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Int. J. Mol. Sci. 2022, 23, 13175. https://doi.org/10.3390/ijms232113175 www.mdpi.com/journal/ijms
Article
Polysaccharides of Salsola passerina: Extraction, Structural
Characterization and Antioxidant Activity
Victoria Golovchenko
1
, Sergey Popov
1,
*, Vasily Smirnov
1
, Victor Khlopin
1
, Fedor Vityazev
1
,
Shinen Naranmandakh
2
, Andrey S. Dmitrenok
3
and Alexander S. Shashkov
3
1
Institute of Physiology of Federal Research Centre “Komi Science Centre of the Urals Branch of the Russian
Academy of Sciences”, 50 Pervomaiskaya Str., 167982 Syktyvkar, Russia
2
School of Arts and Sciences, National University of Mongolia, Baga Toiruu 47, Ulaanbaatar 14201, Mongolia
3
N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 47, Leninsky Prospect,
119991 Moscow, Russia
* Correspondence: s.v.popov@inbox.ru; Tel./Fax: +78-21-224-1001
Abstract: The above-ground part of the Salsola passerine was found to contain ~13% (w/w) of poly-
saccharides extractable with water and aqueous solutions of ammonium oxalate and sodium car-
bonate. The fractions extracted with aqueous sodium carbonate solutions had the highest yield. The
polysaccharides of majority fractions are characterized by similar monosaccharide composition;
namely, galacturonic acid and arabinose residues are the principal components of their carbohy-
drate chains. The present study focused on the determination of antioxidant activity of the extracted
polysaccharide fractions and elucidation of the structure of polysaccharides using nuclear magnetic
resonance (NMR) spectroscopy. Homogalacturonan (HG), consisting of 1,4-linked residues of α-D-
galactopyranosyluronic acid (GalpA), rhamnogalacturonan-I (RG-I), which contains a diglycosyl
repeating unit with a strictly alternating sequence of 1,4-linked D-GalpA and 1,2-linked L-rhamno-
pyranose (Rhap) residues in the backbone, and arabinan, were identified as the structural units of
the obtained polysaccharides. HMBC spectra showed that arabinan consisted of alternating regions
formed by 3,5-substituted and 1,5-linked arabinofuranose residues, but there was no alternation of
these residues in the arabinan structure. Polysaccharide fractions scavenged the 1,1-diphenyl-2-pic-
rylhydrazyl (DPPH) radical at 0.2–1.8 mg/mL. The correlation analysis showed that the DPPH scav-
enging activity of polysaccharide fractions was associated with the content of phenolic compounds
(PCs).
Keywords: pectin; polysaccharides; NMR spectroscopy; arabinan; galacturonan (HG); rham-
nogalacturonan-I (RG-I); DPPH radical scavenging; phenolic compounds (PCs)
1. Introduction
Plants of the Amaranthaceae family are associated with noxious garden weeds and
ruderal plants. Perennial or annual herbaceous flowering plants of various species of the
Сhenopodium genus, known as the goosefoots, grow almost everywhere in the world and
are among the most common cosmopolitan weeds. However, this family also contains
valuable, useful plants. The genus of halophyte plant Salsola L. is one of the largest in the
family Amaranthaceae. Plants of this genus are characterized by rapid regeneration, the
ability to grow large biomass, resistance to high environmental temperature, tolerance to
soil salinity and to extended drought conditions. Therefore, the role of plants of this genus
is great in saline, arid regions of various countries with developed distant pastures. Over
150 species of the genus Salsola L., including annual semi-dwarf and dwarf shrubs and
woody trees, are distributed in arid and semi-arid regions of the Middle East, Asia, Eu-
rope and Africa [1].
Citation: Golovchenko, V.; Popov,
S.; Smirnov, V.; Khlopin, V.;
Vityazev, F.; Naranmandakh, S.;
Dmitrenok, A.; Shashkov, A.
Polysaccharides of Salsola passerina:
Extraction, Structural
Characterization and Antioxidant
Activity. Int. J. Mol. Sci. 2022, 23,
13175. https://doi.org/10.3390/
ijms232113175
Academic Editors: Claudiu T.
Supuran and Clemente Capasso
Received: 3 October 2022
Accepted: 25 October 2022
Published: 29 October 2022
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Copyright: © 2022 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://cre-
ativecommons.org/licenses/by/4.0/).
Int. J. Mol. Sci. 2022, 23, 13175 2 of 18
Extracts and decoctions of plants of this genus are used in world folk medicine to
treat bacterial and viral, cardiovascular, skin diseases, coughs and flu, and in cosmetics
[2]. Previously, several biologically active compounds were isolated from different types
of Salsola: flavonoids, phenolic acids, saponins, triterpenes, lignans, sterols, fatty acids,
alcohols, alkaloids, coumarins, as well as nitrogenous cyanogenic, isoprenoid and sul-
phur-containing compounds [3–7]. Most of these studies focus on phenolic compounds
(PCs), which attract a lot of attention because of the great antioxidant activity.
Pectiс polysaccharides are a family of complex polysaccharides present in all plant
primary cell walls [8]. The irregular block sugar chains and various macromolecular seg-
ments of the linear and ramified regions characterize the complicated structure of pectic
polysaccharides. The final model of the primary structure of the pectin macromolecule
and the model of its biosynthesis have not been developed to date. The studies spanning
the last 100 years have made it possible to establish the structure but not interlinking of
the main domains of pectins. Homogalacturonan (HG) forms linear regions and the back-
bone of the substituted galacturonans (rhamnogalacturonan II, xylogalacturonan and api-
ogalacturonan). Rhamnogalacturonan (RG) forms the backbone of the RG-I, in which the
arabinans, the galactans and/or the arabinogalactans form the side chains [9].
Pectin is resistant in the human stomach and small intestine and has to be fermented
in the large bowel by colonic bacteria. Therefore, pectin belongs to dietary fibers and pos-
sesses good prebiotic properties [10]. Pectin is highly valued as a functional food ingredi-
ent because of hypolipidemic, hypoglycemic, satiating, antibacterial and antitumor effects
[11]. In particular, pectic polysaccharides from various sources show antioxidant activity
[12–16]. Various authors suggest that galacturonic acid (GalA) scavenges free radicals
[17,18]; therefore, the HG domain mediates antioxidant activity [18,19]. Moreover, the an-
tioxidant activity of pectins may also be associated with RG-I [20]. Additionally, the side
chains of pectin may be feruloylated in certain cases, which might explain its considerable
antioxidant potential [21].
Based on the wide distribution and ability to produce a large biomass, it is of interest
to isolate pectin polysaccharides from plants of the genus Salsola L., since pectins make
up the bulk of the plant cell and are its biologically active compounds. In this study, we
assume pectins can be biologically active substances of saltwort Salsola passerine, which
remains unexplored both in terms of polysaccharide composition and low-weight molec-
ular phytochemicals and polyphenols. S. passerina, like other plant species of the genus
Salsola, grows effectively in salt soil areas and can gain high biomass in semi-arid desert
conditions, providing food for camels, sheep, goats, cattle. S. passerina is one of the most
common species in Mongolia.
The present study aims to determine the structure and antioxidant activity of poly-
saccharides isolated from the semi-shrubs of Salsola gemmascens ssp. passerina (Bunge)
Botschantz (main name—Salsola passerina Bunge) and to identify the component respon-
sible for antioxidant activity.
2. Results and Discussion
2.1. Isolation and Characterization of Polysaccharides
Five extractants—cold water, hot water, water acidified to pH 2.0, 0.7% aqueous so-
lutions of ammonium oxalate and 0.5% aqueous solutions of sodium carbonate—were
successively used to extract polysaccharides from S. passerine (Figure 1). We performed
the extraction with each extractant out until there were no sugars in the corresponding
extract. As a result, eleven polysaccharide fractions were obtained. Fractions extracted
with the sodium carbonate solution had the highest yield and those extracted with cold
water—the lowest. Polysaccharides isolated with cold water containing a significant
amount of Man, GalA and Ara residues were the principal components of polysaccharides
extracted by other extractants.
Int. J. Mol. Sci. 2022, 23, 13175 3 of 18
HW1, AC, OK1, SO1 were fractionated using a DEAE-cellulose (OH-) column. As a
result, three polysaccharide fractions were obtained from each fraction by elution with
0.01, 0.1 and 0.2 M NaCl. The polysaccharides of the obtained fractions had a similar mon-
osaccharide composition. The GalA and Ara residues were the principal components of
their carbohydrate chains. The exception was the HW1-1 fraction whose polysaccharides,
similar to the polysaccharides of fractions extracted by cold water, were characterized by
a significant content of Man and Glc residues (Table 1). This indicates that the extraction
with cold water was incomplete, and a small part of the polysaccharides was extracted
with hot water in the next step.
All parent fractions included protein components. Fractions extracted with sodium
carbonate included the largest amount of protein (up to 39%). The largest part of the pro-
teins was not connected to polysaccharides because it was removed during separation on
DEAE-cellulose. However, a small part of the co-eluted protein seemed to be connected
to polysaccharides.
Figure 1. Scheme of isolation of polysaccharides from the S. passerine.
Table 1. Chemical characteristics of pectic polysaccharides from the S. passerine.
Fractions Yield, % Content, % Mw,
kg/mol
Mn,
kg/mol PDI
GalA c Rha
c Ara
c Xyl
c Man
c Glc
c Gal
c TS
d Protein
d PCs
d
CW1 0.40
a 17.46 4.33 13.72 15.45 22.45 13.82 12.77 47.44 2.3 0.8 43.59 15.62 2.79
CW2 0.14
a 31.18 5.81 8.39 10.43 24.93 7.23 12.03 46.70 6.9 1.0 63.09 12.74 4.95
CW3 0.09
a 26.14 6.92 16.13 8.12 25.35 4.67 12.67 38.33 3.7 1.0 52.68 14.04 3.75
HW1 0.88
a 47.40 4.79 11.86 9.02 11.77 7.51 7.65 64.02 3.7 0.7 96.07 27.01 3.56
HW2 0.67
a 50.44 3.62 31.59 3.26 4.12 2.63 4.35 70.27 1.9 0.7 71.44 16.02 4.46
HW1-1 6.93
b 10.12 0.59 23.12 1.03 45.41 14.07 5.65 45.00 0 nd 34.47 14.75 2.34
HW1-2 31.30
b 66.46 2.09 14.71 8.78 0.83 0.57 6.56 56.35 0 nd 56.68 20.96 2.70
HW1-3 17.40
b 79.07 4.60 4.91 5.68 0.42 0.76 4.56 76.46 0.17 nd 124.30 50.74 2.45
AC 1.82
a 47.61 3.99 31.87 6.91 2.18 1.92 5.52 52.05 2.0 0.4 55.31 13.56 4.08
AC-1 17.22
b 40.02 6.90 14.75 10.90 3.12 3.81 20.50 59.25 0 nd - - -
AC-2 50.92
b 58.46 8.28 10.50 12.83 1.51 1.64 6.77 46.92 0 nd 80.06 34.55 2.32
Int. J. Mol. Sci. 2022, 23, 13175 4 of 18
OK1 1.04
a 56.99 4.65 23.00 6.51 2.03 2.11 4.71 76.01 1.8 0.6 87.52 22.38 3.91
OK2 0.30
a 59.43 4.52 25.62 2.05 1.93 3.05 3.41 64.17 2.9 0.6 107.17 25.88 4.14
OK3 0.45
a 51.78 5.20 27.80 5.09 2.75 2.97 4.41 75.14 2.9 0.6 124.26 27.95 4.45
OK1-1 9.44
b 32.51 10.27 47.13 0.00 1.43 1.94 6.73 43.46 2.25 nd 293.36 40.02 7.33
OK1-2 25.97
b 29.00 6.00 55.24 1.41 0.18 1.46 6.71 81.05 0 nd 75.11 21.65 3.47
OK1-3 29.98
b 77.92 3.64 11.72 1.53 0.00 0.73 4.47 85.83 0 nd 92.28 44.98 2.05
SO1 7.32
a 55.48 6.84 19.42 4.31 0.00 1.83 12.12 57.84 15.6 1.2 259.19 58.09 4.46
SO2 0.12
a 52.51 7.46 24.72 1.93 0.00 3.39 10.00 62.91 18.2 1.1 239.29 30.43 7.86
SO1-1 33.78
b 21.41 8.09 60.16 1.70 0.03 0.99 7.63 88.51 10.90 0.3 444.41 319.55 1.39
SO1-2 27.97
b 33.72 4.18 47.40 3.94 0.29 1.65 8.82 77.84 0.02 0.3 93.30 33.35 2.77
SO1-3 7.88
b 60.71 6.65 19.54 5.63 0.23 0.75 6.50 68.31 5.08 0.4 81.93 40.37 2.03
a—air-dried; b—in relation to the sample applied to the column; c—data were calculated as molar %;
d—data were calculated as mass %; TS—total sugar content; PCs—phenolic compounds; nd—not
determined; number (Mn) and weight (Mw) average relative molar masses and polydispersity in-
dices (PDI) were measured using pullulan standards; the data are presented in the table as a mean
of three experiments.
2.2. NMR Spectroscopic Study
Information about the structure of the main polysaccharides from S. passerines was
obtained by a combined analysis of the NMR spectra of SO1-1, SO1-2 and SO1-3. The
NMR spectra of the three samples were similar (Figures 2–5). The 13C NMR spectra of the
samples (Figure 2) were assigned using 1H, 13C heteronuclear single quantum coherence
spectroscopy (HSQC) spectra. Analysis of the 1H, 13C HSQC spectra (Figures 3–5, Table 2)
revealed substitutions in the monosaccharide residues based on the comparison of their
13C chemical shifts with those of the parent pyranoses and furanoses [22] and considering
the glycosylation effects in the 13C NMR spectra of the carbohydrates [23,24], as well as
data from our previous NMR studies of pectins [25]. The correlated spectroscopy (COSY),
total correlation spectroscopy (TOCSY), rotating frame Overhauser effect spectroscopy
(ROESY) and heteronuclear multiple bond correlation (HMBC) spectra revealed residues
of α-D-galactopiranoside uronic acid (GA in Table 2), α-L-rhamnopyranose (R) and α-
arabinofuranose (A) in all three samples. Conclusions regarding monosaccharide compo-
sition, ring size and anomeric configuration were drawn based on the comparison of vis-
ible coupling constants and chemical shifts of the sugar residues and corresponding py-
ranoses [26,27] and furanoses [28,29].
Int. J. Mol. Sci. 2022, 23, 13175 5 of 18
Figure 2. 13C spectra of the SO1-1 (a), SO1-2 (b) and SO1-3 (c).
Figure 3. Parts of 1H, 13C HSQC spectrum of the SO1-1. The corresponding parts of the 1H and 13C
NMR spectra are shown along the X and Y axes, respectively. Arabic numerals refer to the carbon
atoms in the residues, as designated in Table 2.
Int. J. Mol. Sci. 2022, 23, 13175 6 of 18
Figure 4. Parts of 1H, 13C HSQC spectrum of the SO1-2. The corresponding parts of the 1H and 13C
NMR spectra are shown along the X and Y axes, respectively. Arabic numerals refer to the carbon
atoms in the residues, as designated in Table 2.
Figure 5. Parts of 1H, 13C HSQC spectrum of the SO1-3. The corresponding parts of the 1H and 13C
NMR spectra are shown along the X and Y axes, respectively. Arabic numerals refer to the carbon
atoms in the residues, as designated in Table 2.
Int. J. Mol. Sci. 2022, 23, 13175 7 of 18
Table 2. Chemical shifts of the signals in the 1H and 13С NMR spectra of the SO1-1, SO1-2 and SO1-
3 (323 K, D2O, TSP, δH 0.0, δC -1.6).
Residue
13С NMR Chemical Shifts (δС) and 1Н (δН, Italic), ppm
C-1
H-1
C-2
H-2
C-3
H-3
C-4
H-4
C-5
H-5,5′
C-6
H-6
→4)-α-D-GalpA-(1→
GA
100.3
5.08
69.6
3.77
70.2
4.00
79.3
4.44
72.6
4.75 176.1
→2)-α-Rhap-(1→4
R
99.5
5.26
77.6
4.12
68.8
3.89
74.1
3.40
69.6
3.82
17.90
1.25
→2,4)-α-Rhap-(1→4
R′
99.5
5.26
77.2
4.12
71.4
4.10
81.8
3.70
70.9
3.90
18.1
1.31
α-L-Araf-(1→3
AT
108.5
5.16
82.8
4.14
77.9
3.97
85.2
4.05
62.5
3.83; 3.73
→5)-α-L-Araf-(1→
AL
108.5
5.09
82.2
4.14
78.2
4.01
83.7
4.21
68.3
3.89; 3.81
→3,5)-α-L-Araf-(1→
AS
108.8
5.12
80.5
4.29
83.8
4.10
82.9
4.30
67.9
3.94; 3.84
TSP—trimethylsilylpropanoic acid.
The occurrence of 1,2-linked (label R) and 2,4-substituted (label R′) α-L-rhamnose
residues in polysaccharides was confirmed by cross peaks at 1.25/17.9 ppm and 1.31/18.1
ppm in the 1H, 13C HSQC spectra (Figures 3–5) and HMBC spectra (Figures 6 and S2). The
ROESY spectrum of SO1-3 (Figure 7) included an inter-residue correlation peak of the
anomeric proton of Rha residues and H-4 of GalA residues at δH/H 5.26/4.44 ppm, confirm-
ing the RG-I regions in polysaccharides.
Figure 6. Part of 1H, 1H ROESY spectrum of the SO1-1. The corresponding part of the 1H NMR
spectrum is shown along the axes. Slashes refer to inter-residue correlation peaks, as designated in
Table 2.
Int. J. Mol. Sci. 2022, 23, 13175 8 of 18
Figure 7. Part of 1H, 13C HMBC spectrum of the SO1-1. The corresponding parts of the 1H and 13C
NMR spectra are shown along the X and Y axes, respectively. Arabic numerals before slash refer to
the protons, and those after slash refer to carbon atoms in the corresponding residues.
Three intense signals at δH 5.16, 5.12 and 5.09 ppm belonging to terminal nonreduc-
tion arabinose residues (label AT), 3,5-substituted arabinose residues (label AS) and 1,5-
linked arabinose residues (label AL), respectively, were found in the anomeric region of
1H NMR spectra of SO1-1 and SO1-2 (Figure S1). In the anomeric region, the 1H NMR
spectrum of SO1-3 signals of 3,5-Ara and 1,5-Ara overlaps the intense signal belonging to
the 1,4-linked D-galacturonic acid residues (label GA) at δH 5.08 ppm.
The resonance of C-6 at δC 176.0 ppm indicated the predominance of non-methyl-
esterified α-1,4-linked D-GalA residues in the structure of polysaccharides SO1-3 (Figure
2c), but the signal of low intensity at δH/C 3.86/54.4 ppm confirmed that some GalA residues
were methyl esterified.
The following inter-residue correlations H-1(glycosylating GA)/H-4(glycosylated
GA) at δH/H 5.08/4.44 ppm in the ROESY spectrum of SO1-3 (Figure 6) and H-4(glycosyl-
ated GA)/C-1(glycosylating GA) at δH/C 4.44/100.3 ppm in the HMBC spectrum of SO1-3
(Figure S2) indicated on the galacturonan (HG) in the studied polysaccharides.
The correlation peak at δН/С 2.09/21.56 ppm in the HSQC spectrum of SO1-3 (Figure
5) confirmed the O-acetylated residues in the structure of polysaccharides from S. passer-
ina. No clear evidence was obtained for the attachment of the O-acetyl group to specific
residues, since the intensity of their signals was low. Rha and GalA residues may be acetyl
esterified [30]. The signals of O-acetyl groups are present only in the spectrum of sample
SO1-3, which included polysaccharides with a high content of GalA, which may indirectly
indicate the O-acetylation of GalA residues.
The resonance of C-6 at δC 176.0 ppm indicated the predominance of non-methyl-
esterified α-1,4-linked D-GalA residues in the structure of polysaccharides (Figure 2).
The sequence of Ara residues in the repeating units was determined using the H/C
correlations in the HMBC spectra and the H/H correlations in the ROESY spectra.
The inter-residue correlation peaks—H-1(glycosylating AS)/C-5(glycosylated AS) and
H-1(glycosylating AL)/C-5(glycosylated AL) in HMBC spectra (Figures 7 and S2) and H-
Int. J. Mol. Sci. 2022, 23, 13175 9 of 18
1(AT)/H-3(AS) in ROESY spectra (Figures 6, S3 and S4)—showed the side chains formed
by single arabinose residues.
The average length of branches in the arabinan side chains, derived from the relative
amounts of terminal and branched arabinose residues, was equal to one, confirming that
the branches in the arabinan side chains comprised a single arabinose residue.
The ratio of AT, AS and AL was approximately 1:1:4 in the 1H NMR spectrum of SO1-
1 and indicated that the lengths of the linear regions were four times the lengths of the
branched regions.
The ratio of Ara residues in the spectra of SO1-2 and SO1-3 was not determined be-
cause of the overlap of the signal of the anomeric proton AL with the signal of the anomeric
proton of GA in the 1H NMR spectrum (Figure S1).
Clear inter-residue correlation peaks in the HMBC spectra (Figures 7 and S2) at δH/C
5.09/68.3 and 5.12/67.9 ppm for H-1(glycosylating AL)/C-5(glycosylated AL) and H-1(gly-
cosylating AS)/C-5(glycosylated AS) mainly indicated that 3,5-Ara substituted 3,5-Ara, and
1,5-Ara substituted 1,5-Ara.
A possible structure of the repeating unit of the arabinan chain of polysaccharides
from S. passerines is proposed below (Scheme), where the lengths of the structural regions
are arbitrary.
Scheme. A possible structure of the repeating unit of the arabinan from S. passerines.
In addition, the following low-intensity peaks were found in the anomeric region of
the 1H, 13C HSQC spectra: at δC/H 98.78/5.26 ppm belonging to Rha residues in the RG-I
regions, at δC/H 103.40/4.54, 103.80/4.49, 104.68/4.48 ppm belonging to Gal residues, respec-
tively (Figures 3 and 4).
The occurrence of 1,2-linked (label R) and 2,4-substituted (label R’) α-L-rhamnose
residues in polysaccharides was confirmed by the cross peaks at 1.25/17.9 ppm and
1.31/18.1 ppm in the 1H, 13C HSQC spectra (Figures 3–5) and the HMBC spectra (Figures
7 and S2). The ROESY spectrum of SO1-3 (Figure 6) included an inter-residue correlation
peak of the anomeric proton of Rha residues, and H-4 of the GalA residues at δH/H
5.26/4.44 ppm confirmed the RG-I regions in polysaccharides.
Thus, three structural domains were identified in the polysaccharides isolated from
S. passerine: arabinan, HG and RG-I. Considering the intensity of signals in the NMR spec-
trum, SO1-1 is dominated by the arabinan units, while SO1-3 is dominated by the galac-
turonan units. In the present study, no links between them were established. Nonetheless,
it is possible that they represent domains of a complex pectin macromolecule.
Arabinans have been found in the cell wall of several plants and are believed to form
RG-I side chains [31]. However, most of the evidence is based on co-extraction and/or co-
elution of RG-I and arabinans [32–34]. Only a few studies found that the L-Ara residues
are covalently attached to rhamnose residues at the O-4 position of the RG-I backbone
[35,36].
1,5-linked residues of α-L-arabinofuranose form both the backbone and the side
chains of most of the arabinans studied [37]. Backbone residues are usually substituted at
O-2 and/or O-3 and/or at both positions, with O-3 substitutions predominating [32]. How-
ever, several arabinans with a high percentage of substitution at the O-2 position have
also been found [38,39]. Other structures of arabinans have also been described. For ex-
ample, in arabinans, both the furanose and pyranose forms of arabinose were found [40].
Int. J. Mol. Sci. 2022, 23, 13175 10 of 18
Terminal β-arabinofuranose residues may glycosylate 1,5-linked α-arabinofuranose resi-
dues of the backbone at position O-5 [41]. Various degrees of branching have been found,
including single, linear and branched oligomeric and polymeric chains, with different
linkage types. The almost linear 1,5-arabinan associated with protein was isolated from
red wine [42]. The arabinans in pectins often have single substituted side chains [32,43].
Arabinans from soybean [44], apple [45], the inner bark of Norway spruce [46] were found
to have a highly branched structure. Arabinan-rich pectins, which constituted 50% of the
total pectic polysaccharides, have been obtained from pea Pisum sativum L. [47].
The roles of arabinans in plant cell walls remain unclear. It was established that arabi-
nans can be substituted by terminal phenolic esters, particularly feruloyl or coumaroyl
esters. Ferulic acid groups may be ester linked to O-2 of the arabinose residues [48,49].
Feruloyl esters may determine guard cell wall flexibility by providing the cross-links be-
tween arabinans and other wall polymers; this testifies a unique role for arabinans in de-
termining the physical and functional properties of guard cell walls [50].
2.3. DPPH Radical-Scavenging Activity
Polysaccharide fractions from S. passerine scavenged the DPPH radical at concentra-
tions of 0.2–1.8 mg/mL. The half-maximal DPPH inhibitory concentration (IC50) of them is
given in Table 3. CW2, CW3, SO1 and SO2 demonstrated the highest activity, which ex-
ceeded 2.51–2.96 times that of commercial apple pectin (AP) activity. CW2, CW3, SO1 and
SO2 scavenged 67, 69, 55 and 67% of DPPH radicals at a concentration of 1 mg/mL. Other
fractions were less effective and scavenged only 31–48% of DPPH radicals at a concentra-
tion of 1 mg/mL.
Table 3. DPPH scavenging effect of polysaccharide fractions from S. passerine.
Pectin IC50 (mg/mL)
CW1 1.14 ± 0.02 cd
CW2 0.69 ± 0.07 bc
CW3 0.66 ± 0.07 b
HW1 1.47 ± 0.22 d
HW2 1.58 ± 0.24 d
AC1 2.20 ± 0.41 e
OK1 1.63 ± 0.26 d
OK2 1.84 ± 0.03 de
OK3 1.64 ± 0.11 d
SO1 0.78 ± 0.06 bc
SO2 0.68 ± 0.08 b
AP 1.96 ± 0.36 de
Trolox 0.006 ± 0.001 a
Data are presented as the mean ± SD of three independent experiments. Different capital letters (a–
e) show the significant differences (p < 0.05, LSD test).
The DPPH radical scavenging assay is widely used to evaluate the antioxidant prop-
erty of plant polysaccharides. The activity of CW2, CW3, SO1 and SO2 seems to be com-
parable to that of polysaccharides from cantaloupe rinds [51], hawthorn wine pomace
[52], fruit bodies of Tremella fuciformis [53] and apple pomace [54]. It should be noted that
some other polysaccharides demonstrated the same level of DPPH scavenging activity at
lower concentrations. These include pectins from Chaenomeles sinensis fruits [55], Epilobium
angustifolium L. [56], Thymus quinquecostatus Celak. leaves [57], Gardenia jasminoides J. Ellis
flowers [58] and Ziziphus jujuba cv. Muzao [59].
The DPPH radical scavenging activity of the fractions obtained by DEAE-cellulose
elution was compared with the activity of the parent fraction SO1 (Figure 8). The polysac-
charides SO1-1, SO1-2 and SO1-3 obtained were less effective (p < 0.05) than the parent
Int. J. Mol. Sci. 2022, 23, 13175 11 of 18
fraction SO1, exhibiting IC50 equal to 3.64 ± 0.18, 5.70 ± 0.92 and 5.70 ± 1.30 mg/mL, respec-
tively.
Figure 8. The DPPH radical scavenging activity of SO1 and of its components obtained by separa-
tion on DEAE-cellulose column. Data are presented as the mean ± SD of three independent experi-
ments.
On the basis of the yield and content of PCs in SO1-1, SO1-2, SO1-3 and SO1, most
of the PCs providing antioxidant activity were removed by anion exchange chromatog-
raphy, assuming that they were not bound to the polysaccharide chains. The sum contents
of PCs in polysaccharides SO1-1, SO1-2, SO1-3 included about 18% from the content of
PCs in the parent fraction SO1. It was detected that three fractions obtained on DEAE-
cellulose provided only 24% of the DPPH radical scavenging activity of SO1, although
they represented about 70% of the parent pectin (Table 1). This suggests that the antioxi-
dant activity of SO1 was mainly provided by the associated PCs but not by polysaccha-
rides.
The relationship between the chemical characteristics of polysaccharides and DPPH
scavenging ability was further investigated using correlation analysis. The total content
of sugars and the (Ara + Gal)/Xyl ratio correlated negatively, whereas the content of PCs,
Gal and Man, as well as PDI correlated positively with DPPH scavenging activity (Table
4).
Table 4. The Pearson correlation coefficients between the DPPH scavenging activity and the chem-
ical characteristics of polysaccharide fractions from S. passerine (n = 14).
Second Variable R p Second Variable R p
PCs −0.89 0.000 Protein −0.51 0.062
Gal −0.59 0.026 Glc −0.46 0.098
PDI −0.58 0.029 Rha −0.34 0.236
Man −0.55 0.040 Xyl/GalA −0.30 0.290
(Ara + Gal)/Rha 0.58 0.028 Xyl −0.27 0.342
Total sugar 0.67 0.009 Rha/GalA −0.28 0.335
Mw −0.11 0.713
Mn 0.12 0.671
Ara/Xyl 0.22 0.455
GalA 0.23 0.430
GalA/NM 0.25 0.384
Int. J. Mol. Sci. 2022, 23, 13175 12 of 18
GalA-Rha 0.26 0.377
2Rha + Ara + Gal 0.30 0.295
Ara 0.52 0.059
We tested the five regression models, subsequently removing the less significant fac-
tors (according to the p-value). The linear regression, including the contents of PCs and
Man as independent variables, resulted in the best model for prediction (adj. R2 = 0.82, p =
0.000) (Table 5). The content of PCs was the only factor contributing significantly to DPPH
scavenging activity (p = 0.000, β = 0.79).
Table 5. Multiple regression analysis of the DPPH scavenging activity.
Variable β Standard
Error of β
Parameter
Estimate
Standard
Error p-Value
Dependent Independent
DPPH scavenging
activity
PCs −0.79 0.13 −2.02 0.32 0.000
Man −0.26 0.13 −0.02 0.01 0.062
Regression results: R2 = 0.847, adjusted R2 = 0.819, F2.11 = 30.438, p < 0.000, Standard estimate error =
0.31.
Thus, the correlation analysis showed that the DPPH scavenging activity of the sam-
ple from S. passerine is associated with the content of PCs. This is consistent with the re-
sults of Ref [54], whose authors evaluated the activity of apple pectins, and our previous
study on fireweed pectins [56]. It is known that PCs may bind covalently to the side chains
of RG I through the Ara and Gal residues and may be involved in the cross-linking of
macromolecules [60].
It was shown that the removal of PCs from polysaccharides reduces the antioxidant
activity but does not completely abolish it [60]. Several authors suggest that the antioxi-
dant activity of pectins may be due to the hydroxyl and carboxyl groups of GalA residues
[52,61]. Previously, we showed that the antioxidant activity of fireweed pectins is partly
related to the xylogalacturonan chains [56]. However, in the present study, we failed to
find the polysaccharide chains responsible for the DPPH radical scavenging activity of
Salsola pectins. The small sample size (n = 14), which determines the statistical power of
multiple regression [62], may be the reason we failed to identify the polysaccharide chains
that contribute to the antioxidant activity of Salsola pectins.
3. Materials and Methods
3.1. Materials
Biological material: plant material, consisting of yellow-green annual branches with
spherical dwarf leaves, was collected in August 2019 from the semi-shrubs of Salsola gem-
mascens ssp. passerina (Bunge) Botschantz. = Caroxylon passerinum (Bunge) Akhani et E.H.
Roalson (main name—Salsola passerina Bunge) growing in Mandal-ovoo soum, Ömnö-
Govi province, Mongolia. They were identified by Prof. B.Oyuntsetseg (School of Arts and
Sciences, National University of Mongolia). The plant material was washed with distilled
water and dried with filter paper.
The chemicals used are described in the Supplementary (Appendix A).
3.2. Isolation of Polysaccharides of S. passerina
Polysaccharides from the plant material were sequentially extracted, as described be-
low; the extraction scheme is shown in Figure 1. At each stage, an exhaustive extraction
of polysaccharides was carried out until the absence of reaction of the extract to the car-
bohydrate; the extraction mixtures were mixed in a mechanical stirrer.
Freshly picked plant material (234 g) was milled in a blender, distilled water (1 L)
was added, and the resulting mixture was stirred in a mechanical mixer at 20 °C for 3 h.
The mixture was centrifuged, and the residue of the plant material was treated again; the
Int. J. Mol. Sci. 2022, 23, 13175 13 of 18
treatment was repeated three times. In the next stage, polysaccharides from the residues
of plant materials were extracted with hot water at 80 °C for 3 h. The extraction was re-
peated twice (each time, the volume of added water was 1 L). Finally, the five aqueous
extracts (three obtained with cold water (CW1, CW2, CW3) and two with hot water (HW1,
HW2)) were obtained. Next, polysaccharides were extracted with acidified water (pH 2.0,
1 L) at 50 °C for 3 h. As a result, one extract (AC) was obtained. Next, polysaccharides
were extracted with aqueous solutions of ammonium oxalate (0.7% w/v) at 70 °C for 6 h.
The extraction was repeated three times (the first volume of salt solution added was 2 L;
the second and third volumes were 1 L). Finally, the three extracts (OK1, OK2, OK3) were
obtained. Next, polysaccharides were extracted with aqueous solutions of Na2CO3 (0.5%
w/v) containing NaBH4 at 70 °C for 3 h. The extraction was repeated twice (the first volume
of soda solution added was 3 L, the second—2 L). The two extracts (SO1, SO2) were ob-
tained.
The carbohydrate content of each extract was detected using a phenol-sulfuric acid
assay [63].
All extracts were dialyzed against distilled water for 48 h at 10 °C. Extracts SO1, SO2
were previously acidified with a diluted solution of acetic acid to pH 5.6. The dialyzed
extracts were concentrated on a Heidolph 4002 rotary evaporator (Germany) under re-
duced pressure at 40 °C.
Polysaccharides were precipitated from the extracts with a four-fold volume of 95%
ethanol, centrifuged, washed twice with 95% ethanol, dissolved in distilled water, frozen
and lyophilized. The yields of the polysaccharide fractions obtained are expressed in %
(w/w) of mass of dry plant material and are presented in Table 1.
3.3. Ion Exchange Chromatography of Polysaccharide Fractions
The major polysaccharide fractions HW1, AC, OK1, SO1 were separated on a DEAE-
cellulose (OH-) column (2.5 cm × 40 cm). Each polysaccharide fraction (100 mg) was dis-
solved in 5 mL of 0.01 M NaCl, and the solution was applied to the column. The column
was stepwise eluted with 0.01, 0.1, 0.2, 0.3, 0.5 and 1.0 M NaCl solution (400 mL of each
eluent) at a flow rate of 0.9 mL/min. The fractions were collected at 12 min intervals using
a low-pressure system Pharmacia Biotech (Sweden) with a FRAC-100 fraction collector,
P-50 pump. The carbohydrate content in each tube was determined by the phenol–sulfuric
acid method [63]. When separating each of the HW1, OK1, SO1, three major polysaccha-
ride fractions were obtained (eluted with 0.01, 0.1 and 0.2 M NaCl). When separating AC,
the fraction eluted 0.2 M NaCl was obtained as minor. In addition, minor fractions were
obtained from all fractions by elution with 0.3, 0.5 and 1.0 M NaCl.
The separation procedure was repeated twice for HW1, OK1 and four times for AC,
SO1. Data on the monosaccharide composition and the yield of the fractions are presented
in Table 1 as a mean of these experiments.
3.4. General Analytical Methods
The content of uronic acids was determined as described earlier [64,65]. The quanti-
tative determination of protein was calculated using the Bradford method [66]. The quan-
titative determination of phenolics was performed with the Folin–Ciocalteu reagent using
gallic acid as a standard [67]. The content of neutral monosaccharides was determined by
gas–liquid chromatography (GLC), as described earlier in detail [68]. The sugar concen-
tration was determined at 490 nm using the phenol–sulfuric acid assay [63].
The relative molar mass distributions (RMM) (including Mn, Mw and PDI) of the
polysaccharide samples were determined by size exclusion chromatography with high-
performance liquid chromatography (HPSEC); the procedure was described in detail ear-
lier [69].
Int. J. Mol. Sci. 2022, 23, 13175 14 of 18
3.5. Nuclear Magnetic Resonance Spectroscopy
All homo- and heteronuclear NMR experiments of the samples were carried out on
a Bruker Avance 600 spectrometer (Germany) at a probe temperature of 303, 313 and 318
K, which provided a minimum overlap of the signal of deuterated water with the polymer
signals. The procedures for preparing the polysaccharide samples and the conditions of
the NMR experiments were described earlier [69].
3.6. Antioxidant Activity
The DPPH solution (0.2 mM, in ethanol) was added to the pectin solution (0.4–3.6
mg/mL water) in equal proportions (v/v) and mixed. After incubation at 25 °C for 1 h, the
absorbance of the sample was measured at 517 nm. The scavenging activity of the pectins
was measured at four different concentrations, and the half-maximal inhibitory concen-
tration (IC50, mg/mL) values were calculated based on a polynomial regression curve [70].
3.7. Statistical Analysis
The significance of the difference among the means in determining the antioxidant
activity was estimated with one-way analysis of variance (ANOVA) and Fisher’s least sig-
nificant difference (LSD) post hoc test at p < 0.05. The relationship between the chemical
characteristics and activity of polysaccharide fractions was evaluated by the calculation
of the Pearson correlation coefficients and multiple linear regression analysis. All calcula-
tions were performed using the statistical package Statistica 10.0 (StatSoft, Inc., USA). The
data were expressed as the means ± s.d. of three independent experiments.
4. Conclusions
Polysaccharide fractions isolated from S. passerine with water and aqueous solutions
of ammonium oxalate and sodium carbonate were characterized by a similar composition,
including polysaccharides, protein and PCs. HG, RG-I and arabinan with regions formed
by 3,5-substituted and by 1,5-linked arabinose residues were identified as the principal
units of the polysaccharides obtained. Polysaccharide fractions of S. passerine demon-
strated a moderate antioxidant potential. Fractions isolated with cold water and sodium
carbonate scavenged the DPPH radical in vitro to a much greater extent than commercial
apple pectin. The correlation analysis of the composition and activity of polysaccharide
fractions obtained by anionic exchange chromatography revealed that the antioxidant ca-
pacity of polysaccharides of S. passerine is mainly due to the associated PCs.
Supplementary Materials: The following supporting information can be downloaded at:
https://www.mdpi.com/article/10.3390/ijms232113175/s1.
Author Contributions: Study design, conceptualization, polysaccharide isolation, V.G.; writing—
review and editing, project administration, S.P.; measurement of antioxidant activity, V.S.; separa-
tion of polysaccharide fractions on an anion exchange resin, V.K.; collecting plant material and its
primary treatment by organic solvents, F.V.; collecting plant material and its primary treatment by
organic solvents, S.N.; experiments and analysis of NMR data, A.S.S. and A.S.D. All authors have
read and agreed to the published version of the manuscript.
Funding: This study was supported by the Russian Science Foundation (grant number 21-73-20005).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data that support the findings of this study are available from the
corresponding author upon reasonable request.
Acknowledgments: NMR spectra were registered on the equipment of the Zelinsky Institute of Or-
ganic Chemistry Shared Research Facility.
Conflicts of Interest: The authors declare no conflicts of interest.
Int. J. Mol. Sci. 2022, 23, 13175 15 of 18
Appendix A
Chemical reagents: ethyl alcohol, ethanol, C2H5OH (96%, JSC Kirov Pharmaceutical
Factory, Russia); methyl alcohol, methanol, CH3OH (99.9%, Reakhim, Russia); sodium hy-
droxide, NaOH (98%, Fluka, Germany); sodium chloride, NaCl (99%, Sigma-Aldrich,
USA); chloroform, CHCl3 (>99.9%, Ekos-1, Russia); ammonium hydroxide solution,
NH4OH ((≥25 NH3 in H2O, >99.9%, Ekos-1, Russia); trifluoroacetic acid, CF3COOH (99%,
Acros organics, USA); pyridine, C5H5N (99%, Ekos-1, Russia); acetic acid, CH3COOH
(99.9%, Khimreactive, Russia); sodium borohydride, NaBH4 (>98.5%, Sigma-Aldrich,
USA); D2O (99.9 atom % D, Sigma-Aldrich, USA); toluene, C6H5-CH3 (99%, Ekos-1, Rus-
sia); sulfuric acid, H2SO4 (>99.9%, Vekton, Russia); phenol, C6H5OH (99%, Reakhim, Rus-
sia); 3,5-dimethylphenol, (CH3)2C6H3OH (≥99%, Sigma-Aldrich, USA); 1,4-α-D-polygalac-
turonase (Sigma, exo- and endo-activity 690 units/g); 1,1-diphenyl-2-picrylhydrazyl radi-
cal (Sigma-Aldrich, USA); the Folin and Ciocalteu’s phenol reagent (Sigma-Aldrich, USA).
For the ion exchange chromatography, we used DEAE-cellulose (Sigma-Aldrich,
USA).
The bovine serum albumin (≥96%, Sigma-Aldrich, USA); myo-inositol (≥99%, Sigma-
Aldrich, USA), L-(+)-Rhap, L-(+)-Araf, D-(+)-Galp, D-(+)-Manp, D-(+)-xylose and D-(+)-Glcp
(≥99%, Sigma-Aldrich, USA); D-(+)-GalpA monohydrate (≥97%, Sigma-Aldrich, USA); gal-
lic acid (MP Biomedicals, USA) were used as standards.
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