Antioxidant and free radical-scavenging activity of constituents from two Scorzonera species

Article abstract of DOI:10.1016/j.foodchem.2014.03.097

The aim of this study was to investigate the secondary metabolites content of Scorzonera papposa DC., an edible plant eaten in the desert region of Jordan and to assess its antioxidant and free radical-scavenging activity. By using this bioassay-oriented

Full text of DOI:10.1016/j.foodchem.2014.03.097

Accepted Manuscript
Antioxidant and free radical-scavenging activity of constituents from two Scor-
zonera species
Luigi Milella, Ammar Bader, Nunziatina De Tommasi, Daniela Russo,
Alessandra Braca
PII:
S0308-8146(14)00500-7
DOI:
http://dx.doi.org/10.1016/j.foodchem.2014.03.097
FOCH 15627
Reference:
Food Chemistry
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20 December 2013
17 March 2014
19 March 2014
Please cite this article as: Milella, L., Bader, A., De Tommasi, N., Russo, D., Braca, A., Antioxidant and free radical-
scavenging activity of constituents from two Scorzonera species, Food Chemistry (2014), doi: http://dx.doi.org/
10.1016/j.foodchem.2014.03.097
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Running Title: Antioxidant activity of constituents from Scorzonera species
Antioxidant and free radical-scavenging activity of constituents from two Scorzonera species
Luigi Milella a, Ammar Bader b , Nunziatina De Tommasi c,*, Daniela Russo a, Alessandra Braca d
a
Dipartimento di Scienze, Università degli Studi della Basilicata, Viale dell’Ateneo Lucano 10,
85100 Potenza, Italy
b Department of Pharmacognosy, Faculty of Pharmacy, Umm Al-Qura University, P.O. Box 13174,
21955 Makkah, Saudi Arabia
c Dipartimento di Farmacia, Università di Salerno, via Ponte Don Melillo, 84084 Fisciano, Italy
d Dipartimento di Farmacia, Università di Pisa, via Bonanno 33, 56126 Pisa, Italy
* Corresponding author. Tel.: +39-089-969754; fax: +39-089-969602.
E-mail address: detommasi@unisa.it (N. De Tommasi)
21

Abstract
The aim of this study was to investigate the secondary metabolites content of Scorzonera papposa
DC., an edible plant eaten in the desert region of Jordan and to assess its antioxidant and free
radical-scavenging activity. By using this bioassay-oriented approach nine compounds, including
the new natural compounds (6-trans-p-coumaroyl)-3-O-β-D-glucopyranosyl-2-deoxy-D-riburonic
acid (1), (6-cis-p-coumaroyl)-3-O-β-D-glucopyranosyl-2-deoxy-D-riburonic acid (2a), (6-trans-p-
coumaroyl)-3-O-β-D-glucopyranosyl-2-deoxy-D-riburonic acid methyl ester (3), and (6-trans-p-
coumaroyl)-3-O-β-D-glucopyranosyl-(5-acetyl)-2-deoxy-D-riburonic acid (4), having the rare
1
deoxy-D-riburonic acid moiety, were isolated. Their structures were elucidated by UV, MS, H and
13C NMR and 2D NMR. The antioxidant activity of the S. papposa pure compounds and of related
derivatives isolated from another Scorzonera species (S. judaica Eig.) was also tested. The Relative
Antioxidant Capacity Index (RACI) was applied as an integrated method to compare the
antioxidant activities obtained using different chemical methods.
Keywords: Scorzonera papposa DC.; Scorzonera judaica Eig.; glycosides; antioxidant activity
22

1. Introduction
Recently, wild edible plants attract a new attention since they represent an important source of
food, beverages and natural remedies for several ailments. People of the rural societies are in
contact with natural sources more than the urban ones. In the Middle East a narrow number of
Bedouins, living in the desert and depending mainly on the pasture of goats and camels, have a
good knowledge about edible (Dal Piaz, Vassallo, Lepore, Tosco, Bader, & De Tommasi, 2009;
Malafronte, Vassallo, Dal Piaz, Bader, Braca, & De Tommasi, 2012), medicinal (Bader, Braca, De
Tommasi, & Morelli, 2003) and aromatic plants (Flamini, Cioni, Morelli, & Bader, 2007). The
genus Scorzonera includes about 170 species distributed worldwide; it belongs to the family
Asteraceae many of which are edible plants. This genus has attracted the attention of researchers
due to the many chemical classes of its secondary metabolites, including dihydroisocoumarins,
stilbenes, lignans, phenolic derivatives (Bader, De Tommasi, Cotugno, & Braca, 2011), phtalides
(Sari, Zidorn, & Ellmerer, 2007), coumarins, kavalactones (Jiang, Wang, Lu, & Yue, 2007),
sesquiterpenes (Zidorn, 2008), triterpenes (Wang, Li, Qui, & Guan, 2007), and flavonoids
(Sareedenchai & Zidorn, 2010). Scorzonera papposa DC. is a perennial herb, with cylindrical root,
lanceolate leaves with undulate margin, pink showy flowers (Al-Eisawi, 1998). It grows extensively
in desert, semi-desert and mountain environments particularly after raining season; all parts of this
plant are considered edible and they are eaten raw or cooked (Baily & Danin, 1981; Tukan, Takruri,
& Al-Eisawi, 1998); however no previous phytochemical investigation on this species was carried
out to date. Since the non economic crops constitute an important source for nutrition mainly in
unfavorable condition, the aim of this study was to investigate the secondary metabolites content of
S. papposa growing in Jordan and to evaluate their antioxidant and free radical-scavenging
23

activities. On the basis of obtained results, some related compounds isolated by our group from
another edible Scorzonera species (S. judaica Eig.) were also tested.
2. Materials and methods
2.1. General
Optical rotations were measured on a Perkin-Elmer 241 polarimeter equipped with a sodium lamp
(589 nm) and a 1 dm microcell. UV spectra were recorded on a Perkin-Elmer-Lambda
spectrophotometer. NMR experiments were performed on a Bruker DRX-600 spectrometer at 300
K. All the 2D NMR spectra were acquired in CD3OD in the phase-sensitive mode with the
transmitter set at the solvent resonance and TPPI (Time Proportional Phase Increment) used to
achieve frequency discrimination in the ω1 dimension. Standard pulse sequences and phase cycling
were used for DQF-COSY, TOCSY, HSQC, and HMBC, experiments. HRESIMS were acquired in
the positive ion mode on a Q-TOF premier spectrometer equipped with a nanoelectrospray ion
source (Waters-Milford, MA, USA). ESIMS were obtained from an LCQ Advantage
ThermoFinnigan spectrometer (ThermoFinnigan, USA), equipped with a Xcalibur software.
Column chromatography (CC) was performed over Sephadex LH-20 (40-70 µm, Amersham
Pharmacia Biotech AB, Uppsala, Sweden). HPLC separations were conducted on a Shimadzu LC-
8A series pumping system equipped with a Shimadzu RID-10A refractive index detector and
Shimadzu injector on a C18 µ-Bondapak column (30 cm x 7.8 mm, 10 µm Waters, flow rate 2.0 ml
min-1). TLC analyses were carried out using glass-coated silica gel 60 F254 (0.20 mm thickness)
plates (Merck). GC analyses were performed using a Dani GC 1000 instrument on a L-CP-Chirasil-
Val column (0.32 mm x 25 m).
24

2.2. Chemicals
Sodium acetate trihydrate, 2, 4, 6-tripyridyl-s-triazine (TPTZ), iron (III) chloride (FeCl3 6H2O),
Folin-Ciocalteu reagent, 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical, β-carotene, linoleic acid,
Tween 20, 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic Acid (Trolox) and gallic acid were
purchased from Sigma-Aldrich (Milano-Italy). n-Hexane, chloroform, methanol, hydrochloric acid
and glacial acetic acid were purchased from Carlo Erba (Milano-Italy). All spectrophotometric
measurement were done on a CARY 1E UV-VIS spectrophotometer (Varian, Leini, Italy). All other
chemicals and solvents used in this study were of HPLC grade.
2.3. Plant materials
The aerial parts and the tuberous roots of S. papposa were collected during the flowering stage in
the Dab’a desert reserve (50 km South of Amman), Jordan, during April 2009 and were identified
by one of the authors (A. Bader). A voucher specimen (number Jo-It 2009/2) is deposited in
herbarium of the laboratory of Pharmacognosy, Umm Al-Qura University, Makkah, Saudi Arabia.
2.4. Extraction and isolation
The aerial parts (300 g) and the roots (250 g) of S. papposa were dried and sequentially extracted
with solvents of increasing polarity, n-hexane, CHCl3, CHCl3-MeOH (9:1), and MeOH by
exhaustive maceration (3 × 2 L), to give 4.1, 2.5, 2.7, 18.0 g of the respective residue for the aerial
parts and 7.9, 4.6, 5.2, 24.6 g for the roots respectively. Both MeOH extracts were partitioned
25

between n-BuOH and H2O. The n-BuOH soluble fraction of aerial parts (1.8 g) was separated by
CC using Sephadex LH-20 (3 x 100 cm) with MeOH as eluent at flow rate 0.8 mL/min. Collected
fractions were combined into five groups (A-E), based on their TLC profiles. Fractions B (380.7
mg) and C (279.5 mg) were separately purified by RP-HPLC eluting with MeOH-H2O (3:7) to give
compounds 1 (12 mg, tR = 27 min) and 3 (1.8 mg, tR = 46 min), from fraction B, and compounds
2a-2b (1.0 mg, tR = 21 min) and 1 (3.6 mg, tR = 27 min), from fraction C. Fractions D (190 mg) and
E (264.6 mg) were separately purified by RP-HPLC eluting with MeOH-H2O (2:3) to give
compounds 8 (1.0 mg, tR = 13 min) and 9 (1.1 mg, tR = 18 min), from fraction D, 7 (2.0 mg, tR = 12
min) and 6 (14.6 mg, tR = 13 min), from fraction E, respectively. The CHCl3-MeOH extract of
aerial parts (1.5 g) was separated by CC using Sephadex LH-20 (3 x 100 cm) with MeOH as eluent
at flow rate 0.8 mL/min. Fractions were pooled into four groups (A-D) on the basis of their TLC
results. Fractions B (508.2 mg) and C (122.8 mg) were separately purified by RP-HPLC with
MeOH-H2O (3.5:6.5) to give pure compound 4 (1.5 mg, tR = 37 min), from fraction B and pure
compounds 1 (14 mg, tR = 12 min), 2a-2b (3.3 mg, tR = 16 min), and 4 (3.0 mg, tR = 37 min), from
fraction C, respectively. The n-BuOH soluble fraction of roots (2.6 g) was separated by CC using
Sephadex LH-20 (3 x 100 cm) with MeOH as eluent at flow rate 0.8 mL/min. Collected fractions
were combined into four groups (A-D), based on their TLC profiles. Fraction C (358.8 mg) was
subjected to Biotage Isolera column chromatography (25 g silica SNAP cartridge) eluting with
CHCl3 followed by increasing concentrations of MeOH in CHCl3, at a flow rate 25 mL/min.
Compound 5 (9.6 mg) was eluted with CHCl3-MeOH 7:3.
Thunberginol F (10), hydramacrophyllol B (11), thunberginol F 7-O-β-D-glucopyranoside (12),
hydrangenol 4'-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (13), hydramacrophyllol A (14),
hydrangenol (15), hydrangenol 8-O-β-D-glucopyranoside (16), and 3S-hydrangenol 4'-O-α-L-
rhamnopyranoysl-(1→3)-β-D-glucopyranoside (17), were purified from S. judaica as reported in
our previous paper (Bader et al., 2011).
26

2.4.1. (6-trans-p-coumaroyl)-3-O-β-D-glucopyranosyl-2-deoxy-D-riburonic acid (1)
Amorphous powder; UV (MeOH) λmax (log ε) 229 (3.83), 314 (4.01) nm; HRESIMS: m/z
481.1335 [M+Na]+, calcd. for C20H26O12 m/z 458.1424; ESIMS: m/z 481 [M+Na]+, 457 [M-H]-, 325
[M-H-132]-; 1H and 13C NMR data (CD3OD, 600 MHz) see Table 1.
2.4.2. (6-cis-p-coumaroyl)-3-O-β-D-glucopyranosyl-2-deoxy-D-riburonic acid (2a)
Amorphous powder; UV (MeOH) λmax (log ε) 230 (3.85), 312 (4.01) nm; HRESIMS: m/z
481.1340 [M+Na]+, calcd. for C20H26O12 m/z 458.1424; ESIMS: m/z 481 [M+Na]+, 457 [M-H]-; 1H
and 13C NMR data (CD3OD, 600 MHz) see Table 1.
2.4.3. (6-cis-p-coumaroyl)-3-O-β-D-glucopyranosyl-2-deoxy-D-ribono-γ-lactone (2b)
Amorphous powder; UV (MeOH) λmax (log ε) 230 (3.85), 312 (4.01) nm; HRESIMS: m/z
1
463.1225 [M+Na]+, calcd. for C20H24O11 m/z 440.1319; ESIMS: m/z 439 [M-H]-; H NMR
(CD3OD, 600 MHz): δ 2.74 (2H, overlapped signal, H2-2), 3.21 (1H, dd, J = 9.0, 8.0 Hz, H-2'), 3.32
(1H, t, J = 9.0 Hz, H-4'), 3.36 (1H, t, J = 9.0 Hz, H-3'), 3.53 (1H, m, H-5'), 3.93 (2H, m, H2-5), 4.30
(1H, dd, J = 12.0, 5.0 Hz, H-6'b), 4.40 (1H, d, J = 8.0 Hz, H-1'), 4.48 (1H, dd, J = 12.0, 3.5 Hz, H-
6'a), 4.53 (1H, m, H-3), 4.57 (1H, m, H-4), 5.81 (1H, d, J = 12.0 Hz, H-α), 6.89 (1H, d, J = 12.0
Hz, H-β), 6.77 (2H, d, J = 8.0 Hz, H-3''/H-5''), 7.66 (2H, d, J = 8.0 Hz, H-2''/H-6''); 13C NMR
(CD3OD, 600 MHz): δ 38.3 (C-2), 60.3 (C-5), 64.2 (C-6'), 71.2 (C-4'), 75.1 (C-2'), 75.4 (C-5'), 77.2
27

(C-3), 77.6 (C-3'), 84.6 (C-4), 105.1 (C-1'), 115.6 (C-3''/C-5''), 116.0 (C-α), 128.0 (C-1''), 133.5 (C-
2''/C-6''), 145.2 (C-β), 159.8 (C-4''),168.5 (COO), 177.0 (C-1).
2.4.4. (6-trans-p-coumaroyl)-3-O-β-D-glucopyranosyl-2-deoxy-D-riburonic acid methyl ester (3)
25
[α
]
D
Amorphous powder;
+17.3 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 230 (3.80), 315 (4.05)
nm; HRESIMS: m/z 495.1490 [M+Na]+, calcd. for C21H28O12 m/z 472.1581; ESIMS: m/z 495
[M+Na]+, 471 [M-H]-, 325 [M-H-146]-; 1H and 13C NMR data (CD3OD, 600 MHz) see Table 1.
2.4.5. (6-trans-p-coumaroyl)-3-O-β-D-glucopyranosyl-(5-acetyl)-2-deoxy-D-riburonic acid (4)
Amorphous powder; UV (MeOH) λmax (log ε) 230 (3.80), 315 (4.05) nm; HRESIMS: m/z
523.1443 [M+Na]+, calcd. for C22H28O13 m/z 500.1530; ESIMS: m/z 499 [M-H]-, 325 [M-H-174]-;
1H and 13C NMR data (CD3OD, 600 MHz) see Table 1.
2.5. Acid hydrolysis of compounds 1-4
A solution of each compound (2.0 mg) in 1 N HCl (1 mL) was stirred at 80 °C in a stoppered
reaction vial for 4 h. After cooling, the solution was evaporated under a stream of N2. The residue
was dissolved in 1-(trimethylsilyl)imidazole and pyridine (0.2 mL), and the solution was stirred at
60 °C for 5 min. After drying the solution, the residue was partitioned between H2O and CHCl3.
The CHCl3 layer was analyzed by GC using a L-CP-Chirasil-Val column (0.32 mm x 25 m).
Temperatures of both the injector and detector was 200 °C. A temperature gradient system was used
28

for the oven, starting at 100 °C for 1 min and increasing up to 180 °C at a rate of 5 °C/min. Peak of
the hydrolysate was detected by comparison with retention times of authentic sample of D-glucose
(Sigma Aldrich) after treatment with 1-(trimethylsilyl)imidazole in pyridine.
2.6. Antioxidant activity assays
2.6.1. Ferric reducing antioxidant power (FRAP) assay
The FRAP assay was carried out as described by Russo, Bonomo, Salzano, Martelli & Milella
(2012) with slight modifications. The FRAP reagent was made fresh before each experiment, and it
was prepared by mixing 300 mM acetate buffer in distilled water pH 3.6, 20 mM FeCl3 6H2O in
distilled water and 10 mM TPTZ in 40 mM HCl in a proportion of 10:1:1. For each sample 150 µL
of appropriately diluted sample (150 µL of methanol for the blank) and 1350 µL of FRAP reagent
was added and incubated at 37 °C for 40 min in the dark. Absorbance of resulting solution was
measured at 593 nm. Trolox was used as a reference antioxidant standard. FRAP values were
expressed as mg Trolox equivalents (mg TE)/g of sample. Each reaction was performed in
triplicate.
2.6.2. β-carotene bleaching assay
29

The antioxidant activity was evaluated by the β-carotene-linoleic acid bleaching method (BCB)
(Russo et al., 2012). β-carotene solution (0.2 mg of β-carotene dissolved in 0.2 mL of chloroform),
linoleic acid (20 mg) and Tween 20 (200 mg) were mixed. Chloroform was removed by using
rotary evaporator at room temperature. Distilled water (50 mL) was added with oxygen, then 9.5 ml
of the emulsion were transferred into several tubes containing 0.5 mL of sample (the final
concentration for all tested samples was 0.1mg/mL) or methanol as blank. BHT was used as
positive control. The tubes were placed at 50°C for 3 h. The absorbance was measured at 470 nm at
0’, 30’, 60’, 90’, 120’, 150’ and 180’. Each sample was carried out in triplicate. Results were
expressed as percentage of β-carotene bleaching inhibition and calculated as follows: (Aβ-carotene after
/Ainitial β-carotene) × 100 (AA%).
180min
2.6.3. DPPH radical-scavenging activity
The DPPH assay was used to measure radical scavenging activity of extracts and fractions. The
ability to scavenge the DPPH free radical was monitored according to the method reported by
Fernandes et al. (2013) with slight modifications. All samples were tested individually at different
concentrations by addition to a methanolic solution of DPPH radical (100 µM). For each measure
300 µL appropriately diluted sample was added to 1200 µL of DPPH reagent, the mixtures were
stirred and allowed to stand in the dark at room temperature. In the control 300 µL of methanol,
instead diluted sample, was added to 1200 µL of DPPH solution. The absorbance of the resulting
solutions was measured at 515 nm after 30’. In all experiments Trolox radical scavenging activity
was also determined and used as a reference. Sample activity was expressed as mg TE/g of sample
(Padula et al., 2013).
210

2.6.4. Total polyphenols
Total polyphenolic content (TPC) was determined according to the Folin-Ciocalteu procedure
(Milella, Caruso, Galgano, Favati, Padula & Martelli, 2011) by adding 75 µL of the diluted samples
(in the blank 75 µL of methanol) to 425 µL of distilled water, 500 µL of Folin-Ciocalteu reagent
and 500 µL of a sodium carbonate aqueous solution (10% w/v). The mixture was stirred and left in
the dark for 60 min then absorbance was measured at 723 nm. Gallic acid was used as reference
standard and TPC was expressed as mg gallic acid equivalents (GAE)/g of sample.
3. Results and discussion
3.1. Screening of extract antioxidant activity
The aerial parts (L) and roots (R) of S. papposa were sequentially extracted with solvent of
increasing polarity yielding n-hexane, chloroform, chloroform-methanol 9:1, and methanol extracts,
in the amounts of 4.1, 2.5, 2.7, 18.0 g for aerial parts and 7.9, 4.6, 5.2, 24.6 g for roots, respectively.
The methanol residues were both partitioned between n-BuOH and water, obtaining a n-butanol
fraction. All extracts were submitted to the BCB and FRAP test for a preliminary screening of
antioxidant activity (data not shown). The extracts that demonstrated significant activities were the
211

two n-butanol (BuL and BuR), the CHCl3 (CL) and the CHCl3/MeOH (CML) extracts of the aerial
parts, while no significant effect was observed for all other extracts. Thus, to confirm and deepen
the antioxidant activity of the four active extracts, BCB, FRAP, DPPH, and Folin Ciocalteu test
were performed (Table 1). A new concept, Relative Antioxidant Capacity Index (RACI) was
applied integrating antioxidant capacity data determined by several methods. The data of BCB,
FRAP, DPPH, and TPC were used to calculate the RACI, that allows the comparison of
phytocomplex antioxidant capacity derived from different chemical methods. TPC results were
included in RACI calculation since phenolics can act with other mechanisms (not measurable with
our tests) and can contribute significantly to phytocomplex health promoting value; moreover it was
recently proposed that results obtained by Folin-Ciocalteu procedure could be also interpreted as an
alternative way to measure the total reducing capacity of extracts as the reagent reacts with any
reducing substance (Fernandes et al., 2013). In this way RACI provided a more comprehensive
assessment of the whole antioxidant potential. Results (Fig. 1 and Table 1) showed BuR as the most
active extract, followed by BuL and CML.
3.2. Bioassay-oriented fractionation of the active extracts
The three extracts that showed a significant activity were separated by Sephadex LH-20 column
chromatography collecting four main fractions from BuR, five fractions from BuL, and four
fractions from CML extracts, respectively. Fractions were evaluated for their TPC and antioxidant
activities (Table 1). Results showed that among CML fractions, C demonstrated to be the one with
the highest activity, with FRAP data four times higher than fractions A and B (Table 1). A single
assay cannot determine completely the antioxidant activity of a phytocomplex, thus different
212

approaches are needed to understand the biological activity of complex mixture of secondary
metabolites (Gironés-Vilaplana, Valentão, Andrade, Ferreres, Moreno & Garcia-Viguera, 2012).
For this reason the antioxidant activity was tested by using two complementary systems: DPPH and
β-carotene bleaching assays. Fraction C from CML was two times more active than fractions A and
B in DPPH and BCB tests, confirming the FRAP results. Fraction D showed no significant activity
(data not shown). RACI was used to compare the antioxidant potential of fractions A-C and as
shown in Fig. 3a, fractions B and C, demonstrating values higher than -0.50, were submitted to
HPLC separation. Five fractions were obtained from BuL extract. TPC was significantly different
among fractions, ranging from 80.7 (fraction A) to 535.1 (fraction E) mg GAE/g (Table 1).
Moreover, the obtained antioxidant activity from DPPH, FRAP, and BCB (Table 1) showed
variability in the antioxidant capacities of BuL fractions. This variability could be due to different
mechanism of action of each assay and to the variety of fraction chemical components. Thus, on the
basis of the RACI (higher than -0.5, Fig. 3b) we selected the active fractions for further
phytochemical analysis. Four fractions were obtained from BuR extract. Fraction C showed the
highest DPPH value (1186.3 mg TE/g) and the highest FRAP and TPC values, while fraction D
demonstrated the best results in BCB test (64.3 %). On the basis of RACI (Fig. 3c) the active
fractions C and D were submitted to chromatographic separation by RP-HPLC. Unfortunately, the
chromatographic separation attempts performed to purify fraction D components failed due to the
presence of high polymerized polyphenols. The phytochemical study of S. papposa active fractions
yielded nine compounds (Fig. 2) of which four were new natural products (1-4).
3.3. Chemical compounds identification
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Compound 1 NMR and MS data demonstrated the molecular formula C20H26O12 (HRESIMS at
m/z 481.1335 [M+Na]+). The 1H and 13C NMR spectra (Table 1) showed typical signals of a trans-
double bond [H-α (δ 6.38, 1H, d, J = 16.0 Hz), H-β (δ 7.66, 1H, d, J = 16.0 Hz), C-α (δ 114.7), C-β
(δ 146.6)]. The presence of a symmetrical 1,4-disubstituted phenyl group was deduced from the
proton signals at δ 6.81 (2H, d, J = 8.0 Hz, H-3''/H-5'') and 7.48 (2H, d, J = 8.0 Hz, H-2''/H-6'').
Furthermore, the HMBC correlations of H-2''/C-1'', C-4'', C-5'', and H-β/COO, C-2'', C-3'' revealed
the presence of a trans-p-coumaroyl moiety. The 13C NMR spectrum of 1 (Table 2) showed 11
other carbon signals including one anomeric carbon signal at δ 104.9 suggesting the presence of a
sugar moiety. 1D-TOCSY and DQF-COSY led to establish two spin systems: H-1—H2-6
attributable to a hexose residue and H2-2—H2-5 suggesting a polyalchol moiety. Hydrolysis of 1
with 1N HCl yielded D-glucose as determined by GC of its trimethylsilylated derivatives on a chiral
column. The polyalchol moiety was elucidated as 2-deoxy-D-riburonic acid by studying the
remaining 1H and 13C NMR signals and by the correlations from the HSQC and HMBC spectra; this
was confirmed also by a peak observed in the ESIMS spectra at m/z 325 [M-H-132]- (Hiradate,
Morita, Sugie, Fujii, & Harada, 2004). The substituent position was achieved from the HMBC
correlations of H-6'/COO, H-1'/C-3, and H-4/C-1' suggesting that the trans-p-coumaroyl moiety
was attached at C-6' and the β-D-glucopyranose was established at C-3 of 2-deoxy-D-riburonic acid.
On the basis of the above evidence, the structure of 1 was elucidated as (6-trans-p-coumaroyl)-3-O-
β-D-glucopyranosyl-2-deoxy-D-riburonic acid.
Compound 2a was assigned molecular formula C20H26O12 (HRESIMS at m/z 481.1340 [M+Na]+),
resulting to be an isomer of 1. The spectral data of compound 2a indicated the same skeleton of 1.
Comparison of NMR spectra of 2a with those of 1 showed that 2a differed from 1 only in the
signals due to the ester moiety linked at C-6' being a cis-p-coumaroyl instead of a trans-p-
coumaroyl group. Moreover in the NMR spectra of 2a were present also signals due to a 2-deoxy-
214

D-ribono-1,4-lactone (Table 2 and Materials and Methods) (Dong, Shi, Wu, & Tu, 2007). The
signals of the 2-deoxy-D-riburonic acid were present both in its open form and closed γ-lactone
ring, indicating that both forms were in inseparable mixture. Thus, 2a and 2b were identified as (6-
cis-p-coumaroyl)-3-O-β-D-glucopyranosyl-2-deoxy-D-riburonic acid and (6-cis-p-coumaroyl)-3-O-
β-D-glucopyranosyl-2-deoxy-D-ribono-γ-lactone, respectively.
Compound 3 was obtained as an amorphous powder with molecular formula of C21H28O12 by
analysis of its HRESIMS spectrum (m/z 495.1490 [M+Na]+). Comparison of its NMR spectra
(Table 2) with those of 1 showed that 3 differed in the signals due to the 2-deoxy-D-riburonic acid
moiety. Analysis of spectroscopic data showed the presence of an additional methoxyl group
esterified at C-1. Thus, 3 was identified as (6-trans-p-coumaroyl)-3-O-β-D-glucopyranosyl-2-
deoxy-D-riburonic acid methyl ester.
Compound 4 had the molecular formula C22H28O13 (HRESIMS at m/z 523.1443 [M+Na]+). The
ESIMS spectrum showed an [M-H]- ion at m/z 499 [M-H]- that was 42 uma higher than that of 1
and a fragment ion at m/z 325 [M-H-174]- was also observed. These data were compatible with the
presence of an additional acetyl group in 4. Its 1H NMR spectrum was very similar to that of 1 with
the main difference being a downfield shift of H2-5 (δ 3.71 and 4.24 in 4 versus δ 3.64 and 3.67 in
1) and a signal at δ 2.12 (3H, s) for one acetyl group, indicating that 4 was esterified with an acetyl
group at C-5 (Table 2). Therefore, the structure of 4 was determined to be (6-trans-p-coumaroyl)-3-
O-β-D-glucopyranosyl-(5-acetyl)-2-deoxy-D-riburonic acid.
Compounds 5-9 were identified as thunberginol G (5) (Mandal & Roy, 2008), isoorientin (6) (Kato
& Morita, 1990), orientin (7) (Kato & Morita, 1990), isoschaftoside (8) (Xie, Veitch, Houghton, &
Simmonds, 2003), and swertiajaponin (9) (Borøy, Rayyan, Fossen, Chalberg, & Andersen, 2009)
by comparison of their NMR and MS literature data.
215

3.4. Antioxidant activity of pure compounds
Antioxidant activity and RACI of isolated compounds were measured (Table 3). Compound 5
showed the highest AA and FRAP value (46.1% and 82.6 mg TE/g, respectively) followed by 6
(47.7% and 60.9 mg TE/g, respectively). Among new natural compounds 1-4, only 3 showed a
weak activity on BCB test. It was expected that new compounds were not so efficient on the basis
of their chemical structure, because it was previously demonstrated that on BCB test the affinity of
the antioxidant for the lipid and thus the lipophilic nature of the molecules proved to be the
determining factor (von Gadow, Joubert, & Hansmann, 1997). Compounds 8-9 did not demonstrate
to be the major contributors to S. papposa extracts activity. It is evident that the potential of S.
papposa pure compounds is definitely lower than the fraction from they come from (Tables 1 and
3). Due to the activity showed by compound 5 and few literature data on antioxidant activity of
dihydroisocoumarins we also evaluated the activity of related compounds 10-17 isolated by our
group from edible S. judaica (Bader et al., 2011). Compounds 10-13 showed the highest BCB
values (AA higher than 50 %), while compounds 10-12 were the most active in the FRAP test. On
the basis of RACI the phtalides 10-12 showed the highest value followed by dihydroisocoumarins
13 and 5.
Conclusions
The bioassay oriented protocol supported by relative antioxidant capacity index (RACI) allowed
the isolation from S. papposa polar extracts of nine compounds, of which four were new. The
structure of the new compounds presented a rare deoxy-D-riburonic moiety. The measured
216

antioxidant activity showed different values among diverse methods in accordance with findings
previously obtained (Tabart, Kevers, Pincemail, Defraigne & Dommes, 2009) where it was
demonstrated that different antioxidant methods can give back sensible differences even due to the
multiple reaction mechanism and different phase locations involved in the measuring of antioxidant
capacity of complex plant extracts and derivatives. According to Sun & Tanumihardjo (2007) we
used a RACI as an approach to compare chemical antioxidant assays. The key advantage of RACI
is that it’s a numeric scale integrating multiple chemical method allowing comparison antioxidant
capacity of food, extracts, fractions, and pure compounds. We could hypothesize that the
antioxidant activity of S. papposa extracts and fractions may be due to the presence of a
combination of compounds acting synergistically or as vehicle enhancing the biological activity.
However, the antioxidant activity of phtalides and dihydroisocoumarins suggest these classes of
compounds for further investigations.
Acknowledgements
Authors are grateful to Immacolata Faraone, Domenico Frescura and Giovanna Imbrenda for their
skillful assistance.
217

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220

Fig. 1 RACI of S. papposa active extracts: CHCl3 aerial parts (CL); CHCl3/MeOH
aerial parts (CML); n-BuOH aerial parts (BuL); n-BuOH roots (BuR)
24

OH
HO
O
O
O
O
O
HO
HO
OR
OH
O
CH2OH
OH
HO
HO
OR
OH
1 R =
3 R =
4 R =
COOH
CH2OH
OH
CH2OH
OH
2a R =
COOH
COOCH3
CH2OH
O
CH2OAc
OH
COOH
2b R =
O
R'''
R'
OH
OR''
R''
R'O
R
O
O
OH
O
OR
O
5
R = R'' = H R' = OH
6 R = glc R' = R'' = H R''' = OH
7 R = R' = H R'' = glc R''' = OH
8 R = R'' = glc R' = H R''' = OH
9 R = glc R' = CH3 R''= H R''' = OH
13 R = R' = H R'' = apio-(1-6)-glc
15 R = R' = R'' = H
16 R = glc R' = R'' = H
17 R = R' = H R'' = rha-(1-3)-glc
OH
OH
OH
R
O
O
O
O
OH
OR
OH
OH
11 R =
14 R =
10 R = H
12 R = glc
Fig. 2. Chemical structure of compounds isolated from S. papposa and S. judaica.
25

Fig. 3 RACI of S. papposa fractions coming from: (a) CHCl3/MeOH aerial parts
extract (CML); (b) n-BuOH aerial parts extract (BuL); (c) n-BuOH roots extracts
(BuR).
26

Table 1.
Total phenolic content and antioxidant activity of S. papposa extracts and fractions
using DPPH, BCB, FRAP, and Folin assays.
Sample
Extracts
Test
DPPH
mgTE/g*
5.2±0.7
BCB
%AA**
40.4±2.4
FRAP
Folin
mgGAE/g***
54.5±3.5
mgTE/g*
10.3±0.9
CHCl3 aerial parts (CL)
CHCl3/MeOH aerial parts (CML)
37.0±02.4
57.9±3.7
11.2±1.2
65.3±4.7
n-BuOH aerial parts (BuL)
58.3±03.5
52.4±2.6
59.6±3.2
53.2±5.7
94.0±8.1
n-BuOH roots (BuR)
261.5±10.5
158.9±9.8
101.2±8.7
BuL fractions
A
15.8±1.1
64.9±4.5
55.2±2.9
53.2±2.4
50.7±2.6
43.9±1.9
55.4±3.1
20.7±3.1
60.9±4.7
80.7±5.4
238.0±9.8
533.9±21.5
276.9±12.5
535.1±25.8
B
C
171.6±8.7
249.4±10.8
439.5±21.4
205.0±11.2
157.7±9.1
418.8±25.4
D
E
CML fractions
A
6.5±0.5
14.4±1.1
16.8±0.8
34.0±1.6
32.6±1.9
50.6±3.5
5.5±0.5
5.6±0.6
19.5±1.1
51.1±3.4
38.9±3.1
116.1±7.8
B
C
BuR fractions
A
B
C
D
319.9±23.4
545.5±41.2
1186.3±71.7
1080.5±75.8
60.2±3.3
49.9±2.8
315.6±18.7
594.6±30.9
836.0±48.2
570.9±42.6
724.8±41.2
573.9±36.8
1480.1±81.5
360.8±18.5
61.5±3.9
642.7±29.9
Values are the mean of three determinations (P < 0.05). *Milligrams of Trolox
Equivalents per g of Extract/Fraction; **Antioxidant activity; *** Milligrams of Gallic
Acid Equivalents per g of Extract/Fraction.
21

Table 2
1H and 13C NMR data of compounds 1-4 (CD3OD. 600 MHz)a
1
2
3
4
H
H
H
H
C
173.4
C
174.0
C
172.0
C
173.0
1
2
3
4
5a
2.72b
4.23 m
3.73 m
3.67b
39.0
78.8
74.3
63.4
2.74b
4.28 m
3.65 m
3.58b
38.3
78.0
74.2
64.3
2.75b
4.22 m
3.65 m
3.70b
38.2
78.0
74.0
63.0
2.74b
4.55 m
3.60 m
4.24b
38.0
77.3
74.1
63.4
3.64b
3.55b
3.60b
3.71b
5b
3.55 s
51.9
OMe
172.0
20.6
COOCH3
COOCH3
2.12 s
Glc 1'
2'
3'
4'
5'
6'a
6'b
4.48 d (7.8)
3.25 dd (9.0. 7.8)
3.39 t (9.0)
3.37 t (9.0)
3.50 m
104.9
74.5
77.2
71.4
75.2
64.3
4.36 d (8.0)
3.20 dd (9.0. 8.0)
3.33 t (9.0)
3.34 t (9.0)
3.49 m
105.1
74.9
77.5
71.0
75.0
64.2
4.44 d (8.0)
3.22 dd (9.0. 8.0)
3.37 t (9.0)
3.34 t (9.0)
3.47 m
105.3
75.0
77.3
71.4
75.1
64.2
4.44 d (8.0)
3.22 dd (9.0. 8.0)
3.37 t (9.0)
3.32 t (9.0)
3.54 m
105.0
74.7
74.4
71.3
75.2
64.2
4.51 dd (12.0. 3.0)
4.35 dd (12.0. 5.0)
4.46 dd (12.0. 3.5)
4.29 dd (12.0. 5.0)
4.46 dd (12.0. 3.5)
4.33 dd (12.0. 5.0)
4.53 dd (12.0. 3.5)
4.34 dd (12.0. 5.0)
p-coumaroyl
1''
2''/6''
3''/5''
4''
127.6
130.9
116.4
160.2
128.0
133.5
115.6
159.8
127.9
131.1
116.6
160.2
126.7
130.9
116.6
161.1
7.48 d (8.0)
6.81 d (8.0)
7.66 d (8.0)
6.77 d (8.0)
7.49 d (8.0)
6.84 d (8.0)
7.48 d (8.0)
6.84 d (8.0)
6.38 d (16.0)
7.66 d (16.0)
114.7
146.6
169.2
5.81 d (12.0)
6.89 d (12.0)
116.0
145.2
168.5
6.36 d (12.0)
7.66 d (12.0)
114.6
146.4
168.8
6.40 d (12.0)
7.68 d (12.0)
114.5
146.0
169.0


COO
a J values are in parentheses and reported in Hz; chemical shifts are given in ppm; assignments were confirmed by DQF-COSY, 1D-TOCSY, HSQC,
and HMBC experiments.
b overlapped signal.
22

Table 3
Antioxidant activity of pure compounds using BCB and FRAP assays and RACI.
test
BCB
%AA*
FRAP
mgTE/g**
RACI
-0.27
71.0
0.12
-0.23
-0.71
-0.93
1.93
1.16
0.62
71.0
-0.15
-0.40
-0.46
-1.31
compound
43.6±3.1
46.1±2.9
47.7±2.5
42.8±2.7
30.4±1.7
18.9±1.2
52.7±4.1
62.4±4.1
51.8±2.4
55.3±2.7
46.2±2.5
40.5±2.1
28.3±2.0
11.6±1.1
11.6±0.9
82.6±5.1
60.9±4.5
26.9±1.9
16.0±1.3
51.9±1.9
383.8±11.2
165.0±7.5
130.4±5.2
7.4±0.5
3
5
6
7
8
9
10
11
12
31
14
15
16
17
19.0±0.9
9.2±0.7
80.9±5.1
0.3±0.0
Values are the mean of three determinations (P < 0.05).
* Antioxidant activity; ** Milligrams of Trolox Equivalents per g of pure compound.
23

Highlights
A bioassay-oriented approach was used to investigate Scorzonera papposa extracts.
Nine compounds including four new derivatives were identified.
The antioxidant activity of pure compounds was tested and RACI method applied.
29