Extraction and structural determination of pectin from pumkin Cucurbita moschata

Cite this paper: Vietnam J. Chem., 2020, 58(5), 592-596 Article 
DOI: 10.1002/vjch.201900181 
592 Wiley Online Library © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH 
Extraction and structural determination of pectin from pumkin 
Cucurbita moschata 
Thanh Thi Thu Thuy
1*
, Do Thi Bien
2
, Quach Thi Minh Thu
1
, Do Thi Thanh Xuan
1
, 
Bui Van Nguyen
3
, Ngo Van Quang
1
, Ho Duc Cuong
2
1
Institute of Chemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, 
Hanoi 10000, Viet Nam 
2
Hanoi University of Science and Technology, 1 Dai Co Viet, Hai Ba Trung, Hanoi 10000, Viet Nam 
4
University of Khanh Hoa, Nha Trang, Khanh Hoa, Viet Nam 
Submitted December 2, 2019; Accepted December 10, 2019 
Abstract 
Pectin is a heterogeneous complex polysaccharide found in the primary cell wall of most cells. In this work, low-
methoxyl pectin was extracted from pumpkin (Cucurbita moschata). The obtained pectin was characterized by GPC 
FTIR and NMR spectra. The obtain results showed that structural feature of the pectin is a main chain of poly-α(1→4)-
D-galacturonic acid and occasionally interrupted by α(1→2)-linked α-L-rhamnopyranose residues and branches Galp 
is linked to GalpA at C-2 position. 
Keywords. Pectin, pumpkin, Cucurbita moschata, structure. 
1. INTRODUCTION 
Pectin is one of the most complex plant 
polysaccharides. It consists mostly of galacturonic 
acid, significant amounts of rhamnose, arabinose 
and galactose as well as 13 other different 
monosaccharides.
[1]
 Pectins are typically extracted 
from citrus fruits and apple pomace.
[2]
 In 
combination with water and some other substances, 
it can act as a thickener, gelling agent, stabilizer, 
emulsifier, cation-binding agent, etc.
[3]
 Pectins offer 
health benefits to consumers, for example, they are 
being recognized as important precursors of 
substrates for gastrointestinal functions and 
structures. Pectins rich in fiber are usually 
recommended for diabetics, because they are able to 
reduce the glycemic response and thus reduce the 
need for insulin.
[4]
 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.
[5,6]
Pumpkin, a member of Cucurbitaceae family, is 
known to contain several bioactive molecules 
including proteins, peptides, polysaccharides, sterols 
and para-aminobenzoic acid. These components can 
be found in seeds, flesh of the fruit and the leaves 
and has been regarded as a functional food.
[7]
 The 
fruit of pumpkin is one of the most important 
vegetables in traditional agricultural systems in the 
world. Cucurbita moschata was known as butternut 
squash, has been used as a traditional medicine and 
health food in Asian contries for many years, as it is 
believed to have specific therapeutic properties 
including anti-inflammatory, anti-tumour, 
cholesterol lowering, hypertensive, anti-parasitic and 
anti-diabetic effects. Moreover, it is also a great 
source of natural and low-cost pectin.
[8,9]
In this work, pectin was extracted from pumpkin 
Cucurbita moschata species. The structure of pectin 
was studied by GPC, FT-IR and NMR spectra. The 
degree of esterification (DE), which affects on the 
application of petin, was determined by FT-IR 
spectroscopy. 
2. MATERIAL AND METHOD 
2.1. Plan material 
Pumpkin (Cucurbita moschata) was purchased from 
the local market in the north part of Vietnam and 
selected for their uniformity in shape, weight and 
color. The pumpkin was peeled, seeded and sliced. 
The slices were dried under sun light until 
completely dry then were ground into powder. 
Vietnam Journal of Chemistry Thanh Thi Thu Thuy et al. 
© 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 593 
2.2. Extraction and purification of pectin from 
pumpkin 
100 ml of 0.1 M HCl solution was stabilized at 65 
°C and after that, 10 g pumpkin powder obtained 
above was added to the solution and let extracting 
for 2 hours. After precipitation and washing with 
ethanol PA (1:4 = solution:ethanol), filtration and 
freeze-drying, it was obtained 1.2 g of pectin. Crude 
pectin after purification by dialysis membranes 
remained 0.69 g of pure pectin (named PP) which 
was used for further analysis (yield calculated from 
pumpkin dry powder is 6.9 %). 
2.3. Chemical analysis 
Neutral monosaccharide compositions were 
elucidated by the method of Bilan et al.
[10]
 Alditol 
acetate derivatives were prepared by hydrolysis of 
ulvan sample in 2 M CF3COOH (TFA), at 100 °C for 
8 hr, and analyzed by 17AAFW Shimadzu GC-FID. 
Uronic acid content was determined following 
the carbazole method using D-glucuronic acid as a 
standard.
[11]
 2.4. Gel permeation chromatography (GPC) 
GPC measurement was performed on an HPLC 
Agilent 1100 with a refractive index detector RID at 
30 
o
C. The eluent was 1.2 N NaNO3 with flow rate 
of 1.0 mL/min. The sample concentration was 
1mg/ml. Pullulan was used as a standard sample 
2.5. IR spectrum 
FT-IR spectrum of the solid sample in the form of 
KBr pellets was recorded on a FTIR Affinity-1S 
Shimadzu spectrometer in the range of 400 cm
-1
 and 
4000 cm
-1
. 
2.6. NMR spectrum 
1D and 2D NMR spectra were recorded on a Bruker 
AVANCE III 500 MHz spectrometer at 70 °C. The 
concentration of polymer was 3 mg/ml in a solvent 
of D2O+1 % CD3COOD. DSS was used as an 
internal standard. The water signal was suppressed 
by a presaturation sequence at the water signal 
frequency. 
3. RESULTS AND DISCUSSION 
The weight averaged molecular weight (Mw) and the 
number averaged molecular weight (Mn) of PP 
determined by GPC. Like other native 
polysaccharides, the molecular weight distribution 
of PP is high polydisperse with Mw/Mn = 2.08 and 
Mw = 2.4 104 g/mol. Published molar mass values 
for pectins from citrus fruits and apple pomace 
ranges from 1.4 105 to 2.3 105 g/mol, So, the molar 
mass of our pumkin pectin is lower than that of 
pectin from other resources.
[12,13]
The results of chemical analysis showed that the 
pectin was composed of rhamnose, galacturonic 
acid, galactose, arabinose and xylose with a weight 
ratio of GalA:Gal:Rham:Ara:Xyl = 45.5:30.2:17.2: 
4.1:3.0. Thus, galacturonic acid was the most 
abundant in the pectin followed by galactose and 
rhamnose. 
Figure 1: FTIR spectrum of polysaccharide from 
pumpkin 
The FT-IR spectrum (figure 1) of PP showed the 
characteristic absorption bands of pectin. The strong 
and broad absorption area at 3319 cm
-1 
was 
attributed to the O-H stretching vibration due to 
intermolecular and intramolecular hydrogen bonds 
The absorption at 2927 cm
-1
 was assigned to the C-H 
stretching vibration.The absorption at 1739 cm
-1
 was 
assigned to methyl ester group. A band at 1597 cm
-1 
coupled with another band at 1406 cm
-1 
were from 
the asymmetric and symmetric stretching vibration 
of the carboxylate groups, respectively, confirming 
the presence of uronic acid. Specific band in the 
1200-1000 cm
-1 
region is dominated by ring 
vibrations overlapped with stretching vibrations of 
(C-OH) side groups and the (C-O-C) glycosidic 
band vibration, which is unique to pectin 
polysaccharide.
[14,15]
Pectins are classified as low methoxyl (LM) or 
high methoxyl (HM) according to their degree of 
esterification. The degree of esterification (DE) 
affects on the application of petin and it is defined as 
follows: (number of esterified carboxylic 
groups/number of total carboxylic groups) x 100. In 
IR spectrum, DE is inferred from the ratio of the 
area of the band at 1739 cm
-1 
(corresponding to the 
number of esterified carboxylic groups) over the 
sum of the areas of the bands at 1739 cm
-1
 and 1597 
Vietnam Journal of Chemistry Extraction and structural determination of 
 © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 594 
cm
-1 
(corresponding to the number of total 
carboxylic groups).
[14]
 For our PP sample, the DE 
was estimated from IR spectrum equal 23.3 %. 
NMR spectroscopy of PP and the literature data 
[16-18]
 were applied to identify the signals of sugar 
ring and carbon/hydrogen of the pumpkin pectin. 
In 
1
H spectrum of PP (figure 2a), a very strong 
signal at 3.81 ppm related methyl groups binding to 
carboxyl groups of galacturonic acid. The peaks at 
the high field (1.24-1.15 ppm) were assigned to the 
CH3(C6) of rhamnose. 
In 
13
C NMR spectrum (figure 2b), a signal at 
about 57.96 ppm was assigned to methyl groups 
attached to carboxylic groups of galacturonic acid 
and a signal at 172.9 ppm was attributed to 
carboxylic groups linked to methyl groups. 
Rhamnose gave the clear resonance for CH3(C6) at 
17.4 ppm. In addition, the peak at 61.23 ppm 
confirmed the presence of galactose in the pectin. 
Figure 2: (a) 
1
H NMR and (b) 
13
C NMR spectrum 
In HSQC spectrum (figure 3), GalpA gave two 
anomeric carbon/hydrogen signals at 100.1/5.35 
ppm and 98.55/5.27 ppm for 1,4-GalpA and 1,4-
GalpA connected with1,2-Rhap, respectively. 
Rhamnose gave clear resonance signals of C-1/H-
1and C-6/H-6 in HSQC spectrum (figure 3): C-1/H-
1signal for 1,2-linked Rhap appeared at 99.5/5.08 
ppm, C-6/H-6 chemical shifts at 17.40/1.15 ppm and 
17.40/1.24 ppm. The C-1/H-1 signal at 99.0/4.95 
ppm and C-6/H-6 signal at 61.23/3.83 ppm indicated 
the presence of D-Galp. 
The cross peak in HMBC spectrum (figure 4) at 
5.35/74.57 ppm (H-1 of GalpA and C-2 of Rhap) 
confirmed the linkage between Rhap and GalpA, 
which is →4)-GalpA-(1→2)-Rhap-(1→. The 1,4-
linkage between GalpA in the main chain was 
confirmed by a cross peak of H-1 and C-4 at 
5.35/77.92 ppm. The peaks at 4.95/72.4 ppm (H-1 of 
Galp and C-2 of GalpA) and at 4.95/72.1 ppm (H-1 
of Galp and C-2 of Galp) confirmed the 1,2-linkage 
of Galp in the side chain. 
Figure 3: HSQC spectrum 
Figure 4: HMBC spectrum 
Vietnam Journal of Chemistry Thanh Thi Thu Thuy et al. 
© 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 595 
Based on all the results above, structure of PP 
can be proposed (figure 5). It contains a main chain 
of poly-α(1→4)-D-galacturonic acid and interrupted 
by α(1→2)-linked α-L-rhamnopyranose residues and 
branches Galp is linked to GalpA at C-2 position. 
Figure 5: Dominant structure of pectin from pumkin 
Cucurbita moschata 
4. CONCLUSION 
The current research characterized chemical 
composition and structure of a pectin extracted from 
pumpkin Cucurbita moschata. The obtained results 
indicated that dominant structural characteristics of 
the pumpkin pectin is a main chain of poly-α(1→4)-
D-galacturonic acid interrupted by α(1→2)-linked 
rhamnopyranose residues. Branches Galp are linked 
to GalpA at C-2 position. This pectin was 
characterized as low-methoxyl pectin with DE = 
23.3. 
Acknowledgement. This research is funded by 
Vietnam Academy of Science and Technology (grant 
number VAST04.02/19-20). 
REFERENCES 
1. Ngouémazong D. E., Tengweh F. F., Fraeye I., 
Duvetter T., Cardinaels R., Loey A. V., Moldenaers 
P., Hendrickx M. Effect of de-methylesterification on 
network development and nature of Ca
2+
-pectin gels: 
Towards understanding structure-function relations 
of pectin, Food Hydrocolloids, 2012, 26, 89-98. 
2. Waldron K. W., Parker M. L., Smith A. C. Plant cell 
walls and food quality, Comprehensive Reviews in 
Food Science and Food Safety, 2003, 2, 101-119. 
3. Willats W. G. T., Knox J. P., Mikkelsen D. J. Pectin: 
new insights into an old polymer are starting to gel, 
Trends in Food Science & Technology, 2006, 17, 97-
104. 
4. Guillon F., Champ M. Structural and physical 
properties of dietary fibres, and consequences of 
processing on human physiology, Food Research 
International, 2000, 33, 233-245. 
5. Libo Wang, Fangcheng Liu, Aoxue Wang, Zeyuan 
Yu, Yaqin Xu, Yu Yang. Purification, 
characterization and bioactivity determination of a 
novel polysaccharide from pumpkin (Cucurbita 
moschata) seeds, Food Hydrocolloids, 2017, 66, 357-
364. 
6. Hemei Song, Zengxian Sun. Hypolipidaemic and 
hypoglycaemic properties of pumpkin 
polysaccharides, Biotech., 2017, 7, 159-204. 
7. Adams G. G., Imran S., Wang S., Mohammad A., 
Kok, M. S., Gray D. A., Channell G. A., Harding S. 
E. Extraction, isolation and characterisation of oil 
bodies from pumpkin seeds for therapeutic use, Food 
Chemistry, 2012, 134(4),1919-1925. 
8. Zhu Hong-Yan, Chen Guang-Tong, Meng Guo-
Liang, Xu Ji-Liang. Characterization of pumpkin 
polysaccharides and protective effects on 
streptozotocin-damaged islet cells, Chinese Journal 
of Natural Medicines, 2015, 13(3), 199-207. 
9. Shuang Wang, Aoxue Lu, Lu Zhang, Meng 
Shen, Tian Xu, Wangyang Zhan, Hui Jin, 
Yongjun Zhang, Weimin Wang. Extraction and 
purification of pumpkin polysaccharides and their 
hypoglycemic effect, International Journal of 
Biological Macromolecules, 2017, 98, 182-187. 
10. M. I. Bilan, A. A. Grachev, N. E. Ustuzhamina, A. S. 
Shashkov, N. E. Nifantiev, A. I. Usov. Structure of a 
fucoidan from the brown seaweed Fucus evanescens 
C. Ag. Carbohydr. Res., 2002, 337, 719-730. 
11. T. Bitter and H. M. Muir. A modified uronic acid 
carbazole reaction, Anal. Biochem., 1962, 4, 330-334. 
12. Morris G. A., Garcial de al Torre J., Ortega A., 
Castile J., Smith A., Harding S. E. Molecular 
flexibility of citrus pectins by combined 
sedimentation and viscosity analysis, Food 
Hydrocolloids, 2008, 22, 1435-1442. 
13. Yoo S. H., Fishman M. L., Hotchkiss Jr A. T. & Lee 
H. G. Viscometric behavior of high-methoxy and 
low-methoxy pectin solutions, Food Hydrocolloids, 
2006, 20, 62-67. 
14. Chylinska M., Szymanska-Chargot M., Zdune A. FT-
IR and FT-Raman characterization of non-cellulosic 
polysaccharides fractions isolated from plant cell 
wall, Carbohydrate Polymers, 2016, 154, 48-54. 
15. Filipov M. P. Practical infrared spectroscopy of 
pectic substances, Food Hydrocolloids, 1992, 6, 115-
118. 
16. Jing Zhao, Fuming Zhang, Xinyue Liu, Kalib St. 
Ange, Anqiang Zhang, Quanhong Li, Robert J. 
Linhardt. Isolation of a lectin binding 
rhamnogalacturonan-I containing pectic 
Vietnam Journal of Chemistry Extraction and structural determination of 
 © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 596 
polysaccharide from pumpkin, Carbohydrate 
Polymers, 2017, 163, 330-336. 
17. M. V. Marcon, P. I. B. Carneiro, G. Wosiacki, E. 
Beleski-Carneiro, Pectins from Apple Pomace - 
Characterization by 
13
C- and 
1
H-NMR 
Spectroscopy, Ann. Magn. Reson., 2005, 4(3), 56-63. 
18. José RR. de Souza, Jeanny S. Maciel, Edy S. Brito, et 
al. Pectin from Cucurbita moschata Pumpkin 
Mesocarp. Macromol Ind J., 2017, 12(3), 110-121. 
Corresponding author: Thanh Thi Thu Thuy 
Institute of Chemistry, Vietnam Academy of Science and Technology 
 18, Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam 
 E-mail: thuyttt@ich.vast.vn.