Isolation and Characterization of Pumpkin Pectin for Drug

Encapsulation

José R. R. Souza1, Judith P. A. Feitosa1*, Nágila M. P. S Ricardo1, Edy S.

Brito2
1
Departament of Organic and Inorganic Chemistry, Federal University of Ceará –
P. O. Box: 6.021, ZIP-Code: 60455-760, Fortaleza, Ceará, Brazil
2
Embrapa Tropical Agroindustry, R. Dra. Sara Mesquita, 2270, Pici, 60511-110,
Fortaleza, CE, Brazil

Pumpkin is an excellent and low cost resource of carotenoids, precursors of vitamin A. Moreover, it is
also a great source of natural and low-cost pectin. Pectin is a heterogeneous complex polysaccharide
found in the primary cell wall of most cells and its effects on health is receiving growing interest,
especially for applications such as drug encapsulation. In this work, high-methoxyl pectin was isolated
from a regional pumpkin (Cucurbita moschata) by the method of acid hydrolysis. The isolated pectin was
characterized by FTIR, 1H and 13C NMR, GPC, elemental analysis and Rheology.

Keywords: Pectin, pumpkin, NMR, infrared, rheology.

*
Corresponding author. Tel: +55 85 33669365; fax: +55 85 33669978
E-mail address: [email protected] (Judith P. A. Feitosa)

1. Introduction

Pumpkin, a member of Cucurbitaceae family, is an excellent resource of

carotenoids, precursors of vitamin A, and has been regarded as a functional food (Arima
& Rodriguez-Amaya, 1990; Adams et al., 2012). It is also a low cost source of pectin
(Murkovic et al., 2002). Under the dried form, pumpkin can be stored longer and used
in various culinary preparations, contributing with one more food option to combat
hipoavitaminose A, which affects thousands of children in Brazil and in other countries
of the world.
Pectin is a multifunctional abundant component from cell walls of all plants
(Ngouémazong et al., 2012; Willats et al., 2006). Pectic polysaccharides consist mostly
of polymers rich in galacturonic acid, containing significant amounts of rhamnose,
arabinose and galactose as well as 13 other different monosaccharides (Vincken et al.,
2003). The three major polysaccharides are currently defined homogalacturonan,
ramnogalacturonan I and ramnogalacturonan II (Vincken et al., 20003; Waldron et al.,
2003) The composition, structure, and physiological properties of pectin can be
influenced by extraction conditions as well as source, location and many other
environmental factors. The network of pectin must be broken to be extracted. This may
involve extraction with calcium chelating agents, dilute bases or dilute acids.
Alternatively, fragments of pectic polysaccharides can be released through the use of
enzymatic degradation. Pectins are typically extracted from citrus fruits and apple
pomace (Waldron et al., 2003). Pectin is traditionally used as a gelling agent for jellies
and marmalades. In combination with water and some other substances, it can act as a
thickener, gelling agent, stabilizer, emulsifier, cation-binding agent, etc. (Bottger, 1990).
It is listed among the ingredients of many food products (U.S. code, E440). The global
annual consumption is estimated to be around 45,000 metric tonnes (Willats et al.,
2006). One substance having so many separate properties of technological interest
makes pectin a biopolymer especially valuable for medicine, food production as well as
for applications in drug encapsulation (Benjamin et al., 2012; Souza et al., 2009;
Ptichkina et al., 2008). Pectins also offer health benefits to consumers, for example, they
are being increasingly recognized as important precursors of substrates for
gastrointestinal functions and structures. Foods rich in fiber are usually recommended
for diabetics, because they are able to reduce the glycemic response and thus reduce the
need for insulin (Guillon & Champ, 2000). Pectin is also effective on lowering the
cholesterol level in blood, removing heavy metal ions from the body, stabilizing blood
pressure, and restoring intestinal functions (Voragen et al., 1995).
Although the potential stock of these raw materials enables the main pectin
producers (USA, Germany, Denmark) to plan an annual increase of pectin production of
approximately 3.8% (Phillips, 2000), searching for new pectin-containing raw materials
is an important task of science and industry (May, 1990). In this work pectin was
extracted from a regional pumpkin Cucurbita moschata, best known as “jerimum de
leite” and characterized in order to be used as matrix for future encapsulation studies.

2. Experimental

2.1. Extraction of pectin

For extraction of pectin, mass of about 5 kg of pumpkin pulp was pressed in an
expeller press. We obtained two types of residues: a thinner which was obtained by
filtration of the juice (approx. 209g), and a thicker one. The thinner dough was dried in
an oven at 60 ° C for 12 hours forming a paste that was used for the extraction
procedure. For extraction by acid hydrolysis, 2L of 0.1 M HCl solution was stabilized at
65 °C and after that, approx. 200 g pumpkin pulp was added to the solution and let
extracting for 2 hours. After precipitation and washing with ethanol PA (1:10 - solution:
ethanol), filtration and freeze-drying, it was obtained 4.7 g of pectin that after
purification by dialysis membranes remained 2.5 g of pure pectin which was used for
further analysis (yield calculated from pumpkin dry fiber of approx. 6.9 %).

2.2. Infrared Spectroscopy (IR)

The Fourier transform IR spectrum (FT-IR) of pectin was recorded using a
Shimadzu IR spectrophotometer (model 8300) in the range of 400 and 4000 cm −1 as
KBr pellet.

2.3. 1H , 13C Nuclear magnetic resonance (NMR)

NMR spectra of 0.1% (w/v) solutions in D2O were recorded at 70 °C on a Fourier
transform Bruker Avance DRX 500 spectrometer with an inverse multinuclear gradient
probe-head equipped with z-shielded gradient coils, and with Silicon Graphics. Sodium
2,2-dimethylsilapentane-5-sulphonate(DSS) was used as the internal standard (0.00 ppm
for 1H).

2.4. Gel Permeation Chromatography (GPC)

 The peak molar mass (Mpk) of pectin was determined by gel permeation
chromatography (GPC) using a Shimadzu instrument (Ultrahydrogel linear column, 7.8
x 300 mm), at room temperature, flow rate of 0.5 ml/min, polysaccharide concentration
of 0.1% (w/v) and 0.1 M NaNO3 as the solvent. A differential refractometer was used as
detector. The elution volume was corrected by the use of the internal marker ethylene
glycol at 11.25 ml. Pullulan samples (Shodex Denko) of molar mass 5.9 x 10 3, 1.18 x
104, 4.73 x 104, 2.12 x 105, and 7.88 x 105 g/mol were used as standards.

2.5. Elemental analysis – protein content

The elemental analysis of carbon, hydrogen and nitrogen of pumpkin pectin was
performed using a microanalyzer Carlo ERBA EA 1108.

2.6. Rheological Measurements (REO)

All rheological measurements were performed using an Advanced Rheometer
AR550 (DP Union). Geometry cone-and-plate (40 mm diameter and angle of 0 59'' 1')
was used for measurements of continuous flow. The viscosity of continuous flow was
determined at 25 ° C in the shear range of 1-100 s-1.

3. Results and Discussion

3.1. Infrared Spectroscopy
An overview of the IR spectrum of pectin is shown in Figure 1 the "fingerprint"
region of the spectrum (up to approx. 2000 cm -1) includes the region of 1200-1800 cm -1
as shown.
3430 cm-1

1.2

1.0
1110cm-1

0.8
1743cm-1

1010cm-1
1650cm-1
2930cm-1

0.6
Abs

1430cm-1

630cm-1

0.4

0.2

0.0

-0.2
4000 3500 3000 2500 2000 1500 1000 500
-1
wavenumber, cm

Figure 1. FTIR spectrum of pectin from pumpkin.

We can observe the region that characterizes the state of carboxylic groups (approx.
1750-1350 cm-1) (Filipov, 1992). The band at approx. 1743 cm-1 is indicative of the
stretching group C = O of non-ionized carboxylic acid (methylated or protonated). Its
ionization (formation of salt) leads to their disappearance, and the appearance of stretch
modes of COO- in approx. 1600-1650 and 1400-1450cm-1, respectively (Filipov, 1992).
The degree of methylation (DM) is defined as the amount of ester groups compared to
the total amount of acid groups and carboxylic ester and it is observed that the high
intensity of the band at 1743 cm-1 shows that the pectin obtained is of high degree of
methylation.

3.2. NMR analysis: Determination of degree of methylation (DM) by 1H

For the determination of DM, the integrals of H-5 (see Figure 2) adjacent to ester
(ICOOMe) are compared with the sum of integrals of H-5 adjacent to the ester (I COOMe) and
H-5 adjacent to the carboxylate (ICOO-).

O
COOMe

HO
H
OH
O
COOMe
H-4
O
H-1
H
HO H
H
OH

H-5 O
COO-
(COOMe)
O

HO
H
OH
H-5 O
(COO-)

Figure 2. Structure of a fragment of pectin.

Due to close proximity (or overlap) of the signals for H-1 and H-5COOMe, it is only
possible to determine the full combined to H-1 and H-5 COOMe (IH1 + ICOOMe). The value of
DM was calculated at 58% (Figure 3).

H-5
(COO-)

H-1 e H-5 H-4

(COOMe)

Figure 3. 1H NMR spectrum of pectin.

 We can also observe in the spectrum of the polysaccharide, a very large signal at
3.81 ppm related methyl groups binding to carboxyl groups of galacturonic acid.
Signals around 2.1 ppm are related to acetyl groups and were not observed for the
pectin. There are other signals related to D-galacturonic acid: H-1, 5.09 ppm; H-2, 3.76
ppm; H-3, 3.97 ppm; H-4, 4.41 ppm; H-5, 4.68 ppm (Tamaki et al., 2008).
Grasdalen (Oakenfull, 1991) pioneered the determination of DM by 1H NMR. He
performed detailed analysis of the sequence of free galacturonic acid and pectin
fragments of groups tri-and tetrameter. By using this simple method, it is possible to
characterize pectins having a specific DM, and therefore, with specific gelling
properties that are dependent on DM (Oakenfull, 1991; Axelos & Thibault, 1991).

3.3. Characterization of pectin by 13C NMR

The spectrum of 13C nuclear magnetic resonance for the sample of pectin is shown
in Figure 4. In the spectrum of the polysaccharide, a signal at about 53.5 ppm was
assigned to methyl groups attached to carboxylic groups of galacturonic acid (Keenan et
al., 1985) and a signal at 173 ppm was attributed to carboxylic groups linked to methyl
groups (Catoire et al., 1998).

Figure 4. 13C NMR spectrum for sample of pectin.

 In the spectrum of the polysaccharide, major and smaller signals can be observed
between the region of about 60.0 and 110.0 ppm. The major signs are assigned to D-
galacturonic acid while the smaller signs are assigned to D-galactose, as shown in Table
1 (Tamaki et al., 2008, Ha et al., 2005). These chemical shifts are in good agreement
with those related to the pattern of pectin studied by Tamaki et al., (2008). There are
also less intense signal assignments made by Ha et al., (2005) related to arabinan but
these signals are not very intense compared to the noise signals of the spectrum and
therefore not all signals will be shown in the table below. There are also other signals
reported by Ha et al., (2005) related to other galactan carbons that are in the same
situation.

Table 1 - Assignments to the peaks of the 13C spectrum of pectic polysaccharides.

Polymer Carbon Shift (ppm)

Galacturonan C-6 free 176
Galacturonan C-6 esther 173
Galacturonan C-6 esther 171
Arabinan C-1 106
Galacturonan C-1 101
Arabinan C-4 84
Arabinan C-4 83
Arabinan C-2 81
Galacturonan C-4 79
Galactan C-4 78
Arabinan C-3 77
Galacturonan C-3 71
Galacturonan C-5 73
Galacturonan C-2 68
Arabinan C-5 67
Galactan C-6 62
Arabinan C-5 61
Galacturonan OCH3 53.5
Rhamnose CH3 17.5
3.4. Gel permeation chromatography (GPC)
A single and wide peak with a little shoulder is present in the chromatogram of
pectin sample (Figure 5). The peak molar mass (M pk) of polysaccharide was estimated
using pullulan (a neutral polysaccharide) standard plot. Taking into account that pectin
is a polyelectrolyte, it is expected that it elutes at a lower volume than a neutral
macromolecule with the same molar mass. This is due to chain stiffening and extent, as
a consequence of electrostatic repulsion of carboxylate groups.

Pectin
refraction index

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0

elution volume (ml)

Figure 5. GPC of pectin sample.

The estimated Mpk is 9.5 x 105 g/mol. So, the molar mass of pectin is equal or lower
than this value. Published molar mass values for pectins ranges from 1.4 x 10 5 to 2.3 105
g/mol (Yoo et al., 2006; Morris et al., 2008).

3.5. Elemental analysis – protein content

The data of microanalysis of pectin are shown in Table 2. The amount of protein
obtained was 2.7%. The calculation was performed using a conversion factor equal to
5.85 (Azero & Andrade, 2002).

Table 2 - Microanalysis data for pumpkin pectin.

%C 34,24

%N 0,46

%H 6,38
 The instrument automatically determines C, H, and N by combustion of the sample,
separation of the combustion gases and measurement by thermal conductivity detector.

3.6. Rheological properties – flow and oscillatory behavior

The flow curves of pectin solutions at concentrations 1-5 % are shown in Figure 6.
Observe that the solution of 5% pectin showed pseudoplastic flow behavior. For the
flow curve of 1 and 3% pectin solutions, significantly lower values were observed in a
Newtonian flow behavior.

3.5
Pectin
3.0 1%
3%
5%
2.5
viscosity (Pa.s)

2.0

1.5

1.0

0.5

0.0
20 40 60 80 100
-1
shear rate (s )

Figure 6. Flow curves of continuous shear of 1% (■), 3% (●) and 5% (▲) pectin
solutions at 25 °C.
4. Conclusions
IR spectroscopy and 1H NMR were effective to qualify the DM of the pectin sample.
DM was 58% for the sample by 1H, and was characterized as high-methoxyl pectin. By
13
C NMR spectroscopy, different groups of known polymers were identified in the
chains of pectic polysaccharides obtained.
The molecular peak was determined by GPC as a value of 9.5 x 10 5 g / mol. Shear
measurements, with temperature variations for the pectin solution and pectin with sugar
showed the possible formation of stronger interactions between the molecular pectin
chains and sucrose around 45 ° C. The rheological study of continuous shear of pectin
solution showed Newtonian behavior.

5. Acknowledgments
The authors would like to express their thanks to CNPq for financial support, to
CENAUREMN at the Federal University of Ceará for performing the NMR analysis,
and to Prof. Edy S. Brito (Embrapa) for the help with pumpkin processing.

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