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Variation in two families of compounds across stems of western red cedar (Thuja Plicata Donn.). Jiang, Kuo-shii 1968

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VARIATION IN TWO FAMILIES OF COMPOUNDS ACROSS STEMS OF WESTERN RED CEDAR (THUJA PLICATA DONN.) BY Kuo - s h i i J i a n g B.Sc. N a t i o n a l Taiwan U n i v e r s i t y , Taiwan, 1963 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of F o r e s t r y We accept t h i s t h e s i s as conforming to the req u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA AUGUST, 1968. In presenting th is thesis in pa r t ia l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i sh Columbia, I agree that the Library sha l l make i t f reely. ava i lab le for reference and Study. | further agree that permission for extensive copying of this thesis for scholar ly purposes may be granted by the Head of my Department or by hils representat ives. It is understood that copying or publ icat ion of th is thesis for f inanc ia l gain shal l not be allowed without my wri t ten permission. Department The Univers i ty of B r i t i sh Columbia Vancouver 8, Canada ABSTRACT A q u a n t i t a t i v e a n a l y t i c a l method, using column chromatography, was developed f o r determining the t h u y a p l i c i n methyl ethers (T.M.E.) i n acetone e x t r a c t s of western red cedar wood. A q u a n t i t a t i v e method, using g a s - l i q u i d chromatography, was developed f o r hezukone, w h i l e t h u j a p l i c i n s were determined by paper chromatography. These techniques were used to examine d i s t r i b u t i o n of T.M.E. and t h u j a p l i c i n s across western red cedar stems. R e s u l t s support the theory that polyphenols are formed i n s i t u a t the sapwood-heartwood boundary. The f i n d i n g s a l s o i n d i c a t e that biochemical mechanisms operate f o r s e v e r a l years a f t e r v i s i b l e heartwood has been formed. A d d i t i o n a l l y , the increase i n r a t i o s of monohydroxy- and dihydroxy- to nonhydroxylated T.M.E'. w i t h wood ageing i n the tree suggests a h y d r o x y l a t i o n r e a c t i o n as mechanism f o r i n t e r c o n v e r s i o n of some of the l i g n a n s i n western red cedar. However, t h i s c onclusion cannot be made fo r the h y d r o x y l a t i o n of nezukone to j 3 - t h u j a p l i c i n to ( 3 - t h u j a p l i c i n o l . The t h u j a p l i c i n s were shown to be present only i n the heartwood of western red cedar. d. - T h u j a p l i c i n was not found i n the present study, but i t was c e r t a i n l y present i n a previous study on an American grown western red cedar. Some c h a r a c t e r i s t i c chemical p r o p e r t i e s of ( 3 - t h u j a p l i c i n o l , which • „ i . . . . has an a d d i t i o n a l hydroxyl group to the t h u j a p l i c i n s , are studied. I t s f e r r i c chelate was found to be unstable to change i n pH values of s o l u t i o n s , w h i l e t h u j a p l i c i n s complexes are s t a b l e . Some observations on the determination of t h u j a p l i c i n i n a l k a l i n e s o l u t i o n s , as w e l l as methods f o r e x t r a c t i n g these compounds, are made. - i i -ACKNOWLEDGEMENT The w r i t e r wishes to express h i s g r a t i t u d e to Dr. E. P. Swan, Part-time A s s o c i a t e P r o f e s s o r a t the U n i v e r s i t y of B r i t i s h Columbia and Research S c i e n t i s t , Vancouver Forest Products Laboratory, f o r h i s su p e r v i s i o n i n planning the experimental phases of t h i s t h e s i s , as w e l l as h i s i n s p i r i n g guidance during the past two. years. He i s g r a t e f u l to Dr. J.A.F. Gardner, P r o f e s s o r and Dean, Dr. J . W. Wilson, P r o f e s s o r , Dr. D. Haley, A s s i s t a n t P r o f e s s o r and Dr. L. Paszner, Research A s s o c i a t e , F a c u l t y of F o r e s t r y , f o r reviewing t h i s t h e s i s . He i s a l s o indebted to the s t a f f of Wood Chemistry S e c t i o n , Vancouver F o r e s t Products Laboratory, f o r t h e i r a s s i s t a n c e . - i i i -TABLE OF CONTENTS Page TITLE PAGE ABSTRACT i ACKNOWLEDGEMENT i i TABLE OF CONTENTS i i i LIST OF TABLES v i LIST OF FIGURES v i i INTRODUCTION 1 LITERATURE REVIEW • 5 A. Chemical Research on Western Red Cedar Wood 5 1. Lignans 5 a. P l i c a t i c A c i d (V) and P l i c a t i n (VI) 5 b. T h u j a p l i c a t i n s (VII) 5 c. T h u j a p l i c a t i n Methyl Ethers (VIII) 6 2. Tropone and Tropolones 6 a. Nezukone (IV) 7 b. T h u j a p l i c i n s , (I) 7 c. P -Dolabrin ( I I I ) 8 d. - T h u j a p l i c i n o l ( I I ) 8 3. Hy d r o x y l a t i o n Mechanism 9 B. Hy d r o x y l a t i o n i n the P l a n t Kingdom 9 1. The Conversion of Phenylalanine (XI) to Tyrosine (XII) . 9 2. Conversion of Cinnamic A c i d to p-Coumaric A c i d 10 3. Recent Work on the B i o s y n t h e s i s of Flavonoids 11 C. Di s c u s s i o n 12 EXPERIMENTAL 15 - i v -A. M a t e r i a l s 15 B. E x t r a c t i o n 15 C. A n a l y s i s of T. M. E 16 1. P r e p a r a t i o n of Packing M a t e r i a l - D e a c t i v a t e d S i l i c i c A c i d . 16 2. Pr e p a r a t i o n of the S i l i c i c A c i d Column 16 3. Separation of T. M. E 17 4. Q u a n t i t a t i v e Determination of T. M. E 18 D. A n a l y s i s of T h u j a p l i c i n s 18 1. F e r r i c C h l o r i d e E x t r a c t i o n . 18 2. Spectroscopic Attempts . . 19 a. Reactions of ft-Thujaplicin and §-Thujaplicinol w i t h Sodium Hydroxide 19 b. F e r r i c Complexes of ^ - T h u j a p l i c i n and @-Thujaplicinol i n B u f f e r S o l u t i o n s 19 3. Separation of T h u j a p l i c i n s 19 4. Q u a n t i t a t i v e Determination of T h u j a p l i c i n s 20 E. A n a l y s i s of Nezukone 21 1. Steam D i s t i l l a t i o n 21 2. T h i n - l a y e r Chromatography 21 3. Q u a n t i t a t i v e Determination of Nezukone by Gas Chromatography 24 RESULTS • 27 A. Contents of T. M. E. Across Western Red Cedar Stems 27 B. Contents of Nezukone, T h u j a p l i c i n s and P - T h u j a p l i c i n o l Across a Western Red Cedar Stem 27 C. Spectroscopic Determination of P-Thujaplicin and (3-Thuja-p l i c i n o l D e r i v a t i v e s 32 DISCUSSION 36 A. Q u a n t i t a t i v e A n a l y t i c a l Method f o r T. M. E 36 - V -B. E x t r a c t i o n and Determination of T h u j a p l i c i n s 39 C. Spectroscopic Studies of (3-Thujaplicin and ^ - T h u j a p l i c i n o l 40 D. D i s t r i b u t i o n and Formation of T. M. E. and T h u j a p l i c i n s . . 41 CONCLUSIONS 48 REFERENCES 50 - v i -LIST OF TABLES Table Page 1. P h y s i c a l p r o p e r t i e s of T.M.E. found i n western red cedar . . . . 6 2. Tropone and tropolones o c c u r r i n g i n western red cedar heartwood 9 3. Two e l u t i o n systems of column chromatography f o r the separation of T.M.E 18 4. F r a c t i o n a l d i s t i l l a t i o n of n e u t r a l f r a c t i o n s of " t h u j i c a c i d r e s i d u e s " 21 5. Absorptions of ^ - t h u j a p l i c i n and g - t h u j a p l i c i n o l f e r r i c acetate complexes tre a t e d w i t h various b u f f e r s o l u t i o n s of pH values from 6 to 11 34 6. Absorptions of ( 3 - t h u j a p l i c i n and p-thu j a p l i c i n o l f e r r i c c h l o r i d e complexes t r e a t e d w i t h various b u f f e r s o l u t i o n s of pH values from 6 to 11 . .' 34 7. Peak e f f l u e n t volume and re c o v e r i e s of T.M.E . . . . 37 8. Contents of T.M.E. and r a t i o s of monohydroxy- (VHIb) and dihydroxy- (VIIIc) to nonhydroxylated T.M.E. ( V i l l a ) i n the r a d i a l d i r e c t i o n of a western red cedar stem (Tree No.l) . . . . 57 9. Contents of T.M.E. and r a t i o s of monohydroxy- (VHIb) and dihydroxy- (VIIIc) to nonhydroxylated T.M.E. ( V i l l a ) i n the r a d i a l d i r e c t i o n of a western red cedar stem (Tree No.2) 57 10. Contents of nezukone, t h u j a p l i c i n s and ( 3 - t h u j a p l i c i n o l i n the r a d i a l d i r e c t i o n of a western red cedar stem (Tree No.3) . . . . 58 - v i i -LIST OF FIGURES Figure Page 1. S t r u c t u r e s of tropone (IV) and tropolones ( I , I I , I I I ) i n western red cedar heartwood . 3 2. S t r u c t u r e s of western red cedar li g n a n s 4 3. Paper chromatogram showing separation of (Z-,. Y- thu j a p l i c i n and t h u j a p l i c i n o l 22 4. U l t r a v i o l e t absorption spectra of t h u j a p l i c i n (Curve 1), y - t h u j a p l i c i n (Curve 2) and t h u j a p l i c i n o l (Curve 3) i n isopropanol 23 5. Gas chromatogram of F r a c t i o n No.3 25 6. Gas chromatogram of chloroform e x t r a c t s of western red cedar . . (A) not reacted w i t h C H ^ , (B) reacted w i t h C H ^ 26 7. D i s t r i b u t i o n of T.M.E. i n the r a d i a l d i r e c t i o n of a western red cedar stem (Tree No.l) 28 8. D i s t r i b u t i o n of T.M.E. i n the r a d i a l d i r e c t i o n of a western red cedar stem (Tree No.2) 29 9. Changes i n r a t i o s of monohydroxy- (A) and dihydroxy- (B) to nonhydroxylated T.M.E. as Tree No.l aged 30 10. Changes i n r a t i o s of monohydroxy- (A) and dihydroxy- (B) to nonhydroxylated T.M.E. as Tree No.2 aged 30 11. D i s t r i b u t i o n of nezukone ( I V ) , t h u j a p l i c i n s (I) and (3- thu j a p l i c i n o l ( I I ) i n the r a d i a l d i r e c t i o n of a western red cedar stem (Tree No.3)31 12. Abs o r p t i o n spectra of colored chelates produced by r e a c t i o n of (3 - t h u j a p l i c i n ( s o l i d l i n e ) and ^ - t h u j a p l i c i n o l (dashed l i n e ) w i t h NaOH 33 - v i i i -13. Absorption spectra of f e r r i c complexes of (i-thujaplicin ( s o l i d l i n e ) and 0 - t h u j a p l i c i n o l (dashed l i n e ) reacted w i t h Fe(OAc)^ (A) or F e C l 3 (B) . 35 14. Absorption of eluant f r a c t i o n s of acetone e x t r a c t s from a western red cedar stem by GME-2801F UV spectrophotometer at 280 nm . . 38 15. E f f e c t of s o l u t i o n pH values on the f e r r i c complex of (3 - t h u j a p l i c i n o l 42 16. P o s s i b l e formation of d i h y d r o x y t h u j a p l i c a t i n methyl ether . . . 44 INTRODUCTION Western red cedar (Thuja p l i c a t a Donn.) i s a major timber species i n B r i t i s h Columbia and a l s o one of the l a r g e s t trees i n the P a c i f i c Region. I t s well-known n a t u r a l d u r a b i l i t y and high content of extraneous m a t e r i a l s ( f r e q u e n t l y more than 20 per cent i n the outer b u t t heartwood (13) ) has e x c i t e d i n t e r e s t f o r many years i n the hope of p r o v i d i n g new o u t l e t s f o r u t i l i z a t i o n of large volumes of m i l l r e s i d u e , as w e l l as an understanding of i t s s p e c i f i c problems such as c o r r o s i o n of metals and s t a i n i n g of painted surfaces. The reason f o r the high n a t u r a l d u r a b i l i t y of western red cedar heartwood was r e s o l v e d when three isomeric compounds, which are h i g h l y t o x i c to wood-destroying f u n g i , were i s o l a t e d . The three isomers have been named o*. - ( I a ) , (3-(Ib) , and V - t h u j a p l i c i n (Ic) (3, 4, and 5-isopropyltropolone) , r e s p e c t i v e l y (1, 10, 11). L a t e r , another tropolone c o n t a i n i n g an a d d i t i o n a l hydroxyl group v i c i n a l to the keto group, ( 3 - t h u j a p l i c i n o l ( I I ) , was found (15). F u r t h e r , (3-dolabrin ( I I I ) was demonstrated to be present by Gardner and Barton (14). Recently, the presence of nezukone (IV) i n the cedar was a l s o proved (29). I t was very i n t e r e s t i n g , b i o c h e m i c a l l y , that nezukone was found i n company w i t h t h u j a p l i c i n s and ( 3 - t h u j a p l i c i n o l i n nature. The s t r u c t u r e s of these n a t u r a l p r e s e r v a t i v e compounds are shown i n Figure 1. Several l i g n a n s have been i s o l a t e d from the cedar and c h a r a c t e r i z e d i n recent years. Lignans are present i n both sapwood and heartwood, although i n low concentration i n the sapwood (17) . They are p l i c a t i c a c i d (V), p l i c a t i n ( V I ) , the t h u j a p l i c a t i n s e r i e s (VII) and the t h u j a p l i c a t i n methyl ether s e r i e s (VIII) (17-19, 37-40). The t h u j a p l i c a t i n s i n c l u d e two components - 2 -t h u j a p l i c a t i n ( V i l a ) and d i h y d r o x y t h u j a p l i c a t i n ( V l l b ) ; w h i l e the t h u j a p l i -c a t i n methyl ethers i n c l u d e three compounds -- t h u j a p l i c a t i n methyl ether ( V i l l a ) , h y d r o x y t h u j a p l i c a t i n methyl ether ( V l l l b ) and d i h y d r o x y t h u j a p l i c a t i n methyl ether ( V I I I c ) . F i gure 2 gives the s t r u c t u r e s of these l i g n a n s . A r e l a t i o n s h i p seems to e x i s t between p l i c a t i c a c i d , p l i c a t i n and t h u j a p l i c a t i n s as shown by t h e i r s t r u c t u r e s . This r e l a t i o n s h i p was po s t u l a t e d by Gardner ejt al_. (17) , and i t was suggested that h y d r o x y l a t i o n was the b i o -s y n t h e t i c mechanism i n the production of p l i c a t i c a c i d from t h u j a p l i c a t i n v i a p l i c a t i n . From the same viewpoint, the three components of T.M.E., and the occurrence of nezukone together w i t h t h u j a p l i c i n s and ^ - t h u j a p l i c i n o l i n western red cedar are very i n t e r e s t i n g . In t h i s work, i t i s hoped to show that the h y d r o x y l a t i o n pathway that p o s s i b l y c h a r a c t e r i z e d the formation of p l i c a t i c a c i d could a l s o occur w i t h the T.M.E. and (J-thu j a p l i c i n o l . To t h i s end, methods f o r the a n a l y s i s of these compounds had to be developed. Although three components of T.M.E. can be separated by t h i n - l a y e r chromatography on s i l i c a g e l G using benzene-ethanol (9 : 1) developing solvent (38), no q u a n t i t a t i v e a n a l y t i c a l method has been a v a i l a b l e . This research describes the development of the quanti-t a t i v e determination, and i t s a p p l i c a t i o n . A l s o , development and a p p l i c a t i o n of a n a l y t i c a l techniques f o r nezukone and t h u j a p l i c i n s are mentioned. - 3 -0 0 0 (la) (lb) (Ic) e<-Thujaplicin ^-Thujaplicin r-Thujaplicin (II) (III) (IV) p-Thujaplicinol ^-Dolabrin Nezukone Figure 1. Structures of tropone (IV) and tropolones (I, II, III) i n western red cedar heartwood. - 4 -(VII) Thujaplicatins (VIII) Thujaplicatin Methyl Ethers (T.M.E.) (a) R=H Thujaplicatin (a) R1=R2=H Thujaplicatin Methyl Ether (b) R=OH Dihydroxythujaplicatin (b) R 1=oh, Hydroxythujaplicatin Methyl Ether (c) R1=R2=0H Dihydroxythujaplicatin Methyl Ether Figure 2 . Structures of western red cedar lignans. - 5 -LITERATURE REVIEW A. Chemical Research on Western Red Cedar Wood Research on the extraneous components of western red cedar have been performed f o r many years i n Canada, Japan, Sweden and United S t a t e s . The present knowledge of the extraneous components i s o u t l i n e d as f o l l o w s : 1. Lignans Lignan i s the generic term a p p l i e d to a f a m i l y of o p t i c a l l y a c t i v e p l a n t products which are c h a r a c t e r i z e d by the ^, t-dibenzylbutane s k e l e t o n , and which can be w r i t t e n f o r m a l l y by j o i n i n g two phenylpropanoid u n i t s by the P , p-carbon atoms of the n-propyl side chains. The f o l l o w i n g lignans have been i s o l a t e d from western red cedar heartwood and t h e i r s t r u c t u r e s have been e l u c i d a t e d . a. P l i c a t i c A c i d (V) and P l i c a t i n (VI) P l i c a t i c a c i d i s the f i r s t l i g n a n a c i d found i n nature, i t i s a very strong a c i d (pKa=3) (16) because of i t s h i g h l y hydroxylated side chain (19). I t i s heat and l i g h t s e n s i t i v e and as would be expected from i t s s t r u c t u r e , forms a lactone (VI) on heating. The s t r u c t u r e and absolute c o n f i g u r a t i o n were e l u c i d a t e d (19, 57). P l i c a t i c a c i d can be separated from other l i g n a n s because of i t s high s o l u b i l i t y i n water and very poor s o l u b i l i t y i n e t h y l acetate, together w i t h the poor s o l u b i l i t y i n water and the good s o l u b i l i t y i n e t h y l acetate of the other l i g n a n s . b. T h u j a p l i c a t i n s (VII) Although t h u j a p l i c a t i n i t s e l f and d i h y d r o x y t h u j a p l i c a t i n are known - 6 -(37, 38), no h y d r o x y t h u j a p l i c a t i n has been found. Development of adequate t h i n - l a y e r and pre p a r a t i v e chromatography (38) permits the de t e c t i o n and separation of the two components. Their values ( i n the system mentioned above) are 0.21 and 0.15, r e s p e c t i v e l y . c. T h u j a p l i c a t i n Methyl Ethers (VIII) I n a d d i t i o n to the above l i g n a n s , three r e l a t e d components, namely, t h u j a p l i c a t i n methyl ether ( V i l l a ) , h y d r o x y t h u j a p l i c a t i n methyl ether (VEIIb) and d i h y d r o x y t h u j a p l i c a t i n methyl ether ( V I I I c ) , were proved to be major li g n a n s i n the wood (38, 39, 40). They are the f i r s t known examples of a l i g n a n c o n t a i n i n g both g u a i a c y l and s y r i n g y l r i n g s and the f i r s t s y r i n g y l -group l i g n a n i s o l a t e d from a coniferous wood. Table 1 gives some important data on T.M.E. Table 1. P h y s i c a l p r o p e r t i e s of T.M.E. found i n western red cedar. V i l l a VEIIb V I I I c R f ( p . I . e . ) " 0.30 0.25 ( 0.17 A max 279 281 280 l o g e 3.80 3.58 3.36 m.p. (°C) 167-167.5 55-60 95-97 -48.7 -54.8 -97.2 pr e p a r a t i v e - l a y e r chromatography (36) 2. Tropone and Tropolones Tropone i s a group of non-benzenoid aromatic compounds having the nucleus of 2,4,6-cycloheptatrien-l-one ( I X ) . A s p e c i a l place among i t s d e r i v a t i v e s belongs to the 2-hydroxy-2,4,6-cycloheptatrien-l-ones (X), which were f i r s t recognised as a c l a s s of aromatic compounds by Dewar (7) i n 1945, who proposed the name "tropolones". Since 1945 dozens of a d d i t i o n a l n a t u r a l products were found to have a s t r u c t u r e based on the tropolone skeleton. I n - 7 -the heartwood of western red cedar, and sev e r a l other species of Cupressaceae, one tropone and sev e r a l tropolones have been found. 0 0 II II ,0H o (IX) (X) a. Nezukone (IV) The occurrence of 4-isopropyltropolone, named nezukone by Hiro s e e_t a l . (30), i n the wood of-Thuja s t a n d i s h i i Carr. was the f i r s t case of tropones being i s o l a t e d from n a t u r a l sources. Recently, H i r o s e (29) proved that nezukone was a l s o present i n western red cedar heartwood by means of gas chromatography and i n f r a r e d spectrophotometry. b. T h u j a p l i c i n s (I) from the s t e a m - v o l a t i l e s of western red cedar heartwood by Anderson and Sherrard ( 2 ) , who termed i t a "phenol". I t s unusual s t r u c t u r e was not e l u c i d a t e d u n t i l 1948 by the degradation studies of Erdtman and Gripenberg (10). Using a s i m i l a r technique, Gripenberg (20) and Anderson and Gripenberg (1) demonstrated that two other isomers, m.p. 34 and 51-52°C were 3- and 4-is o p r o p y l t r o p o l o n e , oC- and ( 3 - t h u j a p l i c i n , r e s p e c t i v e l y . d - T h u j a p l i c i n a l s o has been obtained i n another c r y s t a l l i n e form of m.p. 26°C (46) . (3. - T h u j a p l i c i n was a l s o known as h i n o k i t i o l (44) because of i t s occurrence i n the e s s e n t i a l o i l of "Taiwan H i n o k i " (Chamaecyparis taiwanensis Masam. et Suzuki). The i d e n t i t y of ( 3 - t h u j a p l i c i n and h i n o k i t i o l was confirmed by d i r e c t comparison Y - T h u j a p l i c i n ( 5 - i s o p r o p y l t r o p o l o n e ) , m.p. 82°C, was f i r s t i s o l a t e d (45). - 8 -T h u j a p l i c i n s are h i g h l y t o x i c to wood-deteriorating f u n g i , t h e i r t o x i c i t y being of the same order as that of sodium pentachlorophenate (3) . They are thought to c o n t r i b u t e to the n a t u r a l d u r a b i l i t y of the cedar. c. (i - Dolabrin ( I I I ) p - D o l a b r i n , m.p. 56-57°C, was f i r s t obtained from Thujopsis dolabrata S i e . et Zucc. by Npzoe e_t al_. (47) who determined i t to be 4-isopropenyltro-polone. I t gave ( J - t h u j a p l i c i n on hydrogenation over palladium-carbon (51). I t s isomers, d- and V-dolabrin have not been found i n nature, but have been synthesized (50). (3 -Dolabrin was reported present i n western red cedar only i n trace q u a n t i t i e s (0.0003%) (14). d. p - T h u j a p l i c i n o l ( I I ) p - T h u j a p l i c i n o l (7-hydroxy- £-thujaplicin, 7-hydroxy-4-isopropyl-tro p o l o n e ) , m.p. 58°C, i s an i n t e r e s t i n g tropolone c o n t a i n i n g two hydroxyl groups, both of them*are adjacent to the carbonyl group. I t was i s o l a t e d from western red cedar by Gardner e_t aJL. (15) who a l s o proved the s t r u c t u r e . I t corresponds to the hydroxy d e r i v a t i v e s of (3- or V- t h u j a p l i c i n . The synthesis of ( 3 - t h u j a p l i c i n o l was achieved by persulphate o x i d a t i o n of T-thuja-p l i c i n (15) or ^ - t h u j a p l i c i n (49). The n a t u r a l substance i s i d e n t i c a l w i t h the 7 - h y d r o x y h i n o k i t i o l obtained from the diazonium s a l t of 7- a m i n o h i n o k i t i o l (48). No c < - t h u j a p l i c i n o l has been found i n western red cedar although i t was present i n Cupressus pygmaea L. up to about 0.47o of oven-dry wood (62). ^ - T h u j a p l i c i n o l , w i t h one more hydroxyl group than the t h u j a p l i c i n s , has some c h a r a c t e r i s t i c p r o p e r t i e s . I t formed a red chelate w i t h excess f e r r i c i o n rat h e r than a green chelate i n the aqueous l a y e r as was the case w i t h t h u j a p l i c i n s (15). I t a l s o e x h i b i t e d increased r e a c t i v i t y toward copper to / - 9 -give a green colored chelate (15). Table 2. Tropone and tropolones o c c u r r i n g i n western red cedar heartwood. Nezukone T h u j a p l i c i n s ^ - D o l a b r i n / S - T h u j a p l i c i n o l <X- p- T-m.p. (°C) 174-176 26,34 51-52 82 56-57 58 Content ( 7 o on wood basis) . + 0.01 0.3 0.2 0.0003 0.07 + i n d i c a t e s that presence has been detected 3. H y d r o x y l a t i o n Mechanism I n a previous paper (17) the d i s t r i b u t i o n of p l i c a t i c a c i d , p l i c a t i n , t h u j a p l i c a t i n s and T.M.E. across stems of two western red cedar was examined w i t h paper chromatography. The r e s u l t s suggested that s i d e - c h a i n h y d r o x y l a t i o n was a p o s s i b l e mechanism f o r i n t e r c o n v e r s i o n between some of the l i g n a n s . The sequence of formation was considered to be t h u j a p l i c a t i n to d i h y d r o x y 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 a c i d . The h y d r o x y l a t i o n of the s i d e - c h a i n occurred at the ^ - p o s i t i o n s , then at one of the ^ - p o s i t i o n s to form the t e t r a l i n r i n g , and then at the appropriate' T - p o s i t i o n to form p l i c a t i c a c i d i t s e l f . B. H y d r o x y l a t i o n i n the P l a n t Kingdom Hy d r o x y l a t i o n occurs w i d e l y i n b i o s y n t h e s i s of metabolites i n the P l a n t Kingdom. Rather than attempting to survey the whole f i e l d , a few f a m i l i a r h y d r o x y l a t i o n r e a c t i o n s w i l l be considered. 1. The Conversion of Phenylalanine (XI) to Tyrosine (XII) The r e l a t i o n s h i p between phenylalanine and t y r o s i n e i s of considerable i n t e r e s t . Phenylalanine i s an e s s e n t i a l amino a c i d , whereas t y r o s i n e i s not. Conversion of the aromatic amino a c i d phenylalanine to t y r o s i n e , an i r r e v e r s i b l e r e a c t i o n , i s one of the most studied aerobic h y d r o x y l a t i o n r e a c t i o n s . The enzyme-- 10 -cat a l y z e d h y d r o x y l a t i o n i s i l l u s t r a t e d as f o l l o w s (32): C O O coo + W H , - C - H + N H , - C - H I + N A D P H I?k£2Zi ai a2i n2_ hydroxylase O H + H 2 0 + N A D P * ( X I ) (XII) The h y d r o x y l a t i o n almost always r e q u i r e s the cooperation of a hydrogen donor, 18 reduced nicotinamide-adenine d i n u c l e o t i d e phosphate (NADPH). Using 0^ , i t was determined that one atom of the oxygen molecule i s i n s e r t e d i n t o phenyl-a l a n i n e w h i l e the second forms water, thus o x i d i z i n g the hydrogen donor. 2. The Conversion of Cinnamic A c i d to p-Coumaric A c i d p-Coumaric a c i d , l i k e phenylalanine, i s a good precursor of l i g n i n i n a l l p l a n t s (43). Two routes are p o s t u l a t e d f o r formation of p-coumaric a c i d : from t y r o s i n e by deamination, or from cinnamic a c i d by h y d r o x y l a t i o n (43). The former i s w e l l developed i n grasses (of the species examined so f a r ) . Both types of r e a c t i o n s are i r r e v e r s i b l e and enzyme-catalyzed. Hydroxylations of cinnamic a c i d , l i k e phenylalanine to t y r o s i n e , are cat a l y z e d by hydroxylase. An e x t e r n a l e l e c t r o n donor i s a necessary supplement f o r t h i s conversion (32). - 11 -3. Recent Work on the Bi o s y n t h e s i s of Flavonoids I n recent years considerable progress has been made i n e l u c i d a t i o n of the b i o s y n t h e s i s of fl a v o n o i d s ( X I I I ) , w i t h the a i d of radiochemicals. I t was w e l l e s t a b l i s h e d that r i n g A of the fl a v o n o i d s a r i s e s by h e a d - t o - t a i l condensation of three a c e t y l u n i t s , w h i le r i n g B and carbon atoms of the u n i t s come from an i n t a c t phenylpropane u n i t (24). Both cinnamic a c i d and phenylalanine were e x c e l l e n t f l a v o n o i d percursors (33). The f i r s t s t a b l e intermediate formed i n f l a v o n o i d b i o s y n t h e s i s has been p o s t u l a t e d to be chalcone (XIV) . ( X I I I ) ( X I V ) Hydroxylation,. together w i t h methylation and g l y c o s y l a t i o n , presumably occurred toward the end of synthesis because t h i s process a f f e c t e d some flav o n o i d s but not others (24). The f a c t that chalcone was incorporated i n t o both c y a n i d i n and qu e r c e t i n i n d i c a t e d that a change i n the o x i d a t i o n stage of the chain, as w e l l as the i n t r o d u c t i o n of an a d d i t i o n a l hydroxyl group ortho to the one already present i n r i n g B can take place a t , or a f t e r , the l e v e l of C^^ intermediate. This assumption has been corroborated by Grisebach and Grambow (22) . The i n t e r e s t i n g conversion of flavones (XV) i n t o 3-hydroxyflavanones (XVI) has been studied. The p o s s i b l e pathway was p o s t u l a t e d as. 3-hydroxy-flavanones formed from flavones by an oxygenase which replaces the hydrogen - 12 -atom at C-3 of the f lavanone by a hydroxyl group (2.1) : HO. OR 0 OR n H o »l OH o OH OH R * g l u c o s e (XV) (XVI) A l s o , H i l l i s and I s o i (28) studying Eucalyptus spp. obtained evidence which i n d i c a t e d that q u e r c e t i n (XVII) was formed by h y d r o x y l a t i o n of kaempferol C. D i s c u s s i o n There has been great i n t e r e s t i n the b i o s y n t h e s i s and the s i t e of formation of wood e x t r a c t i v e s , p a r t i c u l a r l y of phenolic compounds. D i e t r i c h s (9) pointed out that a f t e r the a d d i t i o n of l a b e l l e d sucrose to European beech (Fagus s y l v a t i c a L.) sapwood, l a b e l l e d s h i k i m i c a c i d was present i n the parenchyma of o l d e r sapwood. von R u d l o f f and Jorgensen (58) used the same technique and showed that p i n o s y l v i n s were present i n wounded branches of red pine (Pinus r e s i n o s a A i t . ) . Thus the phenolic compounds i n the heartwood arose from D-glucose by the s h i k i m i c pathway. S h i k i m i c a c i d was f u r t h e r converted ( X V I I I ) . (XVII) (XVIII) - 13 -i n t o phenylalanine or t y r o s i n e , which served as the precursor of most wood e x t r a c t i v e s . For example, l i g n a n was supposed to be formed from d i m e r i z a t i o n of the Cg-C-j u n i t s . At the present time, the assumption that heartwood e x t r a c t i v e s a r i s e from the sapwood carbohydrate has been w e l l supported. H i l l i s and Garle (27). studied Eucalyptus kino and concluded that the kino ( c o n t a i n i n g polymerized l e u c o d e l p h i n i d i n and traces of l e u c o c y a n i d i n and e l l a g i c acid) was formed i n , s i t u from t r a n s l o c a t e d sugars. Although the mode of formation of heartwood phenolics has not been shown c o n c l u s i v e l y , most evidence suggests that they were formed i n s i t u a t the sapwood-heartwood boundary. The previous study (17) on lignans of western red cedar a l s o supported t h i s p o i n t of view. A l s o i t suggested a h y d r o x y l a t i o n mechanism f o r the i n t e r c o n v e r s i o n of some of the l i g n a n s , as discussed above. Thus h y d r o x y l a t i o n appears to be a route o f . t e r m i n a l o x i d a t i o n . The conversions of phenylalanine to t y r o s i n e , flavones to 3-hydroxyflavanones, and cinnamic a c i d to p-coumaric a c i d , are i r r e v e r s i b l e . L i k e w i s e , hydroxyl-a t i o n was expected to be terminal and i r r e v e r s i b l e i n western red cedar wood. To examine t h i s hypothesis, f u r t h e r evidence had to be c o l l e c t e d . Compounds having s i m i l a r s t r u c t u r e were p r e f e r a b l e f o r t h i s purpose. Thus, the s e r i e s of T.M.E. ( V i l l a , b , c ) and tropolones ( I , I I , I V ) were s e l e c t e d f o r study. Evidence bearing on b i o s y n t h e s i s of a compound can come from several d i f f e r e n t types of i n v e s t i g a t i o n , such as "comparative anatomy", t r a c e r , -genetic, p h y s i o l o g i c a l or enzymic s t u d i e s . The t r a c e r s t u d i e s , i . e . , i s o t o p i c s t u d i e s , has been one of the most u s e f u l methods i n b i o s y n t h e s i s study. One d i f f i c u l t y i n i n t e r p r e t i n g r e s u l t s from t r a c e r studies i s that - 14 -compounds fed a r t i f i c i a l l y i n t o p l a n t s through cut surfaces may be d e t o x i f i e d and metabolized by other than the normal s y n t h e t i c pathway. Using t h i s method for i n v e s t i g a t i n g formation of wood e x t r a c t i v e , w h i l e p o s s i b l e t h e o r e t i c a l l y , depends on the a b i l i t y of a precursor to penetrate to the s i t e of sy n t h e s i s . Although the lignans of western red cedar were synthesized i n the sapwood, the amount may be too small to be determined. I n t h i s study, another approach was adopted. The h y d r o x y l a t i o n hypothesis was explored by determining v a r i a t i o n of r e l a t e d compounds across the stem. A s i m i l a r approach was used by Roux and Paulus (53) who d i d q u a n t i t a t i v e a n a l y s i s on ( + ) - m o l l i s a c a c i d i n (3,3*,4,4',7-pentahydroxyflavane), (rt-)-fustin (7,3' 4' - trihydroxy-2,3-dihydrof lavonol) and f i s e t i n (5,7,3',4'-tet r a h y d r o x y f l a v a n o l ) which occurred i n c r o s s - s e c t i o n s of b l a c k w a t t l e (Acacia m o l l i s s i m a Willd.)heartwood. From d i s t r i b u t i o n patterns of these compounds they suggested a b i o s y n t h e t i c pathway which was that (+)-mollisac-a c i d i n was the precursor of ( + ) - f u s t i n , and f i n a l l y converted i n t o f i s e t i n during ageing of the t r e e . - 15 -EXPERIMENTAL A. M a t e r i a l s Three western red cedar stems were s e l e c t e d f o r t h i s study. Two (Trees No.l and 2) were used f o r the determination of T.M.E. and one (Tree No.3) was used f o r study of tropone and tropolones. Tree No.l was about 90 years o l d , 12 inches i n diameter. I t s average number of r i n g s per inch was 12 and 25 r i n g s per inch before Ring No.30 (which was counted from the periphery of the stem) and 6 a f t e r i t . The sapwood-heartwood boundary was between Rings No.5 and 7. Tree No.2 was more than 400 years o l d , the sap-wood-hear twood t r a n s i t i o n zone was at Rings No.16 and 17. The average r i n g number of Tree No.2 was 22 per i n c h , w i t h r i n g s i n the sapwood very crowded (22-40 per i n c h ) , whereas there were only 8-17 r i n g s per inch i n the heart-wood. Tree No.3 was 260 years o l d , the t r a n s i t i o n zone was located a t Rings No.25-30. I t s average number of r i n g s per inch was 16. Tree No.l was from Haney, B.C., Tree No.2 from U c l u e l e t , B.C., and Tree No.3 from a l o c a l saw m i l l . The r i n g s of the stems were separated by hand w i t h a k n i f e . Wood segments were ground to 40-mesh meal i n a l a b o r a t o r y Wiley m i l l . To avoid l o s s or r e l o c a t i o n of e x t r a c t i v e s , the stems had not been water f l o a t e d . B. ' E x t r a c t i o n The wood meals were e x t r a c t e d i n a Soxhlet f o r e i g h t hours w i t h solvents which v a r i e d according to the p r o p e r t i e s of compounds de s i r e d . Acetone was used f o r exhaustive e x t r a c t i o n of T.M.E. Though tropone and tropolones may be ex t r a c t e d a l s o by acetone, they comprised only a small p o r t i o n of the f i n a l e x t r a c t s , thus making the subsequent paper chromatographic separation d i f f i c u l t . Even when la r g e amounts of the acetone e x t r a c t s were - 16 -spotted on the chromatographic paper, the amount of the i n d i v i d u a l components was s t i l l too small to be detected. Petroleum ether and hexane were not s u i t a b l e solvents because they d i d not e x t r a c t these compounds, e x h a u s t i v e l y , and the amount of t h u j a p l i c i n s i n these e x t r a c t s were much l e s s than these reported i n the l i t e r a t u r e (35). F i n a l l y , chloroform was found to be an e x c e l l e n t solvent f o r t h i s purpose. I n order to minimize experimental e r r o r s , the amount of wood sample was kept s i m i l a r . . The acetone or chloroform s o l u b l e s were concentrated w i t h a r o t a r y evaporator, and then d r i e d under vacuum i n a d e s i c c a t o r . Thus y i e l d s were obtained. The dry e x t r a c t s , e i t h e r . b y acetone or chloroform, were d i s s o l v e d i n known amounts of acetone again f o r subsequent analyses. C. A n a l y s i s of T.M.E. 1. P r e p a r a t i o n of Packing M a t e r i a l --. Deactivated S i l i c i c A c i d S i l i c i c a c i d (I^SiO^-nl^O) , manufactured by F i s h e r S c i e n t i f i c Company, was heated i n an oven a t 130°C f o r two hours. . A f t e r i t was cooled i n a d e s i c c a t o r , " a known amount of d i s t i l l e d water (about 0.5 ml per gram) was added w i t h thorough s t i r r i n g to o b t a i n as homogeneous a mix as p o s s i b l e . Thus the surface of the s i l i c i c a c i d became covered w i t h a f i l m of l i q u i d water, f i r m l y absorbed on the s o l i d . 2. P r e p a r a t i o n of the S i l i c i c A c i d Column The standard survey column was prepared as f o l l o w s : A wad of g l a s s -wool was placed i n the bottom of the chromatographic tube (10 mm i n diameter, 35 cm i n length w i t h a 250-ml so l v e n t r e s e r v o i r on the top) to support the column. The"deactivated s i l i c i c a c i d was s t i r r e d w i t h ethanol-benzene (1:99), the f i r s t e l u t i n g s o l v e n t , to make a smooth s l u r r y . The s l u r r y was added to - 17 -the chromatographic tube i n successive p o r t i o n s . The tube was then tapped to ensure u n i f o r m i t y of the s l u r r y . An a i r pressure of 9 cm Hg was a p p l i e d to remove excess solvent,care being e x e r c i s e d not to allow the solvent l e v e l to f a l l below the column top. A solvent head was always maintained above the s i l i c i c a c i d i n order to prevent c r a c k i n g the column. This procedure gave a uniformly packed column 30 cm long. A f t e r the survey column had been f i n i s h e d , the e x t r a water on the surface of the s i l i c i c a c i d was taken out by a gradual increase of the ethanol content i n the benzene. An abrupt increase of ethanol content made e l u t i o n d i f f i c u l t . 3. Separation of T.M.E. About 5 mg of acetone e x t r a c t s from Trees No.l and 2 were d r i e d and d i s s o l v e d i n 1 ml of 107o ethanol/benzene. The s o l u t i o n was c a r e f u l l y introduced onto the top of the column by means of a p i p e t , and forced i n t o the column under a i r - p r e s s u r e , followed by i n t r o d u c i n g 1 ml of pure benzene. A gradient o f e l u t i n g solvents (Table 3,A) was then added, step by step i n order to overcome t a i l i n g and provide a good separation. The eluant from the survey column was connected to a GME-2801F U l t r a -v i o l e t Spectrophotometer which absorbed a t 280 + 5 nanometers (nm) connected to a Hewlett-Packard recorder. Therefore, those compounds e l u t e d from the column and having Amax i n t h i s range of wavelength show peaks on the recorder. V i l l a , b and c, had Amax at 279, 281 and 280 nm r e s p e c t i v e l y and could be separated according to the curve from the recorder. Each component was c o l l e c t e d by hand i n 25-ml t e s t tubes. The a i r - p r e s s u r e forced the eluent through the column a t the r a t e of 20 ml per hour. The e l u t i o n systems i n - 18 -Table 3 w i l l be discussed l a t e r . Table 3. Two e l u t i o n systems of column chromatography for the separation of T.M.E. A B 17. 100 ml 1.07= 50 ml 27, 50 ml 2.57o 50 ml 37. 50 ml 5.07o 50 ml 47o 50 ml 7.57, 50 ml 57o 50 ml 15.07o 50 ml percentage of ethanol i n benzene 4. Q u a n t i t a t i v e Determination of T.M.E. The three d e s i r e d f r a c t i o n s were d r i e d w i t h an a i r - j e t . A r o t a t o r y evaporator was not s u i t a b l e for t h i s s o r t of q u a n t i t a t i v e a n a l y s i s . The dry m a t e r i a l s were then d i s s o l v e d i n known amounts of pure propanol and t h e i r o p t i c a l d e n s i t i e s (absorbances) were determined w i t h a Beckman Spectrophoto-meter, Model DK-2 at 280 nm. The qu a n t i t y of each compound was c a l c u l a t e d from the known e x t i n c t i o n c o e f f i c i e n t (38, 39, 40). D. A n a l y s i s of T h u j a p l i c i n s 1. F e r r i c C h l o r i d e E x t r a c t i o n This method was t r i e d f o r separating p - t h u j a p l i c i n o l from the t h u j a p l i c i n s . Chloroform s o l u t i o n s of t h u j a p l i c i n s and ( j - t h u j a p l i e i n o l were tr e a t e d w i t h aqueous f e r r i c acetate s o l u t i o n s , made up w i t h sodium acetate and f e r r i c c h l o r i d e to pH 4.3 (34). Both the t h u j a p l i c i n s and t h u j a p l i c i n o l gave a red c o l o r i n the organic l a y e r . The red chelate i s c h a r a c t e r i s t i c f o r t h u j a p l i c i n s and other tropolones (52). With excess f e r r i c i o n (17, f e r r i c c h l o r i d e s o l u t i o n ) , the red f e r r i c chelates were s t a b l e i n chloroform. A green c o l o r was present when ether was used to e x t r a c t the chelate of t h u j a p l -i c i n s , i n s t e a d of chloroform. - 19 -2. Spectroscopic Attempts a. Reactions of ^ - T h u j a p l i c i n and ^ - T h u j a p l i c i n o l w i t h Sodium Hydroxide S o l i d sodium hydroxide was added to a mixture of equal amount of water and ethanol to make up aQ 0.01 N s o l u t i o n . E t h a n o l i c s o l u t i o n s of (3 - t h u j a p l i c i n or | 3 - t h u j a p l i c i n o l were added to the sodium hydroxide s o l u t i o n . A f t e r a few minutes, the co l o r e d s o l u t i o n s were examined w i t h the Backman DK-2 spectrophotometer. The spectrum was determined by measuring the absorbance of the sodium hydroxide s o l u t i o n of the compounds r e l a t i v e to that of the sodium hydroxide s o l u t i o n only which was placed i n the reference c e l l of the spectrophotometer. b. F e r r i c Complexes of p - T h u j a p l i c i n and ^ - T h u j a p l i c i n o l i n Buffe r S o l u t i o n s S i x b u f f e r s o l u t i o n s , w i t h pH 6, 7, 8, 9, 10 and 11 r e s p e c t i v e l y , were prepared from 0.1 N potassium hydroxide and 0.1 N potassium dihydrogen phosphate aqueous s o l u t i o n s . Equimolar q u a n t i t i e s of sodium acetate and f e r r i c c h l o r i d e were d i s s o l v e d i n s u f f i c i e n t d i s t i l l e d water to make a f i n a l s o l u t i o n of 17o i n i r o n a t pH 4.3. To 100 ml of p - t h u j a p l i c i n or ( 3 - t h u j a p l i -c i n o l i n chloroform, 50 ml of f e r r i c acetate s o l u t i o n was added i n a separatory fu n n e l . The sample was shaken f o r 5 minutes on a shaker, then the organic l a y e r was f i l t e r e d . The aqueous l a y e r was washed w i t h 5 ml of a d d i t i o n a l chloroform. S i x 5-ml p o r t i o n s of t h i s organic s o l u t i o n were added to each 5-ml b u f f e r s o l u t i o n r e s p e c t i v e l y and shaken f o r 5 minutes. The organic l a y e r was taken, and i t s absorbance was measured w i t h the Beckman DK-2. With the same procedures, a 17„ f e r r i c c h l o r i d e aqueous s o l u t i o n was added i n s t e a d of the f e r r i c acetate s o l u t i o n . 3. Separation of T h u j a p l i c i n s The paper chromatographic procedures of Wickberg (60) and Wachtmeister - 20 -and Wickberg (59) were used to separate o(-, (3-, V - t h u j a p l i c i n and (3-t h u j a p l i c i n o l . S t r i p s of Whatman No.l paper were f i r s t soaked i n an aqueous s o l u t i o n c o n t a i n i n g 0.015 moles of disodium ethylenediaminetetraactate (EDTA) and 0.005 moles EDTA per l i t e r . The 0.015 moles of disodium s a l t of EDTA was made up w i t h 5.7 grams of EDTA and 1.2 grams of sodium hydroxide i n one l i t e r of d i s t i l l e d water and s t i r r e d f o r an hour on a Mag-Mix s t i r r e r . The wet paper s t r i p s then were removed and the suspended s t r i p s were allowed to a i r -dry. A f t e r the moisture content of the s t r i p s came to e q u i l i b r i u m , they were impregnated twice w i t h a 257, (v/v) s o l u t i o n of dimethyl s u l f o x i d e (DMS) i n toluene. For impregnation, the chromatography papers were passed at even r a t e through the DMS-toluene s o l u t i o n , uniformly b l o t t e d l a y e r s of f i l t e r paper and then d r i e d i n an oven at 60°C f o r 1 minute to remove the toluene. This process was repeated once. As soon as the second impregnation was done, the t r e a t e d s t r i p s were immediately covered w i t h two glass p l a t e s to prevent absorption of atmospheric moisture u n t i l j u s t p r i o r to use. Known amounts of chloroform e x t r a c t s from Tree No.3 were spotted on the chromatographic paper and developed w i t h cyclohexane, using the descending procedure. The absorption of atmospheric moisture during development was avoided by p l a c i n g two trays of anhydrous s i l i c a g e l i n the chromatographic chamber. A f t e r developing for four hours, the paper chromatograph was removed and the solvent f r o n t was marked. The s t r i p s were a i r - d r i e d f o r a minute, i n d i v i d u a l t h u j a p l i c i n s were l o c a t e d under u l t r a v i o l e t l i g h t , and each compound was excised. A l s o , t h u j a p l i c i n s were revealed by spraying w i t h 27o f e r r i c c h l o r i d e s o l u t i o n (Figure 3). 4. Q u a n t i t a t i v e Determination of T h u j a p l i c i n s The excised paper chromatographic s t r i p s were washed w i t h known amounts of isopropanol. The o p t i c a l d e n s i t y of the r e s u l t i n g s o l u t i o n s was obtained w i t h the Beckman DK-2 spectrophotometer. The q u a n t i t y of each t h u j a p l i c i n was estimated from the standard curve of known compounds t r e a t e d w i t h the same procedures. Readings were taken a t 350 nm fo r t h u j a p l i c i n s and 378 nm f o r ^ - t h u j a p l i c i n o l . These maxima were found from t r i a l runs w i t h pure compounds (Figure 4) . E. A n a l y s i s of Nezukone 1. Steam D i s t i l l a t i o n To o b t a i n some nezukone f o r s t a n d a r d i z i n g t h i s a n a l y s i s , s e v e r a l grams of " t h u j i c a c i d r e s i d u e s " (obtained i n 1957) was shaken w i t h 57o potassium hydroxide s o l u t i o n to remove the a c i d i c p o r t i o n . The n e u t r a l f r a c t i o n was then d i s t i l l e d i n vacuo to give three f r a c t i o n s as l i s t e d i n Table 4. Table 4. F r a c t i o n a l d i s t i l l a t i o n of n e u t r a l f r a c t i o n s of " t h u j i c a c i d r e s i d u e s " F r a c t i o n b.p. °C/1.5 mm 1 70-80 2 80-90 3 90-115 2. Thin-layer Chromatography The three f r a c t i o n s were spotted on a s i l i c a g e l G t h i n - l a y e r p l a t e using benzene as the developing s o l v e n t . Nezukone ( I ) , c o n t a i n i n g a keto group, was revealed by spraying w i t h 2,4-dinitrophenylhydrazine s o l u t i o n . The yellow c o l o r e d spot of a keto-compound at R^ 0.32 i n d i c a t e d that F r a c t i o n No.3 contained nezukone. The prep a r a t i o n of the spray reagent i s described elsewhere(55). 0 -Thuja- r-Thujapli- 0-Thujapli- Chloroform p l l c i n c i n c i n o l Extract 0 0 0 0 0 o Figure 3- Paper chromatogram showing separation of 0-, r-t h u j a p l i c i n and 0-thujaplicinol. .50 • * * • i » • • 300 310 320 330 340 350 360 370 380 390 400 Wavelength (nm) Figure 4 « Ultraviolet spectra of p-thujaplicin (Curve 1 ) , r-thujaplicin (Curve 2), and p~thujaplicinol (Curve 3) in isopropanol. - 2 4 3. Q u a n t i t a t i v e Determination of Nezukone by Gas Chromatography The gas chromatogram of F r a c t i o n No.3 i s shown i n Figure 5, which was obtained under these c o n d i t i o n s : column, high vacuum s i l i c o n e grease o 25% SE-30 on Gas Chrom Q; column temperature, 130 C; flow r a t e , m 20 ml/min.; column le n g t h , 5 f e e t ; column diameter, 1/8 i n c h ; chart speed, 5 mm/min. The peak having a r e t e n t i o n time of 8 minutes was i d e n t i f i e d as methyl th u j a t e . The main peak w i t h a r e t e n t i o n time of 15 minutes was supposed to be nezukone from i t s r e l a t i v e r e t e n t i o n time w i t h that of methyl thujate (29) . By assuming that a l l of these components have same- response w i t h the flame i o n i z a t i o n d etector, the percentage of nezukone i n the mixture was obtained. Thus the amount of nezukone corresponding to the peak area was used as standard f o r the a n a l y s i s of the chloroform e x t r a c t of western red cedar. Chloroform e x t r a c t s of 9 r a d i a l f r a c t i o n s of western red cedar stem No.3 were run through the gas chromatography under the same co n d i t i o n s as above. I t was found that nezukone overlapped another component, which was proved to be t h u j i c a c i d by a d i r e c t comparison (Figure 6 A ) . The peak of t h u j i c a c i d was removed by r e a c t i n g the chloroform e x t r a c t s w i t h diazomethane (CE^^) to. form methyl t h u j a t e . Thus an i s o l a t e d peak of nezukone was obtained (Figure 6 B). A l s o , residue components from the previous i n j e c t i o n i n t e r r u p t e d the a n a l y s i s . This i n t e r r u p t i o n was avoided by i n c r e a s i n g the o column temperature to 170 C f o r 10 minutes a f t e r every run. - 25 -B 1 0 5 10 15 Figure 5. Gas chromatogram of Fraction No. 3. (A - methyl thujate, B - nezukone.) 2C5 Retention Time (min.) - 26 -0 5 10 15 20 25 Retention Time (min.) Figure 6. Gas chromatogram of chloroform extracts of western red cedar. (A) not reacted with CHj^, (B) reacted with CH^. RESULTS A. Contents of T.M.E. across Western Red Cedar Stems There were s e n s i t i v e c o l o r i m e t r i c methods f o r q u a n t i t a t i v e determin-a t i o n of phenols, but they were such more time-consuming than d i r e c t a bsorption measurement, and l e s s accurate. The amount of each component was estimated from the d i r e c t measurement of the absorbance w i t h the Beckman DK-2 a t 280 nm. The equation used f o r c a l c u l a t i o n was: A = ebc where: A = absorbance 6 = molecular e x t i n c t i o n c o e f f i c i e n t b = c e l l l e ngth c = sample concentration The amounts of T.M.E., i n terms of per cent y i e l d s on oven-dry wood, across stems of Trees No.l and 2 are given i n Tables 8 and 9, r e s p e c t i v e l y . The r e l a t i o n s h i p of T.M.E. contents w i t h p o s i t i o n across the stems i s presented g r a p h i c a l l y as Figures 7 and 8. A l s o , Tables 8 and 9 give the r a t i o s of monohydroxy- and dihydroxy- to nonhydroxylated T.M.E., Figures 9 and 10 present these data g r a p h i c a l l y . B. Contents of Nezukone, T h u j a p l i c i n s and (3-Thujaplicinol across a Western Red Cedar Stem Q u a n t i t i e s of t h u j a p l i c i n s were obtained by measuring the absorption of the compounds d i r e c t l y . Data were based on c a l i b r a t i o n curves of standard compounds. As noted above, the amounts of nezukone were not absolute. Table 10 gives the contents of nezukone and t h u j a p l i c i n s of 9 f r a c t i o n s from Tree No.3. Figure 11 presents these data g r a p h i c a l l y . - 28 -• UOr SapwoodJ • 30h T3 O O Q O o •20L .10L Heartwood ^ V i l l a VIIIc 10 20 30 AO R i n g Number f r o m P e r i p h e r y 5 0 6 0 6 5 F i g u r e 7. D i s t r i b u t i o n o f T.M.E. i n t h e r a d i a l d i r e c t i o n o f a w e s t e r n r e d c e d a r stem (Tree No.1). - 29 -.60 .50 .40 .30 L .20 .10 Sapwood Heartwood \ V I I I c VHIb V i l l a 10 20 30 40 50 60 70 Ring Number from Periphery Figure 8. D i s t r i b u t i o n of T.M.E. i n the r a d i a l d i r e c t i o n of a western red cedar stem (Tree No.2). 80 90 100 - 30 -1.50 1.00--p 0.50-10 20 30 40 R i n g Number f r o m P e r i p h e r y 50 60 65 F i g u r e 9. Changes i n r a t i o s o f monohydroxy- (A) and d i h y d r o x y - (B) t o n o n h y d r o x y l a t e d T.M.E. as Tree No.1 aged. 1.50 1.00 0.50 _ Sapwood Heartwood 10 20 30 40 50 60 R i n g Number f r o m P e r i p h e r y 70 80 F i g u r e 10. Changes i n r a t i o s o f monohydroxy- (A) and d i h y d r o x y - (B) t o n o n h y d r o x y l a t e d T.M.E. as Tree No.2 aged. 90 Sapwooq Heartwood 50 100 150 R i n g Number fr o m P e r i p h e r y 200 250 F i g u r e 11. D i s t r i b u t i o n . o f nezukone ( IV) , t h u j a p l i c i n s (I) and p - t h u j a p l i c i n o l ( I I ) i n t h e r a d i a l d i r e c t i o n o f a w e s t e r n r e d c e d a r stem (Tree No.3). - 32 -C. Spectroscopic Determination of (3-Thujaplicin and (i-Thu j a p l i c i n o l D e r i v a t i v e s The u l t r a v i o l e t spectra of the colored s o l u t i o n s which were obtained when 0.1 N NaOH was added to the t h u j a p l i c i n s , as taken w i t h the Beckman DK-2 spectrophotometer, are shown i n Figure 12. The maximum absorptions were at 393 and 415 nm, r e s p e c t i v e l y , f o r ( 3 - t h u j a p l i c i n and p - t h u j a p l i c i n o l . The r e a c t i o n of both compounds w i t h f e r r i c acetate s o l u t i o n gave red chelates i n the organic l a y e r which had maxima at 418 and 525 nm, r e s p e c t i v e l y , f o r ( 5 - t h u j a p l i c i n and ft-thujaplicinol. The changes of absorption, a f t e r shaking w i t h the b u f f e r s o l u t i o n s , are given i n Table 5 as r e l a t i v e value of the o r i g i n a l . S i m i l a r l y , Table 6 gives the absorptions of t h e i r f e r r i c complexes a f t e r r e a c t i n g w i t h 1% f e r r i c c h l o r i d e s o l u t i o n . While the f e r r i c complex of | ? - t h u j a p l i c i n had only a maximum at 418 nm, that of (3-thu j a p l i c i n o l had two, which v a r i e d according to s o l u t i o n pH (Figure 13). .50 .40 -8 5 on o . 30 a tf £> u o m .20 .10 310 320 330 340 350 425 360 370 380 390 400 Wavelength (nm) . Figure 12. Absorption spectra of colored chelates produced by reaction of t h u j a p l i c i n ( s o l i d l i n e ) and p-thujaplicinol (dashed l i n e ) with NaOH. 450 - 34 -Table 5. Absorption?*, of | J - t h u j a p l i c i n and ^ - t h u j a p l i c i n o l f e r r i c acetate complexes treated w i t h various b u f f e r s o l u t i o n s of pH values from 6 to 11. pH (5>-thujaplicin ( 3 - t h u j a p l i c i n o l A =418 7\ 525 max max 6 90 54 7 92 56 8 91 52 9 95 50 10 92 53 11 92 50 * Data were obtained on percentage of the o r i g i n a l f e r r i c acetate complex s o l u t i o n . • Table 6. Absorptions * of ^ - t h u j a p l i c i n and ( 3 - t h u j a p l i c i n o l f e r r i c c h l o r i d e complexes tr e a t e d w i t h various b u f f e r s o l u t i o n s of pH values from 6 to 11. pH ( 3 - t h u j a p l i c i n ^ - t h u j a p l i c i n o l A = 418 X 445 X = 5 2 5 max max ' max 6 98 97 96 7 101 103 102 8 96 101 102 9 96 99 106 10 103 74 111 11 105 26 47 - 35 -450 475 500 550 600 Wavelength (nm) Figure 13- Absorption spectra of f e r r i c complexes of P- thu jap l ic in ( so l i d l i ne ) and ^ - t hu jap l i c i no l (dashed l i ne ) reacted with Fe(OAc)-} (A) or FeCl-j (B). - 36 -DISCUSSION A. Q u a n t i t a t i v e A n a l y t i c a l Method f o r T.M.E. The s t r u c t u r a l skeleton of the three components of T.M.E. (VIII) are s i m i l a r except f o r the functi o n s a t and R^. They can be w e l l separated by t h i n - l a y e r chromatography as mentioned before. Although the t h i n - l a y e r chromatographic technique gave a p e r f e c t q u a l i t a t i v e a n a l y s i s , no q u a n t i t a t i v e method was a v a i l a b l e . An attempted q u a n t i t a t i v e a n a l y s i s (by spraying the t h i n - l a y e r p l a t e w i t h d i a z o t i z e d s u l p h a n i l i c a c i d (56) and ex-amining w i t h an i n t e g r a t o r ) was not su c c e s s f u l because of the d i f f i c u l t y i n o b t a i n i n g uniform spraying. The s i l i c i c a c i d column chromatographic technique used i n the present study was developed on the b a s i s of the same p r i n c i p l e as that of the t h i n - l a y e r chromatography. With t h i s method, the e l u t i o n system was the c r i t i c a l f a c t o r of the separation. The mixture of ethanol and benzene (1:9) f o r the development of t h i n - l a y e r was undesirable. Even using a constant composition s o l v e n t , such as one w i t h more than 5% ethanol i n benzene, brought out the three components a t the same time. Figure 14 shows the e f f e c t of e l u t i o n system on .the separation. Using System B as shown i n Table 3, overlap of VITIb and V I I I c occurred. With System A a s l i g h t l y f l a t t e r slope of another compound e x i s t e d between V l l l b and V I I I c , but i t d i d not d i s t u r b the a n a l y s i s . T.M.E. were i d e n t i f i e d from t h e i r peak e f f l u e n t volumes, defined by Marvel and Rands (41) as that "volume of e f f l u e n t c o l l e c t e d i n which a given compound moves from the top to the bottom of the column, and i s measured where the greatest concentration of the compound i s e l u t e d . " I t i s apparent that - 37 -such a f a c t o r depends on the dimensions of the column, solvent used, and the change i n p o l a r i t y of the eluant. When these v a r i a b l e s are h e l d constant, each compound has a c h a r a c t e r i s t i c peak e f f l u e n t volume. A knowledge of these constants can be used i n the t e n t a t i v e i d e n t i f i c a t i o n of the T.M.E. i n ex-t r a c t i v e s . Table 7 l i s t s the peak e f f l u e n t volumes and rec o v e r i e s of T.M.E. Table 7. Peak e f f l u e n t volumes and re c o v e r i e s of T.M.E. Peak e f f l u e n t Recovery volume (ml) (7.) V i l l a 160 98.2 VTIIb 195 101.6 VI I I c 225 101.0 U s u a l l y , f o r good separation a t l e a s t a 20-ml d i f f e r e n c e i n the peak e f f l u e n t volume must be present between two successive compounds. The separation was suc c e s s f u l i n the present approach, but the experiment should be watched w i t h care to reduce any i n t e r r u p t i o n caused by i m p u r i t i e s . The manner i n which the e x t r a c t i v e s were added to the column was very c r i t i c a l f o r t h i s separation. To be sure that T.M.E. was d i s s o l v e d , 1 ml of 10% ethanol i n benzene was f i r s t added, followed by 1 ml of pure benzene, then the e l u t i o n system. Another f a c t o r which may a f f e c t -the e f f i c i e n c y of separation i s the s i l i c i c a c i d used. D i f f e r e n t batches of s i l i c i c a c i d u s u a l l y give d i f f e r e n t peak e f f l u e n t volumes. I n the present work, only one l o t of s i l i c i c a c i d was used. - 38 -V i l l a 120 U0 240 260 160 180 200 220 Effluent Volume (ml) Figure 14. Absorption of eluent fractions of acetone extracts from a western red cedar stem by GME-2801F 0V spectrophotometer at 280 nm. (above: elution system B, below:elution system A) - 39 -B. E x t r a c t i o n and Determination of T h u j a p l i c i n s The best solvent f o r e x t r a c t i o n i s one that e x t r a c t s the component(s) d e s i r e d e x h a u s t i v e l y w i t h l e a s t amount of contaminating other components. For e x t r a c t i n g t h u j a p l i c i n s from wood, acetone i s not s u i t a b l e since many other compounds accompany them. I t was known from the work of MacLean and Gardner (34) that n-hexane removes a l l the t h u j a p l i c i n s from aqueous s o l u t i o n s . n-Hexane was t r i e d to e x t r a c t t h u j a p l i c i n s from the wood but the y i e l d was poor. Thus, although n-hexane i s good solvent for the t h u j a p l i c i n s , i t i s a poor medium f o r d i r e c t l y e x t r a c t i n g them from the wood, because a l a r g e p o r t i o n of the content i s apparently r e t a i n e d i n the wood by the n-hexane-isoluble "membrane substances" (10). Among the three isomeric t h u j a p l i c i n s (Table 10), T - t h u j a p l i c i n was obtained i n much higher concentration than (S-thu j a p l i c i n . According to Gardner and Barton's paper (14) , the content of |3-thu j a p l i c i n was higher than that of V-thu j a p l i c i n : 0.3 and 0.2?o of wood, r e s p e c t i v e l y . The present r e s u l t s i n d i c a t e that there can be a l a r g e v a r i a t i o n i n some com-ponents of even the same heartwood. Sometimes a component i s present i n one sample but may be absent i n another sample. For instance, angolensin and p t e r o c a r p i n were i s o l a t e d from Pterocarpus i n d i c u s W i l l d . heartwood by Cooke e_t al_. (6) but were absent i n the study by Bhrara and co-workers (4) . The absence of ot-thu j a p l i c i n i n t h i s study does not mean that no o(-thu j a p l i c i n e x i s t s i n western red cedar heartwood. I t might be that the concentration was too low to be detected by the paper chromatographic technique, the same problem was encountered by Zavarin and Anderson (61) i n the determination of the o ( - t h u j a p l i c i n content i n A t l a n t i c white cedar wood. The presence of o( - t h u j a p l i c i n i n American grown western red cedar has been confirmed (61), o r i g i n a l l y i t was thought to be absent (11). - 40 -C. Spectroscopic Studies of ^ - T h u j a p l i c i n and £>-Thu j a p l i c i n o l F igure 10 shows the sodium s a l t s of | 3 - t h u j a p l i c i n and (J-thuja-p l i c i n o l to h a v e . d i f f e r e n t absorption maxima at 393 and 418 nm r e s p e c t i v e l y . At 323 nm, s a l t s of the t h u j a p l i c i n show a maximum wh i l e that of (3-thuja-p l i c i n o l has none. These data i n d i c a t e that a mixture of the two components can be determined q u a n t i t a t i v e l y i n the region of 370 to 450 nm. For the determination of the content of i n d i v i d u a l t h u j a p l i c i n , paper chromatography was used. I n cases where i t i s not necessary to separate isomeric t h u j a -p l i c i n s , the spectroscopic method can give a f a s t e r determination w i t h only a l i t t l e l e s s p r e c i s i o n . T h u j a p l i c i n d e r i v a t i v e s e x h i b i t c h a r a c t e r i s t i c colored s o l u t i o n s w i t h s e v e r a l heavy metal ca t i o n s due to the formation of s t a b l e chelates which are s o l u b l e i n organic s o l v e n t s . Both ( S - t h u j a p l i c i n and £>-thujaplicinol can r e a c t w i t h the f e r r i c i o n to give a red chelate which i s soluble i n chloroform. I t i s not s u r p r i s i n g that ^ - t h u j a p l i c i n o l , having one more hydroxyl group than the t h u j a p l i c i n , e x h i b i t s some a d d i t i o n a l c h a r a c t e r i s t i c p r o p e r t i e s from t h u j a p l i c i n s . Formula XIX shows the f e r r i c chelate of t h u j a p l i c i n , which has absorption maximum at 418 nm. However, f 3 - t h u j a -p l i c i n o l has one a d d i t i o n a l i o n i c form of f e r r i c chelate (XX) besides XXI. The absorption maxima of XX and XXI are 525 and 445 nm, r e s p e c t i v e l y . 0 OH ( X I X ) ( X X ) - 41 -Thus p-thujaplicinol has two maxima i n Figure 13 and 15. XIX was s t a b l e i n s o l u t i o n s a t various pH values (Tables 5 and 6) where no i o n i c form as XX occurred. Table 5 shows the f e r r i c complexes produced from the r e a c t i o n of (3 - thu j a p l i c i n o l and f e r r i c acetate s o l u t i o n (pH=4.3) reduced t h e i r response at 525 nm when exposing to the b u f f e r s o l u t i o n s w i t h pH values ranging from 6 to 11. At the same Amax, the c o l o r e d chelate from f e r r i c c h l o r i d e s o l u t i o n and p>-thujaplicinol remained unchanged u n t i l the s o l u t i o n had a pH value of 11. These r e s u l t s i n d i c a t e d that the XX were more s t a b l e i n the s o l u t i o n s of pH l e s s than 10. A l s o , Table 6 and Figure 15 i n d i c a t e d that i n h i g h l y . a c i d i c s o l u t i o n both XX and XXI were present, although the l a t t e r was more prominent. When the pH value increased, the formation of the former increased i n company w i t h a decrease of the l a t t e r (see the curve at pH 10 i n Figure 15). A l l t h i s evidence suggests that the pH value of a s o l u t i o n a f f e c t s the formation of the f e r r i c chelate of t h u j a p l i c i n o l . In other words, the pH value changed the c o l o r response. I n t h i s connection, the pH value of the reagent s o l u t i o n should be c a r e f u l l y c o n t r o l l e d when using the c o l o r i m e t r i c method f o r the determination of p - t h u j a p l i c i n o l . D. D i s t r i b u t i o n and Formation of T.M.E. and T h u j a p l i c i n s The d i s t r i b u t i o n of wood e x t r a c t i v e s has been w e l l studied. I n general, the concentraction of e x t r a c t i v e s increased w i t h i n c r e a s i n g distance from the p i t h of a tree to the periphery of the heartwood. This p a t t e r n of d i s t r i b u t i o n was found i n the studies of Sherrard e_t a l . (54) on red wood (Sequoia semperivirens (D. Don.) E n d l ) , of Hancock (23) on Douglas f i r (Pseudotsuga m e n z i e s i i (Mirb.) Franco) and of Roux and Evelyn (53) on black w a t t l e . I n western red cedar, MacLean and Gardner (35) demonstrated that t h u j a p l i c i n s and water-soluble contents increased g r a d u a l l y from the p i t h to the heartwood boundary. L a t e r , they studied the n a t u r a l p r e s e r v a t i v e I I I I I I I 400 425 450 . 475 500 550 600 Wavelength (nm) Figure 15. Effect of solution pH values on the f e r r i c complex of (^-thujaplicinol. - 43 -content i n " t a r g e t p a t t e r n " heartwood (36). These analyses i n d i c a t e d , that the i n t e r n a l white r i n g s were chemically r e l a t e d more to sapwood than to heartwood. The d i s t r i b u t i o n of hot water-soluble phenols i n the cross-d i r e c t i o n of such stems was a l s o s i m i l a r to the e a r l i e r work. In t h i s study, a trace of T.M.E. was proved to be present i n the sapwood, however, i t s amount was too small to give a r e l i a b l e y i e l d w i t h t h i s s m a l l - s c a l e technique. I t was evident that the content of T.M.E. increased a b r u p t l y a t the sapwood-heartwood boundary. T h i s , again, suggested that p h e n o l i c e x t r a c t i v e s were formed i n s i t u at the sapwood-heartwood boundary (17). A d d i t i o n a l l y , the d i s t r i b u t i o n of three components of T.M.E. and the change i n the r a t i o s of monohydroxy- and dihydroxy- to nonhydroxylated T.M.E. i n the cross d i r e c t i o n of Tree No.2, together w i t h t h e i r chemical r e l a t i o n s , suggested that h y d r o x y l a t i o n metabolism increased w i t h the ageing of the wood. A p o s s i b l e pathway i s p o s t u l a t e d i n Figure 16. The h y d r o x y l a t i o n may f i r s t occur a t one of the (^-positions of the side chain of VTIIa due to s i t e a c t i v a t i o n by the adjacent carbonyl group. The new hydroxyl group may then a c t i v a t e the other (3>-position to form d i h y d r o x y t h u j a p l i c a t i n methyl ether ( V I I I c ) . The d i s t r i b u t i o n p a t t e r n f o r Tree No.l d i d not give any evidence as shown f o r Tree No.2. I t i s supposed that Tree No.l (90 years old) was s t i l l too young to show the same metabolism as the o l d e r tree (Tree No.2 400 years o l d ) . I I I Figure 16. Possible formation of d ihydroxythujapl icat in methyl ether. (Dashed arrows indicate the pos i t ion fo r entry of the next hydroxyl group). - 45 -The v a r i a t i o n of n a t u r a l p r e s e r v a t i v e s i n western red cedar, shown i n F i gure 11 and Table 10, apparently followed that found by MacLean and Gardner (35). These components were absent i n the sapwood and ab r u p t l y decreased near the p i t h of the stem. From the s i m i l a r p a t t e r n of d i s t r i -butions of these compounds (nezukone and t h u j a p l i c i n s ) i n t h i s work, the same conc l u s i o n of h y d r o x y l a t i o n r e a c t i o n o c c u r r i n g f o r T.M.E. cannot be made. P o s s i b l y , i n western red cedar t h i s type of h y d r o x y l a t i o n operated e a s i e r a t the side chain o f an aromatic nucleus (T.M.E.) than on the nucleus i t s e l f ( T h u j a p l i c i n s ) . Or i t was much f a s t e r on the nucleus and had a l l occurred a t the s i t e of b i o s y n t h e s i s . i I f one defines h y d r o x y l a t i o n as the conversion of a -C-H group to i i a -C-OH group, there are two d i s t i n c t types of b i o l o g i c a l h y d r o x y l a t i o n i r e a c t i o n s , the aerobic and the anaerobic (32). The aerobic h y d r o x y l a t i o n r e a c t i o n i s c h a r a c t e r i z e d by two requirements, the presence of oxygen and an 18 e x t e r n a l e l e c t r o n donor. Using 0^ , i t has been shown that the hydroxyl oxygen atom i s derived from atmospheric oxygen (32). The more f a m i l i a r anaerobic type i s a c t u a l l y a hyd r a t i o n r e a c t i o n , where the hydroxyl oxygen i s d erived from water and re q u i r e s an e l e c t r o n acceptor, which may be oxygen, but a l s o may be a redox dye (32). H y d r o x y l a t i o n i s now suggested as the p o s s i b l e pathway of the synthesis of T.M.E. and p l i c a t i c a c i d . However, sev e r a l questions should be considered f o r f u r t h e r research. F i r s t l y , the question concerning whether the hydroxy-l a t i o n i s a s p e c i f i c (enzymatic) or a n o n s p e c i f i c ( o f t e n nonenzymatic) r e a c t i o n . Although enzymes have been i s o l a t e d from the cambium and sapwood, they have not been i s o l a t e d from the heartwood. Higuchi and Fukazawa (25) found that a c t i v i t y of the enzyme, e s s e n t i a l i n the process l e a d i n g to the formation of - 46 -phenolic heartwood e x t r a c t i v e s , was greatest i n the cambial r e g i o n , l e s s i n the sapwood and intermediate zone, and absent from the heartwood of Cryptomeria japonica D. Don., Chamaecyparis obtusa Sieb. et Zucc. and other i n v e s t i g a t e d species. The work of Matsukuma et a l . (42)confirmed the f i n d i n g s of Chattaway (5) that enzyme a c t i v i t y increased i n the intermediate zone. These statements i n d i c a t e d that enzymes could be absent i n heartwood or could be i n an i n a c t i v e s t a t e . On the c o n t r a r y , D i e t r i c h s 1 (8) study on a European beech stem showed a peroxidase i n the c e l l w a l l s of various t i s s u e s w i t h an i n c r e a s i n g concentration i n the t r a n s i t i o n zone and the heartwood. From D i e t r i c h s 1 f i n d i n g , together w i t h the known presence of n i t r o g e n i n heartwood (26), i t could be expected that enzymes e x i s t e d i n heartwood. However, i f they were s t i l l a c t i v e f o r c e r t a i n r e a c t i o n s was questionable. I n t h i s connection, the h y d r o x y l a t i o n i n western red cedar heartwood may not be enzyme-mediated. Next, i s the question of whether the h y d r o x y l a t i o n i s aerobic or anaerobic. While both types of h y d r o x y l a t i o n need oxygen, the l a t t e r i s c h a r a c t e r i z e d by a requirement f o r water. Kaufman (32) s t a t e d that aerobic h y d r o x y l a t i o n was a r e a c t i o n which appeared to be r e s t r i c t e d to the metabolism of r a t h e r i n e r t molecules because the r e a c t i o n was e n e r g e t i c a l l y expensive. I n f a c t , oxygen and water are present i n the heartwood. Since the r e a c t i o n s i t e of V i l l a can be a c t i v a t e d by the adjacent carbonyl group, then i t may be p o s t u a l t e d that the h y d r o x y l a t i o n i s anaerobic. The metabolic a c t i v i t y i n the t r a n s i t i o n zone has been discussed by many workers, and i t was thought to be r e l a t e d to the formation of wood e x t r a c t i v e s . Chattaway (5) concluded that t r a n s i t i o n wood represents a zone of i n t e n s i f i e d metabolism. Several recent studies have confirmed t h i s - 47 -f i n d i n g . F u r t h e r , Hugentobler (31) found a high metabolic a c t i v i t y ' a t sapwood-heartwood t r a n s i t i o n i n a study of n u c l e i i n a c t i v e ray parenchyma of maple, ash, and other species. Another increase of enzyme a c t i v i t y was observed i n the t r a n s i t i o n zone of Cryptomeria japonica by Matsukuma e_t a l . (42). The observation of i n c r e a s i n g £he peroxide concentration i n the t r a n s i t i o n zone and heartwood by D i e t r i c h s (8) f u r t h e r supported the above f i n d i n g s . On the other hand, several workers have re-examined the observation of Chattaway, w i t h c o n f l i c t i n g r e s u l t s . Frey-Wyssling and Bosshard (12) concluded, from a c y t o l o g i c a l a n a l y s i s of the ray c e l l s of sev e r a l European t r e e s , that the parenchyma c e l l underwent i r r e v e r s i b l e changes a t an appreciable distance from the heartwood boundary, which r e s u l t e d i n degradating proto-plasm of c e l l ' s o x i d i z i n g system. S i m i l a r p r o p e r t i e s have been observed i n trees by Higuchi and Fukazawa (25). They noted that the a c t i v i t y of the enzyme a s s o c i a t i n g w i t h the phenolic formation was great e s t i n the cambial r e g i o n , l e s s i n the sapwood and intermediate zones and absent from the heart-wood. In the present study, the maximum content of T.M.E. and the three n a t u r a l p r e s e r v a t i v e s was not at the v i s i b l e heartwood periphery, but occurred i n s t e a d many r i n g s past i t towards the p i t h . ' This f i n d i n g confirmed the suggestion by Gardner e_t al_. (17) that the biochemcial mechanism was operative w e l l i n t o the v i s i b l e heartwood zone. CONCLUSIONS A rnew method f o r q u a n t i t a t i v e determination of three t h u j a p l i c a t i n -methyl ethers (T.M.E.) i n the acetone e x t r a c t s of western red cedar was developed by using column chromatography on deacti v a t e d s i l i c i c a c i d . The e l u t i o n system was a s e r i e s of mixture of ethanol and benzene (from 17„ to 5%) added stepwise. The separation of the components was revealed using a GME-2801F u l t r a v i o l e t spectrophotometer. The determination of T.M.E. i n western red cedar again demonstrated that the lign a n s were present i n sapwood i n a low concentration, and increased a b r u p t l y a t the heartwood boundary. This f i n d i n g • supported the i n s i t u hypothesis of the formation of heartwood polyphenols. The d i s t r i b u t i o n of T.M.E. and the changes i n r a t i o s of monohydroxy- and dihydroxy- to nonhydroxy-l a t e d T.M.E. across the heartwood, together w i t h t h e i r - s t r u c t u r a l r e l a t i o n s h i p , suggested that the h y d r o x y l a t i o n r e a c t i o n increased w i t h ageing of the wood. On the b a s i s of the h y d r o x y l a t i o n mechanism i n higher p l a n t s , i t was f u r t h e r suggested that t h i s h y d r o x y l a t i o n i s of the nonenzymatic anaerobic type. The p o s s i b l e pathway of the formation of d i h y d r o x y t h u j a p l i c a t i n methyl ether ( V I I I c ) was from VTIIb, which was formed from VTIIa. Chloroform was found to be a good solvent to d i r e c t l y e x t r a c t t h u j a p l i c i n s from wood. n-Hexane, although i t can e x t r a c t t h u j a p l i c i n s completely from an aqueous s o l u t i o n , i s a poor medium f o r the d i r e c t e x t r a c t i o n from wood, p o s s i b l y owing to the e f f e c t of hexane-insoluble "membrane substances". - 49 -The f e r r i c complex of ^ - t h u j a p l i c i n (produced from r e a c t i n g w i t h e i t h e r f e r r i c acetate or f e r r i c c h l o r i d e ) was s t a b l e to b u f f e r s o l u t i o n s having pH values from 6 to 11. On the contrary, when j 3 - t h u j a p l i c i n o l was reacted w i t h f e r r i c acetate s o l u t i o n (pH 4.3), the c o l o r response was reduced to about one h a l f f o r the range of pH examined. However, when ( 3 - t h u j a p l i c i n o l was reacted w i t h 1% f e r r i c c h l o r i d e s o l u t i o n , the c o l o r response remained unchanged from pH 6 to 9. Exposing to a s o l u t i o n of pH 10 or 11, the Amax s h i f t e d from 445 to 525 nm. I t suggested that the f e r r i c complex i s stabl e i n a c i d i c s o l u t i o n s , and that pH c o n t r o l of s o l u t i o n s i s necessary when using the c o l o r i m e t r i c method f o r the a n a l y s i s of thu j a p l i c i n o l . Since T.M.E. and t h u j a p l i c i n s d i d not show maximum concentration at the v i s i b l e sapwood-heartwood boundary, but occurred many r i n g s past i t towards the p i t h , then the biochemical mechanisms must be operative w e l l i n t o the v i s i b l e heartwood zone. N a t u r a l p r e s e r v a t i v e s , nezukone, t h u j a p l i c i n s and ( 3 - t h u j a p l i c i n o l , were absent i n the sapwood. The presence of oL-thujaplicin i n t h i s study was not c e r t a i n , but i t was b e l i e v e d to be present i n another American grown western red cedar. - 50 -REFERENCES 1. Anderson, A.B. and J . Gripenberg. 1948. A n t i b i o t i c substances from the heartwood of Thuja p l i c a t a D. Don. IV. The c o n s t i t u t i o n of j i - t h u j a p l i c i n . Acta Chem. Scand. 2: 644-650. 2. and E.C. Sherrard. 1933. D e h y d r o p e r i l l i c a c i d , an a c i d from western red cedar (Thuja p l i c a t a Don.). J . Am. Chem. Soc. 55: 3813-3819. 3. 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Acta Chem. Scand. 12: 615-623. 61. Zavarin E. and A.B. Anderson. 1956. Paper chromatography of tropolones of Cupressaceae. J . Org. Chem. 21: 332-335. 62. , R.M. Smith and A.B. Anderson. 1959. Paper chromatography of the tropolones of Cupressaceae I I . J . Org. Chem. 24: 1318. - 57 -Table 8. Contents of T.M.E. an d r a t i o s of monohydroxy- ( V l l l b ) and dihydroxy-(VIIIc) to nonhydroxylated T.M.E. ( V i l l a ) i n the r a d i a l d i r e c t i o n of a western red cedar stem (Tree No.l) Ring No. V i l l a from periphery content(%) V l l l b con tent (7.) r a t i o to V i l l a V I I I c con tent (7.) r a t i o V i l l a 3* + + + 7** 0.13 0.14 1.07 0.12 0.92 10 0.16 0.18 1.13 0.18 1.13 20 0.18 0.17 0.94 0.15 0.83 30 0.38 0.17 0.45 0.19 0.50 45 0.32 0.31 0.97 0.34 1.06 65 0.12 * Sapwood 0.16 1.33 0.15 1.25 ** Sapwood-heartwood boundary + I n d i c a t e s presence below measurable amount Table 9. Contents of T.M.E. and r a t i o s of monhydroxy-(Vlllb) and dihydroxy-(VIIIc) to nonhydroxylated T.M.E. ( V i l l a ) i n the r a d i a l d i r e c t i o n of a western red cedar stem i (Tree No.2) Ring No. V i l l a from periphery content(7o) V l l l b content (7o) r a t i o to V i l l a V I I I c content (7„) r a t i o V i l l a 10* + + 0. + 17** 0.53 0.32 0.60 0.30 0.56 30 0.52 0.41 0.79 0.37 0.71 50 0.41 0.50 1.22 0.54 1.32 75 0.41 0.51 1.24 0.63 1.53 100 0.30 0.36 1.20 0.38 1.27 * Sapwood ** Sapwood-heartwood boundary + I n d i c a t e s presence below measurable amount - 58 -TablelQ. Contents of Nezukone, t h u j a p l i c i n s and ( 3 - t h u j a p l i c i n o l i n the r a d i a l d i r e c t i o n of the western red cedar stem (Tree No.3) Ring No. from p e r i -phery 10*. 30** 50 75 100 125 150 200 250 Nezukone T h u j a p l i c i n s * * * &- r -($ -Thu j a p l i c i n o l 0.13 0.26 0.31 0.20 0.24 0.23 0.15 0.08 0.022 0.030 0.041 0.030 0.030 0.032 0.026 0.009 0.17 0.39 0.32 0.26 0.25 0.22 0.10 0.03 0.06 0.15 0.15 0.15 0.10 0.09 0.04 0.02 * Sapwood ** Sapwood-heartwood boundary I n d i c a t e s absence *** - T h u j a p l i c i n can be present i n the heartwood 

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