The neutral fraction of the petroleum-ether-soluble bark extract contains a homologous series of normal alkanes (C19 to C42), free β-sitosterol, behenyl and lignoceryl alcohols, esters of these three compounds with arachidic, behenic, and lignoceric acids, and a major fraction comprising a mixture of behenyl and lignoceryl ferulates. The triterpene lactones cyclograndisolide and epicyclograndisolide are minor components

Grierson, D.

Rogers, I. H.

English

Wood and Fiber Science (E-Journal)

EXTRACTIVES FROM GRAND FIR [ABIES GRANDIS (DOUGL. ) LINDL.] BARK1 I .  H .  Rogers 1)epartment of the Environment, Canadian Forestry Service, Western Forest Products I,aboratory, Vancouver, British Columbia and D. Grierson Chemistry Department. University of British Columbia, Vancouver, British Co1uinbi;i ( Received 23 December 1971 ) ABSTRACT The neutral fraction of the petroleum-ether-soluble bark extract contains a homologous series of normal alkanes (C,, to C4.), free p-sitosterol, behenyl and lignomryl alcohols, esters of these three compounds with arachidic, behenic, and lignoceric acids, and a major fraction con~prising a mixture of behenyl and lignoceryl fenilates. The triterpene lactones cyclograndisolide and epicyclograndisolide are minor conlponents. INTRODUCTION As part of a continuing program of back- ground research at the Western Forest Prod- ucts Laboratory into the composition of the extractives of western Canadian wood species, we have examined the bark of a true fir, grand fir [Ahies grandis (Dougl.) Lindl. 1. Such studies may eventually assist in commercial exploitation of bark residues by virtue of the discovery of extractives with pharmacological application or of mix- tures of extractives with sales potential for waxes, adhesives, etc. Compounds present as bark extractives probablv play a signifi- cant role in the selection of suitable hosts by attacking bark beetles. These substances may help a tree to withstand severe climatic conditions, and their composition is also of interest to chemotaxonomi~ts.~ Previous workers have isolated and struc- turally identified the triterpvne abieslactone [I] present in the barks of amabilis fir [Ahies amabilis (Dougl.) Forb.] and of 'D. Grierson acknowledges with gratitude the financial support of a National Research Council of Canada qummer studentship during the period of this research. 'The referees have pointed out, and the authors A. mariesii Masters native to the mountains of northern Japan (Uyeo et al. 1968). I t  recently has been shown that structure I is incorrect (Kutney et al. 1971) in turns of the location of the double bond. As there are nine species of true firs 11 a t ' ivc to Canada and the United States, and these are divisible into five taxa for botariical reasons (Smedman et al. 1969), it was of chemosystematic interest to examinc, the triterpene fraction of A. grandis bark, wlllich is not in the same botanical subgrou], as A. amabilis. The triterpene fractioii of the petroleum-ether-sol~ible bark extract from A. grandis was donated to a groul) ~ I I  the chemistry department of the Univt rsitv of British Columbia. They have leported (Allen et al. 1971) on the presencc of two new triterpenes, cyclograndisolide [ I1  a] and epicyclograndisolide [I1 b]. The pres- ent paper describes the chemical corn~bosi- tion of the remainder of the petrolcum- ether soluble neutrals fraction of A. grtlradis bark, after removal of the triterpeile frac- tion. Preliminary work on this extract has been described ( Rogers and Grierson 1969). RESULTS agree, that the results here reported are of limited H ycErocarbons value to chemotaxonomists because the bark from only one tree was exalnined and tree-to-tree var- Normal and branched-chain alkanes were iability was therefore not evaluated. separated by the urea-inclusion-conlplex I. 11. ROGERS AND D. GRIEHSON technicluc using "activc" ilrea (Rosenstein and Gorin 1957). Members of the homol- ogous series of normal paraffins from Cl9 to C42 inclusive were dctected with the weighted center of the distribution at C31. Gas liquid chromatography ( g.1.c.) of the l~rancl~cd-chain fraction showed four poorly defined unidentified peak\. nccrystallization of the crude was esters from acetone yielded a white wax. Metha- iiolysis followed by acetylation of the un- saponifiable portion and analysiv showed the presence of behenyl and lignoceryl alcohols in the ratio 1:2. The wax-acid 1nc.thyl (,stel- fraction was eomposed of arachidic (3%) ,  behenic, (37% ) and lig- iioceric (60%) acids. Esters of unsaturated wax acids were also undoubtedly present as oils, but were removed in the process of recrystallization. Sterol esters Wc were unable to separatc fully the wax-cster fraction from the steroid esters by eithcr chromatography or fractional re- crystallization. Saponificntion and addi- tional purification yielded P-sitosterol ac- companied by the ubiquitous campesterol. No other sterols wvre detcctecl. Once again only normal alkanoic acids wcrc found, the conlposition of this fraction being palniitie ( 12%), stcaric (2.59 ), arachidic (6 . l%) ,  l~chcnic (44% ) , and lignoecric ( 34% ) . Un- cloubtcdly a portion of thc. latter two acids was contributed by the wax cxster contami- nant. Wax alcohols The frec wax alcohols were idei~tified by g.1.c. analysis of their acetate del-ivatives. The components present were bc.henyl ( 15%), lignoceryl ( 80%), and ceryl alcohols (5%) .  Free sterols Only B-sitosterol was isolated. It> itlcntity was confirmed by m.p. and mixed m.p., quantitative i.r. (infrared spectroscopy) spectral cornparisoil with an authe~itic- stan- dard, and from the 1l.m.r. (nuclear ~nasnetic resonance spectroscopy ) and mass spvctral data. Wax alcohol ferulates By far thrl most plentiful component (circa 50%) of the neutral fraction of the extract was composed of a mixture oi two alcohol ferulates. Saponification followed by g.1.c. analysis of the acetate deri\-atives identified behenyl (44%) and l~gr~oceryl alcohols ( 56%). Ferulic acid was identified by rn. and n1.m.p. and quantit,~tivc: i.r. spectral comparisons with an ilutbentic standard. The mass-spectral fragnlentation pattern and n.m.r. data were in harmony with this assignment. Wax alcohol fctt~lates have previously been isolated f roln the barks of other conifers, including mc,nlbers of the gcnern Pseudotsuga, Larit., Ahies, and Pinus (Rowc et al. 1969). Since, they have also been observed in two harclwood species, they are apparently wiclely dis- tributed in trc.e barks. EXPEHIMENTAL Prcparativc layer chromatography platcs were prepared from silica gel G (E  hlerck) IZiTI<ACTIVES FROhf GRAND FIR BARK 35 TABLE 1. Column cllromatographic separation of brown war from A. grandis bark -- - Fr'iction solvent volume ( m l )  Weight ( m g )  Components petroleurn ether ( 200 ) ! (100) ,, (200) ,I (200) ,, (500) petroleum cther/benzene (9:  1 ) (500) ( t  t j  (4 :1) (400) /  (7:3)  (400) (2:3)(400) ,, (2 :3) (300)  benzene ( 400 ) ether methanol i ;E i } Total recovery 13.75 g Total applied 26.70 g hydrocarbons hydrocarbons + wax esters wax esters wax + sterol esters sterol esters + 2 unknowns unidentified mixture wax alcohols + triterpenes wax alcohols + p-sitosterol p-sitosterol + wax alcohol ferulates p-sitosterol + ferulic esters + 2 unknowns ferulic esters and <,ither chloroform or niethylcnc dichlo- ride was used as developi~~g solvent. The 1i.m.r. spectra were measured in deuterio- cliloroform with a Varian HA 100 spectrom- eter. Signal positions are quoted in terms of the Tiers' T scale relative* to tetramethyl- silane as internal standard. Mass spectra were recorded at 70 cv on an AEI MS9 double-focusing mass spectrometer. Infra- red spectra were prepared from KBr disks and were recorded on a Perkin Elmer 521 spectrophotometer with positions of absorp- tion maxima quoted in wave numbers ( cnl-' ) . Melting points were measured in a Mettler FPI automatic apparatus and are uncorrected. The g.1.c. analy- ses were performed on a Hewlett-Packard model 7620-A instrument equipped with an HP 3370-A electronic integrator. Thc. columns were 6' x %" O.D. stainless stcel packed with either 3% JXR on 60-80 mcsh Gas-Chron~ Q or 10% EGSS-X on 100-200 mesh Gas-Chrom P. Wax alcohol fractions were converted into their acetate derivatives by reaction with acetic anhydride/pyridine ( 1: 1 ) at room tcrnperature for 24 hr. Ice water was added to destroy excess reagent. The crude ace- tates were filtered off and washed through a short column of alumina in chloroform solution. The recovered acetates were, ana- lyzed by g.1.c. (3% JXR) and compared with the acetates of purchased standard alcclhols ( K and K labs.). Preparation uncl fractionation of crirde extract Whole bark from a healthy 100-year-old specimen of '4, grandis, growing on the University of British Columbia calrlpus, was air-dried and ground in a W ~ l r )  mill to pass a 3-mm screen. A sampl(, of the bark (61.7 grams oven-dry weiglit) was extracted for 24 hr in an all-glass soxhlet apparatus witli petroleum ether ( 6,s-110" ) . A brown wax was recovered (1.480 g, yield 2.4% ) . This process was repeated several times on a larger scale. In a typical sep- aration, a sample of wax (26.7 g )  was applied to a column of deactivated alurnina (400 g, activity 111). Elution conimenced with petroleum ether (65-110°), ancl frac- tions were recovered as sumniarizcd in Table 1. Free resin and fatty acids, and possibly other highly polar conipolmds, were irreversibly adsorbed on the alumina. 36 I, H. ROGERS AND D. GHIERSON fl!jclrocarbon fraction A portion of mixed fractions A and B (600 mg) was saponified for 8 hr in 5N KOH/ethanol under reflux conditions. The unsaponifiable fraction (327 mg) was ap- plied to a column of 22% AgN03/silicic acid (15 g ) .  Flushing with 100 ml petro- leum ethcr yielded 116 mg of white, wax- like hydrocarbons. Treatment with 1 g "activc" urea (Rosenstein and Gorin 1957) in benzene solution for three weeks at room temperature afforded a white crystalline complex, which was filtered, washed with cold benzenc, and dricd. The recovered washings contained the branched-chain al- kanes. Thc urea complex was twice treated with boiling toluene, filtered, washed with hot toluenc, and the filtrate evaporated to recover the normal paraffins. Both fractions were dissolved in warm petroleum ether and washed through short coluinns of alu- mina ( activity 111). Yields of recovered normal and branched alkanes were respec- tively 42 mg and 57 mg. Analysis of the normal alkanes by g.1.c. (3% JXR) and comparison with a purchased standard ( #19254, Applicd Science Labs. ) indicated the presence of 24 members of the homologous series fro111 Clo to C42 in- clusive, with the weighted center of the distribution at C3,. The branched alkane fraction showed four unideiltified peaks. Wax ester fraction Fraction C, a yellow wax, was recrystal- lized from acetone; negative Liebermann- Burchard test, I,,,],, 1730 and 1250 (ester), 722 and 712 ( long-chain mcthylene) . Meth- anolysis (282 mg) in 5N KOH/methanol ( 6  ml) under refluxing conditions for 4 (27 mg).  Analysis of the acetates (3% JXR) showed Czz and C24 in ratio 1:2. The methyl ester fraction (v,,~,,. 1730 and 1255) was analyzed by g.1.c. (10% EGSS- X)  . Comparison with a purchased standard (K 107, Applied Science Labs.) showed the normal alkanoic acids CZo (3% ) , CPL' (:37% ) and CZ4 (60%). Sterol ester fraction Fraction D gave a positive Liebermann- Burchard test. Fractional recrystallization failed to remove the wax ester contaminant. Accordingly the impure ester (238 mg was saponified for 2 hr in 10% KOH/ethanol. Workup by partition of the reaction prod- ucts between ether and the aqueous phase yielded a mixture of wax alcohols and sterols (78 mg) and a fatty acid fraction containing some wax a la~hol  (127 mg).  Separation of the sterols from \vak alco- hols was accomplished by preparative t.1.c. ( CHZ Cla) . White crystals from hIeOH, m. 139-140°C. v , 3430 and 1050 ( OH),  1640 and 795 (trisubstituted double I~ond) .  Identical with p-sitosterol standard or) tluan- titative i.r. comparison. Diagnostic species at m/e 414 ( M ) ,  399 (M-15), 396 (M-IS), 381 (M-33), 329 (M-85), 315 (M-W), 273 ( M-141) and 255 ( M-159); other abundant fragments at m/e 303, 289, 231, 213, 159, 149, 145 and 133. Electron bombardment at 13 ev showed campesterol (M- 400) mixed with p-sitosterol (M+ 414). The fatty acid fraction, after remoaal of unsaponifiables, was esterified with di- azomethane and analyzed by g.1.c. (10% EGSS-X). Only normal alkanoic acids were detected; Cis (12%), CIS (2.5%), CzO (6.1%), CZZ (44%) and C24 (34%). - hr, followed by addition of excess 4% Tritervene luctones HCl/inethanol and further refluxing for Fraction G consisted of a mixture of wax 90 min, yielded a crude product (123 mg) alcohols with two triterpene 1actont.s of which was separated on a column of alu- closely similar Rr, The layer chmmatog- milla (15 g, activity 111). Elution with raphy comparison with an stan- petroleum ether yielded a fatty acid methyl dard showed neither of them to abies- ester fraction (44 mg ) . Benzene/petroleum lactone. Attempts to separate the niixture ether (3:  1) recovered a wax alcohol fraction on columns of deactivated alumina were IZXTRACTIVES FROM GRAND FIR BARK 37 only partially successful. This material was further investigated in thc chemistry de- partment, University of British Columbia (Allen et al. 1971 ) . Wax alcohols A pure wax alcohol fraction was available from a previous column separation of the crude bark extract on deactivated alumina and from fraction H by preparative t.1.c. White, waxlike crystals from acetone, m.p. 75-6°C. u ,,, 3400 and 1050 (OH) .  The i1.m.r. 6.35 ( triplet, CH-20H), 8.75 (singlet, CH:{), 9.15 (triplet, CH3). Major fragment at m/e 336 (M-18). No parent ion. Gas liquid chromatography analysis (3% JXR) showed CZ2 ( IS%),  Cz4 (80%) and CP6 (5%).  Free sterol p-sitosterol was recovered from fractions H, I and J by preparative t.1.c. (CHCI,). White crystals from MeOH, m.p. 139-140°C. ~I,,,;,,. 3430 and 1050 ( OH) ,  1640 and 795 ( trisubstituted double bond). Identical with an authentic standard on quantitative i.r. spectral comparison. The n.m.r. 4.70 ( lH,  multiplet, olefinic) , 6.50 ( IH, mul- l I tiplet, H-C-OH), 8.17 (singlet, OH) ,  9.01 I and 9.34 ' ( c H ~ ) .  Anal. Calcd. for Can H&: M+ 414.386147. Found: 414.386934. Phenolic acid tTster Fraction K yielded white, waxy crystals, 111.p. 78°C from ethyl acetatc. U ,  ,, 3550 ( OH),  1705 and 1260 (ester), 1625 ( con- jugated double bond), 970 and 1310 (trans disubstituted double bontl), 1595, 1585, 1500 and 1450 (aromatic). Saponification (0.230 g )  in 1.25 N NaOH/ethanol under refluv conditions for 2 hr yielded wax alcohols (53 mg) and a phenolic acid fraction. The wax alcohols gave whitc crystals, m.p. 74-6O, from petro- lcum ether. plllr. 3500-3300 (OH) .  Major fragment at m/e 336 ( M-18). The acetate derivative analyzed by g.1.c. (3% JXR) for C-:! (44%) and C24 (56%). Thc phciiolic acid was purified by vac- uum sublimation (twice at 135OC at 0.07 mm Hg).  Yellow needles, m. and mixed m.p. 169-171°C from hot water. h ,,,,,. 234 nm ( r 7050), x,,,~~,, 296 nm (r  6530) and h ,,,. 324 mm ( 6  8430). u ,,,, 35004250 (OH) ,  1690 (a,p unsaturated (:OOH), 1660,1620 and 970 (conjugated trans double bond), 1600, 1587, 1500 and 1450 (aro- matic). Quantitative comparison with au- thentic ferulic acid gave superimposable spectra. The n.m.r. (deuterioacetone) showed a pair of one proton doublets cen- tered at 2.44 and 3.68 derived froni the olefinic protons with the coupling constant ( J  = 16 Hz) indicative of a trans double bond, a one proton singlet at 2.76 (aro- matic Cz) ,  a pair of one proton doul~lets centered at 2.92 and 3.18 (aromatic C6 and C,, respectively) and a three proton singlet at 6.13 (OCHs). Anal. Calcd. for CloHlo04: C 61.85; H 5.19. Found: C 61.87; H 5.18. REFERENCES ALLEN, F. H., J. P. KUTNEY, J. TROTCEI~, AYD N. D WESTCOTT. 1971. The structurea and absolute stereochemistry of cyclogr.indisolide and epicyclograndisolide, novel I rite1 pene lactones from Abies grandis. Tetrahc dron Lett. 283-286. KUTNEY, J. P., N. D. WESTCOTT, F. H .  AILEN, N. W. Iskacs, 0. KENNARD, A N I ~  W. D. MOTHERWELL. 1971. The structure of abies- lactone. Tetrahedron Lett. 3463-3466. ROGERS, I. H., AND D. GRIERSON. 1969. Extra(tivcs from the bark of grand fir [ A b i e ~  gnmdis (Dougl.) Lindl.]. Can. Dep. Fish. For., Bi-Mo. Res. Notes 25(4):33-34. ROSENSTEIN, L. AND M. H. GORIN. 1957. 'Ireat- ment of a mixture of saturated and un- saturated fatty acids with expanded urea. U. S. Patent 2, 800, 466. ROWE, 1. W., C. L. BOWER, AND E. R. ~ ~ ( N E R .  1969. Extractives of jack pine b ~ r b  oc- currence of cis- and trans- pinosylvin dimethyl ether and ferulic acid estera. Plrytochem. 8:235-241. SMEDMAN, L. A., K. SNAJBERK, E. ZAVARI\, AND T. R. MON. 1969. Oxygenated monoterpt~~ioids and sesquiterpenoid hydrocarbons of the cor- tical turpentine from different Abier s~,~~cies. Phytochem. 8 :1471-1479. UYEO, S., J. OKADA, S. MATSUNAGA, ND 1. W. ROWE. 1968. The structure and the stereo- chemistry of abiedactone. Tetrahedron 24: 2859-2880. 

Extractives From Grand Fir [Abies Grandis (DOUGL.) LINDL.] Bark

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Extractives From Grand Fir [Abies Grandis (DOUGL.) LINDL.] Bark

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Wood and Fiber Science (E-Journal)