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Polymer International 44 (1997) 55È60
Characterization of Anadenanthera
macrocarpa Exudate Polysaccharide
Regina C. M. de Paula,1 Peter M. Budd2* & Judith F. Rodrigues1
1 Departamento de Qui mica Orgaünica e Inorgaünica, Universidade Federal do Ceara, CP 12.200, CEP 60455-760,
Fortaleza-Ceara, Brazil
2 Department of Chemistry, University of Manchester, Manchester M13 9PL, UK
(Received 24 December 1996 ; accepted 25 March 1997)
Abstract : Exudate gum from Anadenanthera macrocarpa Benth. trees was puriÐed and fractionated using 0É1 M aq. NaCl/ethanol as a solvent/non-solvent
system. The composition of the polysaccharide was determined as 67% arabinose, 24% galactose, 2% rhamnose and 7% glucuronic acid, by a combination of
high performance liquid chromatography of fully hydrolysed gum and colorimetric analysis of uronic acid. Molecular characteristics of the polysaccharide and its
fractions were investigated by light scattering intensity, dilute solution viscometry and gel permeation chromatography (GPC). The whole gum was shown to
possess a broad molar mass distribution with M1 w \ 3É7 ] 106 g mol~1 and
[g] \ 11 cm3 g~1. Hydrodynamic properties indicated a highly branched structure. Fractions were obtained covering a range of molar masses. The intrinsic
viscosity in 1É0 M aq. NaCl at 25¡C was found to depend on molar mass according to : [g]/cm3 g~1 \ 0É0145 M0Õ44. The hydrodynamic volume parameter [g]M
gave a common GPC calibration for branched polysaccharide fractions and
linear poly(oxyethylene) standards.
Polym. Int. 44, 55È60 (1997)
No. of Figures : 4. No. of Tables : 2. No. of References : 13
Key words : branched polymer, chromatography,
saccharide, polyelectrolyte, viscometry.
light
poly-
and uronic acid were detected and protein and cation
(Na`, K`, Ca2`, Mg2`, Fe3`) contents determined.4
The present contribution describes the characterization
with respect to composition and molar mass of exudate
polysaccharide from Anadenanthera macrocarpa.
INTRODUCTION
Anadenanthera macrocarpa Benth. trees, popularly
known as “AngicoÏ in Brazil, are found scattered in the
wild in most Brazilian states. The bark is used as a
tanning agent for leather and the wood as a construction material. The exudate gum, which has a dark
colour, is employed as an adhesive and alternative
medicine in some regions of Brazil.1 The neutral sugars
present in the gum have been reported as arabinose and
galactose2 and as arabinose, galactose, xylose and
ribose.3 In recent work, arabinose, galactose, rhamnose
EXPERIMENTAL
Origin and purification of gum
Anadenanthera macrocarpa gum was collected as a
natural exudate from native trees in Ceara, Brazil, in
December 1993. The gum was puriÐed as the sodium
* To whom all correspondence should be addressed.
55
( 1997 SCI. Polymer International 0959-8103/97/$17.50
scattering,
Printed in Great Britain
56
salt using the method developed by Silva et al.4 with
some modiÐcations. Ground gum (5 g) was dissolved in
distilled water (100 ml) in the presence of H O (5 ml)
2 2
and NaCl (0É5 g). During dissolution the pH was adjusted to 6É0 with 0É1 M NaOH. The solution was Ðltered
and the gum precipitated with ethanol AR. The gum
was redissolved in water, dialysed against distilled water
(2 ] 24 h) and reprecipitated with ethanol AR (yield
70%).
Sugar composition
PuriÐed gum (200 mg) was hydrolysed with 4 M triÑuoroacetic acid (TFA) (4 ml) for various times (2È5 h)
at 100¡C in a sealed ampoule. After hydrolysis, solutions were washed with methanol (5 ] 10 ml) to eliminate the acid, and dried in a rotary evaporator.
Paper chromatography was carried out on Whatman
No. 1 paper with n-butanol : pyridine : water (6 : 4 : 3
by volume) as solvent. The chromatogram was developed with diphenylamine/aniline/phosphoric acid
reagent.
High performance liquid chromatography (HPLC)
was carried out with a Waters Associate System. A
Spherisorb 5 amino column (25 cm ] 4É6 mm) was used
with acetonitrile : water (85/15 v/v) as solvent at room
temperature and a Ñow rate of 1É5 ml min~1 to detect
arabinose and rhamnose. A Phenomenex Rezex RPM
monosaccharide Pb2` column (30 cm ] 7É8 mm) was
used with water as solvent at 80¡C and a Ñow rate of
0É6 ml min~1 to monitor the degree of hydrolysis and to
check for glucose. A di†erential refractometer was used
as detector and the chromatograms were analysed by a
Shimadzu Chromatopac C-R2. The relative amounts of
neutral sugars were calculated as percentages of the
total area.
Uronic acid determination
Total uronic acid was determined using the metahydroxydiphenyl method.5 Sodium tetraborate (2É4 ml)
in 0É0125 M H SO was added to c. 10 kg of sample dis2 4
solved in 0É4 ml water and the tubes cooled in crushed
ice. The mixture was shaken in a vortex mixer and the
tubes heated in a water bath at 100¡C for 5 min. After
cooling in a waterÈice bath, 40 kl of 0É15% metahydroxydiphenyl solution in 0É5% NaOH was added
and the mixture shaken. The absorbance was measured
at 520 nm after 2É5 min. As the carbohydrate produces a
pinkish chromogen with sodium tetraborate solution at
100¡C, a blank sample was run without addition of
metahydroxydiphenyl, which was replaced by 40 kl of
0É5% NaOH. The absorbance of the blank sample was
subtracted from the total absorbance. The percentage of
uronic acid was calculated from a calibration curve of
absorbance against glucuronic acid concentration,
obtained using the same procedure.
R. C. M. de Paula, P. M. Budd, J. F. Rodrigues
Fractionation
Anadenanthera macrocarpa polysaccharide was fractionated using 0É1 M aq. NaCl/ethanol as a solvent/nonsolvent system. Polysaccharide (5É04 g) was dissolved in
0É1 M aq. NaCl (500 ml) and the solution Ðltered into a
5 dm3 pear-shaped fractionation vessel. The Ñask was
thermostatted at 25É0¡C and analar ethanol was slowly
added with vigorous stirring until the solution became
turbid. To clear the solution, the temperature was
increased to 45¡C and then allowed to drop slowly back
to 25¡C. After 24 h a white precipitate had settled on the
side and bottom of the Ñask. The supernatant liquid
was siphoned into a second Ñask and the above procedure repeated. The precipitate was dissolved in water
and freeze-dried. Ten fractions were collected in all,
fractions 3 to 7 being separated as a concentrated liquid
phase.
Light scattering
Low angle laser light scattering (LALLS) measurements
were performed at room temperature with linearly polarized light of j \ 633 nm using a Chromatix KMX-6
photometer (scattering angle h B 4É96¡). Samples were
studied in 0É1 M and in 1É0 M aq. NaCl. Solutions were
continuously Ðltered through a 0É45 km Millipore membrane. For each sample, measurements were made for
Ðve di†erent polysaccharide concentrations and for the
solvent.
The Rayleigh factor R was determined using :
h
I
R \ D(pl)~1
h I
0
where I is the averaged light intensity scattered by the
sample at a low angle h, I is the intensity of the inci0
dent beam and D and (pl)~1 are instrumental factors
determined from the known geometry of the photometer. The excess Rayleigh factor *R \ R (solution)
h
h
[ R (solvent) was evaluated for each polysaccharide
h
concentration c. Weight-average molar masses M1 and
w
second virial coefficients A were determined from the
2
intercept and slope, respectively, of plots of Kc/*R
h
versus c (in g ml~1), since at low angle :
A
B
1
Kc
] 2A c ] . . . .
\
2
M1
*R
w
h
where K is an optical constant which depends on the
refractive index of the solvent, n , and on the refractive
0
index increment, (dn/dc) :
K \ 408 ] 10~8n2(dn/dc)2
0
The refractive index increment was determined at
633 nm using a Brice-Phoenix di†erential refractometer,
calibrated with KCl. Values of dn/dc were measured for
polysaccharide dissolved in 0É1 M and 1É0 M aq. NaCl
POLYMER INTERNATIONAL VOL. 44, NO. 1, 1997
Characterization of polysaccharide
and for solutions dialysed to equilibrium against
1É0 M aq. NaCl.
Dilute solution viscometry
Dilute solution viscometry was carried out on solutions
of the whole polysaccharide and representative fractions
in 1É0 M aq. NaCl using an Ubbelohde capillary viscometer mounted in a thermostat bath controlled at
25É0 ^ 0É1¡C. The Ñow time t was measured at several
concentrations c and the relative viscosity determined
from g \ t/t , where t is the Ñow time for pure
r
0
0
solvent. The intrinsic viscosity (limiting viscosity
number) [g] was evaluated as the common intercept at
zero concentration of plots of (g [ 1)/c and (lng )/c
r
r
versus c.
Gel permeation chromatography
Solutions (0É3% w/v in 0É1 M NaNO ) of the poly3
saccharide and its fractions were analysed by gel permeation (size exclusion) chromatography (GPC) using a
Waters Associates instrument operating at 30¡C. Two
TSK columns (PW , 4000 and 3000) were employed
x
with 0É1 M aq. NaNO as eluant and a Ñow rate of
3
0É5 ml min~1. A di†erential refractometer was used as
detector and the elution volume was corrected to the
internal marker of ethylene glycol at 19É75 ml. The GPC
system was calibrated with fractions of Anadenanthera
macrocarpa polysaccharide and with poly(oxyethylene)
standards. The poly(oxyethylene) calibration was
carried out using pure water as eluant, as irreproducible
results were obtained for poly(oxyethylene) in the presence of salt. For the polysaccharide, a moderate salt
concentration was necessary to reduce polyelectrolyte
e†ects.
RESULTS AND DISCUSSION
Composition
Paper chromatography of Anadenanthera macrocarpa
gum hydrolysates indicated that galactose (R \ 0É53),
f
arabinose (R \ 0É62) and glucuronic acid (R \ 0É115)
f
f
were present. Xylose and ribose, proposed by Rosenthal
to be constituents of angico gum,3 were not detected.
HPLC using an amino column conÐrmed the presence of arabinose (as the major component) and galactose, together with a small amount of rhamnose. HPLC
using a cation exchange column of gum hydrolysed for
2 h gave the relative amounts of neutral sugars as :
66É1% arabinose ; 25É8% galactose and rhamnose ; 8É0%
non-hydrolysed material. Similar Ðgures were obtained
for gum hydrolysed for di†erent times.
The glucuronic acid content was determined as 7É0%
by the metahydroxydiphenyl colorimetric method.5
Silva et al.4 have obtained a slightly lower value of
POLYMER INTERNATIONAL VOL. 44, NO. 1, 1997
57
5É9% for the content of uronic acid in angico gum, utilizing a conductometric method based on titration with
0É01 M NaOH.6,7
Taking into account the ratio between neutral sugars
obtained by HPLC and the amount of uronic acid
determined by the colorimetric method, the composition of Anadenanthera macrocarpa polysaccharide is
found to be : 67 ^ 3 wt% arabinose ; 24 ^ 3 wt% galactose ; 2 ^ 0É5 wt% rhamnose ; 7 ^ 1 wt% glucuronic
acid.
Fractionation
Anadenanthera macrocarpa polysaccharide was fractionated in order to obtain samples with narrow molar
mass distributions. Ten fractions were isolated, a solid/
liquid separation occurring for fractions 1, 2 and 8È10,
and a liquid/liquid separation for fractions 3È7. The
yield of each fraction is given in Table 1. Fraction 1 was
shown by microanalysis to be just 50% carbohydrate ;
the remainder appeared to be inorganic impurity.
Analysis of representative fractions by HPLC and 13C
NMR indicated only small variations in polysaccharide
composition and structure.
Light scattering
Refractive index increments for Anadenanthera macrocarpa polysaccharide dissolved in 0É1 M aq. NaCl and
1É0 M aq. NaCl were determined as 0É129 ^ 0É004 and
0É126 ^ 0É003 ml g~1, respectively. Since the polysaccharide contains uronic acid groups and is therefore
a polyelectrolyte, a more appropriate increment for
light scattering studies is that for the condition of constant chemical potential of di†usible components.8 This
was determined, for solutions of Anadenanthera macrocarpa polysaccharide dialysed to equilibrium against
1É0 M aq. NaCl, as 0É1185 ^ 0É0013 ml g~1.
Plots of Kc/*R versus c for the whole polyh
saccharide and for four fractions in 0É1 M aq. NaCl are
shown in Fig. 1. It can be seen that for the lower molar
mass fractions (fractions 7 and 9, Fig. 1(a)) the slope of
the plot is negative at low polymer concentrations. This
behaviour is typical of a polyelectrolyte and indicates
that the charges on the polymer are insufficiently
screened at this salt concentration. At higher salt concentration (1É0 M aq. NaCl) linear plots are obtained
with positive slopes for all samples, as can be seen in
Fig. 2. Weight-average molar masses M1 and second
w
virial coefficients A determined from the data in Fig. 2
2
are given in Table 2.
Dilute solution viscometry
Intrinsic viscosities [g] for the whole polysaccharide
and representative fractions are given in Table 2. A plot
of log[g] versus log M1
for the fractions gave the
w
R. C. M. de Paula, P. M. Budd, J. F. Rodrigues
58
TABLE 1. Yield, GPC peak elution volume and peak molar mass
for fractions of Anadenanthera macrocarpa polysaccharide
Fraction
Yield (%)
Elution volume (ml)
1
2
3
4
5
6
7
8
9
10
2·4
6·1
16·9
14·5
12·5
17·9
7·4
5·2
4·8
2·0
11·75, 13·00
10·59a, 12·36, 13·23a
10·65
10·93
10·95
11·55
12·00
12·80
13·30
13·70
M
pk
(106 g molÉ1)
1·13, 0·30
3·66, 0·59, 0·24
4·86
3·35
3·26
1·47
0·81
0·37
0·22
0·15
a Shoulder.
parameters in the MarkÈHouwink relationship,
[g] \ KMa, as K \ 0É0145 ml g~1 and a \ 0É44 for
Anadenanthera macrocarpa polysaccharide in 1É0 M aq.
NaCl at 25¡C. The low value of the exponent a is
typical of a highly branched polymer.9
Gel permeation chromatography
Fig. 1. Light scattering results for Anadenanthera macrocarpa
polysaccharide in 0É1 M aq. NaCl : (a) fractions 7 (L) and 9
(|) ; (b) puriÐed gum (…) and fractions 3 (È) and 5 (K).
Fig. 2. Light scattering results for Anadenanthera macrocarpa
polysaccharide in 1 M aq. NaCl : (a) fractions 7 (L) and 9 (|) ;
(b) puriÐed gum (…) and fractions 3 (È) and 5 (K).
The GPC curve for Anadenanthera macrocarpa gum
(Fig. 3) is very broad, with a main peak at 11É70 ml and
two shoulders at 9É2 and 12É8 ml. The shoulder at 9É2 ml
probably arises from exclusion. Multimodal behaviour
POLYMER INTERNATIONAL VOL. 44, NO. 1, 1997
Characterization of polysaccharide
59
TABLE 2. Results from light scattering and dilute solution viscometry for Anadenanthera macrocarpa polysaccharide and representative fractions in 1·0 M aq. NaCl at 25ÄC
Sample
M1 (106 g molÉ1)
w
A (10É4 ml mol gÉ2)
2
ÍiË (ml gÉ1)
Whole gum
Fraction 3
Fraction 5
Fraction 7
Fraction 9
3·71 À 0·17
5·10 À 0·15
3·26 À 0·19
0·80 À 0·01
0·23 À 0·01
0·15 À 0·69
0·06 À 0·28
3·98 À 1·69
0·14 À 0·06
1·91 À 0·52
11·0 À 0·95
13·8 À 0·60
10·3 À 0·11
5·8 À 0·66
3·4 À 0·8
has been observed by Vandevelde and Fenyo10 for gum
from Acacia senegal (gum arabic) and by Paula and
Rodrigues11 for gum from Anacardium occidentale
(cashew nut tree gum). Silva et al.4 have investigated
Anadenanthera macrocarpa gum by GPC with both
refractive index and ultra-violet detection, and concluded that it is a three-component system. The Ðrst
peak they attributed to a polysaccharideÈprotein
complex, as has also been proposed for gum arabic12
and cashew nut tree gum.11 Using dextran for calibration they obtained apparent peak molar masses of
7É9 ] 105, 8É3 ] 104 and 2É2 ] 104 g mol~1. In the
present work, similar molar masses (8É6 ] 105,
9É9 ] 104 and 3É8 ] 104 g mol~1) were obtained with
poly(oxyethylene) calibrants. However, light scattering
(Table 2) gives very much higher molar masses for the
polysaccharide and its fractions than is obtained by
GPC using linear calibrants. This is further evidence
that Anadenanthera macrocarpa polysaccharide is highly
branched.
The Ðrst two fractions isolated from Anadenanthera
macrocarpa polysaccharide were shown by GPC to
possess broad molar mass distributions. Subsequent
Fig. 3. Gel permeation chromatography curves for Anadenanthera macrocarpa polysaccharide : puriÐed gum (ÈÈ) and
fractions 4 (È È È) and 8 (- - -).
POLYMER INTERNATIONAL VOL. 44, NO. 1, 1997
fractions exhibited much narrower distributions than
the starting material, indicating that fractionation by
molar mass had been achieved. GPC curves for fractions 4 and 8 are included in Fig. 3.
A GPC calibration constructed using fractions 3, 5, 7
and 9, for which values of M1 were determined by light
w
scattering (Table 2), is shown in Fig. 4(a) to di†er signiÐcantly from a poly(oxyethylene) calibration. In Fig. 4(b)
the data are plotted in terms of the hydrodynamic
volume parameter [g]M \ KM1`a, utilizing the K and
Fig. 4. Gel permeation chromatography calibration data for
poly(oxyethylene) (L) and Anadenanthera macrocarpa polysaccharide (|) : (a) log M versus V ; (b) universal calibration of
log(KM1`a) versus V .
R. C. M. de Paula, P. M. Budd, J. F. Rodrigues
60
a values for Anadenanthera macrocarpa polysaccharide
obtained above and values for poly(oxyethylene) in
water at 30¡C of K \ 0É0125 ml g~1 and a \ 0É78.13
This universal calibration is seen to be valid for the
branched polysaccharide samples. A slight kink in the
calibration curve arises from the use of two GPC
columns in series. The data in Fig. 4(b) can be Ðtted to
the following relationships :
V \ 12 ml :
log(KM1`a) \ 16É652 [ 0É83197V
V [ 12 ml :
log(KM1`a) \ 14É451 [ 0É64578V
where V is the elution volume.
Peak elution volumes are given for all fractions in
Table 1, together with peak molar masses, M , deterpk
mined using the universal calibration. For the whole
polysaccharide, this calibration gives molar masses of
1É2 ] 106 and 0É37 ] 106 g mol~1 for the main peak at
11É7 ml and the shoulder at 12É8 ml, respectively.
ACKNOWLEDGEMENTS
We are grateful to J. C. Feitosa for supplying the specimen of gum, to the Conselho Nacional de Desenvolvimento CientiÐco e Tecnologico (CNPq-Brazil) for
Ðnancial support and to K. Nixon for assistance with
GPC.
REFERENCES
1 Correüa, M. M., Diciona rio das Plantas UŠ teis do Brasil e das
Exo ticas Cultivadas. Imprensa Nacional, Rio de Janeiro, Brasil,
1926.
2 Rangel, J. L., Goma do Angico. Instituto Nacional de Tecnologia,
Rio de Janeiro, Brasil, 1943.
3 Rosenthal, F. R. T., Revista de Qu• mica Industrial, 24 (1955) 17.
4 Silva, A. G., M.Sc. thesis, Universidade Federal do Ceara, Brazil
(1992).
5 Blumenkrantz, N. & Asboe-Hansen, G., Anal. Biochem., 54 (1973)
484.
6 Casu, B. & Gennaro, U., Carbohydr. Polym., 39 (1995) 168.
7 Pal, M. K. & Bhattacharyya, A. K., Makromol. Chem., 185 (1984)
2241.
8 Budd, P. M., In Comprehensive Polymer Science, ed. G. Allen, J. C.
Bevington, C. Booth, C. Price. Pergamon, Oxford, 1989, Vol. 1,
Ch. 11.
9 Granath, K. A., J. Colloid Sci., 13 (1958) 308.
10 Vandevelde, M. C. & Fenyo, J. C., Carbohydr. Polym., 5 (1985)
251.
11 Paula, R. C. M. & Rodrigues, J. F., Carbohydr. Polym., 26 (1995)
177.
12 Fincher, G. B., Stone, B. A. & Clarke, A. E., Ann. Rev. Plant.
Physiol., 34 (1983) 47.
13 Bailey, F. E., Jr., Kucera, J. L. & Imhof, L. G., J. Polym. Sci., 32
(1958) 517.
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