Phytochemistry and Antioxidant Activity of Aerial Parts of Phagnalon sordidum L 1

Article abstract of DOI:10.1055/a-0953-5984

One new natural monoterpene, 5-O-β-d-glucopyranosyl-2-hydroxy-p-cymene (1), and 11 known compounds were isolated through a biologically oriented approach from the aerial parts of Phagnalon sordidum L. The most active extract and fractions were selected using 3 complementary antioxidant activity assays. Results and the different methods were compared by relative antioxidant capacity index. In addition, the most active extract of P. sordidum was subjected to liquid chromatography coupled with electrospray ionization hybrid linear ion trap quadrupole Orbitrap mass spectrometry to quantify secondary metabolites. Antioxidant activities of ethyl acetate extract, and purified 3,4-dihydroxyacetophenone (3) and nebrodenside A (7) were demonstrated by in vitro cell free model assays, and their protective effect against H ₂ O ₂-induced oxidative stress in a HepG2 (human hepatocellular carcinoma) cell line was established.

Full text of DOI:10.1055/a-0953-5984

Original Papers
Phytochemistry and Antioxidant Activity of Aerial Parts
1
of Phagnalon sordidum L.
Authors
Hanene Cherchar *, Immacolata Faraone *, Massimiliano DʼAmbola , Chiara Sinisgalli2, 3, Fabrizio Dal Piaz5,
Patrizia Oliva , Ahmed Kabouche , Zahia Kabouche , Luigi Milella2, 3, Antonio Vassallo2
1
2, 3
4
6
1
1
Affiliations
Correspondence
1
Université des Frères Mentouri-Constantine 1, Laboratoire
Prof. Luigi Milella
dʼObtention de Substances Thérapeutiques Constantine
Dipartimento di Scienze, Università degli Studi della Basilicata
viale dellʼAteneo lucano 10, 85100, Potenza (Pz), Italy
Phone: + 390971205525, Fax: + 390971205503
luigi.milella@unibas.it
(
Algeria)
2
3
4
5
6
Dipartimento di Scienze, Università degli Studi della
Basilicata, Potenza (PZ), Italy
Spinoff BioActiPlant s. r. l., Dipartimento di Scienze,
Università degli Studi della Basilicata, Potenza (Pz), Italy
Dipartimento di Farmacia, Università di Salerno, Fisciano
Supporting information available online at
http://www.thieme-connect.de/products
(
SA), Italy
Dipartimento di Medicina, Università di Salerno, Fisciano
SA), Italy
Dipartimento di Chimica, Università di Salerno, Fisciano
SA), Italy
ABSTRACT
(
One new natural monoterpene, 5-O-β-D-glucopyranosyl-2-hy-
droxy-p-cymene (1), and 11 known compounds were isolated
through a biologically oriented approach from the aerial parts
of Phagnalon sordidum L. The most active extract and fractions
were selected using 3 complementary antioxidant activity as-
says. Results and the different methods were compared by
relative antioxidant capacity index. In addition, the most ac-
tive extract of P. sordidum was subjected to liquid chromatog-
raphy coupled with electrospray ionization hybrid linear ion
trap quadrupole Orbitrap mass spectrometry to quantify sec-
ondary metabolites. Antioxidant activities of ethyl acetate ex-
tract, and purified 3,4-dihydroxyacetophenone (3) and nebro-
denside A (7) were demonstrated by in vitro cell free model
assays, and their protective effect against H O -induced oxi-
(
Key words
Phagnalon sordidum, Asteraceae, phenolic derivatives,
antioxidant activity, mass spectrometry
received February 15, 2019
revised
June 6, 2019
accepted June 7, 2019
Bibliography
DOI https://doi.org/10.1055/a-0953-5984
Published online | Planta Med © Georg Thieme Verlag KG
Stuttgart · New York | ISSN 0032‑0943
2
2
dative stress in a HepG2 (human hepatocellular carcinoma)
cell line was established.
Natural sources rich in antioxidant phenolic compounds have
been attracting increasing attention since they represent an im-
portant source of food supplements, beverages, and natural rem-
edies for several ailments. Nowadays, it is important to discover
new natural sources of safe and inexpensive antioxidants [3,4],
since some synthetic antioxidants show potential health risks and
toxicity [5]. The genus Phagnalon includes annual and perennial
herbaceous plants belonging to the family Asteraceae, including
about 200 species spread all over the world [6,7]. Phagnalon spp.
have found wide use, mainly in folk medicine, and in particular for
Introduction
In recent decades, the medicinal value of plant species has gained
more interest due to the discovery of new secondary metabolites
and evidence that they are effective as antioxidants [1]. Antioxi-
dants are vital substances with the ability to protect the body
from damage caused by free radical–induced oxidative stress,
such as in atherosclerosis, dementia, inflammation, coronary
heart disease, ageing, and cancer [2,3].
1
Dedicated to Professor Dr. Cosimo Pizza on his 70th birthday in recogni-
*
Equally contributing authors.
tion of his outstanding contribution to natural product research.
Cherchar H et al. Phytochemistry and Antioxidant … Planta Med

Original Papers
the treatment of hypertension and respiratory and gastric dis-
eases [8, 9]. Biological studies have also reported several other
properties, such as antibacterial and antifungal, expectorant and
antitussive, insect antifeedant, anti-hypouricemic, antidiabetic,
antioxidant, anti-inflammatory, and cytotoxic activity [6]. Pre-
vious phytochemical investigations of the genus have led to the
identification of about 125 chemical constituents, including flavo-
noids, anthraquinones, phytosterols, sesquiterpenes, diterpenes,
triterpenes, and caffeoylquinic acid derivatives [6].
Phagnalon sordidum L. is a perennial plant species distributed
throughout the Mediterranean region, growing in rocky environ-
ments [10]. Phytochemical profiling of P. sordidum has shown the
presence of several secondary metabolites, such as terpenes and
flavonoids and particularly caffeoylquinic chlorogenic acid deriva-
tives [11, 12]. In this study, an antioxidant-oriented approach was
carried out, leading to the isolation and chemical characterization
of 1 new natural compound, 5-O-β-D-glucopyranosyl-2-hydroxy-p-
cymene (1), together with 11 known phenolic derivatives (2–12),
from P. sordidum aerial parts. In order to more fully characterize
the phytochemical profile of P. sordidum, liquid chromatography-
high resolution electrospray ionization mass spectrometry (LC-
HRESIMS) and tandem MS (LC-HRESIMS/MS) analyses were also
performed. The antioxidant activity of extracts, fractions, and pure
compounds was tested using 3 different complementary assays:
▶
Fig. 1 RACI of fractions derived from the EtOAc extract of
P. sordidum.
As the EtOAc extract showed the strongest antioxidant activity,
it was further analyzed. Sephadex LH-20 column chromatography
(CC) was used for the preliminary fractionation, and 7 major frac-
tions (A–G) were collected and evaluated for their antioxidant ac-
tivities (▶ Fig. 1, Table 1S). The fraction A was inactive in the anti-
oxidation assays (data not shown). In the ABTS assay, fractions F
and C were the most active, with the highest radical-scavenging
activity (Table 1S). These results correlated with those of the
DPPH method. In fact, fractions D and F showed the highest radi-
cal-scavenging activities against the DPPH radical, followed by
fraction C (Table 1S). These results suggested that the most ac-
tive samples with regard inhibiting lipid peroxidation were frac-
tions F, G, D, and C (Table 1S). The lowest activity was found in
fraction E for all the assays.
2
,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-
diphenyl-1-picrylhydrazyl (DPPH), and inhibition of lipid peroxida-
tion by the β-carotene bleaching (BCB) test. In addition, the relative
antioxidant capacity indexes (RACI) were calculated; as dimension-
less values they were useful in comparing outcomes from the dif-
ferent assays. The protective effect of extracts and pure com-
pounds against H O -induced oxidative stress in the HepG2 (hu-
2
2
man hepatocellular carcinoma) cell line was also evaluated.
The data from the ABTS, DPPH, and BCB assays were used to
calculate RACI. This recently introduced statistical approach was
used to compare antioxidant capacities measured by different
chemical methods, which, as in this study, were often expressed
in different units [13]. This dimensionless index measured the dis-
tance between the average of the results obtained and the raw
data expressed in standard deviation units. The interpretation of
the values obtained with the RACI is presented in ▶ Fig. 1, with
higher values representing greater antioxidant capacity.
Fraction F showed the highest anti oxidation rate (1.14), fol-
lowed by fractions D (0.54) and C (0.45). By contrast, fraction E
presented the lowest index (− 1.42) and, therefore, the smallest
antioxidant activity. The results identified P. sordidum fractions
B–G as active for antioxidation, and these were further analyzed.
Reverse phase HPLC was used for the isolation of pure com-
pounds, which might possibly be responsible for the antioxidant
activity of the fraction (▶ Fig. 2, Table 1).
Results and Discussion
In this study the aerial parts of P. sordidum were extracted using
solvents of increasing polarity. All extracts were subjected to the
ABTS, DPPH, and BCB assays to screen for antioxidant activity.
ABTS radical cation decolorization test and DPPH (neutral radical)
test were both spectrophotometric methods, applicable for both
lipophilic and hydrophilic compounds, and widely used for the as-
sessment of radical scavenging activity. Results from the ABTS
and DPPH assays were expressed as milligrams of 6-hydroxy-
2
,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) equiva-
lents per gram of extract/fraction (mg TE/g). In addition, the BCB
test measured the effectiveness of antioxidant compounds
present in the samples in binding the radical derived from linoleic
acid and preventing the destruction of the conjugated system. β-
carotene was used for comparative purposes, with results being
expressed as percentage of antioxidant activity (%AA) at a final
sample concentration of 0.1 mg/mL.
Compound 1 was obtained as a yellow powder. IHRESIMS anal-
+
ysis exhibited a molecular ion at m/z 367.1360 [M + Na] , indicat-
The petroleum ether and chloroform extracts were inactive
data not shown). By contrast, the ethyl acetate (EtOAc) extract
showed antioxidant activity (ABTS assay, 1485.00 ± 36.78 mg TE/
g; DPPH assay, 999.02 ± 3.76 mg TE/g; and AA in BCB assay
8.89 ± 0.29%) and was a good source of phenolics followed by
ing a molecular formula C16H24O . A product ion detected by its
8
+
(
ESIMS spectrum at m/z 205 [M + Na – 162] suggested the pres-
ence of a hexose unit. The H NMR, 13C NMR spectra (▶ Table 1)
of compound 1 indicated, besides signals attributable to 1 sugar
moiety, the presence of an 1,2,4,5 tretrasubstituted aromatic
1
1
3
the n-butanol extract (Table 1S).
ring, and 4 sp carbons, including 2 methyls, 1 hydroxymethylene,
Cherchar H et al. Phytochemistry and Antioxidant… Planta Med

▶
Table 1 1H and 13C NMR data of compound 1 (MeOH-d4,
a
6
00 MHz) .
Position
1
δH
δC
1
2
3
4
5
6
7
8
9
–
147.9
139.4
113.0
150.6
125.5
116.0
26.0
23.0
23.0
60.0
104.0
74.0
77.0
70.0
78.8
61.4
–
6.70; s
–
–
7.12; s
3.50; m
1.19; d (6.5)
1.20; d (6.5)
4.60; s
1
0
1
2
3
4
5
6
6
′
4.76; d (8.0)
3.42; br t (8.5)
3.37; t (9.0)
3.42; t (9.0)
3.46; m
′
′
▶
Fig. 2 Chemical structure of pure compounds 1–12.
′
′
a'
b’
3.72; dd (11.5, 5.0)
3.92; dd (11.5, 3.0)
and 1 methine carbon. Double-quantum filtered correlation spec-
troscopy, selective 1D-TOCSY, and heteronuclear single quantum
coherence spectroscopy (HSQC) analysis of compound 1 estab-
lished the presence of an isopropyl group showing connectivities,
H-7—Me-9 and H-7—Me-8, indicative of an aromatic monoterpe-
noid. The cymene skeleton was confirmed by the presence in the
1H NMR spectrum of signals at δ 3.50 (1H, m,), 1.19 (3H, d,
J = 6.5 Hz), and 1.20 (3H, d, J = 6.5 Hz), characteristic of a isopropyl
ring [14]. Chemical shifts, signal multiplicities, and J-values in the
1H NMR spectrum and 13C NMR chemical shifts indicated the pres-
ence of a β-glucopyranosyl moiety. The sugar moiety was deter-
mined to be a D enantiomer by hydrolysis, trimethylsilylation,
and GC analysis [15]. The chemical shift assignments of the car-
bon atoms were established from the HSQC and HMBC spectra.
Key HMBC correlations were observed between H-7—C-1, H-7—
C-1, H-7—C-9; H-3—C-1, H-3—C-5; H-6—C-10, H-6—C-1; H-6—C-
a
Data assignments were confirmed by DQF-COSY, 1D-TOCSY, HSQC,
and HMBC experiments.
All pure compounds were investigated using the same antioxi-
dation assays described above; however, the newly identified com-
pound 1 was not tested as it was isolated in very small amounts.
Compound 11 in fraction G presented the highest RACI value
(1.03), followed by compounds 10, 5, 12, and 7 (▶ Fig. 3). These
results explained the antioxidant activity of their original fractions.
Compounds 10 and 12 came from antioxidant fraction F, which had
the highest activity, while compound 7 came from fraction D and
compound 5 from fraction C, consistent with previous results [21,
30, 31]. By comparison, compound 9 had the lowest index (− 1.28)
and came from the fraction with the lowest activity, E.
4
; and H-1glc—C-1 and located the glucopyranosyl moiety at C-1.
Therefore, the structure of compound 1 was 5-O-β-D-glucopyra-
nosyl-2-hydroxy-p-cymene, a new natural product. In addition,
With its phytochemical profile and antioxidant activity, the
EtOAc extract of P. sordidum was assessed in a cell model. There
was no cytotoxicity for a HepG2 cell line (▶ Fig. 4a), even at the
highest concentration of the EtOAc extract after 48 h of treat-
ment. Moreover, to confirm the results obtained from the in vitro
antioxidation assays, intracellular levels of reactive oxygen species
(ROS) were measured. For this test, 2′,7′–dichlorofluorescin
diacetate (DCFH‑DA), a nonfluorescent cell permeable probe,
was used. In the presence of intracellular ROS, DCF‑DA was oxi-
dized, with conversion to DCFH, the florescence of which was pro-
portional to the concentration of ROS in the cells. H O treatment
significantly increased ROS in HepG2 cells; however, treatment
with the EtOAc extract decreased their levels to approximately
those of the control (CTRL, ▶ Fig. 4b).
Most of the identified compounds had previously been demon-
strated to have antioxidant activity on cells [30, 32–39]; however,
the current study was the first report of the effect of compounds 3
3
[
-O-feruloyl quinic acid (2) [16], 3,4-dihydroxyacetophenone (3)
17], 3-O-caffeoyl quinic acid (4) [18], caffeic acid (5) [19], gna-
phaliol 3-O-β-D-glucopyranoside (6) [20], nebrodenside A (7)
21, 22], ergiside B (8) [23], picein (9) [24], eryodictiol-7-O-meth-
[
yl ether (10) [25], eryodictiol (11) [25], and 3,4-O-dicaffeoyl
quinic acid (12) [25] were isolated and characterized.
Analyses of the liquid chromatography coupled with electro-
spray ionization hybrid linear ion trap quadrupole Orbitrap mass
spectrometry (LC‑ESI/LTQOrbitrap/MS) profile of the metabolites
identified in the initial EtOAc extract of P. sordidum is reported in
2
2
▶
Table 2. This revealed the presence of 12 compounds (1–12),
identified by comparing their m/z values in the total ion current
with those described in literature [26–29]. ▶ Table 3 reports the
content of phenolic compounds (mg/kg) found in the EtOAc ex-
tract of the aerial parts of P. sordidum.
Cherchar H et al. Phytochemistry and Antioxidant … Planta Med

Original Papers
▶
Table 2 Metabolites identified in P. sordidum EtOAc extract by LC‑ESI/LTQOrbitrap/MS and LC‑ESI/LTQOrbitrap/MS/MS analysis.
n
Retention time (min)
Molecular Formula
C14H18O7
Precursor ion (m/z)
297.2687
Polarity
Negative
Positive
Product ions (m/z)
279.06; 177.01
205.14
Identity
1
1.24
1.83
picein (9)
2
C16H24O8
367.1360
5-O-β-D-glucopyranosyl-2-
hydroxy-p-cymene (1)
3
2.75
2.86
C9H8O4
179.0389
367.1718
Negative
Negative
161.3; 135.1;
caffeic acid (5)
1
9
33.2; 116.91;
7.02
4
C17H20O9
349.11; 323.10;
3-O-feruloyl quinic acid (2)
2
1
91.17; 193.02;
32.99
5
6
4.48
5.4
C17H24O7
C16H18O9
339.2287
353.2336
Negative
Negative
320.95; 177.06;
60.99
nebrodenside A (7)
1
191.02; 179.05;
3-O-caffeoyl quinic acid (4)
1
1
73.03;
60.6;134.9
7
5.47
C25H24O12
515.1786
Negative
497.10; 353.10;
3,4-O-dicaffeoyl quinic acid (12)
1
1
91.01; 178.98;
61.12
8
9
0
8.31
8.6
C12H22O6
C8H8O3
261.1952
151.0666
287.2121
Negative
Negative
Negative
n. s.
ergiside B (8)
n. s.
3,4-dihydroxyacetophenone (3)
eriodictyol (11)
1
1
1
8.66
C15H12O6
269.02; 243.01;
2
1
02.89; 161.08;
50.92; 106.89
1
2
8.92
C16H14O6
C19H26O10
301.1706
413.2795
Negative
Negative
283.01; 239.02;
eriodictyol-7-methyl ether (10)
1
1
93.02; 178.94;
50.94
10.36
n. s.
gnaphaliol 3-O-β-D-glucopyrano-
side (6)
n.s.: no signal
and 7 on HepG2 cells. Compounds 3 and 7 were tested at differ-
ent concentrations (▶ Fig. 5a and Fig. 5b, respectively). Only the
highest concentration of the tested pure compounds showed cy-
totoxicity after 48 h of treatment. Notably, HepG2 treated with
these pure compounds also showed a significant decrease in intra-
cellular ROS (▶ Fig. 5c). For in vitro antioxidant activity, com-
pound 7 had a higher RACI than compound 3 (▶ Fig. 3). The full
procedure for the analysis of antioxidation in extracts from the
aerial parts of P. sordidum is given in ▶ Fig. 6. Taken together, the
data indicated that fractions and pure compounds from P. sordi-
dum might prevent the formation of ROS in cells.
▶
Table 3 Compound contents (expressed as mg/kg) in the EtOAc
extract from P. sordidum.
Compound
Mean ± SD
78.4 ± 3.3
27.3 ± 1.1
81.8 ± 2.6
215.8 ±11.2
179.1 ±9.1
121.7 ±4.8
868.4 ±13.1
55.5 ± 2.7
35.6 ± 1.5
33.0 ± 1.4
picein (9)
5
-O-β-D-glucopyranosyl-2-hydroxy-p-cymene (1)
caffeic acid (5)
-O-feruloyl quinic acid (2)
3
nebrodenside A (7)
3-O-caffeoyl quinic acid (4)
1H NMR, HSQC, HMBC, 1D-TOCSY, and HRESIMS spectra of
compound 1, the LC–MS profile of the EtOAc extract, and anti-
oxidant activities of P. sordidum extracts, fractions, and pure com-
pounds are available as Supporting Information.
3
,4-O-dicaffeoyl quinic acid (12)
ergiside B (8)
,4-dihydroxyacetophenone (3)
3
eriodictyol (11)
eriodictyol-7-methyl ether (10)
gnaphaliol 3-O-β-D-glucopyranoside (6)
29.8 ± 1.1
Materials and Methods
tracea
a
Minimum value not calculated.
Chemicals and reagents
Solvents used for LC‑ESI/LTQOrbitrap/MS, and for the extractions
were purchased from VWR, while acetonitrile and formic acid
were purchased from Merck. Chloroform, MeOH, EtOAc, n-buta-
nol, isopropanol, DMSO, and orthophosphoric acid were pur-
Cherchar H et al. Phytochemistry and Antioxidant… Planta Med

▶
Fig. 3 RACI of pure compounds derived from P. sordidum.
chased from Carlo Erba Reagents. The following were purchased
from Sigma-Aldrich: ABTS, potassium persulfate, DPPH, β-caro-
tene, linoleic acid, Tween 20, DCFH‑DA, DMEM, glutamine, peni-
cillin, streptomycin, hydrogen peroxide, PBS, MTT, Trolox, ascor-
bic acid, butylhydroxytoluene (BHT), D-glucose, FBS, and 1-(tri-
methylsilyl)imidazole and pyridine.
General experimental procedures
▶
Fig. 4 a Cell viability, evaluated by MTT assay, of HepG2 cells
treated for 24 and 48 h with different concentrations of EtOAc ex-
tract from P. sordidum aerial parts. Data are expressed as the
mean ± SD of 3 independent experiments (n = 3). **p < 0.01 vs.
CTRL. b Effect of EtOAc extract on H2O2-induced intracellular ROS
generation in HepG2 cells stained with DCFH‑DA and analyzed by
flow cytometry. Data are expressed as the mean ± SD of 3 indepen-
dent experiments (n = 3). # p < 0.001 vs. CTRL; *p < 0.05 and
Optical rotations were measured on an Atago AP-300 digital po-
larimeter equipped with a sodium lamp (589 nm) and a 1-dm mi-
crocell. Briefly, NMR spectroscopy involved recording at 300 K in
CD OD on a Bruker DRX-500 spectrometer (Bruker BioSpin
3
GmBH) [19]. HRESIMS was performed in a positive ion mode on a
quadrupole time-of-flight premier mass spectrometer (Waters).
ESI‑MS spectra were obtained from an LCQ Advantage Thermo-
Finnigan spectrometer (ThermoFinnigan), equipped with Xcalibur
software. CC was carried out over Sephadex LH-20 (40–70 µm;
Pharmacia). Reverse-phase HPLC (RP-HPLC) was conducted on a
Shimadzu LC-8A series pumping system, equipped with a
Shimadzu RID-10A refractive index detector and Shimadzu injec-
tor on a C-18 Luna column (250 × 10 mm, 10 µm; Phenomenex).
TLC was with silica gel 60 F254 (0.20 mm thickness) plates (Merck)
and cerium sulphate (Sigma-Aldrich) as the spray reagent [19].
LC‑ESI/LTQOrbitrap/MS analyses were performed on a Accela 600
HPLC system (Thermo Scientific) coupled to a LTQ Orbitrap XL
mass spectrometer (Bremen). Separation was achieved using a
Luna 2.50 µm C18 (100 mm × 2.10 mm) column (Phenomenex).
HepG2 cells were analyzed on a FACS Canto II flow cytometer
##
*
*p < 0.01 vs. H2O2-treated cells.
Extraction and isolation
The powdered dried aerial parts (1.3 kg), dried at room tempera-
ture, of P. sordidum were macerated at room temperature in EtOH
and water (8:2). The extract was concentrated under low pres-
sure, and the residue (380.0 g) was dissolved with water (1 L). It
was then filtered and successively extracted with petroleum ether,
chloroform, EtOAc, and n-butanol (3 × 300 mL each) to yield 0.8,
1.8, 11.0, and 20.0 g of the respective residues. The EtOAc extract
(2.5 g) was then submitted to a Sephadex LH-20 CC, using MeOH
as eluent, obtaining 7 fractions (A–G) grouped by TLC. Fraction C
(320.1 mg) was purified by RP-HPLC using MeOH‑H O (30:70) to
2
(
BD Pharmingen). All spectrophotometric measurements were
give compounds 2 (1.9 mg, t 14.0 min), 3 (1.4 mg, t 20.0 min), 4
(2.0 mg, tR 42.0 min), and 5 (1.6 mg, tR 49.0 min). Fraction D
(815.0 mg) was subjected to RP-HPLC on a C-8 Luna column
R
R
taken in 96-well microplates on a SPECTROstarNano UV/Vis spec-
trophotometer (BMG Labtech), and each reaction was performed
in triplicate.
(250 × 10 mm, 10 µm) with MeOH‑H O (2:3) as eluent to give
2
compounds 1 (1.2 mg, tR 16.5 min), 6 (2.1 mg, tR 31.0 min), 7
Plant material
(
7.2 mg, tR 45.0 min), and 8 (2.1 mg, tR 70.0 min). RP-HPLC of
The aerial parts of P. sordidum were collected in May 2016 from
Guelma (eastern Algeria). The plant was authenticated by Mr.
Kamel Kabouche. A voucher specimen (LOST Phs05/16) was
deposited at the herbarium of the Laboratory of Therapeutic
Substances (LOST), Faculty of Sciences, Université des Frères
Mentouri-Constantine 1, Algeria.
fraction E (289.1 mg) on a C-8 Luna column (250 × 10 mm,
10 µm), with MeOH‑H O (3:7) as eluent yielded compound 9
2
(4.2 mg, t 45.0 min). Fraction F (163.6 mg) underwent RP-HPLC
R
on a C-18 Luna column (250 × 10 mm, 10 µm) with MeOH‑H O
2
(3:7) as eluent to give compounds 10 (2.5 mg, t 25.0 min) and
R
Cherchar H et al. Phytochemistry and Antioxidant … Planta Med

Original Papers
▶
Fig. 6 Procedure followed to analyze the aerial parts of P. sordi-
dum.
comparison of retention time with an authenticated sample of D-
glucose (Sigma Aldrich), after treatment with 1-(trimethylsilyl)
imidazole in pyridine.
LC‑ESI/LTQOrbitrap/MS
To analyze the EtOAc extract of P. sordidum, an in-house HPLC
method coupled with a mass spectrometer, associated with linear
trap quadrupole and an Orbitrap mass analyzer, was used. LC‑ESI/
LTQOrbitrap/MS analyses were performed in positive and negative
ion mode using an Accela 600 HPLC system (Thermo Scientific)
coupled to an LTQ Orbitrap XL mass spectrometer. Separation
was achieved using a Luna 2.5 µm C18 (100 mm × 2.10 mm) col-
umn (Phenomenex).
The mobile phases used were water + 0.1% formic acid (solvent
A) and acetonitrile (solvent B). The flow rate was 0.200 mL/min
and the gradient was 20% of B from 0 min to 1 min, 80% at
2
3
1 min, 95% at 22 min until 30 min, returning at 20% of B at
1 min to 35 min.
For MS in positive ion mode, source voltage was 3 kV, capillary
▶
Fig. 5 a Cell viability, evaluated by MTT assay, of HepG2 cells
treated for 24 and 48 h with different concentrations of pure com-
pound 3 from P. sordidum aerial parts. Data are expressed as the
mean ± SD of 3 independent experiments (n = 3). *p < 0.05,
voltage 49 V, and tube lens voltage120 V; while in negative ion
mode, source voltage was 5 kV, capillary voltage − 48 V, and tube
lens voltage − 176.47. Capillary temperature for both positive and
negative ion modes was 280°C. MS spectra were acquired by full
range acquisition covering m/z 150–1000.
Data were acquired using Xcalibur software version 2.1, and
for fragmentation studies a data dependent scan experiment
was carried out selecting precursor ions as the most intensive
peak in LC‑MS analysis. Identification of compounds was based
on retention times, accurate mass measurements, MS/MS data,
exploration of specific spectral libraries and public repositories
for MS-based metabolomic analysis [27, 29], and comparison with
data reported in the literature [21,22, 26,28, 41].
*
**p < 0.001 vs. CTRL. b Cell viability, evaluated by MTT assay, of
HepG2 cells treated for 24 and 48 h with different concentration of
pure compound 7 from P. sordidum aerial parts. Data are expressed
as the mean ± SD of 3 independent experiments (n = 3). *p < 0.01
vs. CTRL. c Effects of pure compounds, 3 and 7, on H2O2-induced
intracellular ROS generation in HepG2 cells stained with DCFH‑DA
and analyzed by flow cytometry. Data are expressed as the mean ±
SD of 3 independent experiments (n = 3). # p < 0.001 vs. CTRL;
##
*
**p < 0.001 vs. H2O2-treated cells.
1
2 (1.8 mg, t 35.0). Finally, fraction G (30.0 mg) did not undergo
R
Antioxidant activity
further fractionation and was identified as compound 11.
DPPH assay
Compound 1: yellow amorphous powder; [α] − 23.1 (c 0.1,
D
1
13
MeOH); H and C NMR data, see ▶ Table 1; ESIMS m/z 367
The radical scavenging activity of the samples was evaluated in vi-
tro using the neutral radical DPPH and Trolox as a standard. Fifty
microliters of different dilutions of sample or standard were
added to 200 µL of a methanolic solution of DPPH in a 96-well
plate. After 30 min in the dark at room temperature, the absorb-
+
+
+
[
(
M + Na] , 205 [M + Na – 162] ; HRESIMS m/z 367.1360 [M + Na]
calcd. for C16H24O Na, 367.1368).
8
Acid hydrolysis of compound 1 was carried out as previously
described [40]. D-Glucose was identified as the sugar moiety by
Cherchar H et al. Phytochemistry and Antioxidant… Planta Med

ance of the mixtures was measured at 515 nm. The results were
expressed as milligrams of Trolox equivalents per gram of dried
extract (mg TE/g) [42].
different concentrations of fractions and pure compounds 3 and 7
for 24 h and then treated with 2 mM H O for 1 h. Finally, the cells
2
2
were stained with 10 µM DCFH‑DA for 30 min at 37°C in the dark,
and fluorescence was measured with a FACSCanto II flow cytome-
ter (BD Pharmingen) at an excitation wavelength of 485 nm and
emission wavelength of 515–540 nm.
ABTS assay
The antioxidant capacity of samples was also studied using the
cationic 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid dia-
mmonium salt) (ABTS ) radical [42]. Fifteen microliters of each
•
Statistical analysis
sample were added to 235 µL of ABTS+• solution, and the mixture
was incubated in the dark for 2 h. Absorbance was then measured
at 734 nm. Trolox was used as a standard, and results were ex-
pressed as milligrams of Trolox equivalents per gram of dried ex-
tract (mg TE/g).
Data was subjected to analysis of variance (one-way analysis of
variance) followed by Tukeyʼs test, using GraphPad Prism 5 Soft-
ware. A p-value ≤ 0.05 was considered statistically significant. All
assays were performed in triplicate as 3 independent experi-
ments. Data were reported as means ± standard deviation (SD).
2
The R values for all calibration curves were over 0.99.
Inhibition of lipid peroxidation
Supporting Information
The BCB method [13] was used to evaluate the inhibition of lipid
peroxidation. To an Eppendorf tube containing 50 µL of 0.1 mg/
mL sample, 950 µL of β-carotene emulsion was added. From this
mixture, 250 µL was transferred into a 96-well plate and incubated
at 50°C for 3 h. Absorbance was measured at 470 nm at 0′, 30′,
1H NMR, HSQC, HMBC, 1D-TOCSY, and HRESIMS spectra of com-
pound 1, the LC–MS profile of the EtOAc extract, and antioxidant
activities of P. sordidum extracts, fractions, and pure compounds
are available as Supporting Information.
6
0′, 90′, 120′, 150′, and 180′ of incubation, with BHT being used
as a positive control. Results were expressed as percentage of
antioxidant activity (%AA) on the basis of BCB inhibition.
Conflict of Interest
The authors declare that they have no conflict of interest.
Cell culture
The HepG2 cell line has been used as a cellular model for testing
the effectiveness of extracts or pure compounds. Cells were cul-
tured in DMEM supplemented with 10% FBS, 2 mM glutamine,
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