AN ANALYTICAL STUDY OF GUM EXUDATES FROM THE GENUS Araucaria JUSSIEU ~(GYMNOSPERMAE) D. M. W. ANDERSON ANDA. C.
Department of Chemistry, The University, EiIinburgh EH9 3JJ (Gieat Britain) (ReceivedNovember5th, 1968; in revisedform, January30th, 1969)
Analytical data are presented for the gum polysaccharides isolated from the
resinous exudates from five Araucaria species; for three of these, two specimens from different geographical locations have been studied. The data are compared with those available for the only Araucuria species examined previously. The results indicate that the analytical differences between species of this Gymnosperm genus are not so great as are known to occur in genera of the Angiospermae. INTRODUCTION
Chemical studies and comparisons of plant gums have tended, in the past, to reflect an academic interest; their infrequent application in other sciences has indicated that such studies should preferably be conducted and reported on a broader basis. Chemical comparisons and &s&cations of plant gums have been made’* ’ on the basis of their oligosaccharide structural units, and general types of gum structure have been compared with the botanical classification of the parent plant at the level of Order and Family3. This paper presents au analytical comparison of some gum polysaccharides from the genus Araucaria in relation to its botanical divisions: certain correlations on such a basis have recently been proposed4 for Acacia gum exudates. The genus Araucuria (Family, Araucariaceae; Class, Coniferales; Sub-division, Gymnospermae) contains fourteen species ‘. The polysaccharide found in the resinous exudate from Araucaria biziwilliihas received detailed attention6. ‘; the presence of a polysaccharide in this exudate was first noted by Birch’. The presence of a gum polysaccharide in the resin from A. angztszzfiIiag*lo and in a water-soluble fraction of A. araucana resin l1 have also been observed. The terpenoid material in Araucuria resins has received little attention. The taxonomy of the Araucaria genus is shown in Fig. 1; the genus is divided into three sections on the basis5 of the morphoIogical- characters of the species (Fig. 2). The exudates from five Araucaria species, representative of its Colymbea and Eutacta Sections, have been studied and camped in- terms of the accepted Cwk&yn. Res., 11 (1969) 43-51
D. M. W. ANDERSON,
A. C. MUNRO
botanical divisions of the genus. Previous studies 6* ’ have indicated a large variation in the composition of two different Australian samples of A. bidzuillii gum. A specimen of this gum from a different geographical source has therefore been examined. In addition, for each of the species A. araucana, A. cunninghamii, and A.- heterophylla, two specimens from different sources were also obtained for comparison, in an attempt to ascertain whether the extent of the variation reported6- ’ for A. bidwillii gum is a general feature of the genus Araucaria. EXPERIMENTAL
The sources of the exudates studied are detailed below; the full botanical nomenclature involved has been discussed elsewhere’ ‘. A. araucana I: from a single tree, Camperdown Park, Dundee (collected April 1968). A. araucana IT: from four trees, Royal Botanic Gardens, Edinburgh (collected November 1967). A. bidzuillii: from a single tree, Kew Gardens, London (collected November, 1967). A. columnaris: from a single tree, Lushoto Arboretum, Tanganyika (collected October, 1962). A. cunnitzghamii I: from a single tree, Kew Gardens, London (collected November, 1967). A. cunninghamii II: from a single tree, Longuza, Tanganyika (collected March, 1968). A. heterophylla I: from a single tree, Royal Botanic Gardens, Edinburgh (collected February, 1968). A. heterophylla IX from a single tree, Tanga, Tanganyika (collected March, 1968). Extraction and purification of polysaccharides. - Each of the exudates was extracted with cold ethanol (6 x 2-litre portions for A. araucana II and A. columnaris; 6 x 800-ml portions for the other samples)_ The residual material was dried, extracted with cold water (to give a ca. 2% solution), filtered, dialysed for 48 h, and freezedried; in each case, a small proportion of water-insoluble material remained, and it was extracted with 1% aqueous sodium borohydride13 for 24 h. The borohydride extracts were dialysed and freeze-dried; generally, this fraction contained ca. 1% of the total material extracted. For A. araucana, however, the borohydride fraction contained ca. 5% of the total, extractable material; this fraction (denoted A. araucana HI) obtained from A. araucana sample II was studied in addition to the watersoluble polysaccharides. After isolation, several of the polysaccharides gave cloudy, aqueous solutions which could not be clarified by filtration or centrifugation, probably because trace amounts of resin remained from the ethanol extractions; such solutions were clarified by passage through “Millipore” filters (pore-size 1.2 pm). Moisture contents were assessed by drying samples to constant weight at 105”. Nitrogen determinations were carried out by a semi-micro Kjeldahl method. Viscosity measurements were made at 25.0” in suspended-level, Ubbelohde, dilution viscometers with polysaccharide concentrations of 4-2% (sequential dilutions) in hi sodium chloride. Ultracentrifugation (Beckman “ Spinco” Model E ultracentrifuge) was carried out at 44,770 r.p.m. with 0.5% solutions in 0.5~ sodium chloride; photographs were taken every 16 min. Weight-average molecular weights (H,) were determined at 25” and 516 nm Carbohyd. Res., 11 (1969) 43-51
D. M. W. ANDERSON, A. C. MUNRD
COLYMBEA A. angustifolia
Endlicher. (Bertoloni) 0. Kuntze Qyn. A. brasiliensis A. Richard; Parana Pine; Candelabra
Tree). A. araucana (Molina) K. Koch (syn. A. imbricata Pavon; Chile Pine; Monkey Puzzle).
A. 5idwiZZiiHooker (Bunya-bunya).
INTERMEDIA C.T. White. A. kiinkii Lauterbach.
EUTACTA Endlicher. A. balansae Bronguiart and Grisebach (syn. A. elegans Hart.) A. beccarii Warburg [syn. A. cunningbamii Becarrii (non D. Don); A. cunninghamii var. papuana Laut.] A. benderi Buchbolz. A_ biramuiata Buchholz. A. coZumnaris (Forster) Hooker (syn. A. cookii R. Brown ex Lindley). A. cunninghpmii D. Don (Hoop Pine, Moreton Bay Pine). A. heterophyf!a (Salisbury) Franc0 (syn. A. e_t-celsa (Lambert) R. Brown: Norfolk Island
Pine). A. hrrmboldfensis Buchholz.
A. muelleri Brongniart and Grisebach. A. rrdei Ferdinand von Mueller (syn. A. niepratschki Baumann
A. wan gaerfii
Hort. ex Dallirnore).
Fig. 2. Botanical division of the genus Araucaria Jussieus.
Photogoniodiffusometer. The average of three determinations a “SOFICA” was taken for soIutions (0.2, 0.15, and 0.1% in M sodium chloride) ciarified by passage through “Miliipore” filters of pore-sizes 0.45 and 0.22 pm. Molecular-sieve chromatography was carried out on a “Biogel P300” column (47 x 5 cm) precalibrated with dextran fractionsI of known an. Polysaccharides (15 mg in 1 ml of 2M sodium chloride) were applied caremly at the top of the column; lvith
elution was with M sodium chloride. Fractions (2.1 ml) were collected, and examined by the phenol-sulphuric acid method’ 5. The values reported for u,, correspond to the apex of the main peak. Several of the polysaccharides showed a second, smaller peak at or near the void volume of the column; this value is reported as “m, (second peak)“. Because of the method of calibration used, the values obtained for m,
cannot be taken as absolute values. Uranic acid contents, assessed as “hexuronic anhydride”, by acid decarboxylation and an infrared technique’ 6* I’_
Chromatography was carried out on Whatman Nos. 1 and ~MM papers, in the following soIvent systems: (CI) benzene-butyl alcohol-pyridine-water (1:5:3:3, upper layer); (6) ethyl acetate-pyridine-water (l&4:3); (c) acetic acid-ethyl acetateformic acid-water (3: 18: 1:4); (d) butyl alcohol-cthanol-O.1~ phosphoric acid (1:10:5); (e) butyl aIcohol-ethanol-O.lM hydrochloric acid (1:10:5). Systems (d) and (e) were developed from a thin-layer technique’ ’ for the separation of uranic acids; Whatman No. 1 papers which had been pretreated with 0.3~ sodium dihydrogen phosphate and then air-dried were used, and good separations of gIucuronic acid from galacturonic acid were obtained. Chromatograms were developed by spraying with a saturated solution of aniline hydrogen oxalate (ethanol-water, 1: I), and then heating for ca. 5 min at 120°. Carbohyd. Res., 11 (1969) 33-51
Thin-layer electrophoresis was carried out on “Millipore Phoroslides” in 0.05b1 ammonium carbonate and 0.05~1 borate buffers. PoIysaccharide soIutions (OS-1.0% in buffer) were applied as a thin band 2 cm from the cathode-end of the strip. Electrophoresis was carried out for 10 min in a “Phoroslide” cell, in conjunction with a Shandon “VOKAM” power unit (model 2541), at a potential gradient of 50 volts along the strip. The polysaccharide bands were located by a modification of the periodate-rosaniline hydrochloride technique’ ‘.
HydroIyses were effected by heating the polysaccharide (150 &g) with N or 2~ suIphuric acid (40 rnI) for 7.5 h on a boiling water-bath. Th= solutions were cooled, neutralised (BaC03), deionised (Amberlite IR-120 resin, Hf form) and concentrated to syrups. The 2N acid hydrolysates were examined for uranic acids in solvents (c). (Q, and (e); the N acid hydrolysates were examined for neutral sugars in solvents (a), (6), and (c), and for aldobiouronic acids in solvent (c). All of the polysaccharides studied showed chromatographic evidence for galactose, arabinose, rhamnose, small proportions of xylose and 3-0-methylrhamnose, and the acidic disaccharides [email protected]
acid)-D-gaiactose and 6-O-(4-0-methyl-B-D-glucopyranosyluronic acid)-D-galactose. GIucuronic acid and its CO-methyl analogue were the only uranic acids found. Periodate oxidations of solutions of the polysaccharides (1.5% in 0.25~ sodium metaperiodate) were carried out in darkness at room temperature. The formic acid released was estimated by titrating aliquots at intervals with 0.01~ sodium hydroxide; the reduction of periodate was estimated after 96 h by back-titration with standard sodium arsenite”. The analytical data obtained are recorded in Table I. DISCUSSION
There have been few studies of gymnosperm exudates, undoubtedly due to the relatively small number of taxa comprising the Gymnospermae. The exudates from Encephalartos IongifoIius2’, E. latefrons22, and Wehuitschia mirabilis22 (see Fig. 1) have shown a high degree of complexity, and alI contain 3-0-methylrhamnose as a component. The presence of this sugar in A. bidwifliigum was not previously noted6* ‘. This study indicates that 3-0-methylrhamnose is a component of all gymnosperm gums examined to date; since this comparatively rare sugar has not been found in any of the gums from Angiosperms examined so far, it may be a useful taxonomic marker12. The gum exudates from different species of Angiospermae genera, e.g., Acacia4* 23, AIbizia24* 25, and Prunus 2*26*’ 7 frequently vary quite widely in terms of composition and moiecuIar properties. The Araucaria specied studied here are, in comparison, much more closely aiike in terms of their analytical parameters. It is particularly interesting that the specimens of A. heterophylla from Edinburgh and from Tanganyika should be so closely alike; the same holds for the specimens of A. crmninghamiifrom Kew and from Tanganyika.~The main exception to this concerns Curbohyd.Res., 11 (1969) 43-51
5.8 7.6 10.7 5.4 - 1.9 -0.7 l-11,0 -1.7 t11 -9.5 0.81 0.91 2050 0074 5.1 5.9 1680 4.7 16.8 IS.1 24.6 12.6 800 300 1,100 185 300 49 49 60 300 300 CII.130 AS AS AS AS AS 2 2 2 It 1 4.050 3.85 3.95 2.20 3.80 9.18 9.030 8,80 8.53 8.10 10 12 11 10 10 18 66 67 49 62 720 590 33 16 14 14 130 150 I I 1 2 trace 7 8 5’J 80 6 7 lh trdCC trace 2h
10.2 6.1 4.7 6.8 5.1 -6.3 -4.8 - 6.8 - .l.O -8.8 0.48 0.69 0.28 0.52 0.30 1.8 3.1 4.4 3.3 1.9 11.4 13.0 10.5 16.5 9.8 145 120 190 150 173 58 57 60 76 60 ca. 120 cu. 120 S S AS s AS 1 1t It tt 1t 4.95 4.55 4405 3.85 4.45 8.65 8.08 9.10 10.50 9.45 10 IO 11 10 10 63 60 69 64 65 13 17 20 13 13 1 I 1 2 2 8 7 7 5 5 trace trace 7h 6h trace
acorrccted for moisture. bCorrectcdfor protein. CAveragemolecular weight by molecular-sievechromatography with reference to dextran samples of defined ]F3,.*AS, asymmetric; S, symmetric. et, tailing. &,d. = not done. OValucconvcrtcd from that reported in the literature. hApproximate value, based on the calibration curve for L-rhamnose.
Moisture (%) Specificrotationnab(dcgrccs) Nitrogen (%) Protein (%N x 6.25) Intrinsic viscositya(cm3/g) z&lx 103n &XlO3~ nn x 103(2nd peak)0 Ultracentrifuge(boundary shape)d Elcctrophoresis(no. [email protected]
Formic acid relcaseda’b(mmoles/&) Pcriodate reducedfllb(mmoles/g) Uranic anhydrid&J (X) Galactose (%) Ardbinose(O/O) Xylose (X) Rhamnose (%) 3.0-Methylrhamnose (%)
Ethanol fraction Water fraction Borohydridc fraction
ANALYTICALDATA FOR SPECIESIN Colyrnbea AND Eutacla SECTIONSOF w3 OENUS Araucaria
8 ? ?
the composition reported6, ’ for the two Australian specimens of A. bidwillii gum. Paleo-botanical studies indicate 28 that the Aruucaria genus is much older in origin than Angiosperm genera, and there is histological evidencezgT that it may be in an evolutionary decline. This may account for the small size of the genus, and it,may be expected that the surviving species are similar, the more extreme members having become extinct. The ‘low intrinsic viscosity of the polysaccharides in comparison to M, indicates that the molecules are likely to be globular, rather than rod-shaped”? The relatively high ratios of M, to a, indicate a broad range of molecular weight. This is supported by molecular-sieve chromatography, where broad elution peaks were obtained, and several of the samples studied showed small peaks at or near the void volume of the column (denoted “mn second peak” in Table r). Such samples showed siightly asymmetric boundaries on ultracentrifugation, and this second component of higher molecular-weight may be responsible for the higher m, values obtained. Although this evidence tends to indicate the presence of two polysaccharide components, this feature was not shown by the specimens from A. columnaris, A. cunninghamii I, and A. heterophylla I (see Table I). Elution patterns involving 2 peaks have been given 31 by column chomatography of freeze-dried, purified Acacia gums on “Sepharose 4B”; these Acacia gums are considered to be homogeneous’4V 32, and it is significant that the component of apparently higher molecular-weight was not found3 ’ when the gums concerned were examined before freeze-drying. The possible occurrence of a form of molecular aggregation during the purification and freezedrying processes cannot therefore be overlooked. It is of interest, however, that, for A. bidwiilii, A. cunninghamiiII, and A. heterophyila II gums, the higher molecularweight peak occurred within the molecular-sieving range of the column. The borohydride-solubilised material, A. araucanaIII, differs from A. araucana LI in several respects. The large difference in molecular weight observed is in agreement with the effect observed’ ’ previously for Acacia drepanolobium gum. The other main differences between A. araucana II and A. araucana III, involving the arabinose and protein contents, may have arisen from borohydride extraction of the bark and other debris present in the water-insoluble material. Calculations of the ratios of neutral sugars assumed that the uranic acid residues were attached solely to galactose, since &romatography of the N acid hydrolysates in solvent (c) showed components having the same mobility as reference specimens of the aldobiouronic acids 6-0-(8-D-glucopyranosyluronic acid)-D-galactose, and 6-O-(4O-methyl-B-D-glucopyranosyluronic acid)-D-galactose; these acids have been characterised in A. bidwiliii gum6, and, as evidence for other aldobiouronic acids was not found, this assumption was presumed to be valid The possibility that xylose arises as an artefact of hydrolysis and subsequent neutralisation can be discounted; a mixture (in the proportions found) of galactose, arabinose, rhamnose, glucuronic acid, and 4-0-methylglucuronic acid was subjected to the conditions of hydrolysis used, and no xylose resulted. The extent and nature of the heterogeneity previously found6* ’ for A. bidwilfii Carbohpf. Res., 11 (1969) 43-51
D. M. W. ANDERSON, A. C. hfUNR0
=~umwould not be expected to. be revealed by the techniques used here. The thinlayer electrophoresis experiments indicate that A. araucana gum contains two acidic components;-all other gums-showed one component. This basic differentiation of A. araucana gum from -the others studied is difficult to explain, although the other species examined, with the exception of A. coZzmnaris, showed a slight tendency to “tailing”. This phenomenon, under the conditions used by us, has not been observed for Acacia or Prunu.s gums, and, although possibly an experimental artefact, the tailing could begdue to a second, incompletely resolved, minor component_
From a chemotaxonomic point of view, there are no apparent, large differences between the species in the Colymbea and Eufacta sections of the genus Araucaria, although the CuZymbea species show slightly higher specific rotations and protein content, and a smaller release of formic acid on periodate oxidation. Further studies directed to discovering-the nature of any fine structural differences involved are in progress. ACKNOWLEDGMENTS
We thank the Science Research Council for a maintenance award (to A. C. M.). We thank Professor R. Brown, F. R. S., for putting a Spinco Ultracentrifuge at our disposal. We are grateful to Mr. D. R. Lyamuya (Tanga), Mr. R. L. Willans (Lushoto), and the Curators of the Royal Botanic Gardens at Kew and Edinburgh for supplying specimens of Araucaria resins. REFERENCES 1 G. 0. ASPMALL, Acres du Symposium Znrernational de Grenoble, July 1964, pp. 89 and 421. E. L. HIRST, 4th International Congress of Biochemistry, Vienna, 1958, Symposium No. 1, Reprint No. 3. 3 E. A. C’L. E. SCHELPEAND A. M. STEPEIEN. S. African Ind. CJzemist, 18 (1964) 12. 4 D. M. W. ANDERSON AND I.C. M. DEA, Phytochemistry, 8 (1969) 167. 5 W. DAlllhfORE AND A. B. JACKSON, Handbook of Coniferae and Ginkgoaceae, 4th Edn., revised S. G. HARRISON, Arnold, 1966. 6 G. 0. ASPINALL AND R. M. FAIRWEATHER, Carhoh_vd. Res., 1 (1965) 83. 7 G. 0. ASPINALL AND J.P. MCKENNA, CarboJzyd. Res., 7 (1968) 244. 8 A. J. BIRCH, 1. Proc. Rqv. Sot. N. S. WaIes, 71 (1938) 259; Chenz. Abstr., 32 (1938) 7665 (9). 9 I. L. RANJEL AND H. S. SCHNEIDER,Reu. Brasil. CJzim. (Slo Paula), 2 (1936) 261; Chem. Abstr., 31 (1937) 1522 (8). IO J. L. RANJELAND H. S. SCHNEIDER,Ministerio traballro ind. corn., Inst. nacl. tech., Bol. informacoes, 7 (1936); Chem. Abstr., 31 (1937) 2028 (3). I 1. G. WEI~~MANN AND K BRUNS. Nararzuiss~nscJzafen, 52 (1965) 185. 12. D. M. W. ANDERSON AND A. C. MUNRO, Piytochemistry, 8 (1969) 633. 13 D. M. W. ANDERSON AND I. C. M. DEA, Carbohyd. Res., 8 (1958) 440. 14 D. M. W. ANDERSON AND J. F. STODDAXT, Carbohyd. Res., 2 (1966) 104. 13 G. Dur&s, K. A. GIL-, J.R. HAMILTON, P. A. REEERS,AND F. Shurri, Anal. Chem., 28 (1956) 350. 16 D. M. w. ANDERSON, TaIanta, 2 (1959) 73. 17 D. M. W. ANDERSON, S. GARBWIT, AND S. S. H. ZAIDI, Anal. Chim. Acfa, 29 (1963) 39. 18 Y. S. OVODOV, E. V. EUTUSHENKO, V. A. VASKOVSKY, R. G. OVODOVA, AND T. F. SOLOV'EVA, ' 2. Chromazug., 26 (1967) 111.
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R. BELCHER, G. DRYHURST, AND A. M. G. MACDONALD, J. Chem. Sot., (1965) 3964. A. M. STEPHEN AND D. C. DEBRUYN, Carbohyd. Res., 5 (1967) 267. M. KAPLAN, A. M. STEPHEN, AND D. VOGT, S. African Med. f., 40 (1966) 702. D. M. W. ANDERSON AND K. A. -RAMALLA, J. Chem. Sot., (1966) 762. D. M. W. ANDERSON AND I. C. M. DEA, Advan_ Frontiers Plant Sri., 23 (1969) 169. D. M. W. ANDERSON, G. M. CREE, J. J. MARSHALL, AND S. %HIMAN, Carbohyd. Res., 2 (1966) 63. G. GUZMAN, A. SOLER, AND M. MAYOL, Rev. Espan. Fisiol., 16 (1960) 239. J. ROSIK Ahv J. KUBALA, Collection CzecJz. Chem. Commun., 33 (1968) 1946. A. C. SEWARD, Fossil Plants, Vol. 4, Hafner, New York, 1963. F. NYSTERAKIS AND S. QUINTIN, Compt. Rend., 240 (1955) 658. B. JIRGENSONS, Organic Colloids, Hsevier, Amsterdam, 1958, p. 145. D. M. W. ANDERSON, 1. C. M. DEA, AND A. C. MIJNRO, Curbohyd. Res., 9 (1969) 363. 32 D. M. W. ANDERSON, I. C. M. DEA, AND SIR EDhfuw Hr~sr, Curbohyd. Res., 7 (1968) 320. Carbohyd
Res., 11 (1969) 4.7-51