For many years pectins have been the subjects of extensive research.
Pectins have a number of applications in the pharmaceutical, cosmetic
and food industries.
These numerous applications and justify the search for new sources of
pectins. Present research is part of a valorisation project on the water-soluble
polymers extracted from the Tunisian flora. With this objective we have
undertaken a study about the extraction, purification and physico-chemical
characterisation of the polysaccharides from this plant, as to our knowledge
no paper on this topic has been published yet. Inula crithmoides is
a plant from the halophyte group and the family of Compositae. Its period
of flowering is from August at October. It is widespread in Mediterranean
basin, in maritime meadows and salt-water marshes (Pottier Alapettite,
1979) and in Tunisia it is abundant in Tabarka, Carthage, Gabès,
Oasis of Djérid (Clintock et al., 1976).
The research carried out on Inula crithmoides, primarily concerned
the ecology of halophytes in the Mediterranean coast of Damietta, Egypt
(Serag, 1999), pharmacological evaluation of the dichloromethanol extract
from Inula crithmoides (Barrachina et al., 1995), chlorinated
tymol derivatives from Inula (Marco et al., 1993) and edaphic characteristics
of salt meadow vegetations eastern regions of spain (Boira, 1995).
However, no data exist concerning the macromolecular properties of polysaccharides
extracted from Inula crithmoides.
In the present study, extraction, purification of polysaccharides is
reported. Our interest has been focused on the physico-chemical characterisation
using several techniques such as: viscosity in dilute solutions and steric
exclusion chromatography equipped with double detection refractometric
and multiangle laser light scattering (SEC/MALLS). The general aim of
this was to understand the structure we report about the dependence of
the extraction procedure on the chemical and physico-chemical characteristics
of pectic polysaccharide isolated from Inula crithmoides, including
sugar composition, DM and molar mass and intrinsic viscosity.
MATERIALS AND METHODS
Materials: The plant was collected in the area of Soliman
(Cap Bon Tunisian) in March, 2005.
One thousand grams of sheets of finely cut plant was washed for 1 h in
4 L of boiling 95% ethanol and the mixture was then allowed to stir mechanically
overnight at room temperature. After filtration through a G2 sintered
glass funnel, the residue washed with ethanol (5x500 cm3) and
acetone (3x300 cm3)-named AIS - was dried, to be used as stock
material for extraction (Forni et al., 1994).
Water-soluble polymers were obtained from AIS after extraction for 2
h in hot water (1:30 w:w, 80°C) with mechanical stirring. The mixture
was filtered on canvas and then on Celite through a G2 sintered glass
funnel. The residue (R1) will be treated later on during the extraction
with oxalate while the filtrate was precipitate by adding an excess of
ethanol (40:60 v:v). After settling for 12 h at 4°C, the insoluble
material was removed by centrifugation (5000 rpm) for 30 min to yield
the fraction INP was dialysed by ultrafiltration on a Minitan cell (Millipore)
using membranes with a molecular weight cut-off of 100000 in order to
eliminate the remaining salts, as well as polymers (proteins and polysaccharides)
of low molar mass. Polymer in retentate (named INP) was finally obtained
(Joye and Luzio, 2000; Majdoub et al., 2001a, b).
The residue (R1) is treated with the oxalate K2C2O4H2O
(0.5%) at 25°C, after filtration, precipitation and freeze-dried,
we obtained IOPU. The residue (R2) is treated in basic medium with ( NaOH;
pH = 12, t = 2 h, 4°C) to give product IBPU (Fig. 1).
Sugar analysis: Neutral sugars were determined as anhydroglucose
by the phenol-sulfuric acid assay (Dubois et al., 1956). Correction
was made for interference from GalA. Total uronic acids were assayed as
anhydrogalacturonic acid using m-phenylphenol colour reagent. All fractions
(0.5 mg) were methanolysed in 2 M anhydride acid in methanol (24 h, 80°C)
for measurement of individual sugars, using myo-inositol as internal standard.
They were silylated (4°C overnight in 1% trimethylchlorosilane in
N, O-bis trimethylsilyl- fluoroaceaamide) and analysed by gas liquid chromatography
on capillary columns DB 225 (J.W. Instruments) with nitrogen as vector
gas and air-hydrogen mixture as fuel (Goubet et al., 1995).
Degree of methylation: DM was defined as the number of moles of
acetic acid or methanol per 100 mol of GalA.
The obtained DM values were confirmed using a FT-IR method described
previously (Monsoor et al., 2001; Kravtchenko et al., 1992).
AEW determination: The anionic groups of polysaccharide samples
(0.5 g in 100 cm3 of distilled water) were first converted
into their acidic form by percolating onto a strongly acidic ion exchange
resin (Dowex 50X8)and then titrated under nitrogen by NaOH, in the presence
of 0.15 M NaCl to minimise the polyelectrolyte effect by screening the
charges on the chain.
Procedure diagram used for extraction and purification
of water-soluble polysaccharides from Inula crithmoides
The molar mass of the Acid Equivalent Unit (AEW) was determined for each sample
from the potentiometric titration according to:
where, m is the mass of the polymer sample, Ve the NaOH volume
of the endpoint.
Viscosimetric measurements: To determine the intrinsic viscosity
[η] of dilute polysaccharide solutions (C inferior to the overlap
concentration C*), the flow time of the polymer solutions at different
concentrations was measured in 1 M NaCl at 25°C using an viscometer
(TI1, Sematech, France). Preliminary experiments have shown that the flow
time of a given polyelectrolyte did not vary when NaCl concentration was
as large as 1 mol L-1. The reduced viscosity (ηsp/C)
was then plotted against the polymer concentration (Fig.
2). The intrinsic viscosity was derived from the intercept (C = 0)
and the Huggins constant (k`) was inferred from the slope. It was subsequently
checked that the starting concentration C0 was inferior to
the critical concentration C* by calculating its value using the equation
(Morris et al., 1980): C* = 1.5/[η].
Polysaccharide molar mass determination by size exclusion chromatography
(SEC): Solutions were prepared by dissolving the polysaccharide samples overnight in 0.1 M
||Reduced viscosity (ηsp/C) measurements were conducted
in 1 M NaCl at 25°C
The pH was adjusted to the desired value by adding phosphate
buffer (I = 0.025 mol L-1; pH 6.3).
The polymer concentration of solutions (0.1 wt. %) was chosen such that
the overlap concentration of polymer C* was not exceeded (see viscometric
measurements), but large enough to give a measurable light scattering
Analyses of polysaccharides were performed on a chromatographic setup
including a pump (Waters, 515), a TSK-GEL GMPWXL column (Tosohaas) and
a multi-angle laser light scattering (MALLS) detector (Dawn™ DSP,
Wyatt Technology Co), coupled with an interferometric refractive
index detector (Optilab, Wyatt Technology Co). The samples
injected through a 0.1 cm3 loop were eluted at 40°C with
a flow rate of 0.5 cm3 min-1.
The light scattering data were analysed using ASTRA software (Wyatt Technology
RESULTS AND DISCUSSION
Extraction: Extraction of polysaccharides from Inula crithmoides
was carried out using three different techniques (Fig. 1).
The first method was mainly based on the boiling-water treatment of AIS
while the second one consisted in the treatment with oxalate and the third
in basic medium cold. In all cases, the final purification was carried
out by elimination of the remaining low molar mass compounds using the
retention of the polysaccharide fraction on ultrafiltration membranes
with a high molecular weight cut off (100,000).
Table 1 shows the yields of the recovered polymer obtained
via the three techniques of extraction. The yields were calculated in
comparison with the corresponding mass of dry plants.
||Comparison of yields of the procedures used to extract
polysaccharides from Inula crithmoides
||Sugar compositions of polysaccharides extracted from
|SNP: Sucre Non Pectique
of the purified INPU, IOPU and IBPU samples showed that these polymers consist of polysaccharides exempt from proteins as no nitrogen
could be detected in their elemental analysis. This result demonstrated
the efficiency of the purification by ultrafiltration.
The first step was the separation of small organic compounds from polymers
by extraction with hot ethanol. The residue AIS had about the same mass
as the starting plants in the dry state.
About 10% of AIS was water soluble, the remaining part being mainly fibres.
The polysaccharides were then roughly isolated from proteins and salts
by precipitation, giving the INP, IOP and IBP fractions. As can be seen
in Table 1, the polymer yield in these cases was very
similar. On the other hand, the yield of the purified fraction after ultrafiltration
was low especially for (IBPU). The highest yield of pectic substances
was obtained with H2O.
Structural analysis: sugar and functionality composition: The
sugar composition of the isolated polysaccharides reported in Table 2
shows that they were mainly composed of Gal A (33.8 to 64.8%). These results
confirm the pectic nature of the obtained polysaccharides as the mole
fraction of Gal A was higher than 65 mol- % (May, 1990). The ammonium
oxalate fraction was found to consist mainly of pectin, heavily branched
with mostly arabinose and xylose units (65% of the galacturonosyl residues
A significantly higher GalA mole fraction featured the ammonium oxalate
fractions (IOPU) with respect to other fractions INPU and IBPU. Rha (7.1
to 12.9%) and two other neutral sugars specific of pectins, galactose
(15.7 to 35.7%) and arabinose (4.9 to 9.6%), were also found. The presence
of Fucose and Xylose suggests that the structure of INPU is formed by
a mixture of pectin (81.3%) and hemicellulose (18.7%) of xylofucane type.
||Compositions HG and RG-I of polysaccharides extracted
from Inula crithmoides
|aHG = GalU-Rha., bRG-I = (2 x
Rha) + Gal + Ara
||Functional analysis of polysaccharides extracted from
|DM results are expressed as mol-% of GalA
Moreover, the significant rhamnose rate indicates the presence of a rhamnogalacturonane
I (RG-I) (between 34.8 and 60.8% are five times the percentage of rhamnose)
In pectins, GalA units can be partly methylated on C6 and/or bear O-acetyl
groups on C2 or C3 (Rombouts and Thibauet, 1986). Acetylation of pectins
is mostly confined to the RG-1-like region while methyl esterification
mainly occurred on smooth HG blocks (M`sakni et al., 2005). The
polymer properties of pectin strongly depend on charge density (electrostatic
repulsion between GalA units) and on esterification (hydrophobic association).
DM determined for the different samples are reported in Table
3. The obtained DM value was in the range 6 to 22 mol-%. Pectin extracted
in neutral water generally has a DM value higher than 50 mol-%. Such a
lower DM is close to those observed for pectins extracted from citrus
with pH 5 succinate-oxalate buffer and from sugar-beet cell walls with
imidazole or carbonate buffer (Mary et al., 2000).
DM was either determined by titration of GalA acid functions (AEW). As
seen in Table 4, an excellent correlation is observed
between DM and AEW the consistency of the different data obtained in the
structural analysis of the isolated polysaccharides.
Molar mass characterization and conformation of the isolated polysaccharides:
The chromatograms of INPU and IOPU introduce practically only one
elute population respectively at the neighbourhoods of 7 and 7.5 mL. This
population is of strong mass this is explained by the great intensity
of signal of diffusion of the light. These chromatograms show the purity
of these two products extracted starting from Inula in spite of the presence
from trace from composed of low mass.
As can be seen, the function log (molar mass) = f (Ve) calculated from
the experimental values is linear over the major part of the peak with
a deviation for higher elution volumes (Fig. 3).
||Molar mass as a function of the elution volume for INPU
and IOPU samples
||Cumulative molar mass for polysaccharides extracted
from Inula crithmoides
phenomenon is indicative of non-SEC effects under the present experimental
conditions due to partial polymer adsorption on the chromatographic support likely caused
by hydrophobic interaction (Fig. 4).
Therefore, calculation of the weight-average molar mass reported in
Table 5 was done assuming a linear variation of the function (Fig.
5). Mw of polysaccharides extracted in hot water was found to be in
the range of 8.3x105 g mol-1 whereas Mw of the IBPU
sample was much higher (2.76x106 g mol-1).
In SEC experiments macromolecules are separated according to their dimension
(hydrodynamic volume) in a given eluent. For a given elution volume, the
higher the molar mass, the more compact the structure of the macromolecule.
A comparison of the molar mass variation versus the elution volume for
the two samples presented in Fig. 3 clearly shows that
IBPU chains display a more compact conformation than the IOPU ones. To
get a deeper insight into the conformation of the studied polysaccharides,
we plotted the root mean square radius of gyration <rg2>0.5
as a function of molar mass (Fig. 6). This variation
can be represented as a power law:
The slope of the double-logarithmic representation of this function gives
the value of x, a parameter being characteristic of the polymer conformation (x = 0.3 for a sphere, x =
0.5-0.6 for a random coil and x = 1.0 for a rod).
||Relationship between molar mass and volume
Relation ship between root mean square radius of gyration
<rg2>2 and molar mass for polysaccharides
extracted from Inula crithmoides
||Relation ship between radius of gyration and volume
(O`Neill et al.,
1996; Kohn and Malovikova, 1978). As expected, the polysaccharides extracted
from Inula crithmoides exhibited a similar conformation with an
x value corresponding to a sphere (Fig. 7, Table
5). The presence of the kinked RG-I region linked to linear HG might
be responsible for a more flexible complex than pure HG. The low x value
found suggests a very intricate compact conformation.
Macromolecular data for polysaccharides extracted from
Inula crithmoides obtained by SEC-MALLS (0.025 M NaNO3.
pH 6.3. T = 25°C)
||Intrinsic viscosity [η] and overlap concentration
C* of polysaccharides extracted from Inula crithmoides
To check the validity
of this assumption, polysaccharide intrinsic viscosity [η] and Huggins`
constant k` are compared in Table 6. Intrinsic viscosity, a measure of
the hydrodynamic volume occupied by a polymer in a given solvent, depends
on polymer conformation and molar mass while the Huggins constant is indicative
of the chain-chain interaction. Preliminary studies on the influence of
the ionic strength indicated that a minimal salt concentration of 1 mol
dm-3 was necessary for screening the electrostatic repulsion
due to the charges located on the GalA residues.
Polysaccharides obtained from Inula crithmoides exhibit similar
viscometric behaviour with similar intrinsic viscosity (C = 0.7dL g-1)
and values of the Huggins constant (Fig. 2, Table
6). Although IBPU has a higher Mw than the other samples, it displays
a significantly lower intrinsic viscosity. This result is an additional
proof of its high level of chain compactness in 1 M NaCl. The high Huggins
constant found for these polymers is correlated to their associative ability
by hydrophobic inter-actions. A stronger effect was observed for INPU.
The decrease in intrinsic viscosity and the increase of Huggins` constant
both reveal enhanced polymer intra- and inter-molecular associations in
comparison with the two other samples IOPU and IBPU.
Polysaccharides were extracted from Inula crithmoides by
selective extraction under mild condition of Ph and temperature and characterized
in terms of molecular and macromolecular structures. Three kinds of polysaccharides
were extracted Each fraction was purified by precipitation in ethanol
followed by ultrafiltration of recovered solids dissolved in water through
a membrane with a molecular weight cut off of 100,000, are purified by
precipitation with ethanol followed by ultrafiltration to obtain respectively
carbohydrates INPU, IOPU and IBPU. It appears from the polymer characteristics
and the chemical analysis that the structure of the polysaccharides extracted
from Inula crithmoides is more dependent on method of extraction.
These exhibited a sugar composition typical of pectins (GalA > 65 mol-%)
with a value of degree of methylation DM in the range 6 to 22 mol-%. Mw
of polysaccharides extracted in hot water was found to be in the range
of 8.3x105 g mol-1 whereas Mw of the IBPU was much
higher (2.76x106 g mol-1).
The polysaccharides extracted from Inula crithmoides exhibited
a similar conformation with an x value corresponding to a sphere. The
presence of the kinked RG-I region linked to linear HG might be responsible
for a more flexible complex than pure HG.
Further study is now undertaken to obtain more information about their
gelling ability, rheological behaviour in semi dilute solutions, study
of amphiphilic derivatives of the isolated pectins from Inula crithmoides
and physico-chemical properties of aqueous dilute solutions, will be published
in a forthcoming paper.
|IN, IO et IB
||Polysaccharide extracted, respectively, by H2O, K2C2O4 and NaOH
|INP, IOP and IBP
||The corresponding intermediate precipitated fractions
|INPU, IOPU and IBPU
||Precipitated and ultrafiltred
||Rhamnogalacturonan of type I
||Degree of methylation