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UBC Theses and Dissertations

Extractives of western larch (larix occidentalis nutt.) Giwa, Shakiru Adisa Obafemi 1973

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Co "u  EXTRACTIVES OF WESTERN LARCH  (LARIX QCCIDENTALIS NUTT.)  by  SHAKIRU ADISA OBAFEMI GIWA  B.Sc.  ( S p e c i a l ) Chem., U n i v e r s i t y of London, 1965  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE  i n the Department of Forestry  We a c c e p t t h i s t h e s i s as conforming to the required standard.  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1973  In p r e s e n t i n g an  this thesis  advanced degree at  the  Library  I further for  shall  the  of  this thesis  written  representatives.  of B r i t i s h  be  for  Fpre*4»^  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, C a n a d a  IS**  H*w  Columbia  {  913  requirements  Columbia,  for reference  the  It i s understood  for f i n a n c i a l gain  the  shall  Head o f my  that  not  be  I agree and  extensive copying of  g r a n t e d by  permission.  Department o f  Date  University  permission  s c h o l a r l y p u r p o s e s may his  f u l f i l m e n t of  make i t f r e e l y a v a i l a b l e  agree that  by  in partial  that  study.  this  thesis  Department  copying or  for  or  publication  allowed without  my  i  ABSTRACT  Western l a r c h  (Larix  oooidentalis  Nutt.) heartwood meals were  s u c c e s s i v e l y e x t r a c t e d w i t h f i v e s o l v e n t s o f i n c r e a s i n g p o l a r i t y and f i n a l l y w i t h a b s o l u t e e t h a n o l . The t o t a l e x t r a c t a b l e m a t e r i a l c o l l e c t e d was 14.94%, oven d r y (OD) wood b a s i s . Compounds i n each s o l v e n t f r a c t i o n were s e p e r a t e d , mostly by column chromatography over s i l i c a g e l , Sephadex A25 and Sephadex LH20. The i d e n t i t i e s o f i s o l a t e d compounds, a f t e r d e t e r m i n i n g r e l e v a n t p h y s i c a l and c h e m i c a l p r o p e r t i e s were c o n f i r m e d by comprison w i t h s t a n d a r d compounds.  P r e v i o u s l y u n r e p o r t e d compounds i n c l u d e d : Four r e s i n a c i d s  found i n western  l a r c h heartwood  (0.017% t o t a l y i e l d ) as s a n d a r a c o p i m a r i c ,  i s o p i m a r i c , a b i e t i c and d e h y d r o a b i e t i c from the p e t r o l e u m e t h e r (65° -80°) e x t r a c t ; pinocembrin (0.003% y i e l d ) ;  (5,7 d i h d r o x y - f l a v a n o n e ) from the benzene e x t r a c t  and f r e e L - a r a b i n o s e from the water e x t r a c t .  Other compounds found were: 3 - s i t o s t e r o l ,  3-sitosteryl  p a l m i t a t e , t r i s t e a r i n , e s t e r s o f l i n o l e n i c and a r a c h i d i c a c i d s , palmitoleic, linoleic,  palmitic,  l i n o l e n i c and a r a c h i d i c a c i d s . A l s o found were  d i h y d r o k a m p f e r o l , ct-conidendrin, d i h y d r o q u e r c e t i n , q u e r c e t i n and arabinogalactan  (11.10% y i e l d ) .  I s o l a t e s were c h a r a c t e r i z e d by t h e i r IR and NMR chrmatographic b e h a v i o u r s l a n d comparison  spectra, various  w i t h s t a n d a r d s . P h y s i c a l and  c h e m i c a l p r o p e r t i e s o f u n i d e n t i f i e d compounds are r e p o r t e d as a guide f o r future research.  ii  TABLE OF CONTENTS Table ABSTRACT  i  TABLE OF CONTENTS  i i  LIST OF TABLES  i i i  LIST OF FIGURES  iv  ACKNOWLEDGEMENT  v  1.0  INTRODUCTION  1  2.0  LITERATURE REVIEW  4  2.1  Definitions  4  2.2  E x t r a c t i v e s of  3.0  4.0  5.0  MATERIALS  Larix  spp.  6  AND METHODS  9  3.1  Wood Source  9  3.2  E x t r a c t i o n Procedure  9  3.3  Chromatography  10  3.4  Derivatives Preparation  14  3.5  D e g r a d a t i v e Techniques  15  3.6  P a r t i t i o n Between S o l v e n t s  16  3.7  S p e c t r a l Techniques  16  RESULTS  17  4.1  P r e l i m i n a r y Study  17  4.2  Main Study  18  DISCUSSION  34  5.1  E x p e r i m e n t a l Procedures  34  5.2  Extracts  36  6.0  CONCLUSION  7.0  LITERATURE CITED  47 4  9  iii  LIST OF TABLES  Table  Page  1.  R e t e n t i o n times of f a t t y a c i d m e t h y l e s t e r s  22  2.  R e t e n t i o n times and y i e l d r a t i o of r e s i n a c i d methyl e s t e r s  22  3.  PC p r o p e r t i e s of Compounds  27  4.  PC p r o p e r t i e s o f e t h a n o l s o l u b l e s i n water e x t r a c t s  30  5.  PC p r o p e r t i e s of n - b u t a n o l s o l u b l e s i n water e x t r a c t s  31  6.  PC p r o p e r t i e s of water s o l u b l e s i n water e x t r a c t s  32  7.  PC p r o p e r t i e s of f i n a l e t h a n o l e x t r a c t s  32  1^, 1^ and I I  iv  LIST OF FIGURES  Figure  Page  1.  B a s i c C^j. r i n g s t r u c t u r a l s k e l e t o n  of flavonoids  2.  Basic  3.  GLC t r a c e o f f a t t y a c i d methyl e s t e r s  37  4.  GLC t r a c e o f r e s i n a c i d methyl e s t e r s  38  5.  IR spectrum o f p i n o c e m b r i n ( l a )  40  6.  NMR  42  7.  IR spectrum o f u n i d e n t i f i e d Compound XIV  43  8.  NMR  45  9.  IR spectrum o f L - a r a b i n o s e (XX)  s t r u c t u r a l skeleton of r e s i n acids  spectrum o f u n i d e n t i f i e d  Compound XIV  spectrum o f L - a r a b i n o s e (XX)  4 6  46  V  ACKNOWLEDGEMENT  The assisted with Professor  author wishes t o express, h i s g r a t i t u d e t o a l l those p e o p l e who t h i s study.  In p a r t i c u l a r  t o Dr. E.P. Swan, A s s o c i a t e  ( p a r t - t i m e ) , F a c u l t y of F o r e s t r y , U n i v e r s i t y o f B r i t i s h Columbia,  f o r h i s p a t i e n t guidance i n the p l a n n i n g  and e x p e r i m e n t a l  study, and f o r h i s . a s s i s t a n c e i n the w r i t t e n  Particular  of t h i s  preparation.  r e c o g n i t i o n must go t o t h e s t a f f o f t h e Vancouver  Western F o r e s t P r o d u c t s L a b o r a t o r y , a l l experimental  stages  Canada Department o f Environment where  phases of t h i s study were performed.  S p e c i a l acknowledgement  i s extended t o the Wood Chemistry s e c t i o n of t h e Vancouver Western F o r e s t Products Laboratory  The  f o r t h e i r a s s i s t a n c e throughout t h e study.  a u t h o r i s a l s o t h a n k f u l t o Dr. J.W. W i l s o n , P r o f e s s o r ,  of F o r e s t r y , U n i v e r s i t y of B r i t i s h Columbia f o r h i s s u g g e s t i o n s , and  f o r reviewing  Faculty  criticisms  the manuscript.  A p p r e c i a t i o n i s a l s o g i v e n t o t h e N i g e r i a n F e d e r a l Government (through  t h e F e d e r a l F o r e s t Research Department, Ibadan) and the Canadian  I n t e r n a t i o n a l Development Agency f o r p r o v i d i n g funds through the academic program.  F i n a l acknowledgement must go t o my w i f e , Ramota, f o r h e r u n d e r s t a n d i n g and s a c r i f i c e s throughout the program of study.  1  1.0.  INTRODUCTION  Wood c o n s i s t s m a i n l y of c e l l u l o s e , hernicelluloses. and l i g n i n [ 9 ] . The  e x t r a c t i v e s a r e a minor component  but  of c o n s i d e r a b l e importance.  (on average about 5% of whole wood),  Values: of l e s s than 1% and more than 20%,  however, have been r e p o r t e d f o r e x t r a c t i v e s from some s p e c i e s [ 2 2 ] .  E x t r a c t i v e s a f f e c t wood usage i n many ways. towards improved wood u t i l i z a t i o n , i t i s n e c e s s a r y wood users  t o know more about wood c h e m i c a l  u t i l i z a t i o n without  With p r e s e n t  trends  f o r wood s c i e n t i s t s and  constituents.  This  assists  expensive t r i a l and e r r o r p r a c t i c e s .  Wood c o l o u r , odour, t a s t e and d u r a b i l i t y a g a i n s t f u n g a l decay and i n s e c t i n f e s t a t i o n a r e determined o f t e n by e x t r a c t i v e type and q u a n t i t y [ 8 ] . For i n s t a n c e , the t h u j a p l i c i n s of w e s t e r n r e d cedar h i g h l y t o x i c t o wood d e s t r o y i n g f u n g i .  This t o x i c i t y  magnitude as sodium p e n t a c h l o r o p h e n a t e t o x i c i t y west A f r i c a n i r o k o (Chlorophora and  exaelsa  (Thuja  •pliaata  Donn.) a r e  i s of the same  13]. The n a t u r a l r e s i s t a n c e of  Benth. and Hook.) heartwood t o f u n g a l  t e r m i t e a t t a c k has been a t t r i b u t e d t o the presence o f c h l o r o p h o r i n [ 4 3 ] .  O l e o r e s i n exudates from some s p e c i e s , e.g., western w h i t e p i n e (P-inus  monticola  and b l i s t e r i n g .  Dougl.), a f f e c t i t s p a i n t i n g p r o p e r t i e s through d i s c o l o u r a t i o n I n extreme cases  a complete l i f t i n g  Where wood i s used i n c o n t a c t w i t h m e t a l s m e t a l s ) t h e p r e s e n c e of some wood p o l y p h e n o l s and  l o o s e n i n g a t p o i n t s of c o n t a c t .  of p a i n t may occur [ 2 8 ] .  (especially  r e s u l t s i n gradual  ferrous corrosion  Complexes formed by i r o n and p h e n o l i c s  of the c a t e c h i n type a r e s t a b l e and impart  u n d e s i r a b l e d i s c o l o u r a t i o n t o the  lumber w i t h which the i r o n , e.g., b o l t s , n a i l s , i s i n c o n t a c t [ 1 2 ] .  2  In the p u l p and paper i n d u s t r y , e x t r a c t i v e s may decrease pulp increase  equipment c o r r o s i o n , consume chemicals and impart u n d e s i r a b l e  to r e s u l t i n g p u l p s  [14].  Also  the i n h i b i t i o n of p u l p i n g  yield, colour  r e a c t i o n s by  e x t r a c t i v e s may r e s u l t i n reduced p e n e t r a b i l i t y of c o o k i n g l i q u o u r i n t o the wood or r e d u c t i o n of l i g n i n s o l u b i l i t y and decomposition o f cooking or b o t h .  D i f f i c u l t i e s experienced i n s u l p h i t e pulping  (Lavtx leptolepsis  liquour  of Japanese l a r c h  (Sieb and Zucc.) Murr.) have been a t t r i b u t e d t o the  presence of s u l f a t e i o n a r i s i n g from l i q u o u r d e c o m p o s i t i o n caused by dihydroquercetin  and a r a b i n o g a l a c t a n [ 1 4 ] .  Wood e x t r a c t i v e s have been u s e f u l i n many ways. types of some woods have been used as a means of g e n e r i c 32].  Extractives  medicine.  T h i s has l e d to syntheses of c o n t r o l l e d potency drugs which a r e initially  Tannins a r e used e x t e n s i v e l y  from t r e e s and other  plants.  i n the l e a t h e r industry.  They a r e  from the b a r k s of woods, e s p e c i a l l y the Fagaceae, Leguminosae,  M e l i a c e a e , A n a c a r d i a c e a e and Rhizophoraceae f a m i l i e s . which a r e p h e n o l i c  clay casts.  Condensed  i n n a t u r e a r e used t o p r e s e r v e f i s h n e t s ,  v i s c o s i t y i n o i l w e l l d r i l l i n g and to i n c r e a s e  tannins,  t o c o n t r o l mud  t e n s i l e s t r e n g t h o f ceramics  Condensed w i t h formaldehyde, they form r e s i n s which a r e used i n  the wood i n d u s t r y f o r f i n i s h i n g and g l u i n g pine  a l l o c a t i o n [11, 31,  ( e s p e c i a l l y a l k a l o i d s ) from t r e e s have been used i n  r e l a t i v e s of those obtained  obtained  The e x t r a c t i v e  [20, 3 6 ] .  Steam d i s t i l l a t i o n of  (P-Cnus spp.) o l e o r e s i n s y i e l d s t u r p e n t i n e , a s w e l l as p i n e o i l s used i n  perfumery [33].  The d i s c o v e r y  o f j u v e n i l e hormone and i t s mimics i n some  t r e e s p e c i e s may l e a d to a n o n - p o l l u t i n g means of i n s e c t c o n t r o l [ 3 9 ] .  3  V a r i o u s compounds, of d i f f e r e n t c h e m i c a l classes, a r e known t o occur i n western  larch.  (Lari'x oaoidentalis:  Nutt) wood e x t r a c t i v e s .  Larch  a r a h i n o g a l a c t a n has been e x h a u s t i v e l y d e a l t w i t h by many workers. [10, 24, 4 4 ] . The f l a v o n o i d s , d i h y d r o q u e r c e t i n (3, 3', 4', 5, dihydrokampferol C3, 3 , r  7-pentahydroxy-flavanone),  (3, 4', 5, 7 - t e t r a h y d r o x y - f l a v a n o n e ) , and q u e r c e t i n  4 , 5, 7-pentahydroxy-flavanone) r  have been d e s c r i b e d and s t u d i e d i n  l a r c h wood [5, 13, 2 1 ] .  The purpose of the p r e s e n t i n v e s t i g a t i o n was t o i s o l a t e and c h a r a c t e r i z e o t h e r western  l a r c h heartwood  extractives.  4  2.0  As  LITERATURE REVIEW  an a i d t o a p p r e c i a t i n g the e x t r a t i v e v a r i e t y o b t a i n e d  western l a r c h wood, i t i s n e c e s s a r y t o d e f i n e the g e n e r a l compounds found. P r e v i o u s mentioned t o s e r v e Lavi-x  2.1  work on v a r i o u s Lavix  from  classes of  spp. e x t r a c t i v e s i s a l s o  as comparison o f western l a r c h e x t r a c t i v e s w i t h  other  spp.  Definitions  The  general  c l a s s e s o f compounds met w i t h  i n this investigation  were f a t t y a c i d s , f l a v o n o i d s , l i g n a n s , n e u t r a l s , r e s i n a c i d s and polysaccharides.  F a t t y acids are long s t r a i g h t chain a l i p h a t i c monocarboxylic acids which a r e found w i d e l y and  i n nature,  c h i e f l y as a r e s e r v e  food  f o r both plants  animals. F a t t y a c i d s may be e i t h e r s a t u r a t e d o r u n s a t u r a t e d  and double  bonds o f the l a t t e r may be conjugated o r n o t .  Flavonoids  a r e composed o f a C  d i s t i n c t u n i t s as the 0.  - C, fragment  D  carbon s k e l e t o n c o n t a i n i n g two  (Ring B) and the  (Ring A) fragment 6  O  (Fig. 1).  2?  1.  Basic C  y  4  5  Figure  3  15  r i n g s t r u c t u r a l skeleton of flavonoids.  5  Flavonoids  c l a s s i f i c a t i o n are according  oxygen c o n t a i n i n g r i n g or w i t h r e g a r d benzene r i n g s .  Two  to carbon l i n k a g e between the  of the f l a v o n o i d classes, r e l a t i n g to the  i n v e s t i g a t i o n a r e f lavanonb l-s , e.g., ;  ;  d i h y d r o q u e r c e t i n and  f l a v a n o l s , e.g.,  quercetin (II).  a)  = R2 = R^  = H,  b)  R^ = R2 = H, R^  = OH,  pinobanksin  c)  R  = OH,  dihydrokampferol  d)  R^ = R  2  = H, 2  R  2  = R^  Apart two  = R  3  to o x i d a t i o n l e v e l of  the  two  present  others  (I) and  pinocembrin  = OH,  dihydroquercetin  from degree of h y d r o x y l a t i o n , the main d i f f e r e n c e between the  c l a s s e s i s the p r e s e n c e ( I I ) or absence (I) of c o n j u g a t i o n  of the B r i n g  w i t h the c a r b o n y l group i n p o s i t i o n 4.  Among p l a n t s , the m a j o r i t y of f l a v o n o i d s occur m o s t l y mono-glucosides.  as g l y c o s i d e s ,  S o l u b i l i t y c h a r a c t e r i s t i c s a r e a f f e c t e d by  of h y d r o x y l a t i o n , as w e l l as l o c a t i o n and  number of a t t a c h e d  degree  glucose u n i t s  [17].  L i g n a n s a r e o p t i c a l l y a c t i v e phenylpropane dimers. carbon bond i s between the m i d d l e carbon atoms (3 chains.  3')  The  carbon-  of the p r o p y l s i d e  In l i g n a n s the p h e n y l n u c l e i a r e s u b s t i t u t e d , and  s i d e chains  exist  6  in various  s t a t e s of o x i d a t i o n .  further modified  In some cases, the non-benzenoid s k e l e t o n i s  by c y c l i s a t i o n to t e t r a h y d r o f u r a n ,  tetrahydronaphthalene d e r i v a t i v e s  Neutrals  r e s i n a c i d s , f a t t y or t e r p e n e a l c o h o l s and  e s t e r s of f a t t y a c i d s  hydrocarbons.  combined w i t h f a t t y a c i d s i n c l u d e g l y c e r o l ( f a t s ) ,  Resin  acids are diterpene  alcohols  3 - s i t o s t e r o l and  The  a c i d s of the g e n e r a l  a b i e t i c type has  ( F i g . 2 ) , w h i l e the p i m a r i c  substituents  The  and  long  (waxes) [33].  c l a s s i f i e d as two.types. p o s i t i o n 13  or  I19J.  i s a term used to r e p r e s e n t  chain f a t t y alcohols  tetrahydrofurofuran  at t h i s p o s i t i o n  f o r m u l a ^20 30^2' H  an i s o p r o p y l s i d e c h a i n  type has  methyl and  at  vinyl  [33].  20 A b i e t i c type Figure  2.2  2.  E x t r a c t i v e s of  The  genus  Pimaric  Basic  Larix  s t r u c t u r a l s k e l e t o n of r e s i n a c i d s  spp.  Lavix i n c l u d e s about ten s p e c i e s w i d e l y s c a t t e r e d  throughout North America, Europe and  Asia  [23].  Western l a r c h occurs as  l a r g e s t timber volume of the North American l a r c h e s . species  type  It i s a  i n the Columbia r i v e r r e g i o n of B r i t i s h Columbia, and  i n t o Washington, Oregon, Idaho and Montana.  I t s p r i n c i p a l use  the  commercial extends a l s o i s as  lumber.  7  S e v e r a l workers have i n v e s t i g a t e d the e x t r a c t i v e chemistry of various larches.  (Lar-ix dahurioa  In 1952,  T s v e t a e v a et al  140]  e x t r a c t e d weeping  larch  Elwes and Henry) w i t h a m i x t u r e of e t h a n o l and benzene  and a l s o w i t h water.  The y i e l d s o b t a i n e d w i t h these s o l v e n t s were 5.6%  19.9%, r e s p e c t i v e l y .  ' D i s t y l i n ' was  1952  from Japanese l a r c h .  i s o l a t e d by Hasegawa and  T h i s compound was  the two 0.45%  compounds from European l a r c h  yields,  He then  [16]  isolated  (Larix deaidua M i l l . ) i n 0.25%  and  respectively.  The heartwood c o n s t i t u e n t s of New European l a r c h e s were s t u d i e d by Brewerton d i h y d r o q u e r c e t i n and d i h y d r o k a m p f e r o l . d i h y d r o q u e r c e t i n and 0.43% 0.52%  and  S h i r a t o [18] i n  l a t e r shown by G r i p e n b e r g  to be a m i x t u r e of d i h y d r o q u e r c e t i n and d i h y d r o k a m p f e r o l .  1:1  Zealand grown Japanese and [7].  From t h e s e he  isolated  The Japanese l a r c h y i e l d e d  2.07%  d i h y d r o k a m p f e r o l w h i l e the European l a r c h gave  and 0.43%, r e s p e c t i v e l y .  He a l s o i s o l a t e d a f l a v o n o i d of unknown  structure.  V a r i a t i o n i n c o m p o s i t i o n of weeping l a r c h heartwood c o n s t i t u e n t s was  s t u d i e d by T s v e t a e v a et al.  i n heartwood 5.6%;  ethanol-benzene  s o l u b l e e x t r a c t s 10 t o 12% arabinogalactan.  [41] who  e x t r a c t from 1.8  and  Siberian  i t s a r a b i n o g a l a c t a n content as 14.2%.  Larix  spp. was  done by N a i r and  They e x t r a c t e d tamarack (L. laricinia  K. Koch) heartwood w i t h acetone, and d i v i d e d benzene and petroleum  and water  (25] gave the hot water e x t r a c t s of  Work on n e u t r a l s and a l c o h o l s from Von R u d l o f f [34] i n 1959.  t o 6.3%;  resin  (up to 20% i n cases) composed c h i e f l y of  Khutorshchikov  l a r c h heartwood as 18.3%  1:1  found the c o m p o s i t i o n to be:  ether i n s o l u b l e s .  (Duroi)  the e x t r a c t i n t o d i e t h y l k e t o n e ,  They o b t a i n e d d i h y d r o q u e r c e t i n  8  (0.3%) and dihydrokampf e r o l (0.05%), as, w e l l as, t r a c e s of q u e r c e t i n and eicosanyl ferulate. acid  The s o l u b l e f r a c t i o n gave ( a f t e r s a p o n i f i c a t i o n ) p h t h a l i c  (1.9%) and l o n g - c h a i n  eicosanol detected.  f a t t y acids  (31.7%), g - s i t o s t e r o l (19.5%),  (2.2%) and 2-nonanol (3.1%).  No t r o p o l o n e s  o r r e s i n a c i d s were  The same workers i s o l a t e d d i h y d r o q u e r c e t i n  from the acetone e x t r a c t of s u b a l p i n e  l a r c h (L.  and d i h y d r o k a m p f e r o l  lyalli.  P a r i . ) heartwood i n  1.25% and 0.82% y i e l d s , r e s p e c t i v e l y [ 3 5 ] . S m a l l amounts of c o n i d e n d r i n also isolated.  Other compounds o b t a i n e d from the e x t r a c t s were 3 - s i t o s t e r o l ,  2-nonanol and p h t h a l i c , p a l m i t i c , a C^g, s t e a r i c , o l e i c , l i n o l e i c , and  acids.  The  were  Two u n i d e n t i f i e d a l c o h o l s were a l s o  water  linolenic  obtained.  (12%) s o l u b l e e x t r a c t of K u r i l e l a r c h  (L. gmeVini-  Qaponioa (Regel) P i l g e r ) was o b t a i n e d by A n t o n o v s k i i et at. [ 1 ] . I t s arabinogalactan  c o n t e n t was found t o be 11%.  L i s i n a et al.  [29] a n a l y s e d the petroleum ether  D a h u r i c a n l a r c h (L. gmeZ-ini, L i t v . ) acetone e x t r a c t . a n a l y s e d by gas l i q u i d were i d e n t i f i e d :  The c o n s t i t u e n t s  were  chromatography (GLC) and from t h i s the f o l l o w i n g  Pelargonic,  l i n o l e i c , octadecadienic  s o l u b l e f r a c t i o n of  p a l m i t i c , o l e i c , 16 methyl o c t a d e c a n o i c ,  and l i n o l e n i c .  The a l c o h o l s  o b t a i n e d from the  heartwood were epimanool, l a r i x o l ,  ( 3 - s i t o s t e r o l and c y c l o a r t e n o l .  l a r i x y l a c e t a t e were a l s o r e p o r t e d  i n European l a r c h [ 3 7 ] .  Using column chromatography isolated  the f o l l o w i n g l i g n a n s  sibtriaa  Ledeb.):  acids  L a r i x o l and  (CC) over s i l i c a g e l Leptova et at. [27]  from weeping l a r c h and S i b e r i a n l a r c h (L.  Conidendrin, p i n o r e s i n o l , l a r i c i r e s i n o l ,  s e c o i s o l a r i c i r e s i n o l and 3,4 d i v a n i l l y l - t e t r a h y d r o f u r a n .  isolariciresinol  9  3.0  The  3.1  MATERIALS AND METHODS  f o l l o w i n g e x p e r i m e n t a l procedures were adopted i n the a n a l y s i s .  Wood Source  A preliminary  i n v e s t i g a t i o n was done w i t h an a i r - d r y western l a r c h  (Lari-x ooc-Cdentalts Nutt.) veneer sample which had been i n the l a b o r a t o r y f o r over two y e a r s .  The heartwood p a r t was c u t i n p i e c e s and ground w i t h a  medium s i z e d W i l e y m i l l . retained.  Portions  t h a t passed through a 5 mm s i e v e were  M o i s t u r e content was determined on f o u r  samples.  Wood f o r the main i n v e s t i g a t i o n was o b t a i n e d  from t h e t r u n k of an  89 year o l d t r e e grown near Armstrong, B.C., c u t i n 1972 and shipped t o the Western F o r e s t P r o d u c t s L a b o r a t o r y . a x i s and heartwood was separated  from the sapwood. •. - The  p i e c e s , a i r - d r i e d and ground as above. s i e v e were r e t a i n e d .  S e c t i o n s were c u t p e r p e n d i c u l a r  Portions  heartwood was c u t i n t o  t h a t passed through a 5 mm  The wood meal was f u r t h e r a i r - d r i e d w i t h  turning to allow f o r uniform drying.  t o the  occasional  M o i s t u r e content was determined on f o u r  samples.  3.2  E x t r a c t i o n Procedure  Wood meals were weighed and  (819.0 g f o r the p r e l i m i n a r y i n v e s t i g a t i o n  926.6 g f o r the main i n v e s t i g a t i o n ) i n t o a l a r g e c l o t h thimble p l a c e d i n  a 12 L S o x h l e t .  The m a t e r i a l was s u c c e s s i v e l y e x t r a c t e d  8 L of the following solvents  i n the other:  f o r 48 h r each w i t h  10  i. ii. iii. iv. v. vi. Previous  s o l v e n t was  petroleum ether  (65°-80°);  benzene; benzene-ethanol  1:1;  ethanol; d i s t i l l e d water;  and  ethanol.  allowed to dry o f f c o m p l e t e l y b e f o r e  the next e x t r a c t i o n .  A f t e r e x t r a c t i o n , s o l v e n t was  removed by r o t a t o r y e v a p o r a t o r a t reduced  pressure  Solvent  from a t a r e d f l a s k .  removed under vacuum. weight of e x t r a c t .  F l a s k w i t h sample was  3.3  the e v a p o r a t o r  was  then weighed to o b t a i n  the  In the case of water e x t r a c t , water was  continuous admixture w i t h a b s o l u t e vapour p r e s s u r e  not removed by  than water  ethanol  removed by  to form a m i x t u r e w i t h  alone.  Chromatography  T h i n l a y e r chromatography  (TLC) was  of compounds i n column e l u a t e s , determine from e x t r a c t s w i t h s t a n d a r d s .  employed to a s c e r t a i n p u r i t y values,  and  compare compounds  Commercially a v a i l a b l e s i l i c a - g e l  plates  (Quantum I n d u s t r i e s , A n a l y t i c a l , s i l i c a - g e l GF w i t h gypsum b i n d e r Q4F)  s t o r e d i n a 70°  0.25  mm  C oven to p r e v e n t d e a c t i v a t i o n were used.  t h i c k l a y e r were used f o r TLC,  s e p a r a t i o n and  purification.  m i x t u r e to be p u r i f i e d was  higher  and  This l a t t e r  thicker plates  r e q u i r e d compound p l u s s i l i c a g e l was  one  Plates with a  (2.5 mm)  for  scraped o f f the p l a t e .  the s i l i c a - g e l was  concentrated  compound.  the  s i d e of a t h i c k p l a t e .  A f t e r v i e w i n g under UV  d i s s o l v e d the compound and to o b t a i n the  phosphor  procedure i n v o l v e d d e p o s i t i n g  ( i n a solvent) along  developed i n a s u i t a b l e s o l v e n t .  and  filtered off.  light,  Plate  the  Another Filtrate  solvent was  11  The d e v e l o p i n g s o l v e n t s used f o r s i l i c a g e l TLC were: methylene chloride  ( C H C 1 ) ; and benzene-ethanol, 2  2  g i v e n by volume u n l e s s o t h e r w i s e  9:1  (BE). A l l s o l v e n t r a t i o s  are  stated.  F o r l o c a t i o n o f c a r b o n y l compounds and e s t e r s o f f a t t y a c i d s , p l a t e s were sprayed w i t h 2,4 r e a g e n t was  d i n i t r o p h e n y l h y d r a z i n e reagent  (2,4 DNPH). T h i s  p r e p a r e d by adding c o n c e n t r a t e d s u l p h u r i c a c i d  dinitrophenylhydrazine added dropwise  (20 ml)  (4 g). i n a 250 ml c o n i c a l f l a s k . Water  c a r e f u l l y with shaking.  Ethanol  (100 ml  the  ) was  to  2,4  (30 ml)  was  added t o the  warm s o l u t i o n . C a r b o n y l compounds g i v e y e l l o w c o l o u r on warming the p l a t e on a h o t p l a t e .  D i a z o t i s e d . s u l p h a n i l i c a c i d spray p h e n o l i c compounds, such as l i g n a n s on s i l i c a orange t o red-orange  (DSA)  was  used f o r l o c a t i n g  gel plates.  T h i s reagent g i v e s  c o l o u r s w i t h a-hydroxy g u a i a c y l n u c l e u s and y e l l o w c o l o u r  w i t h f l a v o n o i d s []5].  DSA  i s p r e p a r e d by r e a c t i n g s u l p h a n i c a c i d  5% aq sodium n i t r i t e and 20% aq p o t a s s i u m b i c a r b o n a t e (2:1:3). a c i d s o l u t i o n was  made by adding s u l p h a n i l i c a c i d  hydrochloric acid  (90 ml)  procedure  acid f i r s t ,  and then adding p o t a s s i u m  reagent was  used immediately  f l a v o n o i d s , was metal.  3-OH  i d e n t i f i c a t i o n o f 3-OH  s o l u t i o n w i t h the  carbonate  The  sulphanilic  s o l u t i o n a f t e r 5 min.  The  f l a v a n o n e s , as d i s t i n c t from o t h e r  made by d u s t i n g the s i l i c a  A camel h a i r b r u s h was  flavanone [4].  (9 g) t o c o n c e n t r a t e d  f o l l o w i n g p r e p a r a t i o n i n a c o l d room (2° C ) .  concentrated h y d r o c h l o r i c acid. of  Sulphanilic  and making the s o l u t i o n up t o 1 L w i t h water.  i n v o l v e d m i x i n g the sodium n i t r i t e  The  solution,  g e l TLC p l a t e w i t h powdered z i n c  used f o r t h i s . - The p l a t e was  sprayed w i t h  B r i g h t p u r p l e c o l o u r s i g n i f i e d the  presence  12  Also The  c o m m e r c i a l l y a v a i l a b l e t h i n l a y e r c e l l u l o s e p l a t e s were used.  c e l l u l o s e was  0.25  mm  thick.  D e v e l o p i n g s o l v e n t f o r c e l l u l o s e TLC  n - b u t a n o l , a c e t i c a c i d and water, 60:15:25 (BAW^). c e l l u l o s e p l a t e s was  heating  i n an oven (110°  C) f o r 15  Sephadex A25  and  Sephadex LH20O  Separation  on s i l i c a - g e l  of the m a t e r i a l .  amounts of e t h y l a c e t a t e or e t h a n o l  column was  Petroleum ether  TLC  obtained  by a d d i t i o n of  to the main s o l v e n t s .  on s i l i c a - g e l  and  cm column was  e l u t i n g with a l e s s polar  CC w i t h ( f a t t y and  Stepwise varying  cm,  was  a 45 cm  x  the compounds. used f o r p u r i f i c a t i o n ,  solvent.  Sephadex A25;  had  r e s i n ) from n e u t r a l s  s a t u r a t e d w i t h carbon d i o x i d e  been v e r y u s e f u l f o r s e p a r a t i o n of  [46].  of d i e t h y l ether and methanol, 9:1;  The  e l u t i n g solvents are:  d i e t h y l e t h e r and methanol,  (using dry i c e ) ; and  of d i e t h y l e t h e r and methanol 9:1.  The  removed n e u t r a l s from the column, w h i l e acids.  (65°-80°)  F r a c t i o n s were  In some cases,  used to s e p a r a t e  Subsequently, a s m a l l e r column, 28 cm x 1.6  always  paper chromatography were employed  to a s c e r t a i n s e p a r a t i o n of compounds i n e l u a t e s . cm or 35 cm x 2.3  by  the most used means f o r s e p a r a t i n g  benzene were the main s o l v e n t s employed to e l u t e the column.  c o l l e c t e d i n 25 ml.  saturated  min.  i n c r e a s e i n e l u t i n g s o l v e n t p o l a r i t y was  3.7  A  on  Chromatographic media employed were s i l i c a - g e l ,  preceeded by a s i l i c a - g e l TLC and  spray.  sprayed on the p l a t e f o l l o w e d  Column chromatography (CC) was e x t r a c t components.  D e t e c t i o n of compounds  done w i t h p - a n i s i d i n e h y d r o c h l o r i d e  s o l u t i o n of p - a n i s i d i n e i n e t h a n o l was  was  first  a mixture 9:1,  4% f o r m i c a c i d i n a m i x t u r e  eluting solvent  the l a s t  acids  two  normally  e l u t e d r e s i n and  fatty  13  Sephadex LH.2Q was. employed t o s e p a r a t e what was suspected f l a v o n o i d g l y c o s i d e s 138].  The e l u t i n g s o l v e n t was pure e t h a n o l .  S i l v e r n i t r a t e impregnated r e s i n a c i d s methyl e s t e r s . s l u r r y of alumina and  alumina TLC was used f o r the f a t t y and  G l a s s p l a t e s (20 cm x 20 cm) were coated w i t h a  (aluminium  oxide G w i t h b i n d e r , Research  S p e c i a l t i e s Co.)  s i l v e r n i t r a t e s o l u t i o n u s i n g a spreader s e t f o r 0.25 mm t h i c k n e s s .  s o l u t i o n of s i l v e r n i t r a t e  (12 g) i n water  shaking.  A  (20 ml) was d i l u t e d w i t h methanol  (40 ml) and the r e s u l t i n g s o l u t i o n was added t o alumina  (30 g) and mixed by  The p l a t e s were a l l o w e d t o a i r - d r y o v e r n i g h t , then they were d r i e d  at 110° C f o r 30 min b e f o r e use. and  t o be  low b o i l i n g petroleum  The d e v e l o p i n g s o l v e n t was d i e t h y l ether  e t h e r , 1:3.  Concentrated  d i e t h y l e t h e r , 1:4,was used as spray r e a g e n t . oven (110° C) f o r 15 min and then c h a r r e d  Reverse phase TLC (RPC) was used  ether.  The p l a t e was heated  i n an  (200° C) f o r 1 h r [ 4 5 ] .  to amplify r e s u l t obtained  s i l i c a - g e l TLC of $ - s i t o s t e r o l and n e u t r a l s . w i t h 10% N u j o l i n petroleum  s u l p h u r i c a c i d and  from  A c e l l u l o s e p l a t e was washed  The petroleum  ether was a l l o w e d t o  evaporate from the p l a t e s u r f a c e and specimens were then s p o t t e d .  The  d e v e l o p i n g s o l u t i o n was methanol s a t u r a t e d w i t h N u j o l ; w h i l e 2,4 DNPH was used f o r d e t e c t i o n .  Paper chromatography  (PC) was u s e f u l f o r 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 f l a v o n o i d s and l i g n a n s . and u l t r a v i o l e t  R^ v a l u e s , c o l o u r s under v i s i b l e  l i g h t and ease of c o l o u r f o r m a t i o n w i t h reagents make such  compounds r e a d i l y  identifiable.  Descending  one-dimensional  (ID) and two-dimensional  (2D) PC were  used i n these i n v e s t i g a t i o n s w i t h Whatman No. 1 paper i n a Shandon apparatus.  14  Papers of 45 cm l e n g t h and 15 cm t o 45 cm width, were used i n ID chromatography,  w h i l e papers of 45 cm square were used f o r 2D  The s o l v e n t systems employed  f o r development  chromatography.  of papers were:  BAW2,  BAW^;  i . e . , 10:3:7; the top l a y e r of a m i x t u r e c o n s i s t i n g of n - b u t a n o l ,  0 c o n c e n t r a t e d ammonia and water, 20:3:10 (BNW); and 2% a c e t i c a c i d i n water (AW).  D e t e c t i n g r e a g e n t s were DSA  and Bartons r e a g e n t f o r c o n f i r m a t i o n .  Bartons r e a g e n t i s a m i x t u r e of 0.5% aq f e r r i c c h l o r i d e and 0.5% potassium f e r r i c  c y a n i d e (100 ml of each made up t o 1 L ) .  Gas l i q u i d 1/8  chromatography (GLC)on a 10% EGSS-X column  i n ) gave good s e p a r a t i o n s of f a t t y a c i d s and r e s i n a c i d s  and a l s o of n e u t r a l s o b t a i n e d i n the petroleum e t h e r e x t r a c t . of m a t e r i a l  (1 g i n 10 ml) was  injected.  Two  (5 f t x  (Me  esters),  About  1.5 u l  chromatographs were used, an  Aerograph 204 and a Hewlett Packard Research Chromatograph l a t t e r was  aq  7620A.  The  coupled w i t h an i n t e g r a t o r t o o b t a i n d i r e c t r e a d i n g on q u a n t i t i e s  of m a t e r i a l s b e i n g s e p a r a t e d by the  P r e p a r a t i v e GLC was  column.  used i n an attempt to q u a n t i t a t i v e l y s e p a r a t e  n e u t r a l s from the p e t r o l e u m e t h e r e x t r a c t . i n c l u d e d s i n c e t h e r e was  D e t a i l s of the p r o c e d u r e a r e not  not enough m a t e r i a l a v a i l a b l e t o use the  equipment  effectively.  3.4  Derivatives Preparation  Q_-Methyl e s t e r s and 0_-methyl e t h e r s were o b t a i n e d f o r a c i d s and phenols, r e s p e c t i v e l y .  The compound(s) to be m e t h y l a t e d was  i n a m i n i m a l amount of s o l v e n t and c o l d diazomethane  (were) d i s s o l v e d  i n e t h e r was  added.  E f f e r v e s c e n c e i n d i c a t e d presence of e a s i l y m e t h y l a t e d h y d r o x y l groups, as i n acids.  The m i x t u r e i n methanol was  left  i n the c o l d room (2° C) o v e r n i g h t to  15  methylate phenolic hydroxyl  groups.  Excess, diazomethane was removed by  blowing n i t r o g e n gas over t h e m i x t u r e .  R e s i d u a l s o l v e n t was removed u s i n g a  vacuum pump.  Amine s a l t s were used t o s e p a r a t e M i x t u r e c o n t a i n i n g b o t h was t r e a t e d w i t h  f a t t y a c i d s from r e s i n a c i d s .  10% cyclohexamine i n e t h a n o l .  r e a c t i o n m i x t u r e was put i n t h e c o l d room (2° C) o v e r n i g h t . amine s a l t s p r e c i p i t a t e d , w h i l e solution.  The  The r e s i n a c i d  t h e f a t t y a c i d amine s a l t s remained i n  The p r e c i p i t a t e was c o l l e c t e d and washed w i t h e t h a n o l and  p a r t i t i o n e d between c h l o r o f o r m a c i d l a y e r was d i s c a r d e d .  and 1 N h y d r o c h l o r i c a c i d .  The c h l o r o f o r m  The h y d r o c h l o r i c  was evaporated and t h e r e s i d u e was  methylated w i t h diazomethane t o o b t a i n t h e r e s i n a c i d methyl e s t e r s . solution containing between c h l o r o f o r m  The  the f a t t y a c i d amine s a l t s was s i m i l a r l y p a r t i t i o n e d and 1 N h y d r o c h l o r i c a c i d and the c h l o r o f o r m  l a y e r was then  methylated as above to o b t a i n the f a t t y a c i d methyl e s t e r s .  3.5  D e g r a d a t i v e Techniques  S a p o n i f i c a t i o n o f e s t e r s was a p p l i e d t o n e u t r a l s i n order t o convert  them t o f a t t y a c i d s and a l c o h o l .  a f t e r methylation  by u s i n g GLC.  d i s s o l v i n g sodium m e t a l  The f a t t y a c i d s were i d e n t i f i e d  The s a p o n i f i c a t i o n r e a g e n t was made by  (1.6 g) i n a b s o l u t e  added a f t e r d i s s o l u t i o n was complete. s a p o n i f i e d were r e f l u x e d f o r 1 1/2 h r .  ethanol  (50 m l ) .  Water  T h i s reagent and t h e m a t e r i a l t o be The s o l u t i o n was allowed  excess 1 N h y d r o c h l o r i c a c i d was added t o n e u t r a l i s e t h e m i x t u r e . a c i d s were e x t r a c t e d w i t h petroleum e t h e r evaporated t o d r y n e s s .  (5 ml) was  t o c o o l and The f a t t y  (65° - 8 0 ° ) , and t h e s o l v e n t  The c o l l e c t e d f a t t y a c i d s were m e t h y l a t e d as above.  16  3.6  P a r t i t i o n Between S o l v e n t s  From the r e s u l t s o b t a i n e d w i t h PC u s i n g BAW^, b e f o r e s i l i c a g e l CC was p a r t i t i o n between two  performed  s o l u t i o n s were evaporated  3.7  effected  by  Material  e x h a u s t i v e l y e x t r a c t e d w i t h water.  to dryness on the r o t a r y evaporator by  Both constant  with absolute ethanol.  S p e c t r a l Techniques  Infrared spectroscopy  (IR) was  p u r i f i e d samples, e i t h e r from potassium chloride plates. The  T h i s was  i m m i s c i b l e s o l v e n t s , n - b u t a n o l and water.  e x t r a c t e d w i t h n - b u t a n o l , was  admixture  on some mixtures..  i n i t i a l separation  A "-'Ref kin-Elmer  521  performed  to o b t a i n the s p e c t r a of  bromide p e l l e t s o r as smears on sodium  I n f r a r e d Spectrophotometer  s p e c t r a of s t a n d a r d compounds were a l s o taken f o r  N u c l e a r magnetic resonance HA-100, 100 MHz  NMR  spectrometer.  The  d e u t e r o c h l o r o f o r m , d e u t e r a t e d acetone solubility.  T e t r a m e t h y l s i l a n e was  s i g n a l a t r = 10.  (NMR)  was  comparison.  s p e c t r a were o b t a i n e d on a V a r i a n  samples were prepared i n o r heavy water depending on compound  added as an i n t e r n a l s t a n d a r d and  Sample d i l u t i o n was  used.  approximately  lock  15 mg/300 p i of s o l v e n t .  17  4.0  The procedures  4.1  RESULTS  f o l l o w i n g a r e the r e s u l t s obtained i n the experimental  taken i n t h i s  investigation.  P r e l i m i n a r y Study  The veneer  sample m o i s t u r e content was 8.77% and t h e wood meal  weight was 747.2 g (OD b a s i s ) .  The petroleum OD sample. developed  ether  (65° - 80°) e x t r a c t (4.50  Three s i l i c a - g e l TLC were done on t h i s e x t r a c t . w i t h . C ^ C ^ , BE and petroleum  ether  o c c u r r e d i n t h e p l a t e developed w i t h petroleum r e v e a l e d v a r i o u s compounds under UV. and  g) was 0.60% o f the  (65° - 8 0 ° ) . ether.  They were No movement  The other two p l a t e s  One p l a t e was sprayed w i t h 2,4 DNPH  the o t h e r w i t h a 1:1 m i x t u r e o f c o n c e n t r a t e d s u l p h u r i c a c i d and  concentrated n i t r i c a c i d .  The p l a t e sprayed w i t h 2,4 DNPH r e v e a l e d more spots  than t h e one sprayed w i t h mixed a c i d s . ether  A GLC of t h e methylated  petroleum  (65° - 80°) e x t r a c t sample gave f i v e major peaks.  E x t r a c t i o n w i t h benzene gave 1.50 g of m a t e r i a l (0.20% of OD Two  s i l i c a - g e l TLC were developed  i n BE and viewed under UV.  sprayed w i t h 2,4 DNPH and t h e other w i t h DSA. signified  the presence  One p l a t e was  Colour of v a r i o u s spots  of c a r b o n y l and p h e n o l i c compounds.  The benzene-ethanol sample.  sample).  1:1 e x t r a c t (23.12 g) was 3.09% of the OD  A p a i r o f 2D, PC was done w i t h BAW^ and AW.  w i t h DSA and t h e o t h e r w i t h Bartonss  reagent.  One paper was sprayed  Spots s i g n i f y i n g 'the presence  of c a r b o n y l compounds, y e l l o w and b l u e r e s p e c t i v e l y w i t h t h e r e a g e n t s , were  18  observed.  The most conspicuous, spot was, shown to be  The  e t h a n o l e x t r a c t (6,85  d i h y d r o q u e r c e t i n on 2D, 15.7%  of OD  extract  PC w i t h BAW^  0.92%  and AW.  of OD  sample) r e v e a l e d more  The water e x t r a c t (117.30 g  sample) c o n s i s t e d m a i n l y a r a b i n o g a l a c t a n , w h i l e the f i n a l  (5.44  g and  0.73%  of OD  other minor compounds, on 2D,  The  4.2  g and  dihydroquercetin.  and  ethanol  sample) r e v e a l e d more d i h y d r o q u e r c e t i n , among  PC w i t h BAW^  t o t a l amount of e x t r a c t was  and  21.2%  AW.  of the OD  sample.  Main Study  The m o i s t u r e OD weight 819.78 g.  content  of the a i r - d r y wood meal was  11.53%, and  the  R e s u l t s from each s u c c e s s i v e e x t r a c t i o n a r e g i v e n below.  Petroleum e t h e r  (65° - 80°)  e x t r a c t was  5.27  g (0.65%) of the  OD  sample.  Two developed Yellow  i n CR^C^  and  BE.  p l a t e s were s p o t t e d w i t h Afterwards  and p u r p l e spots were noted.  compounds and sprayed  s i l i c a - g e l TLC  esters.  w i t h 2,4  spots were  the p l a t e s were viewed under  This s i g n i f i e d  T h e i r p r e s e n c e was  confirmed  DNPH and warmed on a hot p l a t e .  the p r e s e n c e of  UV. carbonyl  when the p l a t e s were S e v e r a l y e l l o w and  purple  observed.  S e p a r a t i o n of the n e u t r a l s from the a c i d s e f f e c t e d by CC of 1.75  g of the petroleum  ( f a t t y and  resin)  gave 1.22  g of n e u t r a l s .  a c i d s w h i c h came o f f the column ( s a t u r a t i o n of the s o l v e n t w i t h  d i o x i d e , and  a d d i t i o n of 4% f o r m i c a c i d  was  ether e x t r a c t on Sephadex A-2S5  E l u t i o n of the column w i t h d i e t h y l ether-methanol, 9:1 The  the e x t r a c t and  to the s o l v e n t ) were 0.41  g.  carbon  19  A p o r t i o n o f the f i r s t n e u t r a l s f r a c t i o n o f f t h e column ( t o a v o i d contamination  from f a t t y and r e s i n a c i d s which, might be p r e s e n t  f r a c t i o n ) was d i s s o l v e d i n c h l o r o f o r m  i n the l a t t e r  (1 g t o 10 ml) and 1.3 y l of t h i s was  i n j e c t e d i n t o the gas chromatograph o p e r a t i n g under t h e f o l l o w i n g c o n d i t i o n s : i. ii. iii. iv. v. vi. vii.  column, 10% EGSS-X, 1/8 i n x 4 f t ; oven temperature, 175° C i s o t h e r m a l ; c a r r i e r gas, n i t r o g e n ; i o n i s a t i o n gas, hydrogen; flame i o n i s a t i o n d e t e c t o r temperature, 300° C; i n j e c t i o n p o r t temperature, 300° C; and c h a r t speed 1 i n / 5 min.  Juvabione i . e . , (+) - methyl e s t e r o f (+) - todomatuic a c i d [1'  ( (+) - 4 (R) -  (S) - 5' - d i m e t h y l - 3 ' - o x o h e x y l ] - c y c l o h e x - l - e n e - l - c a r b o x y l i c  a c i d ) and  c i s - d i h y d r o j u v a b i o n e were i n j e c t e d b e f o r e and a f t e r f o r comparison.  One of  the peaks gave the same r e t e n t i o n time (38.75 min) as that o b t a i n e d f o r j u v a b i o n e , but none gave the same as i t s c i s d i h y d r o - d e r i v a t i v e ,  A p o r t i o n o f t h e same specimen (0.52 g) was p l a c e d on a s i l i c a - g e l chromatography column and e l u t e d w i t h petroleum  ether  l i t t l e m a t e r i a l came o f f the column w i t h t h i s s o l v e n t . s i m i l a r e l u t i o n w i t h petroleum acetate. and  E l u t i o n continued  e t h y l a c e t a t e 9:1.  until  the s o l v e n t was a m i x t u r e the remaining  washed down w i t h pure e t h y l a c e t a t e .  standards. heated  on a hot p l a t e .  of e t h y l  of petroleum  ether  m a t e r i a l on the column was  TLC of f r a c t i o n s c o l l e c t e d was done on  c i s dihydrojuvabione  The p l a t e s were developed  very  T h i s was f o l l o w e d by  ether.,rcontaining 1% increments  Afterwards  s i l i c a - g e l with juvabione,  (65° - 80°) u n t i l  r  and g - s i t o s t e r y l p a l m i t a t e as  i n CILjCl,,, sprayed w i t h 2,4 DNPH and  20  Spots which, gave the same R^ values, and c o l o u r r e a c t i o n s w i t h 2,4 DNPH ( y e l l o w ) as the j u v a b l o n e and c i s d i h y d r o j u v a b i o n e d i d n o t g i v e the same c o l o u r r e a c t i o n  ( f a i n t y e l l o w ) on h e a t i n g .  As a c o n f i r m a t i o n , t h i s  f r a c t i o n was p u t i n the gas chromatograph and standards were i n j e c t e d b e f o r e and a f t e r f o r comparison.  Retention value obtained f o r t h i s f r a c t i o n  differed  from e i t h e r of the s t a n d a r d s .  Some compounds i n t h e f r a c t i o n s had the same R^ (0.80 and 0.30) as 3 - s i t o s t e r y l p a l m i t a t e and 3 - s i t o s t e r o l , r e s p e c t i v e l y .  A r e v e r s e phase TLC  of these compounds i n the f r a c t i o n s and standards gave R^ v a l u e s of 0.35 and 0.56,  respectively.  Hence 3 - s i t o s t e r y l p a l m i t a t e and 3 - s i t o s t e r o l were b o t h  present.  A f r a c t i o n of t h e n e u t r a l s from the s i l i c a - g e l CC was found t o have a t r a c e component a s s o c i a t e d w i t h i t as seen by TLC. T h i s f r a c t i o n was put a l o n g t h e l e n g t h of a t h i c k s i l i c a - g e l p l a t e which was developed  i n CH^Cl^.  The r e q u i r e d p o r t i o n was scraped o f f , a f t e r d e l i n e a t i n g i t s contours w i t h UV light.  The s c r a p i n g s were e x t r a c t e d w i t h c h l o r o f o r m .  f i l t e r e d and the f i l t r a t e was evaporated  t o dryness.  d e u t e r o c h l o r o f o r m w h i l e the IR was conducted plates.  The c h l o r o f o r m was The NMR was taken i n  as a smear on sodium c h l o r i d e  The s p e c t r a showed t h a t the t r a c e component was t r i s t e a r i n .  proved by comparison  T h i s was  of the s p e c t r a w i t h t h a t o f the s t a n d a r d .  In o r d e r to i d e n t i f y f a t t y a c i d s a t t a c h e d t o a l c o h o l forming  fats  i n the n e u t r a l s , a p o r t i o n (0.17 g) o f the n e u t r a l s was h y d r o l y s e d w i t h a prepared  s a p o n i f i c a t i o n reagent  (20 ml) and r e f l u x e d as p r e v i o u s l y d e s c r i b e d .  The r e s u l t i n g f a t t y a c i d s were methylated,  and 1.5 u l v o f the s t a n d a r d  s o l u t i o n was i n j e c t e d i n t o t h e gas chromatograph.  Standards c o n t a i n i n g  21  ^10'  1 2 ' ^14  C  before  a n C  ^ ^15 ~ ^19 f  a t t  Y  and a f t e r the unknown.  s t e a r i c and two o t h e r s  a  ^  c  m e t h y l e s t e r s were a l s o i n j e c t e d  The f a t t y acids, i d e n t i f i e d were p a l m i t i c ,  beyond C^g, p r o b a b l y e i t h e r l i n o l e i c , p i n o l i c , or  arachidic.  F a t t y a c i d s and r e s i n a c i d s were separated recovery  from the Sephadex . A25  column.  sample b a s i s ) was 0.85 g, i . e . , 0.10%.  TLC  of f a t t y and r e s i n a c i d s  n i t r a t e impregnated alumina p l a t e s . chromatogram, i t was l e f t  as amine s a l t s a f t e r  T o t a l y i e l d of f a t t y a c i d s Resin  (OD  a c i d s y i e l d was 0.14g, 0.017%.  (Me e s t e r s ) was performed on s i l v e r  A f t e r developing  and s p r a y i n g the  i n an oven (110° C) f o r 15 min and then charred f o r  1 hr [45]. The 0.72.  f a t t y acids  L i t e r a t u r e values  l a s t was p a l m i t i c a c i d e s t e r s ) obtained  (Me e s t e r s ) R^ v a l u e s revealed  the f i r s t  (Me e s t e r s )  were 0.06, 0.31, 0.66 and  one was l i n o l e i c a c i d w h i l e the  [ 4 5 ] . R^ v a l u e s  were 0.13, 0.22, 0.50 and 0.63.  of the r e s i n a c i d s  These on comparison w i t h  standards were shown to be i s o p i m a r i c , sandaraeopimaric,. dehydroabietic  The  acids  (Me  a b i e t i c and  (Me e s t e r s ) , r e s p e c t i v e l y .  f a t t y a c i d methyl e s t e r s were d i s s o l v e d i n c h l o r o f o r m  (1 g i n  10 ml) and 1.5 y l of t h i s s o l u t i o n was i n j e c t e d i n t o the gas chromatograph (Aerograph 204 w i t h oven temperature s e t a t 155° C ) . were as g i v e n p r e v i o u s l y . ^10'  ^12' ^14  a n c  ^ ^15 ~ ^19  A l l other  Standard f a t t y a c i d s methyl e s t e r s which a  c  ^  s  w  e  r  e  injected before  contain  and a f t e r the sample.  From comparative r e t e n t i o n times, the f a t t y a c i d s l i s t e d identified.  conditions  i n T a b l e 1 were  22  Compound  R e t e n t i o n time, min.  Identity  III  5.31  Palmitic  IV  6.86  Palmitoleic  V  13.28  Linoleic  VI  17.50  Possibly  linolenic  VII  19.53  Possibly  arachidic  TABLE 1.  R e t e n t i o n times of f a t t y a c i d methyl e s t e r s .  The chromatograph.  r e s i n a c i d methyl e s t e r s  s o l u t i o n was i n j e c t e d i n t o the gas  C o n d i t i o n s were as d e s c r i b e d  f o r the n e u t r a l s  above.  In  a d d i t i o n , an i n t e g r a t o r was connected so as to g i v e a p r i n t o u t f o r c a l c u l a t i n g the r a t i o y i e l d f o r each r e s i n a c i d . e s t e r s m i x t u r e was i n j e c t e d as b e f o r e .  Compound  R e t e n t i o n time, min.  Standard r e s i n a c i d methyl  These d a t a a r e g i v e n i n T a b l e 2.  Identity  R a t i o based on minimum  VIII  13.75  Sandaracopimaric  IX  18.75  Isopimaric  X  25.47  Abietic  4.00  XI  31.72  Dehydroabietic  3.21  TABLE 2.  1 18.29  R e t e n t i o n times and y i e l d r a t i o of r e s i n a c i d methyl e s t e r s .  23 Benzene e x t r a c t s were r e c o v e r e d as. 1.22  Two  s i l i c a - g e l TLC,  sample d i s s o l v e d i n e t h a n o l . w i t h 2,4  DNPH.  developed One  g  i n CH^C^  (0.15% OD  and BE were done on  p l a t e was. sprayed w i t h DSA  Colour of the s p o t s suggested  sample b a s i s ) .  the presence  the  and the o t h e r  of c a r b o n y l  compounds and p h e n o l i c s .  M e t h y l a t i o n w i t h diazomethane d i d not produce n i t r o g e n e f f e r v e s c e n c e and TLC  the m i x t u r e was  of the unmethylated  sample was  developed  atmosphere.  left  o v e r n i g h t i n the c o l d .  sample s p o t t e d s i d e by s i d e w i t h the  i n BE.  sample.  silica-gel  methylated  Spots were l o c a t e d by exposure to an i o d i n e  V a r i o u s spots not p r e s e n t i n the unmethylated  i n the methylated  A  T h i s confirmed  the presence  CC s e p a r a t i o n of the m a t e r i a l (0.51 g) was  of  sample were seen phenols.  done on  silica-gel  f o l l o w i n g s o l u t i o n i n the minimum amount of benzene and e t h a n o l m i x t u r e . The  column was  first  e l u t e d w i t h pure benzene and  25 ml f r a c t i o n s were  c o l l e c t e d , u n t i l v e r y l i t t l e m a t e r i a l came o f f by f u r t h e r e l u t i o n w i t h solvent.  P o l a r i t y of the e l u t i n g s o l v e n t was  e t h y l a c e t a t e i n 2% increments,  and  this  then i n c r e a s e d by a d d i t i o n s of  e l u t i o n was  e t h y l a c e t a t e c o n t e n t of the e l u t i n g s o l v e n t was a f t e r e l u t i o n w i t h 10% e t h y l a c e t a t e i n benzene.  c o n t i n u e d as b e f o r e . i n c r e a s e d by 5%  The  increments  After elution with  e t h y l a c e t a t e i n benzene, the m a t e r i a l s t i l l remaining  40%  on the column  was  washed down w i t h pure e t h y l a c e t a t e f o l l o w e d by pure e t h a n o l .  S i l i c a - g e l TLC of the f r a c t i o n s were made i n d u p l i c a t e and developed 2,4  i n BE.  DNPH and  A f t e r v i e w i n g under UV  the other w i t h DSA.  f o l l o w i n g compounds a r e r e p o r t e d :  light,  one p l a t e was  sprayed  S i m i l a r f r a c t i o n s were combined.  with  The  24  Compound l a was o b t a i n e d when the column was e l u t e d w i t h 10% e t h y l a c e t a t e i n benzene.  I t formed about 2% of the benzene e x t r a c t  (0.003% of t h e OD sample).  I t c r y s t a l l i s e d out of e t h a n o l s o l u t i o n a f t e r  being l e f t  i n t h e c o l d f o r some days.  c o l o u r a t i o n under UV l i g h t .  i n BE was 0.62, w i t h p u r p l e  I t was n o t s e n s i t i v e t o 2,4 DNPH even on  warming the p l a t e , but gave a y e l l o w c o l o u r w i t h DSA and no r e a c t i o n w i t h Zn/HCl.  I t s IR spectrum  as f o l l o w s :  ( F i g . 5) was taken i n a KBr p e l l e t and s i s d e s c r i b e d  3100 cm" , s h o u l d e r ; 1650, 1570, 1490, 1290, v e r y s t r o n g ; 1250 1  weak; 1150 v e r y s t r o n g ; 1070, s t r o n g ; 1050 and 850, weak.  These p r o p e r t i e s were compared w i t h those of compounds l i k e l y t o have t h e same c h a r a c t e r i s t i c s .  Complete agreement was found  with  pinocembrin ( l a ) .  Compound I c was c o l l e c t e d on e l u t i n g e t h y l acetate mixture,  7:3.  the column w i t h benzene and  The compound w i t h i t s a s s o c i a t e d  was 3.6% of the benzene e x t r a c t (0.005% o f the OD sample). s i l i c a - g e l CC d i d n o t remove a l l the contaminants, prevent i d e n t i f i c a t i o n .  contaminants  Repeated  however t h i s d i d n o t  A p u r p l e f l u o r e s c e n c e was observed when i t s spot  ( s i l i c a g e l TLC) was viewed under UV.  R^ i n BE was 0.29 and a r e d d i s h - y e l l o w  c o l o u r developed w i t h 2,4 DNPH, w h i l e a y e l l o w c o l o u r o c c u r r e d w i t h DSA. R e a c t i o n w i t h Zn/HCl gave a p u r p l e c o l o u r . revealed  Comparison w i t h  standards  the compound t o be d i h y d r o k a m p f e r o l ( I c ) .  Compound X I I formed about 1% of the benzene e x t r a c t (0.002 of t h e OD sample).  I t came o f f the column w i t h b e n z e n e - e t h y l  f a i n t p i n k f l u o r e s c e n c e was observed  a c e t a t e (85:15).  under UV when s p o t t e d  and orange c o l o u r w i t h 2,4 DNPH and p i n k c o l o u r w i t h DSA. p u r p l e c o l o u r w i t h Zn/HCl.  The R  A  ( s i l i c a - g e l TLC), I t d i d not give a  i n BE was 0.36 and 0.87 i n BAW .  ^COOH  VIII  X:OOH  IX  26  Comparison with, s t a n d a r d compounds, showed t h a t i t was, a - c o n i d e n d r i n  (XII).  Some other compounds were o b t a i n e d from the s i l i c a - g e l CC of the benzene e x t r a c t .  Low y i e l d s and a s s o c i a t i o n w i t h t r a c e contaminants which  were not removed by repeated  chromatography prevented  positive  identification.  The f o l l o w i n g compounds amongst them were o b t a i n e d i n t h e l a r g e s t  yields:  Compound X I I I formed about 2% of t h e benzene e x t r a c t (0.003% of the OD sample) and was e l u t e d w i t h benzene and e t h y l a c e t a t e m i x t u r e , i n BE was 0.35 and a p i n k f l u o r e s c e n c e o c c u r r e d under UV.  3:1.  Its  The compound  was u n s e n s i t i v e t o 2,4 DNPH, but on warming the p l a t e , a f t e r s p r a y i n g w i t h t h i s r e a g e n t , an orange c o l o u r c o u l d be n o t i c e d . w i t h DSA and d e l a y e d p u r p l e c o l o u r w i t h Zn/HCl. flavonoid structure.  Its  was s l i g h t l y lower  A yellow colour T h i s suggested  a  developed 3-OH  than t h a t o f p i n o b a n k s i n  (0.36) and d i d not g i v e the d u l l y e l l o w f l u o r e s c e n c e o f p i n o b a n k s i n under UV.  O v e r n i g h t m e t h y l a t i o n of t h i s compound i n the c o l d o t h e r compounds of R^ 0.39 and 0.52  (2° C) gave two  ( s i l i c a - g e l TLC, developed  i n BE).  Both  compounds gave p u r p l e c o l o u r under UV, orange and p i n k c o l o u r s , r e s p e c t i v e l y , w i t h DSA.  M e t h y l a t i o n of p i n o b a n k s i n gave o n l y one compound of R^, 0.45,  y e l l o w under UV and orange c o l o u r w i t h DSA.  Compound XIV w i t h a s s o c i a t e d minor compounds formed about 10% of the whole benzene e x t r a c t (0.015% o f OD sample). compounds when the column was f i n a l l y  I t came o f f w i t h o t h e r  eluted with ethanol.  T h i s e l u a t e on  e v a p o r a t i o n o f the e t h a n o l was d i s s o l v e d i n a minimum amount of e t h a n o l and put on a s i l i c a - g e l column chromatograph. e t h a n o l 95:5 y i e l d e d  E l u t i o n of the column w i t h  t h i s unknown w i t h a s s o c i a t e d minor compounds.  ( s i l i c a - g e l TLC, developed  i n BE) was 0.50.  benzene-  I t s R^  The compound was u n s e n s i t i v e to  27  2,4 DNPH but when the p l a t e was warmed, a green c o l o u r was o b t a i n e d w i t h DSA. Repeated s i l i c a  colour r e s u l t e d , a pink  g e l CC d i d n o t remove the  c a r b o n y l compound a s s o c i a t e d w i t h i t .  Its  NMR  bromide p e l l e t , 3400 cm  and IR s p e c t r a were taken i n d e u t e r o c h l o r o f o r m a n t i potassium  r e s p e c t i v e l y . The IR spectrum i s d e s c r i b e d as f o l l o w s :  shoulder;  2900 v e r y s t r o n g ; 1600 weak; 1500 v e r y s t r o n g ; 1250  v e r y s t r o n g ; 1000 and 950, weak.  Benzene-ethanol  (1:1) e x t r a c t e d 10.58 g (1.30% o f the OD  sample).  No immediate r e a c t i o n was n o t i c e d on m e t h y l a t i o n w i t h diazomethane and the mixture and  was l e f t  o v e r n i g h t i n the c o l d . A s i l i c a g e l TLC o f the e x t r a c t  the methylated  sample was developed  i n BE and exposed t o i o d i n e vapour.  Spots n o t p r e s e n t i n the unmethylated sample were observed  i n the methylated  sample.  o  A 2D, PC w i t h BAW  1  and AW,  sprayed w i t h DSA a f t e r v i e w i n g under UV  showed t h r e e major compounds. T h e i r PC p r o p e r t i e s are g i v e n i n T a b l e 3.  Compound  R.^  Colour  Identity  BAW  AW  UV  Ic  0.88  0.57  l.y  y  Dihydrokampferol  Id  0.82  0.37  yy  y  Dihydroquercetin.  II  0.71  0  b.y  y  Quercetin  1  TABLE 3.  DSA  PC p r o p e r t i e s o f compounds I c , I d and I I . b.y = b r i g h t y e l l o w ;  l . y = l i g h t yellow; y = yellow.  28  S i l i c a s - g e l CC of a p o r t i o n of the e x t r a c t was. f i r s t  eluted with  benzene and s u b s e q u e n t l y w i t h a m i x t u r e of benzene and e t h a n o l i n 5% increments of the l a t t e r . in  BE, viewed  under UV  The f i r s t  E l u t i o n was  f o l l o w e d by s i l i c a - g e l TLC,  and sprayed w i t h  DSA.  f r a c t i o n c o l l e c t e d from the column c o n t a i n e d  d e s c r i b e d i n the benzene e x t r a c t .  developed  Dihydrokampferol  XIV,  (Ic),dihydroquercetin  (Id) and q u e r c e t i n ( I I ) were a l s o i d e n t i f i e d from the column e l u t i o n s comparison  w i t h standards  (R^ i n BAW^  compounds i n BE were 0.29, TLC p l a t e was  0.16  and 0.06,  sprayed w i t h Zn/HCl.  gave p u r p l e c o l o u r s .  and BE).  respectively.  silica-gel  PC)  I t seems t o have been masked by a  compound which a l s o gave a y e l l o w c o l o u r w i t h  DSA.  c o l l e c t e d i n an amount of 4.59  g (0.56% of the  sample).  A 2D, PC developed w i t h BAW^ d i h y d r o q u e r c e t i n and spots were a l s o  and AW  r e v e a l e d the presence of  t r a c e s of q u e r c e t i n on s p r a y i n g w i t h DSA.  Two  other  observed.  Dissolving dihydroquercetin.  the e x t r a c t i n e t h a n o l p r e c i p i t a t e d what was  I t was  f i l t e r e d o f f , the f i l t r a t e was  on a s i l i c a - g e l chromatography column. and  Another  Only d i h y d r o k a m p f e r o l and d i h y d r o q u e r c e t i n  gave a n e g a t i v e r e s u l t w i t h Zn/HCl.  OD  The R^ v a l u e s of these  D i h y d r o q u e r c e t i n ( e l u t e d l a t e r and i d e n t i f i e d on  E t h a n o l e x t r a c t was  by  then w i t h 5% increments  d i h y d r o q u e r c e t i n and  of e t h a n o l added.  c o n c e n t r a t e d and  put  f i r s t w i t h pure benzene  Dihydrokampferol,  t r a c e s of q u e r c e t i n were o b t a i n e d .  r e p o r t e d above were a l s o o b t a i n e d . were as g i v e n below.  E l u t i o n was  shown to be  The  two  spots  They were not i d e n t i f i e d but t h e i r  data  29  Compound X I I I had an R y e l l o w c o l o u r w i t h DSA  f  o f 0.51  i n BAW^  and z e r o i n AW.  and a l i g h t p u r p l e f l u o r e s c e n c e under UV  I t gave a  light. Its  o t h e r p r o p e r t i e s were the same as d e s c r i b e d f o r the u n i d e n t i f i e d compound X I I I o f the benzene e x t r a c t . I t i s l i k e l y t h a t they are  Compound XV had l i g h t p u r p l e c o l o u r was i t had  an R  f  o f 0.45,  an R  f  observed  o f 0.82  i n BAW  as was  w i t h DSA,  h i g h e r than t h a t o f a -  comparison, but s i m i l a r t o them under UV  The  1  and  and 0.69  identical.  i n AW.  Under UV  a  s u g g e s t i n g a l i g n a n . In g-  and w i t h  BE,  c o n i d e n d r i n , used as DSA.  e t h a n o l e x t r a c t a l s o c o n t a i n e d low R^  r e s i n o u s m a t e r i a l s which  s t a y e d on the column u n t i l washed w i t h pure e t h a n o l . T h i s m a t e r i a l formed a brown g l a s s y s o l i d on e v a p o r a t i o n o f the s o l v e n t . I t was  about 60% o f the  ethanol extract.  Water e x t r a c t e d m a t e r i a l s were 97.55 g, i . e . , 11.90% o f the sample. The mixture  c o n s i s t e d mostly  o f crude a r a b i n o g a l a c t a n i n 11.10% o f  the OD wood. T h i s p o l y s a c c h a r i d e i s common t o Lavi-x  The  e x t r a c t was  evaporated  d i l u t e d 10 times w i t h e t h a n o l , f i l t e r e d  t o d r y n e s s . The  and the i n s o l u b l e p o r t i o n , which was f i l t r a t e was BAW^  and BNW.  spp.  c o n c e n t r a t e d t o remove as much water as p o s s i b l e .  A p o r t i o n of the c o n c e n t r a t e was the f i l t r a t e  r e s i d u e was  I t was  and  r e d i s s o l v e d i n ethanol  a r a b i n o g a l a c t a n was  c o n c e n t r a t e d and examined by PC.  discarded.  developed  R^ v a l u e s o f the spots are g i v e n i n T a b l e 4.  possibly lignans.  OD  i n 2D  The with  These were  30  Compound  R  Colour with  DSA  BAW  BNW  XVI  0.86  0.06  Pink  XVII  0.64  0.29  L i g h t pink  XVIII  0.83  0.55  Pink  XIX  1.00  0.91  Yellowish pink  1  TABLE 4.  PC p r o p e r t i e s o f e t h a n o l s o l u b l e s i n water e x t r a c t s  F l a v o n o i d g l y c o s i d e s were sought  i n the e t h a n o l s o l u b l e p o r t i o n o f  the water e x t r a c t u s i n g the method o f B i r k o f e r and K a i s e r PC i n BAW^  were made. One  o t h e r w i t h DSA. and 0.69;  The  was  sprayed w i t h c o n c e n t r a t e d ammonia and  ammonia sprayed paper gave t h r e e spots o f R^  the paper was corresponded  l e f t i n the oven  f  0.42  0.90,  yellow, when  None of these c o l o u r s  from g l y c o s i d e s of the f l a v o n o i d s found.  s p r a y i n g the o t h e r chromatogram w i t h DSA, t h r e e gave b r i g h t  0.82  and l i g h t b l u e under UV appeared  (100°C) f o r 5: min.  w i t h those expected  of,ID  the  under UV t h e i r c o l o u r s were y e l l o w i s h b l u e , b l u e and l i g h t  r e s p e c t i v e l y . A f o u r t h s p o t of R  first  [6] . A p a i r  the same f o u r spots appeared.  On  The  orange t o orange c o l o u r s , w h i l e the f o u r t h gave a  l i g h t r e d c o l o u r . P o s s i b l y they were l i g n a n s .  To c o n f i r m the absence of f l a v o n o i d g l y c o s i d e s i n the e t h a n o l s o l u b l e p o r t i o n o f the water e x t r a c t , e x t r a c t specimen was  p l a c e d on a  Sephadex LH20 column and e l u t e d w i t h e t h a n o l . T h i s procedure  has been  used  s u c c e s s f u l l y f o r the i s o l a t i o n o f f l a v o n o i d g l y c o s i d e s [38]. F r a c t i o n s e l u t e d were f o l l o w e d by PC w i t h BAW^ DSA  and the papers were sprayed w i t h  o r ammonia. Most o f the compounds were r e c o v e r e d i n the f i r s t 25  f r a c t i o n . The  second  f r a c t i o n c o l l e c t e d gave a compound which  out o f aqueous e t h a n o l as white n e e d l e s . IR and NMR  either ml  crystallised  s p e c t r a showed i t t o be  31  a c a r b o h y d r a t e , w i t h m.p.  159° C.  a r a b i n o s e as standards i n BAW^  C e l l u l o s e TLC w i t h g a l a c t o s e and  and s p r a y i n g w i t h p^-anisidine h y d r o c h l o r i d e  reagent f o l l o w e d by warming i n the oven (12Q° brown c o l o u r f o r t h i s compound NMR  ( i n D2O)  and m.p.  (XX).  1130,  845 and  785,  C) f o r 5 min  T h i s c o l o u r , R^,  IR  gave a p u r p l e ( i n KBr  d a t a c o i n c i d e d w i t h t h a t of L - a r a b i n o s e .  spectrum i s d e s c r i b e d as f o l l o w s : 1230,  s t r o n g ; 1095,  1055,  3250 cm \  s h o u l d e r ; 1315,  v e r y s t r o n g ; 1005,  weak;  1255, 895,  Each e x t r a c t was ethanol mixture.  PC  developed  and  the other i n BNW.  e l u t e d w i t h benzene and  done i n d u p l i c a t e . The  One  paper  was  chromatograms were sprayed  with  Data f o r the n - b u t a n o l s o l u b l e s o b t a i n e d a r e  5.  Compound  and  DSA.  put on a s i l i c a - g e l CC and  of the e l u a t e s was  a f t e r v i e w i n g under UV.  given i n Table  the  A p r e l i m i n a r y 2D, PC w i t h BAW^  r e v e a l e d s e v e r a l compounds when sprayed w i t h  i n BAW^  by  f r a c t i o n of the water e x t r a c t , these were p a r t i t i o n e d  between water and n - b u t a n o l as d e s c r i b e d .  Colour with  R,  DSA  BAWj^  BNW  XXI  0.93  0.93  Reddish-brown  XXII  0.87  0.41  Purple  XXIII  0.84  0.04  Orange  XXIV  0.88  0.84  Light red  XXV  0.75  0.16  Light yellow  XXVI  0.73  0.05  Light yellow  XXVII  0.81  0.71  Redd i s h - o r a n g e  TABLE 5.  I t s IR  strong.  non-arabinogalactan  DSA  pellets),  s t r o n g ; 945, weak;  Because of d i v e r g e n t R^ v a l u e s e x h i b i t e d i n BAV^  BNW  L-  PC p r o p e r t i e s of n - b u t a n o l s o l u b l e s i n water e x t r a c t s .  Compound X X I I was  compared w i t h f e r u l i e a c i d  BAW  and BNW,  sprayed w i t h DSA) .  0.08  w i t h BNW  and p u r p l e c o l o u r w i t h  1  The water s o l u b l e s had  Compound  F e r u l i e a c i d gave R  f  of 0.86  BAW^  with.  the PC p r o p e r t i e s shown i n T a b l e  R  Colour w i t h  6.  DSA  BNW  XXVIII  0.89  0.88  L i g h t orange  XXIX  0.84  0.79  Reddish-pink  XXX  0.80  0.61  Orange  XXXI  0.79  0.68  Reddish-pink  XXXII  0.61  0.36  Reddish-pink  XXXIII  0.59  0.33  B r i g h t orange  PC p r o p e r t i e s of water s o l u b l e s i n water e x t r a c t s .  F i n a l ethanol extraction yielded OD  with  DSA.  BAW^^  TABLE 6.  (PC behaviour  3.18  g of m a t e r i a l (0.39% of the  sample).  A 2D, PC was  done w i t h BAW^  and AW,  and sprayed w i t h DSA.  were o b t a i n e d as g i v e n i n T a b l e 7.  Compound  R  Colour with  BAW^  BNW  XXXIV  0.87  0  Brownish-red  XXXV  0.76  0.28  Yellow  XXXVI  0.82  0.44  Reddish-pink  XXXVII  0.82  0.54  Reddish-pink  XXXVIII  0.82  0.68  Reddish-pink  XXXIX  0.96  0.85  Yellow  TABLE 7.  PC p r o p e r t i e s of  f i n a l ethanol extracts.  DSA  Data  33  Solubility  t e s t s were performed on p o r t i o n s o f the specimen.  s o l v e n t c o m p l e t e l y d i s s o l v e d the specimen.  Acetone p r e c i p i t a t e d a white  compound which was s o l u b l e i n water and h o t methanol. aqueous e t h a n o l and was i d e n t i f i e d c e l l u l o s e TLC developed i n BAW^  I t crystallised  as L - a r a b i n o s e from IR s p e c t r a , m.p.  (CC) w i t h benzene-ethanol  crystallised  out o f and  and s p r a y e d w i t h p - a n i s i d i n e h y d r o c h l o r i d e .  The r e m a i n i n g acetone s o l u b l e s were chromatographed o v e r gel  No  as the e l u t i n g s o l v e n t .  A compound which  o u t as brown f l a k e s was o b t a i n e d . IR and NMR  impure compound showed t h a t i t was a l i g n a n . The R^ corresponded w i t h t h a t o f compound XXXVII.  silica  s p e c t r a o f the  i n BAW^  and 'AW on PC  34  5.0  DISCUSSION  V a r i o u s o b s e r v a t i o n s were made i n the e x p e r i m e n t a l procedures  of  t h i s i n v e s t i g a t i o n . These r e s u l t s , i n c l u d i n g the p h y s i c a l and c h e m i c a l c h a r a c t e r i s t i c s o f the compounds  ( e s p e c i a l l y those u n r e p o r t e d p r e v i o u s l y )  found i n the wood are here d i s c u s s e d .  5.1  Experimental  Procedures  S u c c e s s i v e e x t r a c t i o n o f the wood meals w i t h s o l v e n t o f i n c r e a s i n g p o l a r i t y was most h e l p f u l f o r p r e l i m i n a r y i s o l a t i o n o f compounds. Compounds from s p e c i f i c c h e m i c a l c l a s s e s were o b t a i n e d i n most o f the d i f f e r e n t e x t r a c t s . F o r i n s t a n c e , f a t t y and r e s i n a c i d s were o b t a i n e d o n l y i n the petroleum  ether extract;  low h y d r o x y l a t e d f l a v o n o i d s appeared o n l y i n the benzene  e x t r a c t ; h i g h e r h y d r o x y l a t e d f l a v o n o i d s were found i n the e t h a n o l e x t r a c t ; and a r a b i n o g a l a c t a n o c c u r e d o n l y i n t h e water e x t r a c t . T h i s a s s i s t e d i s o l a t i o n o f compounds which were p r e s e n t o n l y i n t r a c e amounts.  CC procedures  gave good s e p e r a t i o n s , w i t h a p p r o p r i a t e c h o i c e o f  e l u t i n g s o l v e n t s . Most minor compounds o f low  v a l u e s s t a y e d a t the top o f the  column, e s p e c i a l l y when e l u t i n g w i t h s o l v e n t s o f low p o l a r i t y . Washing t h e column f i n a l l y w i t h a s t r o n g l y p o l a r s o l v e n t removed them. U s i n g a rubber hand pump t o a c t i v a t e CC e l u t i o n , a c u r r e n t procedure  a t the Western F o r e s t Products  L a b o r a t o r y , i n c r e a s e d the flow r a t e c o n s i d e r a b l y . T h i s s o l v e d a problem (low flow r a t e ) o f t e n encountered  Although  w i t h CC t e c h n i q u e .  PC w i t h BAW^  and d e t e c t i o n w i t h DSA were mostly used f o r  the i d e n t i f i c a t i o n o f f l a v o n o i d s and l i g n a n s , the use o f s i l i c a - g e l TLC w i t h BE as the d e v e l o p i n g s o l v e n t and d e t e c t i o n w i t h DSA was s u p e r i o r . Most  35  compounds: t r i e d on the PC system r a n c l o s e t o t h e s o l v e n t f r o n t , in similar  values.  resulting  A c c u r a t e measurement of R^ values: was f u r t h e r  hampered by the l a r g e , uneven spots: r e s u l t i n g from development.  Colour  developed w i t h d e t e c t i n g r e a g e n t s was dependent on the c o n c e n t r a t i o n and p u r i t y of the compound s p o t t e d .  A s s o c i a t e d t r a c e contaminants  (especially  low R^ v a l u e compounds) d i d n o t s t a y a t the o r i g i n and t h i s prevented p u r i f i c a t i o n on t h i c k paper.  In c o n t r a s t , these minor components d i d not  move w i t h the compound i n s i l i c a - g e l TLC.  The problems mentioned and development w i t h BE.  above were n o t observed w i t h s i l i c a - g e l TLC  In a d d i t i o n , a l l TLC d a t a were r e p r o d u c i b l e w i t h  o n l y s l i g h t v a r i a t i o n s i n R^ v a l u e s f o r r e p e a t TLC t r i a l s .  The s m a l l  d i f f e r e n c e s observed might have been due t o temperature v a r i a t i o n or i n a c c u r a t e m i x i n g of the d e v e l o p i n g s o l v e n t s .  The average  time taken t o develop a 20 cm x 20 cm TLC p l a t e (BE)  was 45 min, w h i l e PC took about  15 h r .  f o r f o l l o w i n g e l u t i o n s from columns.  Hence, TLC was found most convenient The i d e n t i f i c a t i o n of sugars on c e l l u l o s e  TLC was a l s o more r e p r o d u c i b l e than PC (both developed i n BAW^).  Bartons reagent was n o t used e x t e n s i v e l y i n t h i s because  i n e a r l i e r experiments  investigation,  i t was found t h a t the b l u e c o l o u r on the  paper g r e a t l y masked the p o s i t i o n of minor components.  T h i s was unacceptable,  s i n c e i n some case o n l y t r a c e amounts were b e i n g i n v e s t i g a t e d .  A l t h o u g h the presence of f a t t y and r e s i n a c i d s was confirmed by comparison w i t h s t a n d a r d s u s i n g GLC, f u r t h e r c o n f i r m a t i o n u s i n g s i l v e r impregnated  TLC d i d n o t proceed as a n t i c i p a t e d  [45],  Spots appeared  b e f o r e the c o m p l e t i o n of the recommended 1 hr c h a r r i n g p e r i o d .  nitrate  even  36  5.2  Extracts.  Petroleum ether  (.65° - 80°) e x t r a c t e d  were e a s i l y i s o l a t e d by CC w i t h Sephadex A—25.  f a t t y and r e s i n a c i d s , they Most p r o b a b l y ,  of r e s i n a c i d s from the r e s t of the p e t r o l e u m ether p e t r o l e u m ether  extract  a c i d s had not been found p r e v i o u s l y  i n t h i s wood.  spp.,  f r e e r e s i n a c i d s have been r e p o r t e d  only i n  f a t t y a c i d s , a l i k e w i s e , had n o t been r e p o r t e d t r a c e s of f r e e f a t t y a c i d s  (VI) and a r a c h i d i c  (VIII), isopimaric  Of a l l the  Larix  (Me e s t e r s ) and f r e e r e s i n a c i d s  (III), palmitoleic (IV), l i n o l e i c (VII).  medium.  Larix  L. si.bi.ri.oa [ 2 ] . F r e e  i n most of  found a r e shown i n F i g s . 3 and 4, r e s p e c t i v e l y .  linolenic  (2.68% of the  e x t r a c t s ) , was due to t h e use of t h i s a b s o r p t i o n  Resin  palmitic  separation  spp.  The GLC  (Me e s t e r s )  The f a t t y a c i d s were  (V) and two o t h e r s ,  possibly  The r e s i n a c i d s were s a n d a r a c o p i m a r i c  ( I X ) , a b i e t i c (X) and d e h y d r o a b i e t i c  (XI).  Retention  times on GLC a r e g i v e n i n T a b l e s 1 and 2.  Benzene e x t r a c t s c o n s i s t e d of many c a r b o n y l  and p h e n o l i c  compounds, as seen from TLC ( s i l i c a - g e l , BE and DSA, 2,4 DNPH).  The s m a l l  amount of m a t e r i a l e x t r a c t e d by t h i s s o l v e n t prevented complete s e p a r a t i o n of the  compounds.  37  VI  t  i  0  5  :  i 10  _ i 15  TIME (mins.) F i g u r e 3.  GLC  t r a c e of f a t t y a c i d methyl e s t e r s .  1 20  - i 25  IX  I  1  I  1  0  5  10  15  -1  20  :  1  25  TIME (mins) Figure  4.  GLC  t r a c e of r e s i n a c i d methyl e s t e r s .  39  Pinocembrin i n 0.003% y i e l d . around 3500. cm 165Q cm  ( l a ) p r e v i o u s l y unreported  I t s IR spectrum  i s shown i n F i g . 5.  shows t h e presence  are aromatic s i g n a l s .  i n t h i s s p e c i e s , was o b t a i n e d The a b s o r p t i o n band  of -OH. groups w h i l e the bands a t 1500 t o  Absence of an -OCH^ group i n the m o l e c u l e i s  proved by l a c k o f a band a t 1400 cm \  The complete IR spectrum  of t h i s  compound c o i n c i d e d w i t h t h a t of the s t a n d a r d compound, e s p e c i a l l y the bands a t 1,000 t o 800 cm  The  (fingerprint region).  R^ i n BE o b t a i n e d f o r the f l a v a n o n e s i n t h i s wood decreased  degree o f h y d r o x y l a t i o n on the f l a v a n o n e r i n g .  Dihydroquercetin with  h y d r o x y l groups had an R^ of 0.16, d i h y d r o k a m p f e r o l had an R^ o f 0.29 and pinocembrin The  R^ of p i n o b a n k s i n  found  five  w i t h f o u r h y d r o x y l groups  w i t h two h y d r o x y l groups had an R^ o f 0.61.  (with t h r e e h y d r o x y l groups) used  T h i s v a l u e was c l o s e t o the R^  with  as s t a n d a r d was 0.36.  (0.35) of the u n i d e n t i f i e d  i n the benzene e x t r a c t and d e s c r i b e d above.  compound X I I I ,  I t a l s o resembled  the  above f l a v a n o n e s by g i v i n g a p u r p l e c o l o u r w i t h Zn/HCl, i n d i c a t i v e of a 3-0H group.  It differed  from p i n o b a n k s i n i n the type of i t s f l u o r e s c e n c e under UV  (a p u r p l e c o l o u r w h i l e p i n o b a n k s i n was d u l l y e l l o w ) . products d i f f e r e d from suggested  those of p i n o b a n k s i n  Further, i t s methylation  (both r e p o r t e d above).  t h a t X I I I i s a t r i h y d r o x y l a t e d , 3-0H f l a v a n o n e .  Iti s  I n order t o d i f f e r  from p i n o b a n k s i n i t c o u l d have one of the two h y d r o x y l groups i n the A r i n g of p i n o b a n k s i n t r a n s f e r r e d t o the B r i n g , i . e . , an isomer  The NMR  of p i n o b a n k s i n .  and IR s p e c t r a of u n i d e n t i f i e d compound XIV a r e shown i n  F i g . 6 and F i g . 7, r e s p e c t i v e l y .  IR s i g n a l s a t 3400 cm ^ and 1400 cm ^ a r e  t y p i c a l of -OH and a r o m a t i c -OCH^ groups r e s p e c t i v e l y .  A c c o r d i n g t o Ludwig  et al. 130], the s i g n a l a t x 6.19 comes from t h e 3-OMe group i n t h i s type of compound.  I n t e g r a t e d v a l u e f o r t h i s peak i n d i c a t e d s i x p r o t o n s , i . e . , two  WAVE NUMBER  F i g u r e 5.  IR spectrum of pinocembrin ( l a ) . o  41  -OCH^  groups..  not w i t h i n  The s p l i t t i n g of this, peak, i n d i c a t e d  the same environment.  and 1,5 p r o t o n s  observed i n F i g . 6. no a l i p h a t i c -OH  M u l t i p l e t s around 3.15  ( l i t e r a t u r e v a l u e s , 3.14  L i t e r a t u r e J30] v a l u e f o r the 4-OH  and 3.18  i s 4.15  T.  the two -OCH^ T a r e from the  as i n l a r i c i r e s i n o l  were 2,6  T ) respectively.  This  s i g n a l can be  The absence of a s i n g l e t between 5.95  indistinctly  and 6.50  indicates  (XL) and s e c o l a r i c i r e s i n o l ( X L I ) .  suggested t h a t XIV i s r e l a t e d to p i n o r e s i n o l  XLI  that  (XLII).  XLII  It is  WAVE NUMBER  F i g u r e 7.  IR spectrum of u n i d e n t i f i e d  Compound XIV.  44  Water e x t r a c t s c o n t a i n e d  free L-arabinose,  but no  D-galactose.  T h i s r a i s e s the p o s s i b i l i t y o f the former coming from incomplete h y d r o l y s i s of arabinogalactan.  On  the o t h e r hand, e x t r a c t i o n c o n d i t i o n s were not  s u f f i c i e n t t o cause h y d r o l y s i s . T e s t s , d e s c r i b e d above, a l s o r u l e out p r e s e n c e o f f l a v o n o i d g l y c o s i d e s i n t h i s e x t r a c t . The been a s s o c i a t e d w i t h and  sophorosides  g l y c o s i d e s t h a t have  f l a v o n o i d s found i n t h i s i n v e s t i g a t i o n , were  [6,22].  Of course,  the  glucosides  the sugar c o u l d a l s o e x i s t f r e e l y i n  the heartwood.  Another p o s s i b i l i t y i s t h a t L - a r a b i n o s e i s the sugar moiety o f e a s i l y hydrolysed glucoside  f l a v o n o i d g l y c o s i d e i n the wood. D i h y d r o q u e r c e t i n - 3  i s the o n l y g l y c o s i d e t h a t has  an  -  1  been r e p o r t e d i n the sapwood o f  western l a r c h [ 6 ] .  The respectively.  NMR  and  IR s p e c t r a of L - a r a b i n o s e are shown i n F i g . 8 and  The  s t r o n g band a t 3300 cm  1  i s t y p i c a l o f sugars and  9,  i s due  to  the oxygen-hydrogen s t r e t c h i n g frequency o f hydrogen bonded hydroxy groups [26].  The  IR spectrum agreed w i t h  by U r b a n s k i et al.  having  spectrum  [42].  F i n a l ethanol (BNL)  the d e s c r i p t i o n o f the L - a r a b i n o s e IR  e x t r a c t p o s s i b l y contained  Braun's n a t i v e  lignin  among the compounds r e p o r t e d . T h i s i s brought about b y the water e x t r a c t i o n broken down the c e l l membrane t o enable BNL  subsequent e t h a n o l  extraction.  t o be e x t r a c t e d  on  1  I  I  •  '  1 . . , ,  2  •  i  i  i  •  ]  r  •  3  i  •  i  i  i  i  t  i  i  i  t  •  '  •  i , 1  r '  i 1  i •  i •  A-  i i —  5 F i g u r e 8.  NMR  6  7  spectrum of L - a r a b i n o s e  (XX).  r  3  •  i  .  .  i  «.  .  .  -  !  r  i  .  .  .  ' .  . . . . . .  9  i  i  i  .  .  47  6.0  The larch  CONCLUSIONS  t o t a l amount of e x t r a c t o b t a i n e d from the heartwood of western  {Lavtx ooovdental'is- N u t t ) by s u c c e s s i v e e x t r a c t i o n w i t h f i v e  solvents  of i n c r e a s i n g p o l a r i t y and f i n a l l y w i t h e t h a n o l was 14.94% (OD wood sample basis).  Petroleum ether  (65° - 80°) e x t r a c t e d  0.64%.  The compounds  found  on a n a l y s i s of t h i s e x t r a c t were 3 - s i t o s t e r o l , 3 - s i t o s t e r y l p a l m i t a t e , t r i s t e a r i n and e s t e r s acids.  ( f a t s ) of two a c i d s , p r o b a b l y l i n o l e n i c and a r a c h i d i c  F r e e f a t t y a c i d s found were p a l m i t i c , p a l m i o l e i c , l i n o l e i c and two  a c i d s , p r o b a b l y l i n o l e n i c and a r a c h i d i c .  F r e e r e s i n a c i d s found were  s a n d a r a c o p i m a r i c , i s o p i m a r i c , a b i e t i c and d e h y d r o a b i e t i c  The  benzene e x t r a c t was 0.15% of the wood.  pinocembrin, dihydrokampferol, a conidendrin  acids.  From t h i s e x t r a c t ,  and two u n i d e n t i f i e d  alcohols  were o b t a i n e d .  The and  benzene-ethanol (1:1) e x t r a c t was 1.29% (OD wood sample b a s i s )  t h i s y i e l d e d dihydrokampferol, dihydroquercetin  Ethanol extracted of d i h y d r o q u e r c e t i n  0.56% of the wood.  and a g l a s s y r e s i n o u s  material.  d i h y d r o k a m p f e r o l and q u e r c e t i n were a l s o found. a flavonoid  quercetin.  extract  consisted  Two u n i d e n t i f i e d compounds, (from i t s  i n this extract.  water e x t r a c t accounted f o r 11.90% the wood and  m a i n l y of crude a r a b i n o g a l a c t a n .  mainly  T r a c e s of  ( a l s o o b t a i n e d i n the benzene e x t r a c t ) and a l i g n a n  c o l o u r r e a c t i o n w i t h DSA) a l s o o c c u r r e d  The  This  and  I t also contained  consisted  L - a r a b i n o s e and  48  unidentified  The  lignans.  f i n a l e t h a n o l e x t r a c t was. 0.39%  m a i n l y of g u a i a c y l lignin.  type compounds such as  of the wood.  lignans  and  It  consisted  p o s s i b l y Braun's  native  49  7.0  LITERATURE CITED  A n t o n o v s k i i , S.D., M.M. 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