gu. symmetric «x—O—C "yclic ethers 6-m. and larger 1140-1070 8.77-9.35 s. gu. 5-m. 1100-1075 9.1-9.3 s. gu. 4-m. 980-970 10.2-10.3 s. gu. Epoxides 1250 S.O s. gu. G'j-disubstituted 890 11.25 s. gu. 7>art5-disubstituted 830 12.05 s.gu. — 0 Double Bonds Ketones 1715 5.8? s. gu. *c—o- unstrained 0"=0 group in acyclic and 6 m ring compounds in carbon tetrachloride solution. (See discussion for effects of conjugation and ring size.) a. ^-unsaturated 1685 5.93 s, gu. »c—o- For the s-cis configuration O u R (C. JZ) the »c—c m a v appear above I60C cm" 1 (below 6.26 u) with an intensity approximately that of the »C—o- The fflflj-configuration O C does not show this enhanced intensity of absorption of theC—C. o- and 0-dikeiones 1720 5.81 s, gu. *c—O- ' * ° bands at higher frequency when in the i-cu con-figuration O li JO c—c 1650 6.06 s. gu. «>c—C 'f enolic ( 0 ~ C ) O H Quinones 1675 5.97 s, gu. » c - o 218 Appendix 13. (continued) Functional group Frequency em-' Ho tlrnglh M Remarks Tropolones •° Aldehydes — C . X H I6S0 1600 1725 6.06 6.26 S.80 *. »C-0 s. ju . »c—0- if intramolecular? hydro-gen bonded as in «-tropolones. i . gu. »c—o (See discussion for effects of conjugation.) Carboxylic acids and derivatives / — C — O H 1710 5.84 t. gu. »c—O' usually as the dimer in nonpolar solvents, monomer absorbs at 1730 cm" 1 (5.78 u ) and may appear as a shoulder in the spectrum of a carboxylic acid: for conjugation ef-fects see the discussion. Esters 1735 5.76 s. gu, r<>.o. acyclic and 6-m. lactones: 1300-1050 7.7-9.5 Anhydrides Acid halide 1820 and 1760 1800 5.48 and 5.68 5.56 Amides 1650 6.06 a. ^-unsaturated 1300 1665 7.7 6.01 Carboxylate to 1610-1550 and 1400 6.2-6.45 and 7.15 Source: Pasto and Johnson, (1969). i , Iu. symmetric ana antisymmetric »C—o—C 8<v'ig 2 bands, indicative of type of ester, for example, formates: 1178 em"' (8.5 u) acetates: 1242 cm"'(8.05 n) methyl esters: 1164 c m " 1 (8.6*) others: 1192cm" 1 (8.4 u) but the distinction generally is not great enough to be of diagnostic value. s. gu. re—O- the intensity and separa-tion of the bands may be quite variable. (See discussion for general effects of conjugation and ring size.) s, gu. » c - o . *cid chlorides and fluor-ides absorb at slightly higher fre-quency while the bromides and iodides absorb at slightly lower fre-quency. (See discussion for effects of conjugation.) "Amide I" band, this fre-for the associated amide t s. gu. » c - o quency is (see COOH) , free amide at 1686 cm (5.93 it) in dilute solution, cyclic amides shift to higher frequency as ring size decreases. s. gu. »c—N . "Amide III*' band, free amide at slightly higher frequency. a, gu. » c - o i . gu antisymmetric and stretching of— symmetric 219 

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