Polysaccharides have a number of applications in the pharmaceutical, cosmetic and food industries. These numerous applications justify the search for new sources of polysaccharide. Therefore, discovery and evaluation of new polysaccharides from plant becomes a hot research spot. In traditional medicine, extracts of polysaccharide containing plants are widely employed for the treatment of skin and epithelium wounds and of mucous membrane irritation (Bedi and Shenefelt, 2002).
Retama raetam, locally named as R'tm, is a wild plant belonging to the Fabaceae family. It is common to North and East Mediterranean regions. In Tunisia, it is largely abundant. Its abundance in arid area makes it a good candidate for industrial utilisation.
The plant flowers from April to May. The molecular and biochemical mechanisms associated with dormancy and drought tolerance in this desert plant have been elucidated. Retama raetam is prescribed for diabetes control and also for treatment of hypertension (Archer and Pyke, 1991; Taylor 1981; Maghrani et al., 2003, 2004).
The research carried out concerned on characterization of root-nodulating bacteria on Retama raetam (Mahdhi et al., 2008). The structural aspects of water-soluble galactomannans isolated from the seeds of Retama raetam have been investigated by Ishurd et al. (2004) while Kassem et al. (2000) focused their search on the extraction of two new flavonoids from Retama raetam. Maghrani et al. (2005) pointed out the effect of aqueous extract of Retama raetam in normal rats.
However, no data exist concerning the macromolecular properties of polysaccharides extracted from Retama raetam. The general aim of this research is to evaluate the impact of different extraction conditions on the yield and some physico-chemical characteristics of pectin isolated from Retama raetam.
MATERIALS AND METHODS
Materials: Retama raetam was harvested in the region of Monastir (Tunisian Sahel) at the flowering period (April). All other chemicals and solvents used were of analytical grade.
Extraction of pectin: The fresh Retama raetam (500 g) 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). They were extracted with hot distilled
water for 4 h at 70°C. The residue (R1) will be treated later on during
the extraction with oxalate ammonium while the filtrate was precipitated by
adding an excess of ethanol (40:60 v:v) 24 h at 4°C. The precipitate was
collected by centrifugation (3000 rpm, 20 min) and respectively washed several
times with 70% ethanol and finally with acetone and then vacuum-dried at, giving
a powder RNU.
||Procedure diagram used for extraction and purification of
water soluble polysaccharides from Retama raetam
The residue (R1) is treated with the oxalate K2C2O4.H2O
(0.5%) at 25°C, after filtration, precipitation and freeze-dried, we obtained
ROU. The residue (R2) is treated in basic medium with (NaOH; pH =
12, t = 2 h, 4°C) to give product RBU (Fig. 1).
Uronic acid and neutral sugars contents: The uronic acid contents of the peels, AIR and extracted pectins were determined colorimetrically by the automated m-phenyl phenol method (Thibault, 1979). Neutral sugars were analysed as their alditol acetate derivatives by Gas Liquid Chromatography (GLC) after acid hydrolysis. The pectin was hydrolysed with 1 M H2SO4 (3 h, 100°C). The peels and AIR were prehydrolysed with 13 M H2SO4 (30 min, 25°C), diluted to 1 M and heated (2 h, 100°C). The individual neutral sugars obtained were reduced with NaBH4, acetylated and analysed by GLC according to (Harris et al., 1983).
Degree of methylation (DM) and acetylation (DAc): The degree of methylation (DM) is defined as the number of moles of methanol per 100 moles of galacturonic acid. The degree of acetylation (DAc) is defined as the percentage of galacturonosyl residues esterified with one acetyl group. DAc is generally low in native pectins, ranging between 3 and 15% (Voragen et al., 1995). The botanic origin and the extraction procedure determine the content of galacturonic acid, DM and DAc in pectins.
SEC-MALLS: Solutions were prepared by dissolving the polysaccharide samples overnight in 0.1 M LiNO3. The pH was adjusted to the desired value by adding phosphate buffer (I = 0.025 mol L1; pH = 6.3). The concentration of solutions (0.1 wt.%) was chosen such that the overlap concentration of polymer C* was not exceeded, but large enough to give a measurable light scattering response.
The samples injected through a 100 μL loop, were eluted on a TSK-GEL GMPWXL
column (Tosohaas) with a flow rate of 0.5 mL min1. The eluent
and samples were filtered prior to use, through a 0.1 and 5 μm hydrophilic
membrane (Millex® SV, Millipore) respectively. A MALLS detector
(Dawn DSP, Wyatt Technology Co) coupled with a refractive index
detector (Optilab, Wyatt Technology Co.) as concentration detector, was used
to obtain on-line determination of the absolute molar mass (M) and of the root
mean square radius of gyration (<rg2>0.5)
for each elution fraction of ca 0.01 mL which allow the molar mass and size
distributions to be calculated for the SEC profile using ASTRA software (Wyatt
Technology Co.) (Podzimek, 1994). Well-adapted representations of experimental
data such as <rg2>0.5 versus M for example,
<rg2>0.5 = K. Mx (Eq.
1) give information about polymer conformation in solution. Zimm plot (first-order
fitting) of the data collected for each slice (Zimm, 1948), observed to be linear
over a large range of molar mass, was used to determine M and <rg2>0.5
from extrapolation of the intercept and the initial slope.
Treatment of the light scattering data and determination of the polysaccharide concentration in each fraction eluted requires an accurate knowledge of the refractive index increment of pectin samples in a given eluent, which were determined at 25°C using the interferometric refractive index detector (Optilab-DSP, Wyatt Technology Co.) equipped with a P2L cell operating at the same wavelength as the light scattering laser (632.8 nm).
Polyelectrolyte behaviour: Titration of the different samples was carried out after conversion of the carboxylic residues under their acidic form to determine the Acid Equivalent Weight (AEW) related to the content of free acid moieties. In fact, a fraction of the carboxylic acid functions present in pectins is naturally esterified with O-methyl groups. So, the discrepancy between the content of galacturonic residues inferred from the sugar composition and that from the potentiometric titrations is indicative of the methylation degree of galacturonic acid functions.
Viscosity measurements: Intrinsic viscosity is another important feature of the polymer behaviour because it is related to the dimension in solution and the molar mass of macromolecules. As the polysaccharides extracted are polyelectrolytes, repulsion between charged segments affects the dimension in solution that is to say that the viscosity will depend strongly on the ionisation degree of the chain. In order to minimise the effect of dilution upon ionisation, measurements were performed at high ionic strength (NaCl) to shield the anionic charges of macromolecules.
RESULTS AND DISCUSSION
Extraction yields: The yields were calculated in comparison with the corresponding mass of dry plants. Lipids and pigments, were removed by the ethanolic treatment (Selvendran and Du Pont, 1980).
From the extraction data in Table 1, it appears that the
yield of Retama pectin extraction varied from 4-12% dry weight of AIR,
depending on the extraction condition used. The highest yields were obtained
with oxalic (ROU) and the lowest with water (RNU). In comparison, yields for
lime (9-30%) (Koubala et al., 2008) were lower under water extraction
Uronic acid and neutral sugars contents: Retama raetam pectins
had galacturonic acid (260-640mg g1) and total neutral sugars
(320-540 mg g1) contents that varied widely, depending on the
extraction method used (Fig. 2). Water-extracted pectins were
particularly rich in uronic acids while ROU and RBU extracted pectins were rich
in total neutral sugars and poorest in uronic acid (Table 2).
Degree of methylation (DM) and acetylation (DAc): Results in Table
3 show that water-extracted and oxalic ammonium extracted pectins were more
methylated (DM 29-31%) than were basic extracted pectins (DM 20%). Compared
to those of Ambarella pectins and the DM of lime pectins were significantly
higher (58-82%). Retama pectins exhibited low degrees of acetylation (2-19%)
for all three methods of extraction. The basic extraction process yields a pectin
of low degree of esterification (2%) as a result of saponification of the ester
groups whereas the oxalic extraction process yields a high degree of esterification
||Yields and extractabilities of pectins obtained under different
extraction conditions from
||Sugar compositions of polysaccharides extracted from Retama
||Sugar composition of different polysaccharide from Retama
||Macromolecular data for polysaccharides extracted obtained
by SEC-MALLS (0.1 M LiNO3, T = 25°C)
||Light scattering response at 90° (dotted line) from SEC
/MALLS analysis of ROU and of RNU in LiNO3 0.1 M at 25°C,
D = 0.5 mL min1
The DAc values of water extraction RNU (6.6%), were similar to Ambarella extracted pectins and to that of lime pectin extracted with water (6.0%) (Koubala et al., 2008).
Macromolecular features: SEC-MALLS experiments were carried out in 0.1 M LiNO3 at a near neutral pH in order to determine molar mass and size information of the biopolymers studied.
The chromatograms of RNU and ROU (Fig. 3) introduce practically
two elute population at the neighbourhoods respectively of 7 and 9.5 mL. These
population is of strong mass this is explained by the great intensity of signal
of diffusion of the light. Figure 3 shows the SEC elution
profile observed for polysaccharides RNU and ROU. 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.
This 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.
The weight average molar mass (Mw) of Retama pectins
varied from 8.93x104 to 3.42x105 g mol1
and their corresponding intrinsic viscosities ([η]) from 23.55-68.57 mL
g1 (Table 3). These results show the important variability
of molar mass and intrinsic viscosity of pectins according to the extraction
conditions used. Macromolecular parameter values depend, not only on the extraction
process used, but also on the plant material used. These differences of molar
masses could also be due to the type of method used for the determination of
molecular parameter. According to Ralet et al. (2002), the water (in)-solubility
and the aggregation of pectin could influence molar mass measurement.
||Average macromolecular characteristics of polysaccharides
extracted from Retama raetam determined by SEC/MALLS (0.1M LiNO3,
40°C, pH 6.3)
||Relation between the RMS and molar mass for the SEC high molar
mass fraction of polysaccharides extracted from Retama (RNU and ROU)
The value of the specific refractive index increment was called 0.14 mL g1 for RNU, ROU and RBU.
Complete dissolution of different samples was difficult and the presence of aggregates could not be avoided even after sonication (hazy solutions) so that the macromolecular features above determined may be only the characteristics of the soluble part. Further investigations on this compound were not undertaken.
The weight-average values of molar mass and root mean square radius of gyration
determined for each fraction are reported in Table 4. It must
be noted that the <rg2>0.5 determination
for the slices of higher elution volume was affected by a retention on the stationary
phase involving a tailing of molecules having high molar masses due to a non-size
exclusion mechanism, resulting in a poor accuracy of the values obtained (Fig.
SEC-MALLS experiments, owing to the simultaneous determination of the mass
and the size for each elution volume, allow one to reach information about the
polymer conformation. The value of the slope x resulting from the log-log plot
of the root mean square radius of gyration as a function of the molar mass (Eq.
1), reveals whether the macromolecule is a sphere (x = 0.33), a random coil
(x = 0.5-0.6) or a rod (x = 1.0) (De Gennes, 1979). As can be seen from the
slope values given in Table 4, both these samples have spherical
structure. In spite of the data scattering, slopes can be estimated to be ca
0.3 for RNU, ROU and RBU, so spherical conformation.
Polyelectrolyte behaviour: Titration of different carbohydrates was carried out after conversion of the carboxylic residues under their acidic form to determine the AEW related to the content of free acid moieties.
AEW1 was determined for each sample from the potentiometric titration
according to Eq. 1:
pKa was determined according to Eq. 2 :
where, m was the mass of the polymer sample, V0, Ve respectively
the polymer and NaOH volume of the endpoint, Ce the concentration
of NaOH (Fig. 5).
In fact, a fraction of the carboxylic acid functions present in pectins is
naturally esterified with O-methyl groups. The results presented in Table
3 show that all of samples are lowly methoxylated (DM<50%).
From the data of Table 5, it was concluded that the polysaccharides
extracted in water (RNU) are low charged anionic polymers whereas the pectin
extracted on NaOH can be viewed as a moderately charged anionic polymer (Fig.
||Potentiometric titrations of polysaccharides extracted from
||Relation between pKa and alpha of different sample
Viscosity measurements: The influence of the NaCl concentration on the
relative viscosity of different sample is shown in Fig. 7.
The repulsion between charged sites prevails in pure water causing the chain
to adopt an extended conformation evidenced by the maximum viscosity. A salt
concentration as low as 0.1 M results in a dramatic decrease of viscosity indicating
decreasing dimensions of the polysaccharide macromolecules related to a weakening
of the electrostatic interactions by shielding of the polyion charges. The viscosity
decrease slowed down in the range of ionic strength 0.1-0.3 M and reached a
limiting value at high salt concentration meaning that the polyelectrolyte character
is minimised in this domain.
The values of intrinsic viscosity reported in Table 6, demonstrate
a similar trend for polymer conformation as that inferred from the SEC-MALLS
data. The large decrease of intrinsic viscosity on going from RBU to ROU (Fig.
8) indicates not only the lowering of molecular weight but also the chain
folding due to a higher content of galacturonic acid units.
||Titration of different polysaccharide from Retama raetam
||Variation of relative viscosity of RNU, ROU and RBU
||Viscometric characteristics of polysaccharides extracted from
|C*: Overlap polymer concentrations
||Variation of reduced viscosity of RNU, ROU and RBU
It is noteworthy that the values of overlap polymer concentrations C*
estimated using equation using the equation: C* = 1.5/[η] (Morris
et al., 1980), confirm the fact that the determination of the
molar mass, root mean square radius and intrinsic viscosity were performed in
the dilute regime.
Different polymers were extracted from Retama raetam by sequential extraction. The results showed that Retama raetam are a source of pectins with high molecular weight. The extraction conditions had a significant impact on the characteristics of the extracted pectins. Ammonium oxalate allowed high extraction yields, the recovered pectins being of high degree of methylation, high molar mass and high intrinsic viscosity. Basic extraction gave low yields of pectins with low uronic acids and DM. Water extraction gave the lowest yields of pectins with high degrees of methylation and high intrinsic viscosity. On the whole, oxalate extraction of pectins from Retama raetam provides good yields, which are biochemically interesting due to their high molar mass.
These investigations revealed that the polysaccharides from Retama raetam have a sugar composition typical of pectin with a high and medium degree of esterification of galacturonic residues respectively. SEC-MALLS experiments gave information of the distribution of the molar mass and root mean square radius. Information on the conformation of these polysaccharides was obtained by applying the relation between the root mean square radius and the molar mass by viscometric measurements. Spherical conformation was found for the former samples.
The authors are very grateful to C. Morvan for helpful discussions and determination of the sugar composition.