Beneficial Health Potential of Algerian Polysaccharides

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Article
Beneficial Health Potential of Algerian Polysaccharides
Extracted from Plantago ciliata Desf. (Septentrional Sahara)
Leaves and Seeds
Noura Addoun 1 , Zakaria Boual 1,2 , Cédric Delattre 3,4 , Toufik Chouana 1 , Christine Gardarin 3 ,
Pascal Dubessay 3 , Fatima Benaoun 1 , Seddik Addaoud 1 , Mohamed Didi Ould El Hadj 1 , Philippe Michaud 3
and Guillaume Pierre 3, *

1 Laboratoire de Protection des Ecosystèmes en Zones Arides et Semi-Arides, Université Kasdi Merbah,
Ouargla 30000, Algeria; nourabio14@gmail.com (N.A.); biozakaria1983@gmail.com (Z.B.);
chouanatoufik@yahoo.fr (T.C.); biodoctorante@gmail.com (F.B.); adseddik@gmail.com (S.A.);
mohameddidi@yahoo.fr (M.D.O.E.H.)
2 Laboratoire de Biologie Médicales IBN ROCHD, Rue Khemisti, 47000 Ghardaïa, Algeria
3 Université Clermont Auvergne, CNRS, SIGMA Clermont, Institut Pascal, 63000 Clermont-Ferrand, France;
cedric.delattre@uca.fr (C.D.); christine.gardarin@uca.fr (C.G.); pascal.dubessay@uca.fr (P.D.);
philippe.michaud@uca.fr (P.M.)
4 Institut Universitaire de France (IUF), 1 rue Descartes, 75005 Paris, France
* Correspondence: guillaume.pierre@uca.fr; Tel.: +33-(0)-473-407-222


Featured Application: This study substantially contributes to raising the understanding, safety,
Citation: Addoun, N.; Boual, Z.; and efficiency of ethnobotanical remedies from Plantago ciliata employed by traditional healers
Delattre, C.; Chouana, T.; Gardarin, in various Algerian health disorders.
C.; Dubessay, P.; Benaoun, F.;
Addaoud, S.; Ould El Hadj, M.D.; Abstract: Today, an ethnobotanical approach makes sense for identifying new active bioactive chemi-
Michaud, P.; et al. Beneficial Health
cals from uses of indigenous plants. Two water-soluble enriched polysaccharide fractions (L-PSPN
Potential of Algerian Polysaccharides
and S-PSPN) were extracted by hot water extraction from the leaves and seeds of Plantago ciliata Desf.
Extracted from Plantago ciliata Desf.
(1798), a Mzab indigenous herb currently used in Algeria by traditional healers. Primary investi-
(Septentrional Sahara) Leaves and
gation was performed for describing the main structural features of these polysaccharides (pectin-
Seeds. Appl. Sci. 2021, 11, 4299.
https://doi.org/10.3390/
and heteroxylan-like compositions) by using colorimetric assays, FTIR spectroscopy, HPAEC/PAD
app11094299 and GC/MS-EI analyses. Some biological activities were also monitored, such as anticomplement,
anti-inflammatory (phagocytic ability, NOX2 and MPO inhibitions) and anti-diabetic (α-amylase
Academic Editor: Ana M. L. Seca and α-glucosidase inhibitions). L-PSPC seems able to moderately modulate innate immune sys-
tem (IC50 around 100 µg/mL) and contribute to wound-healing processes (IC50 close to 217 vs.
Received: 12 April 2021 443 µg/mL for sodium heparin). S-PSPC shows some potential as an anti-hyperglycemic (IC50
Accepted: 30 April 2021 around 4.7 mg/mL) and anti-inflammatory (IC50 ranging from 111 to 203 µg/mL) agent, as well as
Published: 10 May 2021 other (fiber) psyllium-like polysaccharides extracted from Plantago species.

Publisher’s Note: MDPI stays neutral Keywords: Plantago ciliata; polysaccharides; characterization; biological activities; Africa
with regard to jurisdictional claims in
published maps and institutional affil-
iations.

1. Introduction
Herbal medicines have attracted considerable interest as alternative anti-inflammatory
remedies due to their large therapeutic properties, low toxicity and costs [1]. Today,
Copyright: © 2021 by the authors.
involved mechanisms of anti-inflammatory remain unclear [2] and potentiating innate
Licensee MDPI, Basel, Switzerland.
immune system has been recognized as a possible way for inhibiting inflammation growth
This article is an open access article
without harming the host [3].
distributed under the terms and
Polysaccharides from various traditional medicinal herbs have been shown to be im-
conditions of the Creative Commons
Attribution (CC BY) license (https://
munopotentiating, in vitro as well as in vivo [4]. Many reports on the biological activities
creativecommons.org/licenses/by/
of polysaccharides from Plantago highlighted that species belonging to this genus showed
4.0/). anti-inflammatory effects and immune enhancements through specific and non-specific

Appl. Sci. 2021, 11, 4299. https://doi.org/10.3390/app11094299 https://www.mdpi.com/journal/applsci


Appl. Sci. 2021, 11, 4299 2 of 15

immunity, anti-peroxidation of lipids, antidiabetic, and reduction in serum lipids [5].


Arabinogalactan Type II were isolated from the leaves of Plantago major and shown anti-
complementary activity [6]. A highly branched heteroxylan from Plantago asiatica seeds
could induce maturation of murine dendrite cells, promote defecation and have antioxi-
dant activity [7]. Intake of a Plantago ovata husk-supplemented diet prevented endothelial
dysfunction, obesity development, and improved dyslipidemia, abnormal plasma concen-
trations of adiponectin and TNF-α (tumor necrosis factor-α) in obese Zucker rats [8]. The
polysaccharides from Plantago palmata leaves possessed immunomodulatory properties
by stimulating NO (nitric oxide) and TNF-α production by activated macrophages [9].
A rhamnoarabinogalactan extracted from aerial parts of Plantago lanceolata was able to
inhibit the peroxidation of soybean lecithin liposomes with OH (hydroxyl) radicals. This
polysaccharide showed a high antioxidant activity of 19.1% at 0.23 mM, which represented
45.3% of the activity of α-tocopherol standard [10].
Today, identifying, characterizing, and evaluating the potential health benefits of
new polysaccharides from African medicinal plants have become a challenge. Plantago
ciliata, belonging to Plantaginaceae, is a traditional Algerian herbal plant abundantly
distributed in the northeast region of Sahara. It has been reported for having many
supposed ethnopharmacological properties including anti-inflammatory, anti-ulcer, anti-
cough, and anti-diabetic activities [11]. To our knowledge, no studies deal with the chemical
elucidation and some biological benefits of two water-soluble polysaccharides extracted
from Plantago ciliata leaves (L-PSPC) and seeds (S-PSPC).
Preliminary structural features of both fractions were determined by colorimetric
assays, Fourier Transform Infrared (FTIR) spectroscopy, High-Pressure Anion Exchange
Chromatography equipped with Pulsed Amperometric Detection (HPAEC/PAD) and
Gas Chromatography coupled to Mass Spectrometry with Electronic Impact (GC/MS-EI)
analyses. Some biological activities were monitored (anticomplement, anti-inflammatory,
anti-diabetic) regarding the structures of polysaccharides (structure/function relationship)
and current literature.

2. Materials and Methods


2.1. Raw Material and Chemicals
Plantago ciliata Desf. (1798) (Fl. Atlant), abbreviated P. ciliata (Conservatoire et Jardin
botaniques de Genève & South African National Biodiversity Institute, number collection
cc3472, record n◦ 115142 modified 10 April 2008; taxonomic number 1737; nomenclatural
Tela Botanica number: 115142), was freely (not protected) harvested and taxonomically
identified by Z. Boual and A. Chehma from ECOSYS laboratory (Université Kasdi Merbah,
Ouargla) entitled by Algerian Government for botanical exploration and use of Saharan
plants in Algeria. P. ciliata leaves and seeds were collected from Oued nechou (region of
Ghardaïa), in March–April 2015 and April 2017, respectively. The ears of each plant were
cut out and dried at room temperature for three weeks in the dark. The leaves were cut
into smaller parts then air-dried at ambient temperature in an environment with no direct
sunlight and stored in kraft paper bags (room temperature). The seeds were manually
isolated from the dry ears and also stored in kraft paper bags (room temperature).
Zymosan-A (Saccharomyces cerevisiae origin) were purchased from Fluka (Bio Chemika).
Coomassie Brilliant Blue G-250 was purchased from Serva (Canada). Heparin H 108
(sodium salt, from hog intestin mucosa, specific anticoagulant activity 173 IU/mg) was
obtained from SANOFI (Suresnes, France). Clofenal®(diclofenac) was from Saidal group.
The human peripheral blood was obtained from healthy donors ranging from 20 to 35 years
old. Whole blood was collected in a vacutainer consisting of NH sodium Heparin as
anti-coagulant. All other chemicals and buffers were purchased from Sigma-Aldrich and
were of analytical grade.
Appl. Sci. 2021, 11, 4299 3 of 15

2.2. Polysaccharides Extractions


Two different polysaccharidic fractions were extracted from Plantago ciliata leaves
(L-PSPC) and seeds (S-PSPC). First, Plantago ciliata leaves (100 g) were grounded (150–
200 mesh) and exhaustively extracted with 60 mL n-hexane 96% and 60 mL methanol
99% using a solvent extractor (SER 148, Velp Scientifica) to remove hydrophobic and low
molecular weight compounds (LMW). After filtration through a gauze (100 mesh), the
residues were dried at room temperature, and then extracted three times with deionized
water (1:20 w:v) under constant stirring (150 rpm) for 2 h at 60 ◦ C. The preparation was
then centrifuged at 10,000 g and 25 ◦ C for 10 min to remove remaining leaves and obtain a
mucilage enriched fraction. This solution was concentrated to one fourth of the original
volume by vacuum evaporation. Aqueous solution of 5% TCA (trichloroacetic acid) was
used to precipitate the proteins. The mucilage enriched fraction was then dialyzed in
membrane dialysis tubes with a cut-off of 12–14 kDa (Spectra/pore, Spectrum Labs, USA)
for 72 h against distilled water at 4 ◦ C to remove salts and other LMW compounds. The
polysaccharides were precipitated by the addition of absolute ethanol to a final concentra-
tion of 75% (v/v), and recovered by centrifugation at 4000 g and 25 ◦ C for 20 min. Finally,
the crude water-soluble polysaccharides fraction (L-PSPC) was washed with 10 mL acetone
100%, dissolved in 5 mL of deionized water and freeze-dried.
Secondly, Plantago ciliata seeds (211 g) were macerated for 4 h at 60 ◦ C under stirring
(450 rpm) in ultra-pure water (5% w/v). The residues were sequentially filtered (100 mesh
at the end), solubilized at 50 g/L in ultra-pure water (60 ◦ C) then precipitated by adding
three volumes of cold ethanol (96%, −20 ◦ C) under stirring. The pellet was recovered after
centrifugation at 10,000 g and 4 ◦ C for 15 min. The same loop of alcoholic precipitation
was performed three times. The final pellet was washed two times against pure acetone on
a frit glass (16–40 µm) under vacuum. The final enriched fraction (S-PSPC) was crushed
into a fine powder (<3 mm).

2.3. Determining Biochemical Compositions


Total carbohydrate and neutral sugar contents were determined by phenol-sulfuric
acid [12] and 1,3-dihydroxybenzen [13] methods, respectively, using Glc (Glucose) as
standard. Uronic acids were quantified using m-hydroxydiphenyl assay using GlcA (Glu-
curonic acid) as standard [14]. Protein content was estimated by Coomassie Brilliant Blue
G-250 method using bovine serum albumin as standard [15]. Phenolic compounds (gallic
acid equivalents) were determined according to Folin–Ciocalteu assay [16].

2.4. FTIR Footprints


Fourier Transform Infrared (FTIR) spectroscopy experiments were performed on a
VERTEX 70 FTIR apparatus equipped with an ATR A225 diamante. Fifty scans were mea-
sured at laboratory temperature ranging from 4000 to 400 cm−1 . Reference (20 background
scans) was made against air. OPUS 7.2 software (Bruker) was used for treating data.

2.5. Monosaccharide Compositions by HPAEC-PAD en GC/MS-EI


Monosaccharide compositions of both L-PSPC and S-PSPC were conducted by Gas
Chromatography coupled to Mass Spectrometry with Electronic Impact (GC/MS-EI) and
High-Pressure Anion Exchange Chromatography equipped with Pulsed Amperometric
Detection (HPAEC-PAD) to ensure data complementarity. The results were expressed in
molar ratio. All the experiments were done in triplicate.

2.5.1. GC/MS-EI Experiments


The analyses of monosaccharides compositions by GC/MS-EI were conducted accord-
ing to the method of Pierre et al. [17]. Ten mg of polysaccharides were hydrolyzed with
2M TFA (trifluoroacetic acid) for 90 min at 120 ◦ C under stirring. The hydrolysates were
trimethylsilylated using BSTFA: TMCS (N,O-bis[trimethylsilyltrifluoroacetamide] w:1%
trimethylchlorosilane) (99:1), evaporated under dry nitrogen then finally solubilized into
Appl. Sci. 2021, 11, 4299 4 of 15

dichloromethane. This procedure was applied for standards including Ara (Arabinose),
Glc, Gal (Galactose), GalA (Galacturonic acid), Rha (Rhamnose), Xyl (Xylose). GC/MS-
EI system was an Agilent 6890 GC / 5973 Network Mass Selective Detector, equipped
with an OPTIMA-1MS Accent column (Macherey-Nagel; 30 m, 0.32 mm, 0.25 µm), using
the following parameters, for instance, target ion: 40–800 m/z, injector line temperature:
250 ◦ C, trap temperature: 150 ◦ C, split ratio: 50:1, helium pressure: 8.8 psi; helium flow
rate: 2.3 mL/min, ionization: 70 eV, rise in temperature: starting at 100 ◦ C during 3 min,
8 ◦ C/min up to 200 ◦ C for 1 min and then 5 ◦ C/min up to 215 ◦ C. Data were analyzed
with MestReNova 7.1. software (Mestrelab Research, USA).

2.5.2. HPAEC-PAD Experiments


Both fractions were hydrolyzed using the same procedure described for GC/MS-EI
(see Section 2.5.1). pH was neutralized after hydrolysis with ammonium hydroxide (35%
w/v) then filtered (0.22 µm). Samples were injected on a precolumn CarboPac PA1-column
(4 × 50 mm) and an analytical CarboPac PA1-column (4 × 250 mm) equilibrated 15 min
with 18 mM NaOH, using an ICS 3000 System (Dionex Corporation, Sunnyvale (CA),
USA) equipped with PAD and AS 50 autosampler. Samples were eluted isocratically at
1 mL/min and 25 ◦ C with the following conditions: 18 mM NaOH for 25 min, then a
linear gradient between 0 to 0.5 M sodium acetate in 200 mM NaOH for 20 min to elute
acidic monosaccharides. Each run was followed by 15 min washing with 200 mM NaOH.
Same set of monosaccharides were used as standards for quantification with a Dionex
Chromeleon 6.80 software (Sunnywale, CA, USA).

2.6. Biological Activities


Regarding the structural features obtained for L-PSPC and S-PSPC, some biological
activities were monitored (anticomplement, anti-inflammatory, anti-diabetic) according to
the famous structure/function relationship and data described in the literature.

2.6.1. Phagocytotic Activity


Phagocytic ability of polymorphonuclear leukocytes (PMNs) from human peripheral
blood was measured using Candida albicans yeast. Briefly, C. albicans (ATCC 2091) was
inoculated into Sabouraud liquid broth from a stock culture maintained on a Sabouraud
agar slope and left overnight at 30 ◦ C. The culture was washed three times with phosphate-
buffered saline (PBS) and incubated for 1 h at 90 ◦ C to inactive the yeasts. Cell concentra-
tions in PBS is estimated by measuring absorbance at 540 nm. The absorbance is adjusted
to 1.0 which approximately corresponds to 1 × 107 blastoconidia of C. albicans / mL [18,19].
Forty µL of polysaccharides (L-PSPC) were mixed with 200 µL heparinized blood and
incubated in closed shaking water bath at 37 ◦ C for 60 min (60 rpm). After that, tubes were
put on ice to stop the reaction. Mixed samples were added with 40 µL of C. albicans at
0 ◦ C. Samples were incubated in shaking water bath for 10 min at 37 ◦ C, while for negative
control; the samples were put on ice. After incubation, the samples were put on ice to stop
the reaction. Next, samples were washed twice with 3 mL of PBS. Then, samples were
added 2 mL of lysing solution to lyse erythrocytes and incubated at room temperature for
20 min. Zymosan was used as a positive control. After lysing, PMNs were washed with
PBS by centrifugation for 5 min at 1000 g three times and then fixed in ethanol. The fixed
specimens were mounted on the glass slide and stained with May-Grünwald-Giemsa. The
cells with and without phagocytized yeasts out of a total of at least 100 cells were counted
by direct visual enumeration using a light microscope (1000×).

2.6.2. Anti-inflammatory Activity


Immunomodulating potential of S-PCPC was evaluated by determining the capac-
ity to inhibit the enzymatic activities of NADPH oxidase (NOX2) and myeloperoxidase
(MPO). MPO activity was measured using adapted methods from Wanikiat et al. and Mez-
iti et al. [20,21]. Briefly, 175 µL of cell suspension (6 × 106 cells/mL) were pre-incubated
Appl. Sci. 2021, 11, 4299 5 of 15

with 30 µL of S-PSPC at different concentrations (5–100 µg/mL), 30 µL of different dilu-


tions (5–100 µg/mL) of diclofenac (positive control) or 30 µL of PBS (phosphate buffer
saline, negative control) for 10 min at 37 ◦ C, then stimulated with 25 µL of PMA (phor-
bol 12-myristate 13-acetate) (10−6 M) for 10 min at 37 ◦ C. After centrifugation (320 g,
10 min, 4 ◦ C), supernatants were incubated with the reaction mixture: 25 µL of 4-AA
(4-aminoantipyrine) (1 mg/mL) solution in 0.1M PBS (pH 6.0) supplemented with 100 µL
of 0.003% H2 O2 . The reaction was ended after 5 min by adding 50 µL of 4M H2 SO4 . The
absorbances were measured at λ = 546 nm. NADPH oxidase activity was measured using
the method of Boudoukha which was slightly modified [22]. Briefly, 400 µL of cell sus-
pension (6 × 106 cells/mL) was incubated with 100 µL of PSPC (5–100 µg/mL), 100 µL
of different dilutions (5–100 µg/mL) indomethacin (positive control) or 100 µL of PBS
(negative control) for 10 min at 37 ◦ C. The cells were thus stimulated with 100 µL of PMA
solution (10−6 M), then incubated for 10 min at 37 ◦ C. For each tube, 100 µL of a freshly
prepared Cytochrome C solution (0.2 mg/mL) were added followed by incubation for
15 min at 37 ◦ C. The absorbance of the supernatants obtained after centrifugation (400 g,
5 min, 4 ◦ C) were measured at λ = 550 nm. Inhibition activity results were expressed
regarding Equation 1 [20]. All the experiments were done in triplicate.

Inhibition (%) = (1 − (A(sample) )/A(control) )) × 100 (1)

2.6.3. Anti-complement Activity


Anti-complement activity was monitored for L-PSPC. Based on a modified method
from Mayer, normal human serum (NHS) obtained from healthy male donors (mean age
20 years) was used as the complement source, and it was treated with sheep erythrocyte
to remove the anti-sheep erythrocyte antibody. Sheep erythrocytes were washed twice
with 150 mM NaCl and once with gelatin veronal buffer (pH 7.4, containing 0.1% gelatin,
141 µM NaCl, 500 µM MgCl2 , 150 µM CaCl2 , and 1.8 mM Sodium barbital (GVB2+ )). The
NHS was diluted to a concentration giving 50% hemolysis with GVB2+ in the absence
of complement inhibitors. Sensitized erythrocytes were prepared by incubation of sheep
erythrocytes (4.0 × 108 cells/mL) with an equal volume of rabbit anti-sheep erythrocyte
antibody in GVB2+. Heparin served as the positive control. Each sample was dissolved in
GVB2+ . Various dilutions of test samples (100 µL) were pre-incubated with 100 µL of NHS
(1:10) and 200 µL of GVB2+ at 37 ◦ C for 10 min. Then, 200 µL of sensitized erythrocytes
were added, and the mixture was incubated at 37 ◦ C for 30 min. The reaction mixture
was immediately centrifuged at 900 g for 5 min. Absorbance of the supernatant was
measured at 405 nm. Total lysis (100%) was obtained by adding 400 µL of distilled H2 O to
200 µL of sensitized sheep erythrocytes. Samples containing GVB2+ , serum, and sensitized
sheep erythrocytes were used as background controls (A control). The degree of lysis
and inhibition of hemolysis induced by the test samples were calculated according to
Equations (2) and (3), respectively. All the experiments were done in triplicate.

Lysis degree (%) = (A(control) /A(water) ) × 100 (2)

Hemolysis inhibition (%) = ((A(control) − A(sample) )/A(control) ) × 100 (3)

2.6.4. Anti-diabetic Activity


Anti-hyperglycemic activity of S-PSPC and a hydrolyzed fraction (S-PSPCh ) was
investigated by evaluating the inhibition of both α-amylase and α-glucosidase activities.
Inhibition of α-amylase activity was estimated using the methods of Kumar et al. and
Kajaria et al. which were slightly modified [23,24]. Briefly, 180 µL of S-PSPC, acarbose
(positive control) or ultrapure water (negative control) were introduced in dry tubes.
Ninety µL of α-amylase solution (5 IU/L) was added to each tube. The reaction mixtures
were preincubated for 15 min at 37 ◦ C. Then, 500 µL of CNPG3 (2-chloro-p-nitrophenyl-
α-D-maltotrioside) solution (0.5 mg/mL) were added under gentle stirring, followed by
incubation for 10 min at 37 ◦ C. The absorbances were measured at λ = 405 nm. Inhibition
Appl. Sci. 2021, 11, 4299 6 of 15

of α-glucosidase activity was estimated using the methods of Christudas et al., Bisht et al.
and Qian et al., which were also slightly modified [25,26]. Briefly, 500 µL in dry tubes of
α-glucosidase solution (2 UI/L) was introduced with 100 µL of each dilution of S-PSPC or
S-PSPCh , acarbose (positive control) or ultrapure water (negative control). S-PSPCh was
a hydrolyzed version of S-PSPC (H2 SO4 , 50 ◦ C, 30 min) for preliminary investigations of
molar mass effects (data not shown). The mixture was pre-incubated for 15 min at 37 ◦ C.
Then, 100 µL of p-NPG (p-nitrophenyl-α-D-glucopyranoside) solution (4 mM) were added.
The tubes were shaken and incubated for 20 min at 37 ◦ C. One mL of Na2 CO3 (0.2M) was
added to stop the reaction, and the absorbances were measured at λ = 405 nm. Inhibition
activity results were expressed regarding Equation (4) [27]. All the experiments were done
in triplicate.
Inhibition (%) = ((A(control) − A(sample) )/A(control) ) × 100 (4)

2.7. Statistical Analysis


All statistical analyses were run using the statistical software SigmaPlot 12.5 (Systat
Software). Data were expressed as mean ± standard deviation (SD) of three replicate and
evaluate by one-way analysis of variance (ANOVA). Post hoc procedures (Tukey test) were
performed to analyze pairwise differences as well as Holm–Sidak method for multiple
comparisons versus control group. Differences were considered significant for p < 0.05.

3. Results and Discussions


3.1. Structural Characterization of L-PSPC and S-PSPC
3.1.1. Main Biochemical Compositions
The extraction yields of both L-PSPC (leaves) and S-PSPC (seeds) fractions are detailed
in Figure 1, i.e., 46 g/kg (4.60% w/w) and 186 g/kg (18.6% w/w), respectively. Regarding
the procedure of extractions, these values are consistent with the literature. Mass yields of
water-soluble polysaccharides extracted from various parts of Plantago species are ranged
from 1 to 20%, as reported for Plantago major [6], Plantago asiatica [28] or Plantago notata [29].
Obviously, extraction yields are strongly influenced by the methodology, physicochemical
parameters (temperature, pH, reaction time, solid/liquid ratio, stirring, etc.), solvents, type
and concentration of polysaccharides [30]. The higher yields are given for polysaccharides
extracted from Plantago seeds (Psyllium) [31].

Figure 1. Extraction yields (w/w %) of L-PSPC (leaves) and S-PCPC (seeds).

L-PSPC and S-PSPC are mainly composed of carbohydrates (66.6% and 86.5% w/w,
respectively) (Table 1). Water-soluble polysaccharides extracted from Plantago ciliata leaves
contained more uronic acids (24.0% w/w) than those extracted from seeds (7.96% w/w).
Note that L-PSPC and S-PSPC are clearly enriched-polysaccharide fractions since both are
poorly contaminated by proteins (2.30% and 0.35% w/w) and phenol compounds (close
to 0% w/w). Three polysaccharide fractions from Plantago asiatica seeds (PLP-1, -2, -3)
with high levels of total carbohydrates varying between 79.2% and 87% (w/w), and very
low protein contents (around 1% w/w) [32]. PSPN, a heteroxylan extracted from Plantago
notata seeds, was composed of 85.6% of carbohydrates including 78% of neutral sugars [29].
279 both are poorly contaminated by proteins (2.30% and 0.35% w/w) and phenol compounds
280 (close to 0% w/w). Three polysaccharide fractions from Plantago asiatica seeds (PLP-1, -2, -
281 3) with high levels of total carbohydrates varying between 79.2% and 87% (w/w), and very
282 Appl. Sci. 2021, 11, 4299 low protein contents (around 1% w/w) [32]. PSPN, a heteroxylan extracted from Plantago 7 of 15
283 notata seeds, was composed of 85.6% of carbohydrates including 78% of neutral sugars
284 [29]. Craeyveld et al. also described few amounts of uronic acids (< 5% w/w) in a seed
285 husk arabinoxylan extracted from Plantago ovata Forsk [33]. A branched rhamnogalac-
286 Craeyveld
turonan with et al.side
alsochains
described few amounts ofwas
of arabinogalactan, uronic acids (<
extracted in5% w/w)conditions
similar in a seed husk
from
287 arabinoxylan extracted from Plantago ovata Forsk [33]. A branched rhamnogalacturonan
Plantago major leaves and mainly contained uronic acids (59.3% w/w) followed by neutral
288 with
sugarsside chains
(39.1% w/w) of arabinogalactan,
[34]. Homogeneous was extracted
acidic protein-bound conditions from Plantago
in similarheteropolysaccharides com-
289 major leaves and mainly contained uronic acids (59.3%
posed of carbohydrates (89–92% w/w), proteins (3–7% w/w) and uronicw/w) followed by neutral sugars
acids (10–20%
290 (39.1%
w/w) werew/w)extracted
[34]. Homogeneous
from seedsacidic protein-bound
of Plantago depressa heteropolysaccharides composed
[35]. Thus, pectic substances and
291
of carbohydrates (89–92% w/w), proteins (3–7% w/w) and uronic acids (10–20%
mainly heteroxylan are usually identified, respectively, from Plantago leaves or seeds by w/w)
292
were
usingextracted from seeds
water extraction of Plantago
bioprocesses depressa [35]. Thus, pectic substances and mainly
[6,36].
heteroxylan are usually identified, respectively, from Plantago leaves or seeds by using
293 water
Table extraction
1. Biochemicalbioprocesses
composition[6,36].
of L-PSPC (leaves) and S-PCPC (seeds).

Carbohydrate
Table 1. Biochemical composition (w/w
of L-PSPC %) and S-PCPC
(leaves) Proteins
(seeds). Phenols Ash
Fractions
Total Neutral Uronic acids (w/w %) (w/w %) (w/w %)
Carbohydrate (w/w %) Proteins Phenols Ash
L-PSPC
Fractions 66.6 ± 1.42 42.6 ± 1.37 24.0 ± 1.12 2.30 ± 0.09 0.6 ± 0.03 3.65 ± 0.08
Total Neutral Uronic Acids (w/w %) (w/w %) (w/w %)
S-PSPC 86.5 ± 4.32 63.3 ± 3.17 7.96 ± 0.39 0.35 ± 0.02 0 5.12 ± 0.24
L-PSPC 66.6 ± 1.42 42.6 ± 1.37 24.0 ± 1.12 2.30 ± 0.09 0.6 ± 0.03 3.65 ± 0.08

294 S-PSPC
3.1.2. 86.5 ± 4.32
FTIR spectroscopy 63.3 ± 3.17 7.96 ± 0.39 0.35 ± 0.02 0 5.12 ± 0.24

295 FTIR spectra of both (a) L-PSPC and (b) S-PSPC are shown in Figure 2. The peaks at
296
3.1.2. FTIR Spectroscopy
3300–3400 cm-1 were attributed to O-H stretching vibration of both water and carbohy-
297 dratesFTIR spectra
[29]. of both
Aliphatic (a) L-PSPC
bending groupsand (b) S-PSPC
(C-H) are observed
were also shown in close
Figureto2.2900
The cmpeaks at
-1 [37].

3300–3400 − 1
cm were attributed to O-H stretching vibration of both water and carbohy-
298 The characteristic absorption peaks around 1620 cm -1 were attributed to the vibration of
drates [29]. Aliphatic bending groups (C-H) were also observed close to 2900 cm −1 [37].
299 carboxylate groups whereas the ones close to 1416 cm could correspond to ester carbonyl
-1

The characteristic absorption peaksofaround −1 were attributed to the vibration of


300 groups of the carboxylic function uronic1620 cm[34].
acids The main peaks (1034–1100 cm-1)
carboxylate groups whereas the ones close to 1416 cm −1 could correspond to ester carbonyl
301 were attributed to C-O functions of carbohydrates [38]. Assuming these observations, the
302 groups
results of thein
were carboxylic
accordance function of biochemical
with the uronic acidscompositions
[34]. The main peaks
(see (1034–1100
Section cm−1 )
3.1.1.). L-PSPC
303 were
couldattributed to C-Ofraction
be a pectin-like functions of carbohydrates
whereas S-PSPC could [38]. Assuming
correspond to these observations,
a heteroglycan con-
304 the results were in accordance with the biochemical
taining few amounts of uronic acids (pectic contamination). compositions (see Section 3.1.1.). L-
PSPC could be a pectin-like fraction whereas S-PSPC could correspond to a heteroglycan
containing few amounts of uronic acids (pectic contamination).

305
306 Figure2.2.FTIR
Figure FTIRfootprints
footprintsof
of(a)
(a)L-PSPC
L-PSPC(leaves)
(leaves)and
and(b)
(b)S-PCPC
S-PCPC(seeds).
(seeds).

3.1.3. Monosaccharide Compositions and Main Structural Features


Results from HPAEC-PAD and GC/MS-EI experiments are given in Figure 3 for both
(a) L-PSPC and (b) S-PSPC enriched-polysaccharide fractions. Data complementarity be-
tween the two methodologies was confirmed since no significant differences were observed
among the data sets for L-PSPC (p = 0.805) and S-PSPC (p = 0.957).
Appl. Sci. 2021, 11, 4299 8 of 15

Figure 3. Monosaccharide compositions of (a) L-PSPC (leaves) and (b) S-PCPC (seeds). White and black bars, respectively
correspond to the results obtained from HPAEC-PAD or GC/MS-EI experiments.

• Composition of L-PCPC
L-PSPC is composed of six main monosaccharides, highlighting its complexity, i.e.,
GalA (22.4–31.8%), Gal (18.4–20.7%), Glc (20.6–15.9%), Rha (8.93–14.4%), Xyl (13.4–9.58%)
and Ara (14.9–7.6%). Accordingly, the composition is similar to some kinds of pectin.
Significant differences of monosaccharide compositions were described in the litera-
ture, depending on the extraction/purification methodologies, analytical procedures but
also species of Plantaginaceae and obviously plant parts. Polysaccharides from Plantago
major leaves, are often composed of GalA, Gal, Ara and Rha in addition to small amounts of
Glc and Xyl [34,39]. Samuelsen et al. showed that water-soluble polysaccharides extracted
from Plantago major leaves contained two subfractions (PMIa and PMIb). The first one
was composed of Ara (38%), Gal (49%), Rha (6%) and GalA (7%) whereas the second one
consisted of Ara (31%), Gal (32%), Xyl (18%), Glc (7%), Rha (5%) and GalA (8%) [6]. A
water soluble heteroxylan extracted from Plantago notata leaves was composed of Gal (44%),
Rha (20%), Glc (11%), Ara (10%) and GalA (13%) [40]. Neutral rhamnoarabinogalactan
were also extracted from Plantago lanceolata, which were rich in Gal (54%), Ara (35%) and
Rha (11%) [10]. Other water-soluble polysaccharides extracted from Plantago palmata leaves
were composed of Gal (39%), Ara/Xyl (27%), Man (Mannose) (5%), Rha (10%), Glc (5%)
and GalA (14%) [9]. As stated before, these differences may be related to species, cultivation
regions, extraction procedures, analysis methods, and samplings [41].
• Composition of S-PCPC
S-PSPC is easily identified as an arabinoxylan mainly composed of Xyl (85–78% ± 3–
3.75), Ara (11–18% ± 3.45–2.1), Rha (2–3% ± 0.45–0.75) and GalA (1–1% ± 0.47–0.21). With
a Xyl:Ara molar ratio close to 4:1, results are in accordance with the literature concerning the
identification of arabinoxylan or heteroxylan from Plantago seeds. Polysaccharide fractions
(PLP-2 and PLP-3) isolated from Plantago asiatica were defined as arabinoxylan containing,
respectively Xyl (61 and 56%) and Ara (32.2 and 39.6%) residues [32]. An arabinoxylan
extracted from the pericarp of Plantago ovata Forssk. seeds was mainly composed of Xyl
(75%) and Ara (23%) with a Xyl: Ara molar ratio close to 3: 1 [42,43]. Seeds from other
Plantago species could contain heteroxylan as reported for an acidic one from Plantago
Appl. Sci. 2021, 11, 4299 9 of 15

major L., composed of Xyl (40%), Ara (13%), Glc (10%), Gal (3%), Rha (2%), GalA (17%) and
GlcA (16%) [44]. Benaoun et al. identified a neutral heteroxylan Plantago notata Lagasca
seeds. This polysaccharide was mainly composed of Xyl (77%), Rha (9%), Ara (8%), Gal
(3%), Glc (1%) and GalA (2%) [29]. Note that further structural characterization has been
published for S-PSPC [45].

3.2. Potential Health Benefits of L-PSPC and S-PSPC


Few biological activities were analyzed (anticomplement, anti-inflammatory, anti-
diabetic) according to the structure/function relationship and data described in the liter-
ature. Thus, antidiabetic and anti-inflammatory activities were investigated on polysac-
charides from Plantago seeds (S-PSPC) since psyllium-like polysaccharides are described
for this biological potential. Besides, polysaccharides from Plantago leaves seem pos-
sessing specific anti-complement and phagocytotic properties, justifying the focus on
L-PSPC. These choices are also in accordance with the uses of Plantago ciliata formulations
by Nganga and traditional Algerian healers. Remedies from Plantago ciliata seeds are
often used for constipation, stomach/guts pains and inflammatory problems (infusion)
whereas those from leaves are more used for treating wounds (bandage), muscular pain
and fever (decoction).

3.2.1. Biological Activities of L-PSPC


One of the important notable features of polymorphonuclear leukocytes activation
would be an increase in phagocytic activity. To further investigate whether L-PSPC stim-
ulate immune system, phagocytic activity of PMNs from human peripheral blood was
monitored. Phagocytic activity of PMNs cells was examined by the uptake of opsonized C.
albicans (see Section 2.6.1.). As shown in Figure 4a, significant enhancements of phagocytic
Appl. Sci. 2021, 11, x FOR PEER REVIEW
activity were observed in PMNs treated with zymosan as positive control (p < 0.01)10 of 15
and
L-PSPC (p < 0.01) comparing to negative controls (mean 26.7% ± 2.08).

370
371
Figure 4. 4.Biological
Figure Biologicaleffects
effectsof
ofL-PSPC
L-PSPC on
on (a)
(a) phagocytosis activity of
phagocytosis activity ofpolymorphonuclear
polymorphonuclearleukocytes
leukocytesbybythe
theuptake
uptakeof of
op-
372 opsonized
sonized C.C.albicans
albicansand
and(b)
(b)complement
complementfixation.
fixation.

The results also seemed showing a dose-dependent increase in phagocytic activity in


373 The results also seemed showing a dose-dependent increase in phagocytic activity in
PMNs treated with doses ranging from 50 to 150 µg/mL of zymosan or L-PSPC (p = 0.644).
374 PMNs treated with doses ranging from 50 to 150 μg/mL of zymosan or L-PSPC (p = 0.644).
Note that zymosan still showed the highest stimulated activity with an IC50 of 47.35 µg/mL
375 Note that zymosan still showed the highest stimulated activity with an IC50 of 47.35 µ g/mL
against 99.75 µg/mL for L-PSPC.
376 against 99.75 µ g/mL for L-PSPC.
377 Figure 4b showed that L-PSPC had a significant in vitro human complement fixing
378 activity. IC50 for sodium heparin was around 443 µ g/mL, as reported for other works with
379 values ranging from 420 to 750 µ g/mL for sodium heparin [46]. Pure heparin gave better
380 results with IC50 values between 16 to 36 µ g/mL [47] and up to 150 µ g/mL [48]. IC50 for L-
Appl. Sci. 2021, 11, 4299 10 of 15

Figure 4b showed that L-PSPC had a significant in vitro human complement fixing
activity. IC50 for sodium heparin was around 443 µg/mL, as reported for other works
with values ranging from 420 to 750 µg/mL for sodium heparin [46]. Pure heparin gave
better results with IC50 values between 16 to 36 µg/mL [47] and up to 150 µg/mL [48].
IC50 for L-PSPC was 216.7 µg/mL which was lower than for other pectic-like polysac-
charides extracted from Plantago species. Samuelsen et al. reported IC50 between 35
and 60 µg/mL [6]. Accordingly, pectin usually show decent to strong anti-complement
activities below 100 µg/mL due to their anionic character [10]. Heteroxylan from Plantago
seeds (major and asiatica), consisting of a 1,3- and 1,4-linked β-D-Xylp backbone with short
side chains, also showed potent complement activity, as reported with values ranging from
200 to 350 µg/mL [44].
Further experiments could also be done on S-PSPC to check this behavior. It is
noteworthy that polysaccharide fractions with IC50 values higher than 200–400 µg/mL
(sodium heparin) have no chance to emerge as new anti-complement drugs on the market.
These results explained the uses of Plantago ciliatia leaves as remedies for treating wounds
and specific inflammation by traditional healers.

3.2.2. Biological Activities of S-PSPC


• Anti-inflammation potential of S-PCPC
Activation of neutrophils not only results in the activation of membrane NADPH
oxidase (NOX2) and the generation of reactive oxygen species (ROS) but also phenomenon
of degranulation. Cytoplasmic granules fuse with the membrane of the phagosome. Mi-
crobicidal proteins and enzymes, in particular myeloperoxidase (MPO) in the following
oxidative explosion. Various autoimmune diseases and chronic inflammation are observed
due to these mechanisms [49]. Focusing on new ways to modulate the metabolic activ-
ity of neutrophils is today observed in the literature, e.g., through the uses of synthetic
steroidal compounds with unwanted side effects. Various natural metabolites such as poly-
and oligosaccharides [50], flavonoids, tannins, alkaloids, coumarins but also essential oils
could be proposed as an alternative [51]. In vitro anti-inflammatory effects of S-PCPC for
inhibiting MPO and NADPH oxidase activities were analyzed as presented in Figure 5a,b.
Both positive controls (dichlofenac and indomethacin) showed better IC50 values (2.71 and
3.77 µg/mL) than S-PSPC (203 and 111 µg/mL), respectively (p < 0.05). Inhibition of NOX2
by polysaccharides extracted from medicinal plants has shown varying potentials. The
involved polysaccharide extracted from Artemisia tripartita leaves was mainly composed of
Xyl, Ara, Glc, Gal, and GalN (Galactosamine) residues [52]. A water-soluble polysaccharide
extracted from Bletilla striata (BSPb), a medicinal plant used by Chinese ancestors to treat
burns in diabetics, exhibited an inhibitory effect on NOX2. It was of 260 kDa and mainly
composed of α-D-(1,2)-Manp and β-D-(1,4)-Glcp, with a molar ratio of 3:1 [53]. In the
presence of Angiotensin II (Ang II), significant expression of NADPH oxidase was noted
(73.6%). Pretreating the cells with 5 µg/mL of BSPb resulted in an intense reduction in
the expression of NOX2 and consequently the reduction in ROS, up to 30.2%. This result
was in the range of the reduction observed for PSPC at 5 µg/mL (23.4%). A water-soluble
polysaccharide (PLPC) from Plantago asiatica L. seeds showed an anti-inflammatory effect
on experimental colitis caused by dextran sulfate sodium salt (DSS) in BALB / c type
rats [54].
Appl. Sci. 2021, 11, 4299 11 of 15

Figure 5. In vitro anti-inflammatory and anti-hyperglycemic effects of S-PSPC regarding inhibitions


of (a) myeloperoxidase, (b) NADPH oxidase, (c) α-amylase and (d) α-glucosidase activities. S-PSPCh
is a hydrolyzed version of S-PSPC (H2 SO4 , 50 ◦ C, 30 min) for preliminary investigations of molar
mass effects (data not shown).

In this study, PLPC was a heteroxylan of 3870 kDa. Histological observations high-
lighted a significant reduction in the length of colitis in the group treated with DSS com-
pared to the control group, i.e., 64.3 ± 4.1 mm and 85.6 ± 4.5 mm, respectively. Type 2
diabetes is a progressive disease that benefits from a complex therapeutic arsenal. Synthetic
drugs prescribed for diabetics are increasingly numerous and more and more effective,
e.g., hypoglycemic sulfonamides, acarbose, metformin, etc. These products can cause
undesirable effects on human health such as the risk of hypoglycemia, weight gain or
digestive disorders [55,56].
• Antihyperglymic potential of S-PCPC
Today, alternative approaches are currently being targeted as the use of plant mu-
cilages and/or gums with antihyperglycemic potential [57]. One of the regulatory pathways
is to inhibit the functioning of α-amylase and α-glucosidase, thus preventing the cleavage
of carbohydrates into oligosaccharides and their conversion to simple monosaccharides.
Thus, it delays digestion process and prolongs their stay in the jejunum [58]. In vitro anti-
hyperglycemic effects of S-PSPC using acarbose as positive control are reported in Figure 5
c, d. Acarbose showed better IC50 values (0.30 and 0.32 mg/mL) against α-amylase and α-
glucosidase than S-PSPC (3.60 and 10 mg/mL), respectively (p < 0.05). The results observed
Appl. Sci. 2021, 11, 4299 12 of 15

for the positive control were consistent with the literature with values ranging from 0.05
to 0.60 mg/mL [59]. As reported by Chen et al., inhibition of α-glucosidase activity was
inversely proportional to molar mass and S-PSPCh showed an IC50 of 4.71 mg/mL [60].
Note that S-PSPC had a molar mass of 700 kDa [45]. A heteroxylan extracted from Plantago
notata seeds also showed a moderate inhibitory potential at 10 mg/mL (58%) [29]. Varia-
tions in monosaccharide compositions are also classically involved in these differences [61].
Inhibitory capacity of heteroxylan and arabinoxylan are due to Xyl:Ara ratio and degree of
substitution, highly substituted ones presenting the best IC50 values. Arabinoxylan also
uncompetitively inhibit α-glucosidase [62]. Treating diabetic rats with an arabinoxylan
(PLP) isolated from Plantago asiatica seeds leaded to a reduction in blood glucose levels [63].
PLP significantly inhibited α-amylase activity and diffusion of glucose, in vitro. At 2.5
mg/mL, PLP reduced α-amylase activity by 10% compared to the negative control, which
was lower than for S-PSPC at the same concentration (36%). Inhibition of α-amylase could
be due to the number of free carboxylic groups [64]. Note that S-PSPC contained few
uronic acids (8% w/w). Adsorption of polysaccharides to starch remains the most reported
hypothesis, which would prevent the hydrolytic activity of enzymes such as α-amylase [65].
Consuming Plantago ovata fibers (rich in arabinoxylan) improved postprandial glycemic
index and insulin sensitivity in rats [43,66]. Dietary fibers from Plantago ovata may also
reduce blood glucose levels in patients with type II diabetes [67]. Bisht et al. tested in vitro
antihyperglycemic potency of Acacia tortilis polysaccharide (AG), showing its potential as
an effective remedy for treating diabetes mellitus. IC50 of AG was around 0.7 mg/mL for α-
glucosidases extracted from rats and 0.5 mg/mL for those extracted from yeast [25]. More
recently, an arabinan-rich polysaccharide exhibited significant α-glucosidase inhibitory
activities [60,68]. These observations are consistent with traditional uses of Plantago ciliata
seeds in medicine for reducing blood sugar levels [11].
Finally, all these preliminary results assessed the biological potential of both L-PSPC
(pectin-like) and S-PSPC (arabinoxylan-like), giving an understanding as to why Plantago
ciliata leaves and seeds are widely used in Algerian ethnobotany approach to treat diabetes
and diseases often associated with inflammation. Further experiments should be needed in
the future, as instance, the analysis of the antiproliferative and in vitro digestion activities
of both fractions to better comprehend their biological potentials in Nutraceutics and
Inflammation processes. In vivo (cells, animals and clinical) experiments should complete
these results to assess their uses as new healthy drugs (or based-drug compounds).

Author Contributions: Conceptualization, G.P., N.A., Z.B.; methodology, C.D., G.P., N.A., P.D.;
validation, C.D., G.P., N.A., P.D.; formal analysis, C.G., F.B., G.P., N.A., S.A., T.C., Z.B.; investigation,
C.D., F.B., N.A., P.D., S.A., T.C., Z.B.; writing—original draft preparation, G.P., N.A., Z.B.; writing—
review and editing, G.P., Z.B.; supervision, G.P., M.D.O.E.H., P.M., Z.B.; project administration,
M.D.O.E.H., P.M.; funding acquisition, M.D.O.E.H., P.M. All authors have read and agreed to the
published version of the manuscript.
Funding: This work has been sponsored by Campus France and the Hubert Curien Tassili program—
Phase II (15MDU933).
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki, and approved by Ethics Research Committee of Laboratoire de Biologie
Médicales IBN ROCHD (Ghardaïa, Algeria), number 1154/He/Ce/Z/11/LSPSPC, 23 August 2017.
Conflicts of Interest: The authors declare no conflict of interest.

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