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Extractives of western larch (larix occidentalis nutt.) 1973

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Co " u EXTRACTIVES OF WESTERN LARCH (LARIX QCCIDENTALIS NUTT.) by SHAKIRU ADISA OBAFEMI GIWA B.Sc. (Special) Chem., Univ 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 accept t h i s thesis 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 t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed w ithout my w r i t t e n p e r m i s s i o n . Department o f Fpre*4»^ The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date IS** H*w { 9 1 3 i ABSTRACT Western l a r c h (Larix oooidentalis Nutt.) heartwood meals were successively extracted with f i v e solvents of increasing p o l a r i t y and f i n a l l y with absolute ethanol. The t o t a l extractable material c o l l e c t e d was 14.94%, oven dry (OD) wood basi s . Compounds i n each solvent f r a c t i o n were seperated, 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 of i s o l a t e d compounds, a f t e r determining relevant p h y s i c a l and chemical properties were confirmed by comprison with standard compounds. Previously unreported compounds found i n western larch heartwood included: Four r e s i n acids (0.017% t o t a l y i e ld) as sandaracopimaric, isopimaric, a b i e t i c and dehydroabietic from the petroleum ether (65° -80°) extract; pinocembrin (5,7 dihdroxy-flavanone) from the benzene extract (0.003% y i e l d ) ; and free L-arabinose from the water extract. Other compounds found were: 3 - s i t o s t e r o l , 3 - s i t o s t e r y l palmitate, t r i s t e a r i n , esters of l i n o l e n i c and arachidic acids, p a l m i t i c , p a l m i t o l e i c , l i n o l e i c , l i n o l e n i c and arachidic acids. Also found were dihydrokampferol, ct-conidendrin, dihydroquercetin, quercetin and arabinogalactan (11.10% y i e l d ) . Isolates were characterized by t h e i r IR and NMR spectra, various chrmatographic behavioursland comparison with standards. Physical and chemical properties of u n i d e n t i f i e d compounds are reported as a guide f o r future research. i i TABLE OF CONTENTS Table ABSTRACT i TABLE OF CONTENTS i i LIST OF TABLES i i i LIST OF FIGURES i v ACKNOWLEDGEMENT v 1.0 INTRODUCTION 1 2.0 LITERATURE REVIEW 4 2.1 D e f i n i t i o n s 4 2.2 Extractives of Larix spp. 6 3.0 MATERIALS AND METHODS 9 3.1 Wood Source 9 3.2 Extraction Procedure 9 3.3 Chromatography 10 3.4 Derivatives Preparation 14 3.5 Degradative Techniques 15 3.6 P a r t i t i o n Between Solvents 16 3.7 Spectral Techniques 16 4.0 RESULTS 17 4.1 Preliminary Study 17 4.2 Main Study 18 5.0 DISCUSSION 34 5.1 Experimental Procedures 34 5.2 Extracts 36 6.0 CONCLUSION 47 7.0 LITERATURE CITED 4 9 i i i LIST OF TABLES Table Page 1. Retention times of f a t t y acid methyl esters 22 2. Retention times and y i e l d r a t i o of r e s i n a c i d methyl esters 22 3. PC properties of Compounds 1^, 1^ and II 27 4. PC properties of ethanol solubles i n water extracts 30 5. PC properties of n-butanol solubles i n water extracts 31 6. PC properties of water solubles i n water extracts 32 7. PC properties of f i n a l ethanol extracts 32 i v LIST OF FIGURES Figure Page 1. Basic C^j. r i n g s t r u c t u r a l skeleton of flavonoids 4 2. Basic s t r u c t u r a l skeleton of r e s i n acids 6 3. GLC trace of f a t t y a c i d methyl esters 37 4. GLC trace of r e s i n a c i d methyl esters 38 5. IR spectrum of pinocembrin (la) 40 6. NMR spectrum of u n i d e n t i f i e d Compound XIV 42 7. IR spectrum of u n i d e n t i f i e d Compound XIV 43 8. NMR spectrum of L-arabinose (XX) 45 9. IR spectrum of L-arabinose (XX) 46 V ACKNOWLEDGEMENT The author wishes to express, h i s gratitude to a l l those people who assisted with t h i s study. In p a r t i c u l a r to Dr. E.P. Swan, Associate Professor (part-time), Faculty of Forestry, University of B r i t i s h Columbia, for h i s patient guidance i n the planning and experimental stages of t h i s study, and for his.assistance i n the written preparation. P a r t i c u l a r recognition must go to the s t a f f of the Vancouver Western Forest Products Laboratory, Canada Department of Environment where a l l experimental phases of th i s study were performed. Special acknowledgement i s extended to the Wood Chemistry section of the Vancouver Western Forest Products Laboratory f o r t h e i r assistance throughout the study. The author i s also thankful to Dr. J.W. Wilson, Professor, Faculty of Forestry, U n i v e r s i t y of B r i t i s h Columbia for h i s suggestions, c r i t i c i s m s and for reviewing the manuscript. Appreciation i s also given to the Nigerian Federal Government (through the Federal Forest Research Department, Ibadan) and the Canadian International Development Agency f o r providing funds through the academic program. F i n a l acknowledgement must go to my wife, Ramota, f o r her understanding and s a c r i f i c e s throughout the program of study. 1 1.0. INTRODUCTION Wood consists mainly of c e l l u l o s e , hernicelluloses. and l i g n i n [9]. The extractives are a minor component (on average about 5% of whole wood), but of considerable importance. Values: of less than 1% and more than 20%, however, have been reported f o r extractives from some species [22]. Extractives a f f e c t wood usage i n many ways. With present trends towards improved wood u t i l i z a t i o n , i t i s necessary f o r wood s c i e n t i s t s and wood users to know more about wood chemical constituents. This a s s i s t s u t i l i z a t i o n without expensive t r i a l and error p r a c t i c e s . Wood colour, odour, taste and d u r a b i l i t y against fungal decay and insect i n f e s t a t i o n are determined often by extractive type and quantity [8]. For instance, the t h u j a p l i c i n s of western red cedar (Thuja •pliaata Donn.) are highly t o x i c to wood destroying fungi. This t o x i c i t y i s of the same magnitude as sodium pentachlorophenate t o x i c i t y 13]. The natural resistance of west A f r i c a n iroko (Chlorophora exaelsa Benth. and Hook.) heartwood to fungal and termite attack has been a t t r i b u t e d to the presence of chlorophorin [43]. Oleoresin exudates from some species, e.g., western white pine (P-inus monticola Dougl.), a f f e c t i t s painting properties through d i s c o l o u r a t i o n and b l i s t e r i n g . In extreme cases a complete l i f t i n g of paint may occur [28]. Where wood i s used i n contact with metals (especially ferrous metals) the presence of some wood polyphenols r e s u l t s i n gradual corrosion and loosening at points of contact. Complexes formed by i r o n and phenolics of the catechin type are stable and impart undesirable d i s c o l o u r a t i o n to the lumber with which the i r o n , e.g., b o l t s , n a i l s , i s i n contact [12]. 2 In the pulp and paper industry, e x t r a c t i v e s may decrease pulp y i e l d , increase equipment corrosion, consume chemicals and impart undesirable colour to r e s u l t i n g pulps [14]. Also the i n h i b i t i o n of pulping reactions by extractives may r e s u l t i n reduced p e n e t r a b i l i t y of cooking liquour into the wood or reduction of l i g n i n s o l u b i l i t y and decomposition of cooking liquour or both. D i f f i c u l t i e s experienced i n sulphite pulping of Japanese l a r c h (Lavtx leptolepsis (Sieb and Zucc.) Murr.) have been at t r i b u t e d to the presence of s u l f a t e ion a r i s i n g from liquour decomposition caused by dihydroquercetin and arabinogalactan [14]. Wood extractives have been u s e f u l i n many ways. The ext r a c t i v e types of some woods have been used as a means of generic a l l o c a t i o n [11, 31, 32]. Extractives ( e s p e c i a l l y a l k a l o i d s ) from trees have been used i n medicine. This has led to syntheses of controlled potency drugs which are r e l a t i v e s of those obtained i n i t i a l l y from trees and other plants. Tannins are used extensively i n the leather industry. They are obtained from the barks of woods, e s p e c i a l l y the Fagaceae, Leguminosae, Meliaceae, Anacardiaceae and Rhizophoraceae f a m i l i e s . Condensed tannins, which are phenolic i n nature are used to preserve f i s h nets, to control mud 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 increase t e n s i l e strength of ceramics clay casts. Condensed with formaldehyde, they form resins which are used i n the wood industry f o r f i n i s h i n g and gluing [20, 36]. Steam d i s t i l l a t i o n of pine (P-Cnus spp.) oleoresins y i e l d s turpentine,as w e l l as pine o i l s used i n perfumery [33]. The discovery of j u v e n i l e hormone and i t s mimics i n some tree species may lead to a non-polluting means of insect control [39]. 3 Various compounds, of d i f f e r e n t chemical classes, are known to occur i n western larch. (Lari'x oaoidentalis: Nutt) wood ext r a c t i v e s . Larch arahinogalactan has been exhaustively dealt with by many workers. [10, 24, 44]. The flavonoids, dihydroquercetin (3, 3', 4', 5, 7-pentahydroxy-flavanone), dihydrokampferol (3, 4', 5, 7-tetrahydroxy-flavanone), and quercetin C3, 3r, 4 r, 5, 7-pentahydroxy-flavanone) have been described and studied i n la r c h wood [5, 13, 21]. The purpose of the present i n v e s t i g a t i o n was to i s o l a t e and characterize other western l a r c h heartwood extractives. 4 2.0 LITERATURE REVIEW As an a i d to appreciating the extrative v a r i e t y obtained from western l a r c h wood, i t i s necessary to define the general classes of compounds found. Previous work on various Lavix spp. extractives i s also mentioned to serve as comparison of western l a r c h extractives with other Lavi-x spp. 2.1 D e f i n i t i o n s The general classes of compounds met with i n t h i s i n v e s t i g a t i o n were f a t t y acids, flavonoids, lignans, neutrals, r e s i n acids and polysaccharides. which are found widely i n nature, c h i e f l y as a reserve food f o r both plants and animals. Fatty acids may be e i t h e r saturated or unsaturated and double bonds of the l a t t e r may be conjugated or not. Flavonoids are composed of a C carbon skeleton containing two d i s t i n c t units as the 0. - C, fragment (Ring B) and the (Ring A) fragment D O 6 (Fig. 1). Fatty acids are long s t r a i g h t chain a l i p h a t i c monocarboxylic acids 2? 3y 5 4 Figure 1. Basic C 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 to oxidation l e v e l of the oxygen containing r i n g or with regard to carbon linkage between the two benzene r i n g s . Two of the flavonoid classes, r e l a t i n g to the present i n v e s t i g a t i o n are f lavanonb;l-s;, e.g., dihydroquercetin and others (I) and f l a v a n o l s , e.g., quercetin ( I I ) . a) = R2 = R^ = H, pinocembrin b) R^ = R2 = H, R^ = OH, pinobanksin c) R 2 = H, R 2 = R 3 = OH, dihydrokampferol d) R^ = R 2 = R^ = OH, dihydroquercetin Apart from degree of hydroxylation, the main differ e n c e between the two classes i s the presence (II) or absence (I) of conjugation of the B r i n g with the carbonyl group i n p o s i t i o n 4. Among plants, the majority of flavonoids occur as glycosides, mostly mono-glucosides. 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 are affected by degree of hydroxylation, as w e l l as l o c a t i o n and number of attached glucose units [17]. Lignans are o p t i c a l l y a c t i v e phenylpropane dimers. The carbon- carbon bond i s between the middle carbon atoms (3 - 3 ' ) of the propyl side chains. In lignans the phenyl n u c l e i are substituted, and side chains e x i s t 6 i n various states of oxidation. In some cases, the non-benzenoid skeleton i s further modified by c y c l i s a t i o n to tetrahydrofuran, tetrahydrofurofuran or tetrahydronaphthalene d e r i v a t i v e s I19J . Neutrals i s a term used to represent esters of f a t t y acids and r e s i n acids, f a t t y or terpene alcohols and hydrocarbons. The alcohols combined with f a t t y acids include g l y c e r o l ( f a t s ) , 3 - s i t o s t e r o l and long chain f a t t y alcohols (waxes) [33]. Resin acids are diterpene acids of the general formula ^20 H30^2' c l a s s i f i e d as two.types. The a b i e t i c type has an isopropyl side chain at p o s i t i o n 13 (Fig. 2), while the pimaric type has methyl and v i n y l substituents at t h i s p o s i t i o n [33]. 20 A b i e t i c type Pimaric type Figure 2. Basic s t r u c t u r a l skeleton of r e s i n acids 2.2 Extractives of Larix spp. The genus Lavix includes about ten species widely scattered throughout North America, Europe and Asia [23]. Western l a r c h occurs as the large s t timber volume of the North American larches. I t i s a commercial species i n the Columbia r i v e r region of B r i t i s h Columbia, and extends also into Washington, Oregon, Idaho and Montana. Its p r i n c i p a l use i s as lumber. 7 Several workers have investigated the e x t r a c t i v e chemistry of various larches. In 1952, Tsvetaeva et al 140] extracted weeping l a r c h (Lar-ix dahurioa Elwes and Henry) with a mixture of ethanol and benzene 1:1 and also with water. The y i e l d s obtained with these solvents were 5.6% and 19.9%, r e s p e c t i v e l y . ' D i s t y l i n ' was i s o l a t e d by Hasegawa and Shirato [18] i n 1952 from Japanese l a r c h . This compound was l a t e r shown by Gripenberg [16] to be a mixture of dihydroquercetin and dihydrokampferol. He then i s o l a t e d the two compounds from European l a r c h (Larix deaidua M i l l . ) i n 0.25% and 0.45% y i e l d s , r e s p e c t i v e l y . The heartwood constituents of New Zealand grown Japanese and European larches were studied by Brewerton [7]. From these he i s o l a t e d dihydroquercetin and dihydrokampferol. The Japanese l a r c h yielded 2.07% dihydroquercetin and 0.43% dihydrokampferol while the European l a r c h gave 0.52% and 0.43%, r e s p e c t i v e l y . He also i s o l a t e d a flavonoid of unknown structure. V a r i a t i o n i n composition of weeping l a r c h heartwood constituents was studied by Tsvetaeva et al. [41] who found the composition to be: r e s i n i n heartwood 5.6%; ethanol-benzene 1:1 extract from 1.8 to 6.3%; and water soluble extracts 10 to 12% (up to 20% i n cases) composed c h i e f l y of arabinogalactan. Khutorshchikov (25] gave the hot water extracts of Siberian l a r c h heartwood as 18.3% and i t s arabinogalactan content as 14.2%. Work on neutrals and alcohols from Larix spp. was done by Nair and Von Rudloff [34] i n 1959. They extracted tamarack (L. laricinia (Duroi) K. Koch) heartwood with acetone, and divided the extract into diethylketone, benzene and petroleum ether in s o l u b l e s . They obtained dihydroquercetin 8 (0.3%) and dihydrokampf e r o l (0.05%), as, w e l l as, traces of quercetin and eicosanyl f e r u l a t e . The soluble f r a c t i o n gave (after saponification) p h t h a l i c acid (1.9%) and long-chain f a t t y acids (31.7%), g - s i t o s t e r o l (19.5%), eicosanol (2.2%) and 2-nonanol (3.1%). No tropolones or r e s i n acids were detected. The same workers i s o l a t e d dihydroquercetin and dihydrokampferol from the acetone extract of subalpine l a r c h (L. lyalli. Pari.) 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 [35]. Small amounts of conidendrin were also i s o l a t e d . Other compounds obtained from the extracts were 3 - s i t o s t e r o l , 2-nonanol and ph 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 , l i n o l e n i c and acids. Two u n i d e n t i f i e d alcohols were also obtained. The water (12%) soluble extract of K u r i l e l a r c h (L. gmeVini- Qaponioa (Regel) P i l g e r ) was obtained by Antonovskii et at. [1]. Its arabinogalactan content was found to be 11%. L i s i n a et al. [29] analysed the petroleum ether soluble f r a c t i o n of Dahurican l a r c h (L. gmeZ-ini, L i t v . ) acetone extract. The constituents were analysed by gas l i q u i d chromatography (GLC) and from t h i s the following acids were i d e n t i f i e d : Pelargonic, p a l m i t i c , o l e i c , 16 methyl octadecanoic, l i n o l e i c , octadecadienic and l i n o l e n i c . The alcohols obtained from the heartwood were epimanool, l a r i x o l , (3-sitosterol and cycloartenol. L a r i x o l and l a r i x y l acetate were also reported i n European l a r c h [37]. Using column chromatography (CC) over s i l i c a g e l Leptova et at. [27] i s o l a t e d the following lignans from weeping l a r c h and Siberian l a r c h (L. sibtriaa Ledeb.): 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 , i s o 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 . 9 3.0 MATERIALS AND METHODS The following experimental procedures were adopted i n the ana l y s i s . 3.1 Wood Source A preliminary i n v e s t i g a t i o n was done with 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 laboratory f o r over two years. The heartwood part was cut i n pieces and ground with a medium sized Wiley m i l l . Portions that passed through a 5 mm sieve were retained. Moisture content was determined on four samples. Wood for the main i n v e s t i g a t i o n was obtained from the trunk of an 89 year old tree grown near Armstrong, B.C., cut i n 1972 and shipped to the Western Forest Products Laboratory. Sections were cut perpendicular to the axis and heartwood was separated from the sapwood. •. - The heartwood was cut into pieces, a i r - d r i e d and ground as above. Portions that passed through a 5 mm sieve were retained. The wood meal was further a i r - d r i e d with occasional turning to allow f o r uniform drying. Moisture content was determined on four samples. 3.2 Extraction Procedure Wood meals were weighed (819.0 g f o r the preliminary i n v e s t i g a t i o n and 926.6 g for the main inv e s t i g a t i o n ) into a large c l o t h thimble placed i n a 12 L Soxhlet. The material was successively extracted f o r 48 hr each with 8 L of the following solvents i n the other: 10 i . petroleum ether (65°-80°); i i . benzene; i i i . benzene-ethanol 1:1; i v . ethanol; v. d i s t i l l e d water; and v i . ethanol. Previous solvent was allowed to dry off completely before the next extraction. A f t e r extraction, solvent was removed by rotatory evaporator at reduced pressure from a tared f l a s k . Solvent not removed by the evaporator was removed under vacuum. Flask with sample was then weighed to obtain the weight of extract. In the case of water extract, water was removed by continuous admixture with absolute ethanol to form a mixture with higher vapour pressure than water alone. 3.3 Chromatography Thin layer chromatography (TLC) was employed to a s c e r t a i n p u r i t y of compounds i n column eluates, determine values, and compare compounds from extracts with standards. Commercially a v a i l a b l e s i l i c a - g e l plates (Quantum Industries, A n a l y t i c a l , s i l i c a - g e l GF with gypsum binder and phosphor Q4F) stored i n a 70° C oven to prevent deactivation were used. Plates with a 0.25 mm t h i c k layer were used for TLC, and thicker plates (2.5 mm) for separation and p u r i f i c a t i o n . This l a t t e r procedure involved depositing the mixture to be p u r i f i e d (in a solvent) along one side of a thick p l a t e . Plate was developed i n a s u i t a b l e solvent. A f t e r viewing under UV l i g h t , the required compound plus s i l i c a g e l was scraped o f f the p l a t e . Another solvent dissolved the compound and the s i l i c a - g e l was f i l t e r e d o f f . F i l t r a t e was concentrated to obtain the compound. 11 The developing solvents used for s i l i c a gel TLC were: methylene chloride (CH 2C1 2); and benzene-ethanol, 9:1 (BE). A l l solvent r a t i o s are given by volume unless otherwise stated. For l o c a t i o n of carbonyl compounds and esters of f a t t y acids, the plates were sprayed with 2,4 dinitrophenylhydrazine reagent (2,4 DNPH). This reagent was prepared by adding concentrated sulphuric acid (20 ml) to 2,4 dinitrophenylhydrazine (4 g). i n a 250 ml c o n i c a l f l a s k . Water (30 ml) was added dropwise c a r e f u l l y with shaking. Ethanol (100 ml ) was added to the warm s o l u t i o n . Carbonyl compounds give yellow colour on warming the plate on a hot p l a t e . Diazotised. s u l p h a n i l i c a c i d spray (DSA) was used for l o c a t i n g phenolic compounds, such as lignans on s i l i c a gel p l a t e s . This reagent gives orange to red-orange colours with a-hydroxy guaiacyl nucleus and yellow colour with flavonoids []5]. DSA i s prepared by reacting sulphanic acid s o l u t i o n , 5% aq sodium n i t r i t e and 20% aq potassium bicarbonate (2:1:3). S u l p h a n i l i c acid 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 (9 g) to concentrated hydrochloric a c i d (90 ml) and making the s o l u t i o n up to 1 L with water. The procedure involved mixing the sodium n i t r i t e s o l u t i o n with the s u l p h a n i l i c a c i d f i r s t , and then adding potassium carbonate so l u t i o n a f t e r 5 min. The reagent was used immediately following preparation i n a col d room (2° C). The i d e n t i f i c a t i o n of 3-OH flavanones, as d i s t i n c t from other flavonoids, was made by dusting the s i l i c a gel TLC plate with powdered zinc metal. A camel h a i r brush was used f o r t h i s . - The p l a t e was sprayed with concentrated hydrochloric acid. Bright purple colour s i g n i f i e d the presence of 3-OH flavanone [4]. 12 Also commercially a v a i l a b l e t h i n layer c e l l u l o s e plates were used. The c e l l u l o s e was 0.25 mm thick. Developing solvent for c e l l u l o s e TLC was n-butanol, a c e t i c acid and water, 60:15:25 (BAW^). Detection of compounds on c e l l u l o s e plates was done with p-anisidine hydrochloride spray. A saturated s o l u t i o n of p-anisidine i n ethanol was sprayed on the p l a t e followed by heating i n an oven (110° C) for 15 min. Column chromatography (CC) was the most used means f o r separating extract components. Chromatographic media employed were s i l i c a - g e l , Sephadex A25 and Sephadex LH20O Separation on s i l i c a - g e l column was always preceeded by a s i l i c a - g e l TLC of the material. Petroleum ether (65°-80°) and benzene were the main solvents employed to elute the column. Stepwise increase i n e l u t i n g solvent p o l a r i t y was obtained by a d d i t i o n of varying amounts of e t h y l acetate or ethanol to the main solvents. Fractions were co l l e c t e d i n 25 ml. TLC on s i l i c a - g e l and paper chromatography were employed to a s c e r t a i n separation of compounds i n eluates. In some cases, a 45 cm x 3.7 cm or 35 cm x 2.3 cm column was used to separate the compounds. Subsequently, a smaller column, 28 cm x 1.6 cm, was used f o r p u r i f i c a t i o n , e l u t i n g with a l e s s polar solvent. CC with Sephadex A25; had been very u s e f u l f o r separation of acids (fa t t y and resin) from neutrals [46]. The e l u t i n g solvents are: a mixture of d i e t h y l ether and methanol, 9:1; d i e t h y l ether and methanol, 9:1, saturated with carbon dioxide (using dry i c e ) ; and 4% formic a c i d i n a mixture of d i e t h y l ether and methanol 9:1. The f i r s t e l u t i n g solvent normally removed neutrals from the column, while the l a s t two eluted r e s i n and f a t t y acids. 13 Sephadex LH.2Q was. employed to separate what was suspected to be flavonoid glycosides 138]. The elut i n g solvent was pure ethanol. S i l v e r n i t r a t e impregnated alumina TLC was used f o r the f a t t y and r e s i n acids methyl esters. Glass plates (20 cm x 20 cm) were coated with a s l u r r y of alumina (aluminium oxide G with binder, Research S p e c i a l t i e s Co.) and s i l v e r n i t r a t e s o l u t i o n using a spreader set for 0.25 mm thickness. A 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 (20 ml) was d i l u t e d with methanol (40 ml) and the r e s u l t i n g s o l u t i o n was added to alumina (30 g) and mixed by shaking. The plates were allowed to a i r - d r y overnight, then they were dried at 110° C f o r 30 min before use. The developing solvent was d i e t h y l ether and low b o i l i n g petroleum ether, 1:3. Concentrated sulphuric acid and d i e t h y l ether, 1:4,was used as spray reagent. The pl a t e was heated i n an oven (110° C) for 15 min and then charred (200° C) f o r 1 hr[45]. Reverse phase TLC (RPC) was used to amplify r e s u l t obtained from s i l i c a - g e l TLC of $ - s i t o s t e r o l and neutrals. A c e l l u l o s e p l a t e was washed with 10% Nujol i n petroleum ether. The petroleum ether was allowed to evaporate from the pl a t e surface and specimens were then spotted. The developing s o l u t i o n was methanol saturated with Nujol; while 2,4 DNPH was used for detection. 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 flavonoids and lignans. R^ values, colours under v i s i b l e and u l t r a v i o l e t l i g h t and ease of colour formation with reagents make such compounds r e a d i l y i d e n t i f i a b l e . Descending one-dimensional (ID) and two-dimensional (2D) PC were used i n these in v e s t i g a t i o n s with Whatman No. 1 paper i n a Shandon apparatus. 14 Papers of 45 cm length and 15 cm to 45 cm width, were used i n ID chromatography, while papers of 45 cm square were used f o r 2D chromatography. The solvent systems employed f o r development of papers were: BAŴ ; BAW2, i . e . , 10:3:7; the top layer of a mixture consisting of n-butanol, 0 concentrated ammonia and water, 20:3:10 (BNW); and 2% a c e t i c acid i n water (AW). Detecting reagents were DSA and Bartons reagent for confirmation. Bartons reagent i s a mixture of 0.5% aq f e r r i c c h l o r i d e and 0.5% aq potassium f e r r i c cyanide (100 ml of each made up to 1 L ) . Gas l i q u i d chromatography (GLC)on a 10% EGSS-X column (5 f t x 1/8 in) gave good separations of f a t t y acids and r e s i n acids (Me e s t e r s ) , and also of neutrals obtained i n the petroleum ether extract. About 1.5 u l of material (1 g i n 10 ml) was i n j e c t e d . Two chromatographs were used, an Aerograph 204 and a Hewlett Packard Research Chromatograph 7620A. The l a t t e r was coupled with an integrator to obtain d i r e c t reading on quantities of materials being separated by the column. Preparative GLC was used i n an attempt to q u a n t i t a t i v e l y separate neutrals from the petroleum ether extract. D e t a i l s of the procedure are not included since there was not enough material a v a i l a b l e to use the equipment e f f e c t i v e l y . 3.4 Derivatives Preparation Q_-Methyl esters and 0_-methyl ethers were obtained for acids and phenols, r e s p e c t i v e l y . The compound(s) to be methylated was (were) dissolved i n a minimal amount of solvent and cold diazomethane i n ether was added. Effervescence indicated presence of e a s i l y methylated hydroxyl groups, as i n acids. The mixture i n methanol was l e f t i n the cold room (2° C) overnight to 15 methylate phenolic hydroxyl groups. Excess, diazomethane was removed by blowing nitrogen gas over the mixture. Residual solvent was removed using a vacuum pump. Amine s a l t s were used to separate f a t t y acids from r e s i n acids. Mixture containing both was treated with 10% cyclohexamine i n ethanol. The react i o n mixture was put i n the cold room (2° C) overnight. The r e s i n acid amine s a l t s p r e c i p i t a t e d , while the f a t t y a c i d amine s a l t s remained i n sol u t i o 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 with ethanol and pa r t i t i o n e d between chloroform and 1 N hydrochloric a c i d . The hydrochloric acid layer was discarded. The chloroform was evaporated and the residue was methylated with diazomethane to obtain the r e s i n a c i d methyl esters. The so l u t i o n containing the f a t t y acid 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 between chloroform and 1 N hydrochloric a c i d and the chloroform layer was then methylated as above to obtain the f a t t y acid methyl esters. 3.5 Degradative Techniques Saponification of esters was applied to neutrals i n order to convert them to f a t t y acids and alcohol. The f a t t y acids were i d e n t i f i e d a f t e r methylation by using GLC. The s a p o n i f i c a t i o n reagent was made by di s s o l v i n g sodium metal (1.6 g) i n absolute ethanol (50 ml). Water (5 ml) was added a f t e r d i s s o l u t i o n was complete. This reagent and the material to be saponified were refluxed for 1 1/2 hr. The s o l u t i o n was allowed to cool and excess 1 N hydrochloric a c i d was added to ne u t r a l i s e the mixture. The f a t t y acids were extracted with petroleum ether (65° - 80°), and the solvent evaporated to dryness. The c o l l e c t e d f a t t y acids were methylated as above. 16 3.6 P a r t i t i o n Between Solvents From the r e s u l t s obtained with PC using BAŴ , i n i t i a l separation before s i l i c a g e l CC was performed on some mixtures.. This was effected by p a r t i t i o n between two immiscible solvents, n-butanol and water. M a t e r i a l extracted with n-butanol, was exhaustively extracted with water. Both solutions were evaporated to dryness on the rotary evaporator by constant admixture with absolute ethanol. 3.7 Spectral Techniques Infrared spectroscopy (IR) was performed to obtain the spectra of p u r i f i e d samples, either from potassium bromide p e l l e t s or as smears on sodium chloride plates. A "-'Ref kin-Elmer 521 Infrared Spectrophotometer was used. The spectra of standard compounds were also taken for comparison. Nuclear magnetic resonance (NMR) spectra were obtained on a Varian HA-100, 100 MHz NMR spectrometer. The samples were prepared i n deuterochloroform, deuterated acetone or heavy water depending on compound s o l u b i l i t y . Tetramethylsilane was added as an i n t e r n a l standard and lock s i g n a l at r = 10. Sample d i l u t i o n was approximately 15 mg/300 p i of solvent. 17 4.0 RESULTS The following are the r e s u l t s obtained i n the experimental procedures taken i n th i s i n v e s t i g a t i o n . 4.1 Preliminary Study The veneer sample moisture content was 8.77% and the wood meal weight was 747.2 g (OD b a s i s ) . The petroleum ether (65° - 80°) extract (4.50 g) was 0.60% of the OD sample. Three s i l i c a - g e l TLC were done on t h i s extract. They were developed w i t h . C ^ C ^ , BE and petroleum ether (65° - 80°). No movement occurred i n the pl a t e developed with petroleum ether. The other two plates revealed various compounds under UV. One plate was sprayed with 2,4 DNPH and the other with a 1:1 mixture of concentrated sulphuric acid and concentrated n i t r i c a c i d . The plate sprayed with 2,4 DNPH revealed more spots than the one sprayed with mixed acids. A GLC of the methylated petroleum ether (65° - 80°) extract sample gave f i v e major peaks. Ext r a c t i o n with benzene gave 1.50 g of material (0.20% of OD sample). Two s i l i c a - g e l TLC were developed i n BE and viewed under UV. One plate was sprayed with 2,4 DNPH and the other with DSA. Colour of various spots s i g n i f i e d the presence of carbonyl and phenolic compounds. The benzene-ethanol 1:1 extract (23.12 g) was 3.09% of the OD sample. A pair of 2D, PC was done with BAŴ  and AW. One paper was sprayed with DSA and the other with Bartonss reagent. Spots s i g n i f y i n g 'the presence of carbonyl compounds, yellow and blue r e s p e c t i v e l y with the reagents, were 18 observed. The most conspicuous, spot was, shown to be dihydroquercetin. The ethanol extract (6,85 g and 0.92% of OD sample) revealed more dihydroquercetin on 2D, PC with BAŴ  and AW. The water extract (117.30 g and 15.7% of OD sample) consisted mainly arabinogalactan, while the f i n a l ethanol extract (5.44 g and 0.73% of OD sample) revealed more dihydroquercetin, among other minor compounds, on 2D, PC with BAŴ  and AW. The t o t a l amount of extract was 21.2% of the OD sample. 4.2 Main Study The moisture content of the a i r - d r y wood meal was 11.53%, and the OD weight 819.78 g. Results from each successive extraction are given below. Petroleum ether (65° - 80°) extract was 5.27 g (0.65%) of the OD sample. Two s i l i c a - g e l TLC plates were spotted with the extract and developed i n CR^C^ and BE. Afterwards the plates were viewed under UV. Yellow and purple spots were noted. This s i g n i f i e d the presence of carbonyl compounds and esters. Their presence was confirmed when the plates were sprayed with 2,4 DNPH and warmed on a hot p l a t e . Several yellow and purple spots were observed. Separation of the neutrals from the acids (f a t t y and resin) was effected by CC of 1.75 g of the petroleum ether extract on Sephadex A-2S5 El u t i o n of the column with d i e t h y l ether-methanol, 9:1 gave 1.22 g of neutrals. The acids which came o f f the column (saturation of the solvent with carbon dioxide, and a d d i t i o n of 4% formic acid to the solvent) were 0.41 g. 19 A portion of the f i r s t neutrals f r a c t i o n off the column (to avoid contamination from f a t t y and r e s i n acids which, might be present i n the l a t t e r f r a c t i o n ) was dissolved i n chloroform (1 g to 10 ml) and 1.3 y l of th i s was inject e d into the gas chromatograph operating under the following conditions: i . column, 10% EGSS-X, 1/8 i n x 4 f t ; i i . oven temperature, 175° C isothermal; i i i . c a r r i e r gas, nitrogen; i v . i o n i s a t i o n gas, hydrogen; v. flame i o n i s a t i o n detector temperature, 300° C; v i . i n j e c t i o n port temperature, 300° C; and v i i . chart speed 1 in/5 min. Juvabione i . e . , (+) - methyl ester of (+) - todomatuic acid ( (+) - 4 (R) - [1' (S) - 5' - dimethyl-3'-oxohexyl]-cyclohex-l-ene-l-carboxylic acid) and cis-dihydrojuvabione were in j e c t e d before and a f t e r f o r comparison. One of the peaks gave the same retention time (38.75 min) as that obtained f o r juvabione, but none gave the same as i t s c i s dihydro-derivative, A portion of the same specimen (0.52 g) was placed on a s i l i c a - g e l chromatography column and eluted with petroleum ether (65° - 80°) u n t i l very l i t t l e material came off the column with t h i s solvent. This was followed by si m i l a r e l u t i o n with petroleum ether.,rcontaining 1% increments of e t h y l acetate. E l u t i o n continued u n t i l the solvent was a mixture of petroleum ether and e t h y l acetate 9:1. Afterwards the remaining material on the column was washed down with pure ethyl acetate. TLC of f r a c t i o n s c o l l e c t e d was done on s i l i c a - g e l with juvabione, c i s dihydrojuvabione r and g - s i t o s t e r y l palmitate as standards. The plates were developed i n CILjCl,,, sprayed with 2,4 DNPH and heated on a hot pl a t e . 20 Spots which, gave the same R^ values, and colour reactions with 2,4 DNPH (yellow) as the juvablone and c i s dihydrojuvabione did not give the same colour r e a c t i o n ( f a i n t yellow) on heating. As a confirmation, t h i s f r a c t i o n was put i n the gas chromatograph and standards were injec t e d before and a f t e r f or comparison. Retention value obtained f o r t h i s f r a c t i o n d i f f e r e d from either of the standards. Some compounds i n the 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 palmitate 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 reverse phase TLC of these compounds i n the f r a c t i o n s and standards gave R^ values of 0.35 and 0.56, r e s p e c t i v e l y . Hence 3 - s i t o s t e r y l palmitate and 3 - s i t o s t e r o l were both present. A f r a c t i o n of the neutrals from the s i l i c a - g e l CC was found to have a trace component associated with i t as seen by TLC. This f r a c t i o n was put along the length of a thick s i l i c a - g e l plate which was developed i n CH^Cl^. The required portion was scraped o f f , a f t e r d elineating i t s contours with UV l i g h t . The scrapings were extracted with chloroform. The chloroform was f i l t e r e d and the f i l t r a t e was evaporated to dryness. The NMR was taken i n deuterochloroform while the IR was conducted as a smear on sodium chloride plates. The spectra showed that the trace component was t r i s t e a r i n . This was proved by comparison of the spectra with that of the standard. In order to i d e n t i f y f a t t y acids attached to alcohol forming f a t s i n the neutrals, a portion (0.17 g) of the neutrals was hydrolysed with a prepared s a p o n i f i c a t i o n reagent (20 ml) and refluxed as previously described. The r e s u l t i n g f a t t y acids were methylated, and 1.5 ulvof the standard s o l u t i o n was injec t e d into the gas chromatograph. Standards containing 21 ^10' C 1 2 ' ^14 a n C ^ ^15 ~ ^19 f a t t Y a c ^ methyl esters were also i n j e c t e d before and a f t e r the unknown. 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 , s t e a r i c and two others beyond C^g, probably either l i n o l e i c , p i n o l i c , or arachidi c . Fatty acids and r e s i n acids were separated as amine s a l t s a f t e r recovery from the Sephadex . A25 column. To t a l y i e l d of f a t t y acids (OD sample basis) was 0.85 g, i . e . , 0.10%. Resin acids y i e l d was 0.14g, 0.017%. TLC of f a t t y and r e s i n acids (Me esters) was performed on s i l v e r n i t r a t e impregnated alumina pl a t e s . A f t e r developing and spraying the chromatogram, i t was l e f t i n an oven (110° C) f o r 15 min and then charred f o r 1 hr [45]. The f a t t y acids (Me esters) R^ values were 0.06, 0.31, 0.66 and 0.72. L i t e r a t u r e values revealed the f i r s t one was l i n o l e i c acid while the l a s t was p a l m i t i c acid (Me esters) [45]. R^ values of the r e s i n acids (Me esters) obtained were 0.13, 0.22, 0.50 and 0.63. These on comparison with standards were shown to be isopimaric, sandaraeopimaric,. a b i e t i c and dehydroabietic acids (Me e s t e r s ) , r e s p e c t i v e l y . The f a t t y acid methyl esters were dissolved i n chloroform (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 injec t e d into the gas chromatograph (Aerograph 204 with oven temperature set at 155° C). A l l other conditions were as given previously. Standard f a t t y acids methyl esters which contain ^10' ^12' ^14 a n c ^ ^15 ~ ^19 a c ^ s w e r e i n j e c t e d before 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 acids l i s t e d i n Table 1 were i d e n t i f i e d . 22 Compound Retention time, min. Identity I I I 5.31 Pal m i t i c IV 6.86 Pa l m i t o l e i c V 13.28 L i n o l e i c VI 17.50 Possibly l i n o l e n i c VII 19.53 Possibly a r a c h i d i c TABLE 1. Retention times of f a t t y a c i d methyl esters. The r e s i n acid methyl esters s o l u t i o n was injected into the gas chromatograph. Conditions were as described f o r the neutrals above. In addition, an integrator was connected so as to give a printout 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 . Standard r e s i n a c i d methyl esters mixture was injec t e d as before. These data are given i n Table 2. Compound VIII IX X XI Retention time, min. 13.75 18.75 25.47 31.72 Identity Sandaracopimaric Isopimaric A b i e t i c Dehydroabietic Ratio based on minimum 1 18.29 4.00 3.21 TABLE 2. Retention times and y i e l d r a t i o of r e s i n a c i d methyl esters. 23 Benzene extracts were recovered as. 1.22 g (0.15% OD sample b a s i s ) . Two s i l i c a - g e l TLC, developed i n CH^C^ and BE were done on the sample dissolved i n ethanol. One plate was. sprayed with DSA and the other with 2,4 DNPH. Colour of the spots suggested the presence of carbonyl compounds and phenolics. Methylation with diazomethane did not produce nitrogen effervescence and the mixture was l e f t overnight i n the cold. A s i l i c a - g e l TLC of the unmethylated sample spotted side by side with the methylated sample was developed i n BE. Spots were located by exposure to an iodine atmosphere. Various spots not present i n the unmethylated sample were seen i n the methylated sample. This confirmed the presence of phenols. CC separation of the material (0.51 g) was done on s i l i c a - g e l following s o l u t i o n i n the minimum amount of benzene and ethanol mixture. The column was f i r s t eluted with 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 very l i t t l e m a t e rial came off by further e l u t i o n with t h i s solvent. P o l a r i t y of the e l u t i n g solvent was then increased by additions of et h y l acetate i n 2% increments, and e l u t i o n was continued as before. The et h y l acetate content of the e l u t i n g solvent was increased by 5% increments a f t e r e l u t i o n with 10% e t h y l acetate i n benzene. Aft e r e l u t i o n with 40% e t h y l acetate i n benzene, the material s t i l l remaining on the column was washed down with pure et h y l acetate followed by pure ethanol. 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 duplicate and developed i n BE. A f t e r viewing under UV l i g h t , one p l a t e was sprayed with 2,4 DNPH and the other with DSA. Similar f r a c t i o n s were combined. The following compounds are reported: 24 Compound l a was obtained when the column was eluted with 10% ethy l acetate i n benzene. I t formed about 2% of the benzene extract (0.003% of the OD sample). I t c r y s t a l l i s e d out of ethanol s o l u t i o n a f t e r being l e f t i n the cold f o r some days. i n BE was 0.62, with purple colouration under UV l i g h t . I t was not s e n s i t i v e to 2,4 DNPH even on warming the p l a t e , but gave a yellow colour with DSA and no reac t i o n with Zn/HCl. Its IR spectrum (Fig. 5) was taken i n a KBr p e l l e t and s i s described as follows: 3100 cm"1, shoulder; 1650, 1570, 1490, 1290, very strong; 1250 weak; 1150 very strong; 1070, strong; 1050 and 850, weak. These properties were compared with those of compounds l i k e l y to have the 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 Ic was c o l l e c t e d on el u t i n g the column with benzene and ethy l acetate mixture, 7:3. The compound with i t s associated contaminants was 3.6% of the benzene extract (0.005% of the OD sample). Repeated s i l i c a - g e l CC did not remove a l l the contaminants, however t h i s did not prevent i d e n t i f i c a t i o n . A purple fluorescence 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 reddish-yellow colour developed with 2,4 DNPH, while a yellow colour occurred with DSA. Reaction with Zn/HCl gave a purple colour. Comparison with standards revealed the compound to be dihydrokampferol ( I c ) . Compound XII formed about 1% of the benzene extract (0.002 of the OD sample). It came off the column with benzene-ethyl acetate (85:15). A f a i n t pink fluorescence was observed under UV when spotted ( s i l i c a - g e l TLC), and orange colour with 2,4 DNPH and pink colour with DSA. It d i d not give a purple colour with Zn/HCl. The R i n BE was 0.36 and 0.87 i n BAW . ^ C O O H VIII X : O O H IX 26 Comparison with, standard compounds, showed that i t was, a-conidendrin (XII). Some other compounds were obtained from the s i l i c a - g e l CC of the benzene extract. Low y i e l d s and a s s o c i a t i o n with trace contaminants which were not removed by repeated chromatography prevented p o s i t i v e i d e n t i f i c a t i o n . The following compounds amongst them were obtained i n the largest y i e l d s : Compound XIII formed about 2% of the benzene extract (0.003% of the OD sample) and was eluted with benzene and eth y l acetate mixture, 3:1. Its i n BE was 0.35 and a pink fluorescence occurred under UV. The compound was unsensitive to 2,4 DNPH, but on warming the pl a t e , a f t e r spraying with this reagent, an orange colour could be noticed. A yellow colour developed with DSA and delayed purple colour with Zn/HCl. This suggested a 3-OH flavonoid structure. Its was s l i g h t l y lower than that of pinobanksin (0.36) and did not give the d u l l yellow fluorescence of pinobanksin under UV. Overnight methylation of t h i s compound i n the cold (2° C) gave two other compounds of R^ 0.39 and 0.52 ( s i l i c a - g e l TLC, developed i n BE). Both compounds gave purple colour under UV, orange and pink colours, r e s p e c t i v e l y , with DSA. Methylation of pinobanksin gave only one compound of R^, 0.45, yellow under UV and orange colour with DSA. Compound XIV with associated minor compounds formed about 10% of the whole benzene extract (0.015% of OD sample). I t came o f f with other compounds when the column was f i n a l l y eluted with ethanol. This eluate on evaporation of the ethanol was dissolved i n a minimum amount of ethanol and put on a s i l i c a - g e l column chromatograph. E l u t i o n of the column with benzene- ethanol 95:5 yielded t h i s unknown with associated minor compounds. I t s R̂ ( s i l i c a - g e l TLC, developed i n BE) was 0.50. The compound was unsensitive to 27 2,4 DNPH but when the plate was warmed, a green colour resulted, a pink colour was obtained with DSA. Repeated s i l i c a gel CC d i d not remove the carbonyl compound associated with i t . Its NMR and IR spectra were taken i n deuterochloroformanti potassium bromide p e l l e t , r e s p e c t i v e l y . The IR spectrum i s described as follows: 3400 cm shoulder; 2900 very strong; 1600 weak; 1500 very strong; 1250 very strong; 1000 and 950, weak. Benzene-ethanol (1:1) extracted 10.58 g (1.30% of the OD sample). No immediate reaction was noticed on methylation with diazomethane and the mixture was l e f t overnight i n the cold. A s i l i c a g el TLC of the extract and the methylated sample was developed i n BE and exposed to iodine vapour. Spots not present i n the unmethylated sample were observed i n the methylated sample. o A 2D, PC with BAW1 and AW, sprayed with DSA a f t e r viewing under UV showed three major compounds. Their PC properties are given i n Table 3. Compound R.̂  Colour Identity BAW1 AW UV DSA 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 TABLE 3. PC properties of compounds Ic, Id and I I . b.y = b r i g h t yellow; l . y = l i g h t yellow; y = yellow. 2 8 Silicas-gel CC of a portion of the extract was. f i r s t eluted with benzene and subsequently with a mixture of benzene and ethanol i n 5% increments of the l a t t e r . E l u t i o n was followed by s i l i c a - g e l TLC, developed i n BE, viewed under UV and sprayed with DSA. The f i r s t f r a c t i o n c o l l e c t e d from the column contained XIV, described i n the benzene extract. Dihydrokampferol ( I c ) , dihydroquercetin (Id) and quercetin (II) were also i d e n t i f i e d from the column elutions by comparison with standards (R^ i n BAŴ  and BE). The R̂  values of these compounds i n BE were 0.29, 0.16 and 0.06, r e s p e c t i v e l y . Another s i l i c a - g e l TLC p l a t e was sprayed with Zn/HCl. Only dihydrokampferol and dihydroquercetin gave purple colours. Dihydroquercetin (eluted later and i d e n t i f i e d on PC) gave a negative r e s u l t with Zn/HCl. It seems to have been masked by a compound which also gave a yellow colour with DSA. Ethanol extract was c o l l e c t e d i n an amount of 4.59 g (0.56% of the OD sample). A 2D, PC developed with BAŴ  and AW revealed the presence of dihydroquercetin and traces of quercetin on spraying with DSA. Two other spots were also observed. Dissolving the extract i n ethanol p r e c i p i t a t e d what was shown to be dihydroquercetin. I t was f i l t e r e d o f f , the f i l t r a t e was concentrated and put on a s i l i c a - g e l chromatography column. E l u t i o n was f i r s t with pure benzene and then with 5% increments of ethanol added. Dihydrokampferol, dihydroquercetin and traces of quercetin were obtained. The two spots reported above were also obtained. They were not i d e n t i f i e d but t h e i r data were as given below. 29 Compound XIII had an R f of 0.51 i n BAŴ  and zero i n AW. I t gave a yellow colour with DSA and a l i g h t purple fluorescence under UV l i g h t . Its other properties were the same as described for the u n i d e n t i f i e d compound XIII of the benzene extract. I t i s l i k e l y that they are i d e n t i c a l . Compound XV had an R f of 0.82 i n BAW1 and 0.69 i n AW. Under UV a l i g h t purple colour was observed as was with DSA, suggesting a lignan. In BE, i t had an R f of 0.45, higher than that of a - and g - conidendrin, used as comparison, but s i m i l a r to them under UV and with DSA. The ethanol extract also contained low R^ resinous materials which stayed on the column u n t i l washed with pure ethanol. This material formed a brown glassy s o l i d on evaporation of the solvent. I t was about 60% of the ethanol extract. Water extracted materials were 97.55 g, i . e . , 11.90% of the OD sample. The mixture consisted mostly of crude arabinogalactan i n 11.10% of the OD wood. This polysaccharide i s common to Lavi-x spp. The extract was concentrated to remove as much water as p o s s i b l e . A p o r t i o n of the concentrate was d i l u t e d 10 times with ethanol, f i l t e r e d and the f i l t r a t e evaporated to dryness. The residue was redissolved i n ethanol and the i n s o l u b l e p o rtion, which was arabinogalactan was discarded. The f i l t r a t e was concentrated and examined by PC. I t was developed i n 2D with BAW^ and BNW. R^ values of the spots are given i n Table 4. These were poss i b l y lignans. 30 Compound R Colour with DSA BAW1 BNW XVI 0.86 0.06 Pink XVII 0.64 0.29 Light pink XVIII 0.83 0.55 Pink XIX 1.00 0.91 Yellowish pink TABLE 4. PC properties of ethanol solubles i n water extracts Flavonoid glycosides were sought i n the ethanol soluble portion of the water extract using the method of Bir k o f e r and Kaiser [6] . A p a i r of,ID PC i n BAŴ  were made. One was sprayed with concentrated ammonia and the other with DSA. The ammonia sprayed paper gave three spots of R^ 0.90, 0.82 and 0.69; under UV t h e i r colours were yellowish blue, blue and l i g h t yellow, r e s p e c t i v e l y . A fourth spot of R f 0.42 and l i g h t blue under UV appeared when the paper was l e f t i n the oven (100°C) f o r 5: min. None of these colours corresponded with those expected from glycosides of the flavonoids found. On spraying the other chromatogram with DSA, the same four spots appeared. The f i r s t three gave b r i g h t orange to orange colours, while the fourth gave a l i g h t red colour. Possibly they were lignans. To confirm the absence of flavonoid glycosides i n the ethanol soluble p o r t i o n of the water extract, extract specimen was placed on a Sephadex LH20 column and eluted with ethanol. This 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 of flavonoid glycosides [38]. Fractions eluted were followed by PC with BAŴ  and the papers were sprayed with e i t h e r DSA or ammonia. Most of the compounds were recovered i n the f i r s t 25 ml 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 c r y s t a l l i s e d out of aqueous ethanol as white needles. IR and NMR spectra showed i t to be 31 a carbohydrate, w i t h m.p. 159° C. C e l l u l o s e TLC with galactose and L- arabinose as standards i n BAŴ  and spraying with p^-anisidine hydrochloride reagent followed by warming i n the oven (12Q° C) for 5 min gave a purple- brown colour for t h i s compound (XX). This colour, R^, IR ( i n KBr p e l l e t s ) , NMR ( i n D2O) and m.p. data coincided with that of L-arabinose. Its IR spectrum i s described as follows: 3250 cm \ shoulder; 1315, weak; 1255, 1230, 1130, strong; 1095, 1055, very strong; 1005, strong; 945, weak; 895, 845 and 785, strong. Because of divergent R^ values exhibited i n BAV^ by the non-arabinogalactan f r a c t i o n of the water extract, these were p a r t i t i o n e d between water and n-butanol as described. A preliminary 2D, PC with BAŴ  and BNW revealed several compounds when sprayed with DSA. Each extract was put on a s i l i c a - g e l CC and eluted with benzene and ethanol mixture. PC of the eluates was done i n duplicate. One paper was developed i n BAŴ  and the other i n BNW. The chromatograms were sprayed with DSA a f t e r viewing under UV. Data f o r the n-butanol solubles obtained are given i n Table 5. Compound XXI XXII XXIII XXIV XXV XXVI XXVII R, BAWĵ 0.93 0.87 0.84 0.88 0.75 0.73 0.81 BNW 0.93 0.41 0.04 0.84 0.16 0.05 0.71 Colour with DSA Reddish-brown Purple Orange Light red Light yellow Light yellow Redd ish-orange TABLE 5. PC properties of n-butanol solubles i n water extracts. Compound XXII was compared with f e r u l i e acid (PC behaviour with BAW1 and BNW, sprayed w i t h DSA) . F e r u l i e acid gave R f of 0.86 with. BAW^ 0.08 with BNW and purple colour with DSA. The water solubles had the PC properties shown i n Table 6. Compound R Colour with DSA BAŴ ^ BNW XXVIII 0.89 0.88 Light 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 Bright orange TABLE 6. PC properties of water solubles i n water extracts. F i n a l ethanol e x t r a c t i o n yielded 3.18 g of material (0.39% of the OD sample). A 2D, PC was done with BAŴ  and AW, and sprayed with DSA. Data were obtained as given i n Table 7. Compound R Colour with DSA BAŴ  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 properties of f i n a l ethanol extracts. 33 S o l u b i l i t y tests were performed on portions of the specimen. No solvent completely dissolved the specimen. Acetone p r e c i p i t a t e d a white compound which was soluble i n water and hot methanol. I t c r y s t a l l i s e d out of aqueous ethanol and was i d e n t i f i e d as L-arabinose from IR spectra, m.p. and c e l l u l o s e TLC developed i n BAŴ  and sprayed with p-anisidine hydrochloride. The remaining acetone solubles were chromatographed over s i l i c a gel (CC) with benzene-ethanol as the e l u t i n g solvent. A compound which c r y s t a l l i s e d out as brown flakes was obtained. IR and NMR spectra of the impure compound showed that i t was a lignan. The R^ i n BAŴ  and 'AW on PC corresponded with that of compound XXXVII. 34 5.0 DISCUSSION Various observations were made i n the experimental 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 chemical c h a r a c t e r i s t i c s of the compounds (especially those unreported previously) found i n the wood are here discussed. 5.1 Experimental Procedures Successive extraction of the wood meals with solvent of increasing p o l a r i t y was most h e l p f u l f o r preliminary i s o l a t i o n of compounds. Compounds from s p e c i f i c chemical classes were obtained i n most of the d i f f e r e n t extracts. For instance, f a t t y and r e s i n acids were obtained only i n the petroleum ether extract; low hydroxylated flavonoids appeared only i n the benzene extract; higher hydroxylated flavonoids were found i n the ethanol extract; and arabinogalactan occured only i n the water extract. This a s s i s t e d i s o l a t i o n of compounds which were present only i n trace amounts. CC procedures gave good seperations, with appropriate choice of e l u t i n g solvents. Most minor compounds of low values stayed at the top of the column, e s p e c i a l l y when e l u t i n g with solvents of low p o l a r i t y . Washing the column f i n a l l y with a strongly polar solvent removed them. Using a rubber hand pump to activa t e CC e l u t i o n , a current procedure at the Western Forest Products Laboratory, increased the flow rate considerably. This solved a problem (low flow rate) often encountered with CC technique. Although PC with BAŴ  and detection with DSA were mostly used f o r the i d e n t i f i c a t i o n of flavonoids and lignans, the use of s i l i c a - g e l TLC with BE as the developing solvent and detection with DSA was superior. Most 35 compounds: t r i e d on the PC system ran close to the solvent f r o n t , r e s u l t i n g i n s i m i l a r values. Accurate measurement of R^ values: was further hampered by the large, uneven spots: r e s u l t i n g from development. Colour developed with detecting reagents was dependent on the concentration and p u r i t y of the compound spotted. Associated trace contaminants (especially low R^ value compounds) did not stay at 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 thick paper. In contrast, these minor components did not move with the compound i n s i l i c a - g e l TLC. The problems mentioned above were not observed with s i l i c a - g e l TLC and development with BE. In addition, a l l TLC data were reproducible with only s l i g h t v a r i a t i o n s i n R^ values for repeat TLC t r i a l s . The small differences observed might have been due to temperature v a r i a t i o n or inaccurate mixing of the developing solvents. The average time taken to develop a 20 cm x 20 cm TLC pl a t e (BE) was 45 min, while PC took about 15 hr. Hence, TLC was found most convenient f o r following elutions from columns. 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 also more reproducible than PC (both developed i n BAW^). Bartons reagent was not used extensively i n t h i s i n v e s t i g a t i o n , because i n e a r l i e r experiments i t was found that the blue colour on the paper greatly masked the p o s i t i o n of minor components. This was unacceptable, since i n some case only trace amounts were being investigated. Although the presence of f a t t y and r e s i n acids was confirmed by comparison with standards using GLC, further confirmation using s i l v e r n i t r a t e impregnated TLC did not proceed as an t i c i p a t e d [45], Spots appeared even before the completion of the recommended 1 hr charring period. 36 5.2 Extracts. Petroleum ether (.65° - 80°) extracted f a t t y and r e s i n acids, they were e a s i l y i s o l a t e d by CC w i t h Sephadex A—25. Most probably, separation of r e s i n acids from the r e s t of the petroleum ether extract (2.68% of the petroleum ether e x t r a c t s ) , was due to the use of t h i s absorption medium. Resin acids had not been found previously i n t h i s wood. Of a l l the Larix spp., f r e e r e s i n acids have been reported only i n L. si.bi.ri.oa [2]. Free f a t t y acids,alikewise, had not been reported i n most of Larix spp. The GLC traces of free f a t t y acids (Me esters) and free r e s i n acids (Me esters) found are shown i n Figs. 3 and 4, r e s p e c t i v e l y . The f a t t y acids were pal m i t i c ( I I I ) , p a l m i t o l e i c (IV), l i n o l e i c (V) and two others, possibly l i n o l e n i c (VI) and arac h i d i c (VII). The r e s i n acids were sandaracopimaric (VIII), isopimaric (IX), a b i e t i c (X) and dehydroabietic (XI). Retention times on GLC are given i n Tables 1 and 2. Benzene extracts consisted of many carbonyl and phenolic compounds, as seen from TLC ( s i l i c a - g e l , BE and DSA, 2,4 DNPH). The small amount of material extracted by t h i s solvent prevented complete separation of the compounds. 37 VI t i : i _ i 1 - i 0 5 10 15 20 25 TIME (mins.) Figure 3. GLC trace of f a t t y acid methyl esters. IX I 1 I 1 -1 : 1 0 5 10 15 20 25 TIME (mins) Figure 4. GLC trace of r e s i n acid methyl esters. 39 Pinocembrin (la) previously unreported i n t h i s species, was obtained i n 0.003% y i e l d . I t s IR spectrum i s shown i n F i g . 5. The absorption band around 3500. cm shows the presence of -OH. groups while the bands at 1500 to 165Q cm are aromatic s i g n a l s . Absence of an -OCH^ group i n the molecule i s proved by lack of a band at 1400 cm \ The complete IR spectrum of t h i s compound coincided with that of the standard compound, e s p e c i a l l y the bands at 1,000 to 800 cm ( f i n g e r p r i n t region). The R^ i n BE obtained f o r the flavanones i n t h i s wood decreased with degree of hydroxylation on the flavanone r i n g . Dihydroquercetin with f i v e hydroxyl groups had an R^ of 0.16, dihydrokampferol with four hydroxyl groups had an R^ of 0.29 and pinocembrin with two hydroxyl groups had an R^ of 0.61. The R^ of pinobanksin (with three hydroxyl groups) used as standard was 0.36. This value was close to the R^ (0.35) of the u n i d e n t i f i e d compound XIII, found i n the benzene extract and described above. It also resembled the above flavanones by giving a purple colour with Zn/HCl, i n d i c a t i v e of a 3-0H group. I t d i f f e r e d from pinobanksin i n the type of i t s fluorescence under UV (a purple colour while pinobanksin was d u l l yellow). Further, i t s methylation products d i f f e r e d from those of pinobanksin (both reported above). I t i s suggested that XIII i s a trihydroxylated, 3-0H flavanone. In order to d i f f e r from pinobanksin i t could have one of the two hydroxyl groups i n the A r i n g of pinobanksin transferred to the B r i n g , i . e . , an isomer of pinobanksin. The NMR and IR spectra of u n i d e n t i f i e d compound XIV are 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 signals at 3400 cm ^ and 1400 cm ^ are t y p i c a l of -OH and aromatic -OCH^ groups r e s p e c t i v e l y . According to Ludwig et al. 130], the s i g n a l at x 6.19 comes from the 3-OMe group i n this type of compound. Integrated value f o r th i s peak indicated s i x protons, i . e . , two WAVE NUMBER Figure 5. IR spectrum of pinocembrin ( l a ) . o 41 -OCH^ groups.. The s p l i t t i n g of this, peak, indicated that the two -OCH^ were not within the same environment. M u l t i p l e t s around 3.15 T are from the 2,6 and 1,5 protons ( l i t e r a t u r e values, 3.14 and 3.18 T ) r e s p e c t i v e l y . L i t e r a t u r e J30] value f o r the 4-OH i s 4.15 T . This s i g n a l can be i n d i s t i n c t l y observed i n F i g . 6. The absence of a s i n g l e t between 5.95 and 6.50 indicates no a l i p h a t i c -OH as i n l a r i c i r e s i n o l (XL) and s e c o l a r i c i r e s i n o l (XLI). I t i s suggested that XIV i s re l a t e d to p i n o r e s i n o l (XLII). X L I XLII  WAVE NUMBER Figure 7. IR spectrum of un i d e n t i f i e d Compound XIV. 44 Water extracts contained free L-arabinose, but no D-galactose. This raises the p o s s i b i l i t y of the former coming from incomplete hydrolysis of arabinogalactan. On the other hand, extraction conditions were not s u f f i c i e n t to cause hydr o l y s i s . Tests, described above, also rule out the presence of flavonoid glycosides i n t h i s extract. The glycosides that have been associated with flavonoids found i n t h i s i n v e s t i g a t i o n , were glucosides and sophorosides [6,22]. Of course, the sugar could also 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 that L-arabinose i s the sugar moiety of an e a s i l y hydrolysed flavonoid glycoside i n the wood. Dihydroquercetin-3 1 - glucoside i s the only glycoside that has been reported i n the sapwood of western l a r c h [6]. The NMR and IR spectra of L-arabinose are shown i n F i g . 8 and 9, r e s p e c t i v e l y . The strong band at 3300 cm 1 i s t y p i c a l of sugars and i s due to the oxygen-hydrogen s t r e t c h i n g frequency of hydrogen bonded hydroxy groups [26]. The IR spectrum agreed with the d e s c r i p t i o n of the L-arabinose IR spectrum by Urbanski et al. [42]. F i n a l ethanol extract p o s s i b l y contained Braun's native l i g n i n (BNL) among the compounds reported. This i s brought about b y the water ex t r a c t i o n having broken down the c e l l membrane to enable BNL to be extracted on subsequent ethanol extraction. 1 I I • ' • i i i • ] r • i • i i i i t i i i t i , r i i i i • i «. i - i r ' i i 1 . . , , • ' • 1 ' 1 • • i — . . . . ! . . . . . . . . . . i . . 2 3 A - 5  r  6 7 3 9 Figure 8. NMR spectrum of L-arabinose (XX).  47 6.0 CONCLUSIONS The t o t a l amount of extract obtained from the heartwood of western l a r c h {Lavtx ooovdental'is- Nutt) by successive extraction with f i v e solvents of increasing p o l a r i t y and f i n a l l y with ethanol was 14.94% (OD wood sample b a s i s ) . Petroleum ether (65° - 80°) extracted 0.64%. The compounds found on analysis of t h i s extract were 3 - s i t o s t e r o l , 3 - s i t o s t e r y l palmitate, t r i s t e a r i n and esters (fats) of two acids, probably l i n o l e n i c and ar a c h i d i c acids. Free f a t t y acids found were p a l m i t i c , palmioleic, l i n o l e i c and two acids, probably l i n o l e n i c and a r a c h i d i c . Free r e s i n acids found were sandaracopimaric, isopimaric, a b i e t i c and dehydroabietic acids. The benzene extract was 0.15% of the wood. From t h i s extract, pinocembrin, dihydrokampferol, a conidendrin and two u n i d e n t i f i e d alcohols were obtained. The benzene-ethanol (1:1) extract was 1.29% (OD wood sample basis) and t h i s yielded dihydrokampferol, dihydroquercetin and quercetin. Ethanol extracted 0.56% of the wood. This extract consisted mainly of dihydroquercetin and a glassy resinous material. Traces of dihydrokampferol and quercetin were also found. Two u n i d e n t i f i e d compounds, a flavonoid (also obtained i n the benzene extract) and a lignan (from i t s colour r e a c t i o n with DSA) also occurred i n t h i s extract. The water extract accounted f or 11.90% the wood and consisted mainly of crude arabinogalactan. I t also contained L-arabinose and 48 u n i d e n t i f i e d lignans. The f i n a l ethanol extract was. 0.39% of the wood. I t consisted mainly of guaiacyl type compounds such as lignans and p o s s i b l y Braun's native l i g n i n . 49 7.0 LITERATURE CITED Antonovskii, S.D., M.M. Chochieva, and Z.A. Zhdaneeva. 1963. Technical properties of Larix gmelini japonica. Lesa Kamchatki i ikh Lesokhoz. Znachenie. 63:367-370 (not seen, Chem. Abstr. 63:3156). Bardyshev, I.I. and L.I. Ukhova. 1959. Resin acids from the r e s i n of Siberian l a r c h . Sbornik Nauch. Robot, Akad. Nauk Belorus. S.S.R., Inst. Fiz-Org. Khim 7:89-95. (not seen, Chem. Abstr. 54:25803h). Barton, G.M. 1962. Plant phenolics and t h e i r i n d u s t r i a l s i g n i f i c a n c e . V.C. Runeckles, E d i t o r . Proc. Symposium Plant Phenolics Group of North America, Oregon State U n i v e r s i t y , C o r v a l l i s . pp. 56-80. . 1968. Detection of 3-hydroxyflavanones on papergrams and t h i n - l a y e r p l a t e s . J . Chrom. 34:562. 5. and J.A.F. Gardner. 1958. Determination of dihydroquercetin i n Douglas-fir and western l a r c h wood. Anal. Chem. 30:279-281. 6. B i r k o f e r , V.L. and C. Kaiser. 1962. Neue Flavonglykoside aus Petunia hybrida. Z. Naturforschg. 17:359-368. 7. Brewerton, H.V. 1956. Extractives of Larix deoidua and Larix leptolepsis. New Zealand J . S c i . Technol. 37(B):626-632. 8. Buchanan, M.A. 1963. Extraneous components of wood. _In The Chemistry of Wood. B.L. Browning, Ed. Interscience Publishers, New York, pp. 313-367. 9. Dadswell, H.E. and W.E. H i l l i s . 1962. Wood. In Wood Extractives and t h e i r S i g n i f i c a n c e to the Pulp and Paper Industries. W.E. H i l l i s , Ed. Academic Press, London, pp. 3-51. 10. Ekman, K.H. and C. Douglas. 1962. Some physiochemical properties of arabinogalactan from western l a r c h (Larix oaaidentalis). Tappi 45: 477-481. 11. Gardner, J.A.F. 1962. The tropolones. I_n Wood Extractives and t h e i r S i g n i f i c a n c e to the Pulp and Paper Industries. W.E. H i l l i s , Ed. Academic Press, London, pp. 317-330. 12. . 1965. Extractive chemistry of wood and i t s influence on f i n i s h i n g . J . Paint Tech. Eng. 37:698-706. 13. and G.M. Barton. 1960. The d i s t r i b u t i o n of dihydroquercetin i n Douglas-fir and western l a r c h . Forest Prod. J . 10:171-173. 50 14. 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