Antioxidant and Free Radical Scavenging Activity of Phenolics from Bidens humilis

Article abstract of DOI:10.1055/s-0035-1545928

A bioassay-oriented approach led to the isolation of 11 compounds, including three new natural flavonoids, (2S)-isookanin 7-O-α-L-arabinopyranoside (1), (2S)-isookanin 7-O-(2′′-acetyl)-α-L-arabinopyranoside (2), and luteolin 7-O-β-D-glucopyranosyl-(1→6)-β-D-galactopyranoside (6), from Bidens humilis aerial parts. Their structures were determined via spectroscopic analyses including two-dimensional nuclear magnetic resonance. The antioxidant activity of all compounds was also tested by three different assays. The Relative Antioxidant Capacity Index (RACI) is applied herein, from the perspective of statistics, by integrating the antioxidant capacity data determined by these chemical methods.

Full text of DOI:10.1055/s-0035-1545928

Original Papers
Antioxidant and Free Radical Scavenging Activity of
Phenolics from Bidens humilis*
1
2
3
4
Authors
Mariela Beatriz Vera Saltos , Blanca Fabiola Naranjo Puente , Luigi Milella , Nunziatina De Tommasi ,
4
1,5
Fabrizio Dal Piaz , Alessandra Braca
Affiliations
The affiliations are listed at the end of the article
Key words
Abstract
cluding two-dimensional nuclear magnetic reso-
nance. The antioxidant activity of all compounds
was also tested by three different assays. The Rel-
ative Antioxidant Capacity Index (RACI) is applied
herein, from the perspective of statistics, by inte-
grating the antioxidant capacity data determined
by these chemical methods.
"
l Bidens humilis
!
"
l Asteraceae
A bioassay-oriented approach led to the isolation
of 11 compounds, including three new natural
flavonoids, (2S)-isookanin 7-O-α-L-arabinopyra-
noside (1), (2S)-isookanin 7-O-(2′′-acetyl)-α-L-
arabinopyranoside (2), and luteolin 7-O-β-D-glu-
copyranosyl-(1 → 6)-β-D-galactopyranoside (6),
from Bidens humilis aerial parts. Their structures
were determined via spectroscopic analyses in-
"
l flavonoids
"
l antioxidant activity
Supporting information available online at
http://www.thieme-connect.de/products
Introduction
O-α-L-arabinopyranoside (1), (2S)-isookanin 7-
O-(2′′-acetyl)-α-L-arabinopyranoside (2), and lu-
teolin 7-O-β-D-glucopyranosyl-(1 → 6)-β-D-gal-
actopyranoside (6), together with eight known
!
The genus Bidens L. of the Asteraceae family com-
prises about 200 species of annual or perennial
herbs, widely distributed in tropical and subtrop-
ical countries, especially in America. Plants of this
genus are used in many indigenous systems of
medicine as anti-inflammatory, antiallergic, anti-
bacterial, antidiabetic, antimalarial, antiviral,
antihypertensive, and antioxidant remedies [1].
Bidens species are mainly rich in flavonoids,
polyacetylenes, and phenol glycosides [2,3].
Bidens humilis Kunth (syn. Bidens triplinervia
Kunth) is a plant used in Ecuadorian folk medi-
cine for the treatment of headaches, fever, and liv-
er diseases, while the flower infusion is used
against gastric and abdominal pain. Moreover,
the indigenous Salasacas used the yellow flowers
to dye wool and other clothes. Nevertheless, no
phytochemical study on this species has been re-
ported in the literature. In the frame of our re-
search on plants belonging to Ecuadorian flora,
an antioxidant-oriented approach was carried
out on B. humilis aerial part extracts, leading to
the isolation and structural characterization of
three new natural flavonoids, (2S)-isookanin 7-
"
phenolic derivatives (3–5 and 7–11; l Fig. 1). LC-
HRESIMS and LC-HRESIMS/MS analyses of the n-
BuOH extract were also performed. The antioxi-
dant activity of extracts, fractions, and pure com-
pounds was tested by three different assays. Mul-
tiple reaction mechanisms are usually involved in
the antioxidant capacity evaluation of the natural
compounds, therefore the Relative Antioxidant
Capacity Index (RACI) was applied as an inte-
grated approach to compare the results obtained
by different methods.
received
revised
accepted
February 1, 2015
March 12, 2015
March 14, 2015
Bibliography
DOI http://dx.doi.org/
0.1055/s-0035-1545928
Results and Discussion
!
1
Published online
The aerial parts of B. humilis were sequentially ex-
tracted with solvents of increasing polarity giving
n-hexane, chloroform, and methanol residues.
The methanol extract was partitioned between
n-BuOH and water, yielding an n-BuOH fraction.
Since a single assay cannot completely determine
the antioxidant activity of plant extracts, three
different complementary systems were carried
out [4]. Thus, all residues were subjected to the
2,2-diphenyl-2-picrylhydrazyl (DPPH), β-caro-
tene bleaching (BCB), and ferric reducing antioxi-
Planta Med © Georg Thieme
Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
Correspondence
Prof. Nunziatina De Tommasi
Università di Salerno
Dipartimento di Farmacia
Via Giovanni Paolo II 132
8
4084 Fisciano (Salerno)
Italy
Phone: + 39089969754
Fax: + 39089969602
detommasi@unisa.it
*
Dedicated to Professor Dr. Dr. h.c. mult. Adolf Nahrstedt
on the occasion of his 75th birthday.
Vera Saltos MB et al. Antioxidant and Free… Planta Med

Original Papers
Fig. 1 Chemical structures of compounds 1–11.
dant power (FRAP) assays to screen their antioxidant activity. To-
tal phenolic content (TPC) was also measured. It was demonstrat-
ed that the n-BuOH fraction was the one containing the highest
concentration of total phenolics (from 8 to 20 times higher than
ising fractions should have at least one value higher than their
relative extract, fraction A was excluded from further analysis.
All selected fractions were investigated for their phytochemical
content. Fraction F showed the presence of one spot on TLC and
was directly subjected to spectroscopic and spectrometric analy-
ses. Fractions B–E were purified by RP-HPLC; a total of 11 com-
"
the other extracts) (l Table 1). This extract was more active as a
radical scavenger (DPPH) and antioxidant (FRAP) than as antili-
poperoxidative (BCB). The high phenolic content of a plant ex-
tract is usually directly related to its antioxidant activity mea-
sured with DPPH and FRAP tests, while evidences demonstrated
that with the BCB test, it is not the same [5]. This phenomenon
can be explained by the affinity of the antioxidant complex for
the lipids, and thus the lipophilic nature of the BCB test could be
the determining factor [5,6]. RACI was calculated to examine the
obtained results. TPC results were included in the RACI calcula-
tion since phenolics can act with other mechanisms (not measur-
able with our tests) and could contribute to the extractʼs health
promoting value. In this way, RACI provided a more comprehen-
sive assessment of the whole extract antioxidant potential, and
"
pounds (1–11) were characterized (l Fig. 1), of which three were
new natural flavonoids (1, 2, and 6).
A molecular formula of C20H20O10 was assigned to compound 1
−
13
on the basis of its HR ESIMS (m/z 419.0984 [M – H] ) and
C
NMR data. The ESI MS spectrum of 1 showed a quasimolecular
−
ion peak at m/z 419 [M – H] and one peak at m/z 287 [(M –
−
132)-H] , due to the loss of one pentose moiety. Moreover, HR
ESIMS/MS analysis revealed fragment ions at m/z 151.0058
−
−
[C H O -H] and 135.0049 [C H O -H] , diagnostic for flavanoids
7
4
4
7 4 3
carrying two hydroxyl groups on the A ring [7]. The UV absorp-
tion bands at 281 and 327 nm were suggestive of a flavanone
1
13
"
skeleton. The H and C NMR spectra (l Table 2) displayed sig-
nals for an oxygenated methine doublet of doublets at δ 5.46
(1H, dd, J = 12.5, 3.0 Hz, H-2), two methylene doublets of doublets
"
confirmed the n-BuOH fraction as the most active (l Fig. 2a).
The n-BuOH extract was then subjected to Sephadex LH-20 col-
umn chromatography, collecting six major fractions. Also, in this
case, all fractions were evaluated for their TPC and antioxidant
at δ 3.14 (1H, dd, J = 17.0, 12.5 Hz, H-3 ) and 2.82 (1H, dd,
ax
J = 17.0, 3.0 Hz, H-3 ), and five aromatic protons, of which two
eq
"
activities (l Table 1). All fractions showed to be rich in phenolics
were superimposable, that were assigned with the help of 1D
TOCSY and DQF COSY, together with signals of a sugar residue.
The chemical shifts of the two doublets at δ 7.40 (1H, d, J =
8.0 Hz, H-5) and 6.89 (1H, d, J = 8.0 Hz, H-6) suggested a 7,8-dis-
ubstituted A ring, while the three signals at δ 7.01 (1H, d, J =
2.0 Hz, H-2′), 6.89 (1H, dd, J = 8.5, 2.0 Hz, H-6′), and 6.81 (1H, d,
J = 8.5 Hz, H-5′) were in accordance with a 1,2,4-trisubstituted B
with a total content higher than 361.8 mg of gallic acid equiva-
lents per g of the extract (mgGAE/g) (C) to 518.0 mgGAE/g for F,
with the exception of A (90.1 mgGAE/g). Fraction E showed the
highest DPPH and FRAP values [857.3 and 2208.8 mg of Trolox
equivalent per g (mgTE/g) of fraction, respectively], while the
highest BCB value was registred for fraction F. On the basis of
"
13
"
the RACI results (l Fig. 2a) and the evidence that the most prom-
ring. The 20 C NMR resonances (l Table 2) were easily assigned
Vera Saltos MB et al. Antioxidant and Free… Planta Med

Original Papers
Table 1 Antioxidant activity and total phenolic content of B. humilis extracts, fractions and pure compounds using DPPH, BCB, FRAP, and Folin assays.
Sample
Test
DPPH mgTE/g*
BCB %AA**
FRAP mgTE/g*
Folin mgGAE/g***
Extract
n-Hexane
34.9 ± 1.7
37.6 ± 2.4
173.8 ± 6.2
43.6 ± 2.9
37.9 ± 1.9
8.0 ± 0.7
6.5 ± 0.5
31.3 ± 2.1
34.0 ± 2.2
13.3 ± 0.9
CHCl3
n-BuOH
790.1 ± 12.5
261.7 ± 11.0
Fraction
A
27.4 ± 1.0
147.1 ± 11.2
163.8 ± 8.5
296.3 ± 14.5
857.3 ± 18.8
819.5 ± 21.2
13.8 ± 0.4
3.2 ± 0.4
9.4 ± 0.2
4.5 ± 0.5
11.4 ± 0.7
40.6 ± 1.1
115.0 ± 9.2
675.1 ± 23.1
620.7 ± 19.5
954.3 ± 12.7
2208.8 ± 45.2
1442.3 ± 32.1
90.1 ± 5.2
451.0 ± 21.3
361.8 ± 22.2
495.5 ± 24.2
511.1 ± 19.7
518.0 ± 16.5
B
C
D
E
F
Pure compound
1
2
3
4
5
6
7
8
9
513.7 ± 28.7
484.7 ± 21.3
356.5 ± 16.4
255.3 ± 12.9
238.0 ± 7.7
357.8 ± 17.5
819.5 ± 22.5
151.5 ± 3.2
477.1 ± 14.5
317.5 ± 12.2
470.7 ± 21.6
16.8 ± 1.0
10.3 ± 0.8
20.5 ± 1.5
22.3 ± 1.1
23.2 ± 1.1
17.1 ± 1.0
40.6 ± 2.1
9.5 ± 0.7
1404.2 ± 15.6
777.6 ± 12.4
707.4 ± 12.6
1129.7 ± 24.0
421.2 ± 9.2
823.8 ± 11.4
1442.3 ± 17.8
170.5 ± 4.5
21.0 ± 0.9
14.4 ± 0.9
19.3 ± 1.7
75 ± 4.8
770.8 ± 7.0
1
1
0
1
297.5 ± 7.4
953.1 ± 12.9
BHT****
Values are the mean of three determinations (p < 0.05); * Milligrams of Trolox equivalents per g of extract/fraction/pure compound; ** antioxidant activity at 0.05 mg/mL;
** milligrams of gallic acid equivalents per g of extract/fraction/pure compound; **** BHT = butylhydroxytoluen
*
Fig. 2 RACI values of extracts (BH‑C: chloroform;
BH‑E: n-hexane; BH‑B: n-BuOH), Sephadex-LH20
fractions (A–F) (a), and pure compounds (1–11) (b)
obtained from B. humilis aerial parts.
to a flavanone aglycone moiety and to an α-arabinose unit (δC
02.7, 73.1, 71.4, 68.9, and 66.6). These 1D NMR data, in combi-
nation with the observed 2D NMR correlations, suggested that
compound 1 was an isookanin glycoside [8]. The position of the
arabinose residue was deduced by the HMBC correlation be-
tween δ 4.97 (H-1ara) and 151.8 ppm (C-7). Hydrolysis of 1 with
1 N HCl, followed by GC analysis through a chiral column of the
trimethylsilylated sugar, led to the assignment of an arabinose
configuration. The stereochemistry of C-2 was determined as S
on the basis of a negative Cotton effect at 274 nm and a positive
1
Vera Saltos MB et al. Antioxidant and Free… Planta Med

Original Papers
Table 2 1H and 13C‑NMR data of compounds 1, 2, and 6 (CD3OD, 600 MHz, J in Hz).
Position
1
2
6
δH
δC
81.0
δH
δC
81.4
δH
δC
2
5.46 dd (12.5, 3.0)
3.14 dd (17.0, 12.5)
2.82 dd (17.0, 3.0)
5.43 dd (12.8, 3.0)
3.14 dd (17.2, 12.8)
2.77 dd (17.2, 3.0)
166.0
103.1
3
3
4
5
6
7
8
9
0
1
2
3
4
5
6
ax
45.0
44.6
6.62 s
eq
193.0
118.0
110.0
151.8
135.6
155.0
113.9
130.6
114.5
145.0
145.9
115.7
118.9
102.7
71.4
193.3
118.0
111.3
150.6
136.3
155.4
113.0
130.5
114.9
145.0
145.8
115.9
119.2
100.6
73.0
182.9
161.4
101.7
163.0
95.2
7.40 d (8.0)
6.89 d (8.0)
7.38 d (8.2)
6.88 d (8.2)
6.62 d (2.0)
6.83 d (2.0)
157.8
105.9
122.3
113.2
145.9
150.0
115.6
119.5
1
′
′
′
′
′
′
7.01 d (2.0)
7.02 d (2.0)
7.45 d (2.0)
6.81 d (8.5)
6.81 d (8.5)
6.94 d (8.0)
6.89 dd (8.5, 2.0)
4.97 d (7.0)
6.88 dd (8.5, 2.0)
5.17 d (6.8)
7.45 dd (8.0, 2.0)
Ara 1
2
3
4
3.92 dd (9.0, 7.0)
3.70 dd (9.0, 2.5)
3.94 m
5.30 dd (9.0, 6.8)
3.88 dd (9.0, 2.5)
3.94 m
73.1
71.6
68.9
68.6
5
5
a
3.95 dd (12.0, 2.0)
3.74 dd (12.0, 3.5)
66.6
3.98 dd (12.0, 2.0)
3.72 dd (12.0, 3.5)
66.7
b
COCH3
171.3
21.0
COCH3
2.12 s
Gal 1
5.09 d (7.5)
100.5
73.9
76.3
70.3
76.6
69.3
2
3
4
5
3.52 dd (9.0, 7.5)
3.53 dd (9.0, 4.0)
3.47 dd (4.0, 2.5)
3.83 m
6
6
a
4.23 dd (12.0, 2.5)
3.87 dd (12.0, 4.5)
4.39 d (7.8)
b
Glc1
103.9
74.0
77.4
70.6
77.0
62.0
2
3
4
5
3.30 dd (9.0, 7.8)
3.29 t (9.0)
3.45 t (9.0)
3.37 m
6
6
a
3.91 dd (12.0, 3.0)
3.69 dd (12.0, 5.0)
b
Data assignments were confirmed by DQF-COSY, 1D-TOCSY, HSQC, and HMBC experiments
1
13
"
Cotton effect at 306 nm in the CD spectrum of 1. Consequently, 1
was characterized as (2S)-isookanin 7-O-α-L-arabinopyranoside.
The molecular formula of compound 2 (C22H22O11) was estab-
flavone glycoside. Analysis of the H and C NMR spectra (l Ta-
ble 2) of 6 clearly indicated that it was a luteolin derivative. The
1
3
C NMR spectrum allowed for assigning 15 signals to the agly-
1
3
lished by C NMR and HR ESIMS spectra (m/z 461.1090 for [M –
cone moiety and 12 to the sugar residue, consisting of one inner
β-galactose and one terminal β-glucose. The linkage between the
sugar moieties was deduced by the HMBC correlation between
the signals at δ 4.39 (1H, d, J = 7.8 Hz, H-1glc) and 69.3 (C-6gal). Hy-
drolysis of 6 with 1 N HCl, followed by GC analysis through a chi-
ral column of the trimethylsilylated monosaccharides, permitted
the assignment of the sugars configuration. Accordingly, com-
pound 6 was assigned as luteolin 7-O-β-D-glucopyranosyl-
(1 → 6)-β-D-galactopyranoside.
Compounds 3–4 and 7–11 were characterized as isookanin 7-O-
β-D-glucopyranoside (flavanomarein) (3) [10], 8-methoxybutin
(4) [11], butin 7-O-β-D-glucopyranoside (isocoreopsin) (5) [12],
luteolin (7) [8], chrysoeriol 7-O-β-D-glucopyranoside (thermop-
soside) (8) [13], eriodictyol 7-O-β-D-glucopyranoside (9) [8], 6′,
6′′-sucrose ester of (1α,2α,3β,4β)-3,4-bis-(4-hydroxyphenyl)-
1,2-cyclobutanedicarboxilic acid (10) [3], and chlorogenic acid
−
H] ). In the HR ESIMS/MS spectrum, fragments at m/z 401.1067
−
−
−
[
(M – 60)-H] , 287.0865 [(M – 174)-H] , 151.0043 [C H O -H] ,
7 4 4
−
and 135.0041 [C H O -H] were also observed. Its NMR spectral
7
4 3
"
data (l Table 2) suggested that the structure of 2 resembled that
of 1, but differed for the presence of an additional acetyl group.
The acylation site was on C-2 of arabinose as evidenced by the
strong deshielding of H-2ara at δ 5.30 and was confirmed by an
HMBC experiment [9]. The stereochemistry at C-2 and the arabi-
nose configuration were determined as reported for 1. Thus, the
structure of 2 was deduced to be (2S)-isookanin 7-O-(2′′-acetyl)-
α-L-arabinopyranoside.
Compound 6 (C27H30
O
16) showed a quasimolecular ion peak at
−
m/z 609.1461 [M – H] in the negative HR ESIMS. The prominent
fragment ions observed in the ESI MS spectrum at m/z 447 [(M –
−
−
1
62)-H] and 285 [(M – 162–162)-H] , together with the UV ab-
sorption maxima at 270 and 335 nm, suggested the presence of a
Vera Saltos MB et al. Antioxidant and Free… Planta Med

Original Papers
Fig. 3 Total ion current LC-HRESIMS chromato-
grams obtained for the n-BuOH fraction of the aer-
ial parts of B. humilis in both the negative (A) and
positive (B) ion modes. Peak numbers refer to
compounds 1–11; small case letters (a–s) indicate
other compounds detected in the LC‑MS analyses.
MS data and tentative assignments for these com-
"
pounds are reported in l Table 3.
(
11) [14] by spectrometric and spectroscopic data measurements
son between our data and previously published results on the
MS analyses of flavonoids [19,20] and other phenols [21] allowed
us to deduce the main structural features of five further flavones,
one dimeric flavone, two flavanones, two flavonols, and two caf-
feoylquinic derivatives. Finally, one peptide was detected, whose
amino acidic sequence was easily deduced from MS/MS data. The
presence of these additional phenolic compounds could explain
the high antioxidant activity showed by the B. humilis n-BuOH
extract.
and comparison with the literature data.
Consequently, the antioxidant activity of these new and known
compounds using BCB, FRAP, and DPPH tests was measured
(
"
l Table 1). Among the known compounds, luteolin (7) showed
the highest values: 819.5 mgTE/g, 40.6% of antioxidant activity
AA), 1442.3 mgTE/g in DPPH, BCB, and FRAP tests, respectively.
(
These findings are congruent with previous evidences [15,16].
On the other hand, among new natural compounds, 1 showed to
be the most active in DPPH and FRAP tests. On the basis of RACI
"
(
l Fig. 2b), which combines the results obtained from all antiox-
idant tests, it is possible to assess that 7 and 1 are the most active
compounds, followed by 11 and a group formed by compounds 9,
Materials and Methods
!
4
, 3, 6, and 2. This group showed close but slightly descending
RACI values. The lowest RACIs were observed for compounds 5,
, and 10. All of the isolated compounds are flavonoid derivatives
General experimental procedures
Optical rotations were measured on a Perkin-Elmer 241 polarim-
eter equipped with a sodium lamp (589 nm) and a 1 dm micro-
cell. UV spectra were recorded on a Perkin-Elmer-Lambda spec-
trophotometer. CD spectra were measured on a JASCO J-810
spectropolarimeter with a 0.1-cm cell in MeOH at room temper-
ature under the following conditions: speed 50 nm/min, time
constant 1 s, and bandwidth 2.0 nm. NMR experiments were per-
formed on a Bruker DRX-600 spectrometer at 300 K. The stan-
dard pulse sequence and phase cycling were used for DQF-COSY,
TOCSY, HSQC, and HMBC experiments. ESIMS were obtained
using a Finnigan LC‑Q Advantage Termoquest spectrometer,
equipped with Xcalibur software. HRESIMS were acquired in
both the positive and negative ion modes on a Q‑TOF premier
spectrometer (Waters-Milford). Mass spectrometry analyses
were performed on a Q‑TOF premier spectrometer coupled with
an Alliance HPLC module (Waters-Milford). TLC was performed
on precoated Kieselgel 60 F254 plates (Merck); compounds were
detected by spraying with Ce(SO ) /H SO (Sigma-Aldrich) and
8
with the exclusion of 10 and 11, and on the basis of their struc-
tures, it is possible to confirm the importance of the two hydroxyl
groups in the ortho-diphenolic arrangement (compounds 7, 1,
1
1, 9, 4, 3, 6, 2). The loss of activity of compound 8 could be as-
cribed to the presence of a methoxy group on the B ring. This re-
sult is congruent with previous findings where it was demon-
strated that the presence of a methoxy group decreases, sensibly,
the antioxidant capacity of flavonoids [17,18]. Some of the pure
compounds tested exerted interesting antioxidant activity, and
their action could partially explain the activity shown by the frac-
tion they come from. Thus, in order to deepen the polar fraction
metabolome of B. humilis, the n-BuOH extract was subjected to
LC-HRESIMS and LC-HRESIMS/MS analyses both in the positive
"
and in negative ion modes (l Fig. 3). Twenty-four major com-
pounds were detected, including the 11 isolates. On the basis of
accurate molecular weight determination, and taking into ac-
4
2
2
4
count the main fragments observed, the structure of a further
NTS (Naturstoffe reagent)-PEG (Poliethylene glycol 4000) solu-
tions. Column chromatography was performed over Sephadex
LH-20 (Pharmacia); reversed-phase (RP) HPLC separations were
conducted on a Shimadzu LC-8A series pumping system
equipped with a Shimadzu RID10A refractive index detector and
a Shimadzu injector using a C18 µ-Bondapak column (30 cm ×
7.8 mm, 10 µm, Waters-Milford) and a mobile phase consisting
"
13 derivatives was proposed (l Table 3). Compounds 1–11 were
identified by comparing the HRMS and HRMS/MS data achieved
in the LC/MS analyses with those obtained during the structural
characterization on the isolated compounds. In the case of com-
pounds 3 and 9, the MS data were not informative enough to per-
mit an unambiguous identification; therefore, separate injections
of each pure compound were performed. Moreover, a compari-
of MeOH‑H O mixtures at a flow rate of 2 mL/min. GC analyses
2
Vera Saltos MB et al. Antioxidant and Free… Planta Med

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Vera Saltos MB et al. Antioxidant and Free… Planta Med

Original Papers
Vera Saltos MB et al. Antioxidant and Free… Planta Med

Original Papers
−
+
were performed using a Dani GC 1000 instrument. All spectro-
photometric measurements were done in 96-well microplates
and cuvettes on a UV/VIS spectrophotometer SPECTROstar Nano
[(M – 162–162)-H] , 633 [M + Na] ; HR ESIMS m/z 609.1461 [M –
−
H] (calcd. for C27H29O16, 609.1458).
(BMG Labtech).
Acid hydrolysis of compounds 1, 2, and 6
A solution of each compound (2.0 mg) in HCl 1 N (1 mL) was
Chemicals
stirred at 80°C in a stoppered reaction vial for 4 h. After cooling,
Sodium acetate trihydrate, DPPH, 2,4,6-tripyridyl-s-triazine
the solution was evaporated under a stream of N . The residue
2
(
TPTZ), iron (III) chloride (FeCl *6H O), β-carotene, linoleic acid,
was dissolved in 1-(trimethylsilyl)imidazole and pyridine
(0.2 mL), and the solution was stirred at 60°C for 5 min. After dry-
ing the solution, the residue was partitioned between water and
CHCl . The CHCl layer was analyzed by GC using an L-Chirasil-
Val column (0.32 mm × 25 m). Temperatures of the injector and
detector were both 200°C. A temperature gradient system was
used for the oven, starting at 100°C for 1 min and increasing up
to 180°C at a rate of 5°C/min. Peaks of the hydrolysate were de-
tected by comparison with the retention times of authentic sam-
ples of D-galactose, D-glucose, and L-arabinose (Sigma-Aldrich)
after treatment with 1-(trimethylsilyl)imidazole in pyridine.
3
2
Tween 20, butylhydroxytoluen (BHT) (purity grade 99%), Folin-
Ciocalteu reagent, sodium carbonate, 6-hydroxy-2,5,7,8-tetra-
methylchroman-2-carboxylic acid (Trolox) (purity grade 97%),
and gallic acid (purity grade 99%) were purchased from Sigma-
Aldrich. n-Hexane, chloroform, methanol, hydrochloric acid, and
glacial acetic acid were purchased from VWR.
3
3
Plant material
Aerial parts of Bidens humilis Kunth were collected in Tumbaco,
Ecuador, in September 2011. The plant was identified at the Her-
barium of Jardin Botanico de Quito, Quito, Ecuador. A voucher
specimen (N. 9237 Bidens humilis/1) was deposited at Herbari-
um Horti Botanici Pisani, Nuove Acquisizioni, Pisa, Italy.
Liquid Chromatography-High-Resolution Electrospray
Ionization Mass Spectrometry Analyses
Extraction and isolation
!
The dried aerial parts of B. humilis (610 g) were extracted for 48 h
Liquid chromatography separation was achieved using a Luna C18
column (150 × 2.1 mm, 2.5 µm, Phenomenex) and 0.1% formic ac-
id in water (A) and acetonitrile (B). Compound elution was per-
formed using a linear gradient from 1% to 50% of B in 45 min.
Mass spectra were acquired both in the positive and negative
ion modes, over the m/z 200–800 range. The source temperature
was set at 120°C, capillary voltage at 3300 eV, and cone voltage at
50 eV. HRESIMS/MS spectra were acquired for the three most
abundant ions observed in each MS spectrum (dependent scan
mode). When required, HRESIMS/MS spectra were acquired, set-
ting a specific parent ion in the mass spectrometry method.
with solvents of increasing polarity, n-hexane, CHCl , and MeOH
3
by exhaustive maceration (2 L) to give 5.7, 16.0, and 26.8 g of the
respective residues. The MeOH extract was partitioned between
n-BuOH and H O to afford an n-BuOH residue (4.0 g). Part of the
2
n-BuOH extract (2.5 g) was submitted to a Sephadex LH-20 col-
umn (5 × 70 cm, flow rate 1.0 mL/min) using MeOH as the eluent
and collecting 56 fractions of 10 mL that were grouped by TLC in-
to six major fractions (A–F). Fractions B (230 mg, 230–290 mL), C
(122 mg, 300–330 mL), and E (93.6 mg, 390–480 mL) were sub-
jected to RP-HPLC with MeOH–H O (3:7) as the eluent to give
2
pure compounds 5 (2.4 mg, t = 33 min), 10 (3.6 mg, t = 46 min),
R
R
and 6 (3.7 mg, t = 100 min) from fraction B; compounds 3
Antioxidant activity assays
R
(
5
5.1 mg, t = 23 min), 1 (5.3 mg, t = 32 min), and 9 (2.0 mg, t =
2,2-Diphenyl-2-picrylhydrazyl radical scavenging activity: The
ability to scavenge the DPPH free radical was monitored accord-
ing to the method reported by Milella et al. (2014) with slight
modifications [5]. All samples were tested individually at differ-
ent concentrations by being added to a methanol solution of
DPPH radical (100 µM). For each measure, 50 µL of appropriately
diluted sample were added to 200 µL of DPPH reagent. The mix-
tures were stirred and allowed to stand in the dark at room tem-
perature. In the control, 50 µL of methanol, instead of the diluted
sample, were added to 200 µL of DPPH solution. The absorbance
of the resulting solutions was measured at 515 nm after 30 min.
In all experiments, Trolox radical scavenging activity was also de-
termined and used as reference. Sample activity was expressed as
mg of Trolox equivalents per gram of sample (mg TE/g) [22].
Ferric reducing antioxidant power (FRAP) assay: The FRAP assay
was carried out as described by Russo et al. with slight modifica-
tions [23]. The FRAP reagent was made fresh before each experi-
ment and it was prepared by mixing 300 mM acetate buffer in
distilled water (pH 3.6), 20 mM FeCl *6H O in distilled water,
R
R
R
3 min) from fraction C; and compound 11 (2.2 mg, t = 16 min)
R
from fraction E. Fraction D (185 mg, 340–380 mL) was purified
by RP-HPLC with MeOH–H O (2:3) as the eluent to give pure
compounds 2 (1.6 mg, t = 32 min), 4 (2.5 mg, t = 40 min), and 8
2
R
R
(4.0 mg, t = 90 min). Fraction F (490–560 mL) yielded pure com-
R
pound 7 (14.5 mg). All of the compounds met the criteria of ≥ 95%
purity, as inferred by HPLC and NMR analyses.
(2S)-Isookanin 7-O-α‑L-arabinopyranoside (1): yellow amor-
2
5
phous powder; [α]
ε): 281 (4.31), 327 (3.87); CD [θ] (c 0.05, MeOH, nm) − 5020
D
− 22 (c 0.3, MeOH); UV (MeOH) λmax (log
2
5
13
1
"
(274 nm), + 4380 (306 nm); H and C NMR data, see l Table 2;
−
−
−
ESI MS m/z 419 [M – H] , 287 [(M – 132)-H] ; HR ESIMS [M – H]
19.0984 (calcd. for C20H O10, 419.0978), 287.0602 [(M – 132)-
4
19
−
−
−
H] , 151.0058 [C H O -H] , 135.0049 [C H O -H] .
7
4
4
7 4 3
(2S)-Isookanin 7-O-(2′′-acetyl)-α‑l-arabinopyranoside (2): yellow
2
5
amorphous powder; [α]
D
− 29 (c 0.06, MeOH); UV (MeOH) λmax
(log ε): 283 (4.12), 330 (3.98); CD [θ] (c 0.05, MeOH, nm) − 6400
25
13
1
"
(275 nm), + 4560 (307 nm); H and C NMR data, see l Table 2;
3
2
−
−
ESI MS m/z 461 [M – H] ; HR ESIMS m/z 461.1090 [M – H] (calcd.
for C22
1
and 10 mM TPTZ in 40 mM HCl in a proportion of 10:1:1. For
each sample, 25 µL of appropriately diluted sample (25 µL of
methanol for the blank) and 225 µL of FRAP reagent were added
and incubated at 37°C for 40 min in the dark. Absorbance of the
resulting solution was measured at 593 nm. Trolox was used as a
reference antioxidant standard and FRAP values were expressed
as mg Trolox equivalents per gram of sample (mg TE/g).
−
H
22O11, 461.1084), 401.1067 [(M – 60)-H] , 287.0865 [(M –
−
−
−
74)-H] , 151.0043 [C H O -H] , and 135.0041 [C H O -H] .
7 4 4 7 4 3
Luteolin 7-O-β‑D-glucopyranosyl-(1 → 6)-β‑D-galactopyranoside
2
5
(
(
6): yellow amorphous powder; [α]
D
− 45 (c 0.2, MeOH); UV
1
13
MeOH) λ
(log ε): 270 (4.33), 335 (3.96); H and C NMR data,
max
"
−
−
see l Table 2; ESI MS m/z 609 [M – H] , 447 [(M – 162)-H] , 285
Vera Saltos MB et al. Antioxidant and Free… Planta Med

Original Papers
4
Gironés-Vilaplana A, Valentão P, Andrade PB, Ferreres F, Moreno DA, Gar-
cía-Viguera C. Phytochemical profile of a blend of black chokeberry and
lemon juice with cholinesterase inhibitory effect and antioxidant po-
tential. Food Chem 2012; 134: 2090–2096
β-Carotene bleaching (BCB) assay: For this assay, the following re-
agents were mixed: 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). Chloroform was removed by using a rotary
evaporator at room temperature [23]. Distilled water (50 mL)
was added with oxygen and then 950 µL of the emulsion were
transferred into several tubes containing 50 µL of sample (the fi-
nal concentration for all tested samples was 0.05 mg/mL) or
methanol as the blank. BHT was used as positive control. Next,
5 Milella L, Bader A, De Tommasi N, Russo D, Braca A. Antioxidant and free
radical-scavenging activity of constituents from two Scorzonera spe-
cies. Food Chem 2014; 160: 298–304
6
von Gadow A, Joubert E, Hansmann CH. Comparison of the antioxidant
activity of aspalathin with that of other plant phenols of rooibos tea
(Aspalathus linearis), α-Tocopherol, BHT, and BHA. J Agric Food Chem
1997; 45: 632–638
2
50 µL of emulsion-sample solution was transferred (250 µL/
7 de Rijke E, Out P, Niessen WMA, Ariese F, Gooijer C, Brinkman UAT. Ana-
lytical separation and detection methods for flavonoids. J Chromatogr
A 2006; 1112: 31–63
well) to the reaction plate. Since the reaction was temperature
sensitive, close temperature control throughout the plate was es-
sential in this assay; therefore, the outer wells were filled with
8
Agrawal PK. Carbon-13 NMR of flavonoids. Amsterdam: Elsevier;
989: 134, 314
1
2
50 µL of water to provide a large thermal mass [24]. The micro-
9 Sun LR, Qing C, Zhang YL, Jia SY, Li ZR, Pei SJ, Qiu MH, Gross ML, Qiu SX.
Cimicifoetisides A and B, two cytotoxic cycloartane triterpenoid glyco-
sides from the rhizomes of Cimicifuga foetida, inhibit proliferation of
cancer cells. Beilstein J Org Chem 2007; 3: 1–7
0 Shimokoriyama M. Antochlor pigments of Coreopsis tinctoria. J Am
Chem Soc 1957; 79: 214–220
plate was immediately placed at 50°C for 3 h and the absorbance
was measured at 470 nm at 0′, 30′, 60′, 90′, 120′, 150′, and 180′.
Results were expressed as the percentage of BCB inhibition and
calculated as follows: (Aβ-carotene after 180 min/Ainitial β-carotene) × 100
1
(
AA%). BHT was used as a positive control.
11 Okada Y, Okita M, Murai Y, Okano Y, Nomura M. Isolation and identifi-
cation of flavonoids from Coreopsis lanceolata L. petals. Nat Prod Res
Total polyphenolic content (TPC): TPC was determined according
to the Folin-Ciocalteu method [5] by adding 75 µL of the diluted
samples extract (in the blank, 75 µL of methanol) to 425 µL of dis-
tilled water, 500 µL of Folin-Ciocalteu reagent, and 500 µL of a so-
dium carbonate aqueous solution (10% w/v). The mixture was
stirred and left in the dark at room temperature for 60 min and
then the absorbance was measured at 723 nm. Gallic acid was
used as reference standard and TPC was expressed as mg gallic
acid equivalents (mgGAE)/g of sample.
2
014; 28: 201–204
1
2 Gupta SR, Ravindranath B, Seshadri TR. Glucosides of Butea monosper-
ma. Phytochemistry 1970; 9: 2231–2235
13 Romussi G, Fontana N, De Tommasi N. Flavonoids from Cymbalaria mur-
alis Gaernt. Phytother Res 1996; 10 (Suppl. 1): S84–S85
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effects and tyrosinase inhibitory activities of seven hydroxycinnamoyl
derivatives in green coffee beans. J Agric Food Chem 2004; 52: 4893–
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898
15 Choi JS, Islam MN, Ali MY, Kim YM, Park HJ, Sohn HS, Jung HA. The effects
of C-glycosylation of luteolin on its antioxidant, anti-Alzheimerʼs dis-
ease, anti-diabetic, and anti-inflammatory activities. Arch Pharm Res
Statistical analysis
2
014; 37: 1354–1363
For each spectrophotometric test, three independent experi-
ments were carried out. Results are presented as mean ± stan-
dard deviation (mean ± SD) and a value of p < 0.05 was consid-
ered as significant. The RACI calculation, regression analysis, and
statistical analysis were carried out using Microsoft Excel. Cali-
1
1
1
1
2
6 Zhang XL, Guo YS, Wang CH, Li GQ, Xu JJ, Chung HY, Ye WC, Li YL, Wang
GC. Phenolic compounds from Origanum vulgare and their antioxidant
and antiviral activities. Food Chem 2014; 152: 300–306
7 Heim KE, Tagliaferro AR, Bobilya DJ. Flavonoid antioxidants: chemistry,
metabolism and structure-activity relationships. J Nutr Biochem 2002;
1
3: 572–584
2
bration curves of the standards were considered linear if R re-
sulted > 0.99.
8 Rice-Evans CA, Miller NJ, Paganga G. Structure-antioxidant activity rela-
tionships of flavonoids and phenolic acids. Free Radic Biol Med 1996;
2
0: 933–956
9 Wu W, Yan C, Li L, Liu Z, Liu S. Studies on the flavones using liquid chro-
matography-electrospray ionization tandem mass spectrometry.
J Chromatogr A 2004; 1047: 213–220
0 Kuhn F, Oehme M, Romero F, Abou-Mansour E, Tabacchi R. Differentia-
tion of isomeric flavone/isoflavone aglycones by MS2 ion trap mass
spectrometry and a double neutral loss of CO. Rapid Commun Mass
Spectrom 2003; 17: 1941–1949
Supporting information
NMR spectra of compounds 1, 2, and 6 are available as Support-
ing Information.
Conflict of Interest
!
2
2
1 Jaiswal R, Karakoese H, Ruehmann S, Goldner K, Neumueller M, Treutter
D, Kuhnert N. Identification of phenolic compounds in plum fruits (Pru-
nus salicina L. and Prunus domestica L.) by high-performance liquid
chromatography/tandem mass spectrometry and characterization of
varieties by quantitative phenolic fingerprints. J Agric Food Chem
2013; 61: 12020–12031
2 Padula MC, Lepore L, Milella L, Ovesna J, Malafronte N, Martelli G, De
Tommasi N. Cultivar based selection and genetic analysis of strawberry
fruits with high levels of health promoting compounds. Food Chem
The authors declare no conflict of interest.
Affiliations
1
Dipartimento di Farmacia, Università di Pisa, Pisa, Italy
Departamento de Ciencias de la Vida, Universidad de las Fuerzas Armadas,
2
ESPE, Sangolqui, Ecuador
Dipartimento di Scienze, Università degli Studi della Basilicata, Potenza, Italy
Dipartimento di Farmacia, Università di Salerno, Salerno, Italy
Centro Interdipartimentale di Ricerca “Nutraceutica e Alimentazione per la
3
4
5
2
013; 140: 639–646
Salute”, Università di Pisa, Pisa, Italy
2
2
3 Russo D, Bonomo MG, Salzano G, Martelli G, Milella L. Nutraceutical
properties of Citrus clementina juices. Pharmacologyonline 2012; 1:
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