Antioxidant Activity of Compounds Isolated from Elaeagnus umbellata Promotes Human Gingival Fibroblast Well-Being

Article abstract of DOI:10.1021/acs.jnatprod.9b01030

Four new triterpenoid bidesmosidic saponins (1-4) and a sesquiterpenoid glucoside (5), together with nine known phenolic compounds (6-14), were isolated from the fruits of Elaeagnus umbellata. Their structures were elucidated using 1D and 2D NMR spectroscopy and mass spectrometry data. The antioxidant capability of the isolated compounds was evaluated in human gingival fibroblasts. Compound 6 decreased ROS production and promoted cell proliferation. It also counteracted the cell cycle blockade induced by a low concentration of H₂O₂ decreasing the expression of p21 and CDKN2A (p16^INK4A). Compound 6 decreased the expression of inflammatory cytokines (IL-6 and IL-8) in response to inflammatory stimuli, supporting its possible use in periodontitis lesions.

Full text of DOI:10.1021/acs.jnatprod.9b01030

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Article
Antioxidant Activity of Compounds Isolated from Elaeagnus
umbellata Promotes Human Gingival Fibroblast Well-Being
Anna Maria Iannuzzi, Chiara Giacomelli, Marinella De Leo, Deborah Pietrobono, Fabiano Camangi,
Nunziatina De Tommasi,* Claudia Martini, Maria Letizia Trincavelli, and Alessandra Braca
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ABSTRACT: Four new triterpenoid bidesmosidic saponins (1−
4) and a sesquiterpenoid glucoside (5), together with nine known
phenolic compounds (6−14), were isolated from the fruits of
Elaeagnus umbellata. Their structures were elucidated using 1D and
2D NMR spectroscopy and mass spectrometry data. The
antioxidant capability of the isolated compounds was evaluated
in human gingival fibroblasts. Compound 6 decreased ROS
production and promoted cell proliferation. It also counteracted
the cell cycle blockade induced by a low concentration of H2O2
decreasing the expression of p21 and CDKN2A (p16INK4A).
Compound 6 decreased the expression of inflammatory cytokines
(IL-6 and IL-8) in response to inflammatory stimuli, supporting its
possible use in periodontitis lesions.
he genus Elaeagnus (Elaeagnaceae) includes about 90
species spread around the world, particularly in the
fresh, a custom that was imported from Asia.7 Few
phytochemical investigations on E. umbellata berries are
reported describing the presence of bioactive compounds
such as polyphenols and flavonoids, which contribute in part to
the in vitro antioxidant activity of “cardinal olive” extracts,2 and
also alkaloids, steroids, terpenoids, and saponins.8
T
temperate and subtropical regions of Asia, most of which are
considered as folk medicinal plants for their pharmacological
effects.1 Among these species, Elaeagnus umbellata Thunb, also
known as “cardinal olive” or “autumn olive”, is a small
deciduous shrub that grows as a tree 5−6 m tall, native to
Southern Europe and Central Asia and then introduced to
North America as an ornamental plant.2 In Indian traditional
medicine, E. umbellata flowers are used in cardiac and
respiratory diseases, while the seed oil is used for the treatment
of pulmonary infections, coughs, and cardiac ailments.3 In
Japan and China, the leaves are used as a tonic and their
decoction is used to treat bowel disorders.4 The fruits,
produced in great quantities and ripening between September
and November, are small, round, juicy, reddish to pink, dotted
with scales, and carrying a single seed.5 The fruits are edible,
but not widely cultivated and consumed as a food, except for
some areas of Asia, where the fruits are incorporated into the
common diet, due to their benefits against hepatitis, fractures,
injuries, and diarrhea, and used to prepare juices, jams,
preserves, and other food stuffs.6 The red pigmentation of the
berries is due to the presence of a large amount of carotenoids,
especially lycopene (about 5−20 times higher than that of the
ordinary tomato), which is reported to have anticancer,
antioxidant, hepatoprotective, and cardioprotective effects.5,6
Therefore, these fruits have recently been used for the
development of lycopene-rich extracts or powders for food
formulation.5 In Italy, the plant is nonindigenous and is
cultivated for ornamental purposes, while the fruits are eaten
Thus, the present work was carried out to fully investigate
the chemical profile of E. umbellata fruits cultivated in Italy,
leading to the isolation and structural characterization of five
new compounds, including four triterpenoid saponins (1−4)
and one sesquiterpenoid glucoside (5), together with nine
known phenolics (6−14). It is well known that flavonoid
aglycones and corresponding glycosides possess antioxidant
and anti-inflammatory activities.9−11 Oxidative stress and
related inflammatory processes play crucial roles in different
pathologies of the oral cavity such as periodontitis and oral
mucositis. Periodontitis is a common inflammatory disease that
derives from bacterial infection and proceeds with an abnormal
host response. It can result in the destruction of oral soft
tissues and can affect systemic health.12 Oral mucositis is one
of the most common side effects observed during cancer
radiotherapy and chemotherapy treatment.13 The modulation
of reactive oxygen species (ROS) has gained attention due to
Special Issue: Special Issue in Honor of Jon Clardy
Received: October 17, 2019
© XXXX American Chemical Society and
American Society of Pharmacognosy
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their pivotal role in the progression of oral inflammatory
diseases.14 ROS are a group of molecules derived by the
physiological metabolism of oxygen in cells, and intracellular
ROS such as hydrogen peroxide (H2O2) have been widely
investigated for their controversial role. In the host response to
bacterial infection, the activation of neutrophils promotes ROS
production15 that causes the damage of different macro-
molecules, leading to a premature aging of periodontal tissue.16
Similarly, the production of ROS by radiotherapy and
chemotherapy plays a pivotal role in the initiation phase of
oral mucositis.17 Recently, several efforts have been made to
discover synthetic molecules, natural extracts, and compounds
that could locally counteract the production of ROS and the
insurgence of inflammatory processes.18−20 Interestingly, the
healing effects of an aqueous ethanolic extract of E. angustifolia
has been reported in 5-fluorouracil-induced oral mucositis in
male golden hamsters.21 Considering the possibility to
consume the E. umbellata fruits, the antioxidant ability and
the beneficial effects of their extracts and isolated compounds
(2 and 5−14) in human gingival fibroblasts were evaluated for
the first time. Furthermore, the ability of the most promising
derivative, compound 6, to decrease the transcription of
lipopolysaccharide (LPS)-induced pro-inflammatory cytokines
(IL-6 and IL-8) was also assessed.
between EtOAc, n-BuOH, and H2O. The EtOAc and n-BuOH
extracts, subjected to Isolera flash chromatography, Sephadex
LH-20, and RP-HPLC separations, yielded in pure form five
new (1−5) and nine known compounds (6−14). The
chemical profiles of the EtOAc and n-BuOH extracts are
shown in Figure 1.
The molecular formula of compound 1 was determined as
C65H104O32 by HRESIMS, showing a sodium adduct ion at m/
z 1419.6357 [M + Na]+. Analysis of the NMR data (Tables 1
and 2) confirmed the presence of 30 signals attributable to the
aglycone moiety and 35 to the saccharide portion, establishing
the presence of a bidesmosidic triterpenoid saponin. In the
ESIMS data, fragments obtained in the negative and positive
modes at m/z 1249 [M − H − 146]−, 1071 [M − H − 162 −
162]−, 1257 [M + Na − 162]+, and 1095 [M + Na − 162 −
162]+ suggested the presence of a triterpenoid saponin
structure with at least one deoxyhexosyl and two hexosyl
1
moieties. The H NMR data of 1 displayed seven methyl
singlets at δH 0.87, 0.94, 0.99, 1.06, 1.09, 1.10, and 1.27, a
hydroxymethine at δ 3.18 (dd, J = 11.5, 4.3 Hz), and six
anomeric protons at δ 4.33 (d, J = 7.4 Hz), 4.44 (d, J = 6.8
Hz), 4.68 (d, J = 7.7 Hz), 4.77 (1H, d, J = 7.5 Hz), 5.45 (d, J =
8.0 Hz), and 5.61 (d, J = 1.8 Hz). The 13C NMR data
suggested the structural features of 3-hydroxy-19-oxoolean-13-
en-28 oic acid for the aglycone of compound 1 due to the
presence of a hydroxymethine at δC 89.6 and an α,β-
unsaturated carbonyl (δC 134.0, 153.3, and 211.0). This
conclusion was supported by HMBC correlations between H2-
16−C-14, H2-16−C-17, H2-16−C-18, H2-12−C-18, Me-29−
C-19, Me-29−C-20, and Me-29−C-21.21 The structure of the
sugar moieties was deduced using 1D TOCSY, COSY, HSQC,
and HMBC experiments, leading to recognition of four β-
glucopyranosyl, an α-arabinopyranosyl, and an α-rhamnopyr-
anosyl moiety. The chemical shifts of one glucose anomeric
proton at δH 5.45 and δC 95.9 suggested this sugar moiety to
be involved in an ester linkage with the C-28 carboxylic acid
group at δC 174.2, and the HMBC correlation peak between
H-1glc−C-28 corroborated this substitution. Direct evidence of
the sugar sequence at C-3 was derived from the HMBC
correlations between H-1ara−C-3, H-1glcI−C-3ara, H-1rha−C-
2
glcI, H-1glcII−C-3glcI, and H-1glcIII−C-6glcI. The assignment of
the sugar configuration was obtained through hydrolysis of 1
with 1 N HCl followed by GC analysis through a chiral phase
column of the monosaccharides treated with 1-(trimethylsilyl)-
imidazole in pyridine. Thus, compound 1 was identified as 28-
O-β-D-glucopyranosyl 3β-O-[β-D-glucopyranosyl-(1→6)]-[β-
D-glucopyranosyl)-(1→3)]-[α-L-rhamnopyranosyl-(1→2)]-β-
D-glucopyranosyl-(1→3)-α-L-arabinopyranosyl-19-oxoolean-
13-en-28-oate.
Compound 2 showed a sodium adduct ion at m/z
1257.5826 [M + Na]+, differing by 162 Da from 1. In the
ESIMS fragments obtained in the positive ion mode at m/z
1095 [M + Na − 162]+, 1111 [M + Na − 146]+, 933 [M + Na
− 162 − 162]+, 625 [M + Na − 162 − 162 − 146 − 162]+,
and 447 [M + Na − 162 − 162 − 146 − 162 − 132 − 46]+
were consistent with the presence of a triterpenoid saponin
with five sugar moieties comprising three hexosyl, one
deoxyhexosyl, and one pentosyl unit. Analysis of the NMR
data (Table 1) for the aglycone moiety showed the presence of
nine methylenes, seven methyls, four methines (one oxy-
genated), an sp2 methine, seven quaternary carbons (one sp2),
a keto, and a hydroxy carbonyl carbon. The MS and NMR data
displayed for the aglycone moiety eight indices of hydrogen
RESULTS AND DISCUSSION
The fruits of E. umbellata were defatted with n-hexane and then
extracted with MeOH. The MeOH extract was partitioned
■
B
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Figure 1. Chemical profiles of Elaeagnus umbellata fruit extracts. (A) HPLC-ESIMS of the n-BuOH extract registered in the negative ion mode. (B)
HPLC/UV of the EtOAc extract registered at 350 nm. Peak numbers indicate the isolated compounds.
1
deficiency. The H NMR spectrum showed, in addition to
signals corresponding to seven methyls, a hydroxymethine at δ
3.19 (dd, J = 12.0, 4.0 Hz) and an olefinic proton at δ 5.26 (t, J
= 3.0 Hz). The HMBC spectrum showed correlations between
Me-29 (δ 1.22) and C-19, C-20, and C-21 and between H-18
(δ 3.79) and C-12, C-16, C-18, C-19, and C-20. The
spectroscopic features (Table 1) were in agreement with a
19-oxooleanolic acid as the aglycone of compound 2. This
triterpenoid aglycone structure is described here for the first
time. Comparison of the spectroscopic data of the sugar
portion of compounds 2 and 1 (Table 2) showed structural
similarities: in particular, the saccharide chain of 2 differed
from that of 1 only in the absence of the β-glucopyranosyl
moiety linked at C-6 of glc I. The configuration of the
saccharide units was assigned as reported for 1. Thus,
compound 2 was characterized as 28-O-β-D-glucopyranosyl
3β-O-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→2)-
[α-L-rhamnopyranosyl]-(1→3)-α-L-arabinopyranosyl-19-oxoo-
leanolate.
Compound 3 (C59H94O26), isolated in a small amount,
exhibited a sodium adduct ion at m/z 1241.5990 [M + Na]+,
as well as a fragment peak at m/z 1095.6798 [M + Na − 146]+
C
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a
1
Table 1. H and 13C NMR Data for Aglycones of Compounds 1, 2, and 5
1
2
5
position
δH
δC
δH
δC
position
δH
δC
b
b
b
1a
1b
2a
2b
3
4
5
6a
6b
7a
7b
8
1.77
39.9
1.71
1.08
39.0
2
3
4
5
6
7
8
174.0
126.6
140.0
133.8
126.7
80.5
1.07, m
5.91, s
b
1.92
1.76
27.0
2.05, m
26.5
b
b
1.74
7.91, d (16.0)
6.25, d (16.0)
3.18, dd (11.5, 4.3)
89.6
39.1
57.3
19.0
3.19, dd (12.0, 4.0)
89.5
39.0
57.2
19.0
0.83, br d (11.7)
0.84, br d (11.5)
51.0
39.1
b
b
1.64
1.59
9a
9b
10
11a
11b
12
13
14
15
16
Glc-1
2
3
4
5
6a
6b
2.09, dd (14.0, 7.5)
1.93, dd (14.0, 11.0)
4.01, m
2.45, dd (14.4, 6.5)
1.99, dd (14.4, 10.0)
b
b
1.66
34.3
1.68
34.2
72.8
39.0
1.41, m
1.41, m
43.0
52.2
36.9
23.5
40.0
48.5
36.0
24.1
b
b
9
1.61
1.71
87.5
20.1
178.0
14.0
18.2
103.0
74.6
77.4
71.3
77.7
62.2
10
11a
11b
12a
12b
13
14
15a
15b
16a
16b
17
18
19
20
21a
21b
22a
22b
23
24
25
26
27
28
29
30
2.04, s
b
b
1.61
2.00
b
1.98
1.12, s
1.40, s
2.67, br dd (16.0, 12.0)
1.99, m
134.0
5.26, t (3.0)
129.4
4.37, d (7.8)
3.16, br t (9.0)
3.26, t (9.5)
3.30, t (9.5)
3.38, m
153.3
45.0
27.0
133.0
41.8
28.0
b
b
1.92
1.76
1.99
b
1.82, m
b
2.15, ddd (14.3, 6.5, 3.8)
34.3
1.89
27.0
3.86, dd (12.0, 3.0)
3.68, dd (12.0, 5.0)
b
b
1.93
1.62
53.1
50.0
56.8
216.5
44.0
35.2
134.0
211.0
45.5
3.79, s
b
1.85, ddd (17.0, 14.3, 3.8)
36.6
1.93
b
b
1.65
1.77
2.30, ddd (14.3, 6.6, 3.3)
32.0
2.26, ddd (15.0, 11.0, 4.0)
31.0
b
b
1.63
1.80
1.06, s
0.87, s
0.99, s
0.94, s
1.27, s
28.0
17.0
17.0
14.0
21.0
174.2
26.5
25.5
1.07, s
0.89, s
1.00, s
0.85, s
0.97, s
28.1
17.0
16.0
17.5
23.0
175.0
27.0
25.6
1.09, s
1.10, s
1.22, s
1.11, s
a
Spectra were recorded in methanol-d4 at 600 MHz (1H) and 150 MHz (13C). J values are in parentheses and reported in Hz; chemical shifts are
b
given in ppm; assignments were confirmed by 1D-TOCSY, COSY, HSQC, and HMBC experiments. Overlapped signal.
corresponding to the loss of a deoxyhexosyl unit. Analysis of its
NMR spectroscopic data (Table 2) and comparison with those
of saponin 2 showed that the aglycone moiety and the sugar
chain at C-3 of 3 were the same as those of 2, while the sugar
moiety at C-28 was different. The presence of an anomeric
proton at δH 6.00 and at δC 95.5 belonging to a terminal
rhamnopyranosyl moiety suggested this sugar to be linked at
C-28. This assumption was substantiated by the HMBC
correlation between H-1rha (δH 6.00) and C-28 (δC 175.0).
The configuration of the saccharide units was determined as
reported for 1. The structure of 3 was thus established as 28-O-
α-L-rhamnopyranosyl 3β-O-D-glucopyranosyl-(1→3)-β-D-glu-
copyranosyl-(1→2)-[α-L-rhamnopyranosyl]-(1→3)-α-L-arabi-
nopyranosyl-19-oxooleanolate.
1419.6390, corresponding to a molecular formula of
C65H104O32, and hence was shown to be an isomer of 1. The
HRESIMS/MS data revealed the presence of fragments at m/z
1257.7781 [M + Na − 162]+ and 1095.6188 [M + Na − 162 −
162]+ due to the subsequent loss of two hexosyl moieties.
Comparison of its NMR spectroscopic data with those of
saponins 1−3 showed 4 to have the same aglycone as 2, while
its saccharidic chains at C-3 and C-28 were identical to those
of 1. The aglycone moiety of compound 1 could be an artifact
of 4. The double-bond rearrangement could be due to the
formation of a conjugated α,β-unsaturated carbonyl system.
Therefore, the structure 28-O-β-D-glucopyranosyl 3-O-[β-D-
glucopyranosyl-(1→6)]-[β-D-glucopyranosyl)-(1→3)]-[α-L-
rhamnopyranosyl-(1→2)]-β-D-glucopyranosyl-(1→3)-α-L-ara-
binopyranosyl-19-oxooleanolate was assigned to compound 4.
Compound 4 was isolated in a trace amount. Its HRESIMS
data in the positive ion mode showed an [M + Na]+ ion at m/z
D
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a
1
Table 2. H and 13C NMR Data for Sugar Moieties of Compounds 1−3
1
2
3
position
δH
δC
δH
δC
δH
δC
ara-1 at C-3
2
3
4
5a
5b
glc I-1
2
3
4
5
6a
6b
rha-1
2
3
4.44, d (6.8)
105.7
75.0
83.0
70.0
66.2
4.44, d (6.7)
105.8
75.0
82.7
70.0
66.0
4.44, d (6.8)
106.0
75.1
82.3
71.0
66.5
3.91, dd (9.0, 6.8)
3.95, dd (9.0, 2.0)
4.08, m
3.93, dd (9.0, 6.7)
3.91, dd (9.0, 3.0)
4.08, m
3.93, dd (9.0, 6.8)
3.91, dd (9.0, 2.5)
4.07, m
3.90, dd (12.0, 2.0)
3.62, dd (12.0, 4.5)
4.68, d (8.0)
3.60, dd (9.5, 8.0)
3.66, t (9.5)
3.39, t (9.5)
3.88, dd (12.0, 2.0)
3.65, dd (12.0, 4.0)
4.68, d (7.7)
3.87, dd (12.0, 2.5)
3.61, dd (12.0, 4.0)
4.66, d (7.5)
103.0
77.0
83.4
71.0
76.7
70.0
102.8
77.8
83.3
70.6
77.9
62.0
102.9
77.8
83.6
71.0
78.0
62.4
b
b
3.66
3.64
b
b
3.65
3.65
3.28, t (9.5)
3.55, m
3.40, t (9.5)
3.33, m
3.31, m
4.20, dd (12.0, 3.0)
3.72, dd (12.0, 5.0)
5.61, d (1.8)
3.95, dd (3.0, 1.8)
3.73, dd (9.0, 3.0)
3.45, t (9.0)
4.07, m
1.26, d (6.5)
4.77, d (7.5)
3.38, dd (9.5, 7.5)
3.34, t (9.5)
3.88, dd (12.0, 3.5)
3.72, dd (12.0, 5.0)
5.62, d (1.5)
3.96, dd (3.0, 1.5)
3.74, dd (9.0, 3.0)
3.45, t (9.0)
3.88, dd (12.0, 3.0)
3.72, dd (12.0, 4.5)
5.64, d (1.8)
3.96, dd (3.0, 1.8)
3.72, dd (9.0, 3.0)
3.47, t (9.0)
4.09, m
1.28, d (6.5)
4.79, d (7.5)
3.39, dd (9.0, 7.5)
3.44, t (9.0)
101.0
72.0
72.0
74.0
70.0
17.0
105.7
75.0
78.0
70.0
77.0
62.0
100.7
72.0
71.8
73.5
70.0
17.4
105.3
75.0
78.0
71.0
77.9
62.1
100.0
72.1
72.0
73.0
70.5
18.0
105.6
75.6
78.0
71.0
78.3
62.1
4
5
6
4.10, m
1.25, d (6.8)
4.78, d (7.5)
glc II-1
2
3
4
5
6a
6b
glc III-1
2
3
4
5
6a
6b
glc-1 at C-28
2
3
4
5
6a
6b
b
3.40
3.42, t (9.0)
b
3.40, t (9.5)
3.36, m
3.40
3.43, t (9.0)
3.31, m
3.93, dd (12.0, 3.0)
3.77, dd (12.0, 5.0)
3.33, m
3.90, dd (12.0, 2.5)
3.80, dd (12.0, 4.5)
3.93, dd (12.0, 3.0)
3.75, dd (12.0, 4.5)
4.33, d (7.4)
3.23, dd (9.0, 7.4)
3.35, t (9.0)
3.28, t (9.0)
3.41, m
3.90, dd (12.0, 3.0)
3.69, dd (12.0, 5.0)
5.45, d (8.0)
104.9
75.0
77.0
71.0
77.0
62.0
95.9
75.0
77.7
70.0
77.0
62.0
5.46, d (8.0)
95.7
73.4
77.9
70.6
78.0
62.0
3.29, dd (9.5, 8.0)
3.35, dd (9.5, 8.0)
3.33, t (9.5)
b
3.41
b
b
3.40
3.40
b
3.36, m
3.83, dd (12.0, 3.0)
3.70, dd (12.0, 4.5)
3.40
3.86, dd (12.0, 2.5)
3.72, dd (12.0, 5.0)
rha-1 at C-28
6.00, d (1.8)
3.96, dd (3.0, 1.8)
3.72, dd (9.5, 3.0)
3.44, t (9.5)
4.09, m
1.24, d (6.0)
95.5
72.1
72.0
73.0
70.5
18.0
2
3
4
5
6
a
Spectra were recorded in methanol-d4 at 600 MHz (1H) and 150 MHz (13C). J values are in parentheses and reported in Hz; chemical shifts are
b
given in ppm; assignments were confirmed by COSY, 1D-TOCSY, HSQC, and HMBC experiments. Overlapped signal.
Compound 5 had the molecular formula C21H30O11 as
determined by HRESIMS (m/z 481.1666 [M + Na]+). Its
ESIMS data recorded in the negative ion mode displayed
fragments at m/z 413 [M − H − 44]−, 295 [M − H − 162]−,
251 [M − H − 162 − 44]−, 233 [M − H − 162 − 18 − 44]−,
sesquiterpenoid moiety and six to the sugar portion. The
signals of the aglycone moiety were sorted into three methyls,
two methylenes, three sp2 methines, a hydroxymethine, two
quaternary carbons, two oxygenate tertiary carbons, a
hydroxycarbonyl carbon, and an ester carbonyl. The 1H
NMR data (Table 1) displayed three methyl singlets at δ 1.12,
1.40, and 2.04, the latter having a chemical shift typical of an
allylic methyl, together with signals of three olefinic protons at
δH 5.91 (s), 6.25 (d, J = 16.0 Hz), and 7.91 (d, J = 16.0 Hz), a
hydroxymethine (δ 4.01, m), and an anomeric proton (δ 4.37,
and 189 [M − H − 162 − 18 − 44 − 44]− due to the
,
subsequent loss of CO2, a hexosyl unit, H2O, and a second
CO2 molecule. These data supported the presence of two
carboxylic groups in the structure of 5. The 13C NMR data of 5
(Table 1) showed 21 signals, of which 15 were assigned to a
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Figure 2. Protective effects of Elaeagnus umbellata extracts and isolated compounds on hGF. Human GF cells were treated with different
concentrations of Ex EtOAc, Ex BuOH, compounds 2, 5, and 6−14, quercetin (Q), kaempferol (K), or NAC (1 mM) in the absence (A) or
presence (B) of H2O2 (200 μM) for 72 h in complete medium. In the end, the MTS assay was performed. Data are expressed as percentage of cell
proliferation compared to the control, which was set to 100%. Each bar represents the mean SD of three replicates from two independent
experiments. The significance of the differences was determined by one-way ANOVA, which was followed by Bonferroni’s post-test: *p < 0.05, **p
#
< 0.01, ***p < 0.001, vs CTRL; p < 0.05, ##p < 0.01, ###p < 0.001, vs H2O2.
d, J = 7.8 Hz). The COSY experiment indicated two spin
systems for the aglycone corresponding to the sequence
−CH2−CHOH−CH2− and −CHCH−C−CH−, respec-
tively. The HSQC experiment allowed the assignments of all
protons linked to the respective carbons, to identify the β-
glucopyranosyl moiety, while the HMBC spectrum was crucial
to identify the aglycone as amygdalactone.22 The HMBC
correlation between H-1glc at δH 4.37 and C-10 at δC 72.8
confirmed the glucosyl substitution site. On the basis of these
data, compound 5 was identified as 10-O-β-D-glucopyranosy-
lamygdalactone (5). The aglycone amygdalactone was isolated
before only from Prunus amygdalus22 and Cinnamomum
cassia.23
Compounds 6−14 were also purified and characterized as
kaempferol 3-O-β-D-glucopyranosyl-(1→2)-β-D-galactopyrano-
side-7-O-α-L-rhamnopyranoside (6),24 2-hydroxynaringenin 7-
O-β-D-glucopyranoside (7),25 1-O-(trans-sinapoyl)-β-D-gluco-
pyranoside (8),26 isorhamnetin 3-O-α-L-xylopyranosyl-(1→2)-
β-D-glucopyranoside (9),27 tiliroside (10),28 kaempferol 3-O-β-
D-glucopyranoside (11),29 quercetin 3-O-α-L-xylopyranosyl-
(1→2)-β-D-galactopyranoside (12),30 kaempferol 3-O-β-D-
glucopyranosyl-(1→2)-β-D-galactopyranoside (13),31 and
quercetin 3-O-β-D-glucopyranosyl-(1→2)-β-D-galactopyrano-
side-7-O-α-L-rhamnopyranoside (14),27 by comparison of
their NMR and MS data to literature data.
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Figure 3. Effect of compound 6 on ROS production, cell death, and cell cycle progression. hGF cells were treated with different concentrations of
compound 6, quercetin (Q), kaempferol (K), or NAC as indicated, alone or in the presence of H2O2 (200 μM) for 72 h in complete medium. The
ROS levels were evaluated by H2DCFDA (A). Data are expressed as the percentage of ROS with respect to the amount in the CTRL set to 100%.
Each bar represents the mean SD of three replicates from two independent experiments. (B) Flow cytometry analysis of cell death was performed
as described in the Experimental Section. Data are reported as the number of cells/mL and as percentage of cell death. Each bar represents the
mean SD of two replicates from two independent experiments. Representative histograms are shown. (C) Flow cytometry analysis of the cell
cycle was performed as described in the Experimental Section. Data are reported as the percentage of cells in each cell phase. Each bar represents
the mean SD of two replicates from two independent experiments. Representative histograms are shown. The significance of the differences was
#
determined by one-way ANOVA, which was followed by Bonferroni’s post-test: *p < 0.05, **p < 0.01, ***p < 0.001, vs CTRL; p < 0.05, ##p <
0.01, ###p < 0.001 vs H2O2.
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Figure 4. Compound 6 modulation of senescence-associated and cytokine gene expression in hGF. hGF cells were treated with different
concentrations of compound 6 alone or in the presence of H2O2 (200 μM) or LPS (1 μg/mL) for 72 h in complete medium. Then, the mRNA
levels of p53 (A), p21 (B), p16INK4A (C), IL-6 (D), IL-8 (E), and IL-10 (F) were quantified using real-time RT-PCR analysis. Data are expressed as
the fold change versus the CTRL, which was set to 1, and are presented as the mean values SD of two independent experiments performed in
duplicate. The significance of the differences was determined by one-way ANOVA, which was followed by Bonferroni’s post-test: *p < 0.05, **p <
#
0.01, ***p < 0.001 vs CTRL; p < 0.05, ##p < 0.01, ###p < 0.001 vs H2O2 or LPS.
Compound 2, as a representative saponin, compounds 5−
14, and the EtOAc (Ex EtOAc) and n-BuOH (Ex BuOH)
extracts were screened for their potential proliferative effects
on human gingival fibroblast (hGF) using the MTS assay
(Figure 2A). Among the compounds, 13 and 14 produced a
significant decrease in cell proliferation when used at
micromolar concentrations. Conversely, Ex BuOH and
compound 6 (1 μM) were able to significantly increase the
significantly decreased the proliferation (50.9
4.0%, p <
0.001 vs CTRL), and these effects were counteracted by N-
acetylcysteine (NAC) tested as reference antioxidant molecule
(74.1 12.6%, p < 0.001 vs CTRL).34 Interestingly, Ex BuOH
counteracted the decrease of cell proliferation (62.5 7.5%, p
< 0.05 vs CTRL), and compound 6 produced a more robust
effect (68.8 4.4%, p < 0.01 vs CTRL).
Successively, we extensively investigated the effects of
compound 6 on hGF as a new potential biological activity of
a rare flavonoid glycoside. The correct balance of intracellular
ROS level positively regulates physiological functions,
including signal transduction, gene expression, and prolifer-
ation, favoring adaptive responses and longevity. In contrast,
the uncontrolled production of ROS induces ineffective
adaptive responses, contributing to aging phenomena.35 In
this respect, the antioxidant ability of compound 6 was
investigated in the absence or presence of H2O2 using
H2DCFDA, which is the reduced form of fluorescein used as
an indicator of intracellular ROS levels (Figure 3A).
Compound 6 alone decreased the intracellular ROS concen-
tration (p < 0.05). Despite the fact that this effect was similar
to that obtained with K, a low concentration of Q produced a
more robust effect, in accordance with its antioxidant effects
reported in other cell lines.36 Interestingly, compound 6
significantly counteracted the ROS production induced by
H2O2 (181.9 7.4% H2O2, 124.2 11.3% 6, p < 0.001).37,38
This effect was similar to that obtained for NAC (109.7
hGF proliferation to 122.3
4.8% and 125.6
9.1%,
respectively, showing a beneficial effect of compound 6 on
hGF. Challenging the fibroblast with low concentrations of
quercetin (Q) and kaempferol (K) did not affect the
proliferation of hGF. These data are in accordance with the
ability of kaempferol glycosides to increase keratinocyte
proliferation more than kaempferol itself.32 Conversely, a
high concentration (50 μM) of Q and K caused a significant
decrease in cell proliferation. The treatment with a high
concentration of H2O2 (millimolar range) causes cell death in
different cellular models. However, the use of a micromolar
concentration of H2O2 for a prolonged period induces DNA
damage, cell cycle arrest, and the expression of senescence-
associate protein (p53, p21, and p16).33 Herein, we used a low
concentration of H2O2 (200 μM) that better reproduces the
effects of ROS in periodontitis and mucositis. The ability of all
the tested compounds to counteract the decrease of
proliferation induced by H2O2 was evaluated using the MTS
assay (Figure 2B). Challenging hGF cells with H2O2 for 72 h
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9.4%, p < 0.001 vs H2O2). Overall, these results indicated that
the flavonol derivate of E. umbellata slightly alters the balance
of intracellular ROS in physiological conditions, but could
effectively counteract the stress effects produced by the
external stimuli.
The modification of cell proliferation could be due to the
induction of cell death or a blockade of the cell cycle. In order
to extensively investigate the well-being activity of compound
6, a live/death cell count assay was performed (Figure 3B).
The results demonstrated that treatment with H2O2 did not
induce significant cell death, in accordance with the use of a
low concentration. Similarly, the treatment with compound 6
alone or in combination with H2O2 did not alter the number of
dead cells. Compound 6 alone did not produce a significant
increase in cell number (322 400 39431, CTRL; 358 800
53942); however, it significantly counteracted the decrease of
Compound 6 has recently been reported as a dipeptidyl
peptidase IV (DPP-IV) inhibitor,42 and different kaempferol
glycosides have been reported to increase keratinocyte
proliferation32 and protect red blood cells and neutrophils
from DNA damage;36,43 however, its effect on gingival tissue
has not yet been reported.
Compound 6 counteracted the negative effects of oxidative
stress and decreased the gene expression of pro-inflammatory
cytokines. Poor absorption of some flavonoids and flavonoid
glycosides is a major limitation for use of flavonoids as
systemic nutraceuticals.44 However, the ability of a flavonoid to
act from the extracellular compartment modifying the
membrane composition and interacting with different mem-
brane receptors has recently been reported.45 The exact
mechanism of action of compound 6 is still unclear; however
we could speculate that the glycoside or its metabolites could
interact with extracellular cell components, modifying the hGF
oxidative state and decreasing the inflammatory status. In
conclusion, these results highlight a new property of a
flavonoid glycoside as a possible local protective agent against
oxidative stress and inflammatory stimuli in gingival tissue.
cell number induced by H2O2 (175 200
11 777, H2O2;
209 500 23502, 6 + H2O2; p < 0.05), in accordance with the
results obtained for cell proliferation (Figure 2). Next, a flow
cytometry analysis was performed to investigate the cell cycle
progression (Figure 3C). hGF cells in the absence of H2O2
treatment showed a typical cell cycle distribution with most
cells in G0/G1 phase (78.7 4.9%). The treatment with H2O2
significantly increased the number of cells in the S and G2/M
phases (from 11.1 1.2% to 34.3 8.1%; p < 0.05), as well as
decreased the number of cells in the G0/G1 phase (48.6
7.9%; p < 0.001), in accordance with the reported ability of
ROS to produce a G2/M cell cycle blockade.39 This
modification was almost completely counteracted by NAC
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured by an Atago AP-300 digital polarimeter with a 1 dm
microcell and a sodium lamp (589 nm). NMR experiments were
recorded on a Bruker DRX-600 spectrometer at 300 K (Bruker
BioSpin, Germany), acquiring the spectra in methanol-d4.46
HRESIMS data were obtained in the positive and negative ion
mode on an LTQ Orbitrap XL mass spectrometer (Thermo Fisher
Scientific) and Q-TOF Premier spectrometer equipped with a
nanospray ion source (Waters, USA). ESIMS data were acquired on
an LCQ Advantage ThermoFinnigan spectrometer (ThermoFinnigan,
USA), equipped with Xcalibur software. LC-PDA/UVvis/ESIMS/MS
analyses were performed on a Synergi Fusion-RP column (4.6 × 150
mm, 4 μm, flow rate 0.8 mL/min, Phenomenex, Italy), eluting with a
mixture of MeOH (solvent A) and formic acid in water 0.1% v/v
(solvent B) and using a linear gradient of increasing 5% to 75%
MeOH within 70 min. MS parameters were optimized as previously
reported.47 Columns chromatography was performed over Sephadex
LH-20 and an Isolera Biotage flash purification system (flash silica gel
60 SNAP cartridge). HPLC separations were carried out using a
Shimadzu LC-8A series pumping system equipped with a Shimadzu
RID-10A refractive index detector and Shimadzu injector (Shimadzu
Corporation, Japan) on a C18 μ-Bondapak column (30 cm × 7.8 mm,
10 mm, flow rate 2.0 mL/min). TLC separations were conducted
using silica gel 60 F254 (0.20 mm thickness) plates (Merck, Germany)
and Ce(SO4)2−H2SO4 as spray reagent (Sigma-Aldrich, Italy). GC
analysis was performed using a Dani GC 1000 instrument on a L-CP-
Chirasil-Val column (0.32 mm × 25 m), working with the following
temperature program: 100 °C for 1 min, ramp of 5 °C/min up to 180
°C; injector and detector temperature 200 °C; carrier gas N2 (2 mL/
min); detector dual FID; split ratio 1:30; injection 5 μL.
(70.6
2.5%, G0/G1; p < 0.01; 19.5
1.2%, G2/M; p <
0.05). Interestingly, compound 6 counteracted the cell cycle
blockade, increased the cells in the G0/G1 phase (66.6
10.9%; p < 0.05), and reduced those in the G2/M phase (18.5
7.1%; p < 0.05).
So far, oxidative stress has been associated with senescence
and growth arrest, and it has been shown that these
phenomena depend functionally on the expression of different
proteins such as p16INK4, the cyclin-dependent kinase (CDK)
inhibitor p21, and tumor suppressor p53.40 In this respect, the
gene expression of these proteins was evaluated by means of
real-time reverse transcription (RT)-PCR (Figure 4A−C).
Compound 6 “per se” did not alter the p21 and p53
transcription and produced only a slight decrease of p16 in
accordance with its ability to promote the hGF cell
proliferation. The oxidative stress caused a significant increase
of p53, p21, and p16 gene expression, in accordance with the
H2O2-induced cell blockage. Compound 6 counteracted the
effects of H2O2, decreasing the transcription of all the proteins.
Periodontitis and mucositis are also characterized by the
presence of high levels of pro-inflammatory cytokines that are
released in response to extracellular stimuli (e.g., bacteria) by
different types of cells such as fibroblasts.41 Thus, to further
characterize the beneficial activity of compound 6 on gingival
cells, the gene expression of pro-inflammatory (interleukin-6,
IL6, and interleukin-8, IL-8) and anti-inflammatory (inter-
leukin-10, IL-10) cytokines was evaluated using a real-time
RT-PCR analysis (Figure 4D−F). Challenging cells with LPS
(1 μg/mL) promoted the transcription of IL-6 and IL-8 and
the decrease of IL-10. Compound 6 restored the balance of the
released cytokines in favor of a lower inflammatory status; in
fact, it reduced the expression of IL-6 and IL-8 and increased
the expression of IL-10.
Plant Material. The ripe fruits of Elaeagnus umbellata Thunb.
were collected in October 2016 in Livorno (Italy) and identified by
Prof. Fabiano Camangi. A voucher specimen (N.A. 5472 Elaeagnus
umbellata/026677) was deposited at Herbarium Horti Botanici Pisani
(Pisa, Italy).
Extraction and Isolation. Dried and powdered whole berries of
E. umbellata (400.0 g) were defatted with n-hexane and extracted for
48 h with MeOH by exhaustive maceration (3 × 2.5 L), to give 137.7
g of extract. Dried MeOH extract was dissolved in water and
partitioned with EtOAc and n-BuOH, successively, to yield 5.0 and
1.8 g of the respective residues. The n-BuOH extract was subjected to
Sephadex LH-20 column chromatography (3 × 70 cm) using MeOH
as eluent (flow rate 0.8 mL/min), collecting fractions of 8 mL that
were grouped by thin-layer chromatography (TLC) into 10 major
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fractions (A1−J1). Fractions A1 (25.5 mg), B1 (35.5 mg), and C1 (65.0
mg) were separately subjected to RP-HPLC with MeOH−H2O (3:2)
to yield compounds 4 (0.8 mg, tR 16 min) and 1 (1.8 mg, tR 41 min)
from fraction A1, compounds 2 (0.7 mg, tR 35 min) and 1 (1.2 mg, tR
41 min) from fraction B1, and compounds 2 (1.8 mg, tR 35 min) and
3 (1.1 mg, tR 55 min) from fraction C1. Fractions E1 (115.0 mg) and
G1 (104.0 mg) were individually chromatographed by RP-HPLC with
MeOH−H2O (3.5:6.5) to give compound 5 (1.9 mg, tR 8 min) from
fraction E1 and compounds 8 (1.0 mg, tR 12 min), 7 (3.2 mg, tR 16
min), 14 (4.5 mg, tR 31 min), and 13 (3.7 mg, tR 57 min) from
fraction G1. Fractions F1 (63.2 mg) and I1 (25.0 mg) were separately
purified by RP-HPLC with MeOH−H2O (2:3) to afford compound 6
(3.8 mg, tR 16 min) from fraction F1 and compound 9 (1.8 mg, tR 44
min) from fraction I1. Fraction J1 (67.1 mg) contained pure
compound 12.
for 6 h. Then, cells were treated with test extracts and compounds at
different concentrations (5 nM to 50 μM) in the absence or in the
presence of H2O2 (200 μM) for 72 h. Extracts and compounds were
solubilized in DMSO, and the final concentration of DMSO was 0.5%.
Cell proliferation was evaluated using the MTS assay (CellTiter 96
AQueous One Solution cell proliferation assay kit; Promega)
according to the manufacturer’s instruction. The absorbance at 490
nm was measured with an automated plate reader (EnSight,
PerkinElmer).
ROS Production. The intracellular ROS level was determined
using the fluorogenic probe DCFH2-DA (Molecular Probes,
Invitrogen) as was previously reported.48 Briefly, hGF cells were
seeded in a 96-well microplate (5000 cells/well) and treated with
compound 6, Q, or K (1−5 μM) in the absence or in the presence of
H2O2 (200 μM) for 72 h. Then, cells were incubated in phosphate-
buffered saline (PBS)−glucose containing 50 μM DCFH2-DA for 30
min. The medium was removed and replaced with PBS/glucose. The
fluorescence intensity of DCF was measured with an automated plate
reader (EnSight, PerkinElmer), with wavelengths of 485 nm
(excitation) and 520 nm (emission). The cells were fixed with 3%
paraformaldehyde for 20 min, washed with PBS, and incubated with
crystal violet for 20 min. Finally, cells were washed, a solution of
sodium dodecyl sulfate (1%) was added for 1 h, and the absorbance at
595 nm was quantified. The fluorescence intensity was normalized to
the absorbance at 595 nm and related to the control value.
hGF Live/Dead Cell Count and Cell Cycle Analyses. For cell
live/dead cell count hGF were seeded in a six-well microplate (80 000
cells/well) and were treated in complete medium with compound 6
(1−5 μM) in the absence or in the presence of H2O2 (200 μM) for 72
h. The number of live cells and the percentage of dead cells were
quantified and analyzed by Muse Cell Analyzer (Merck KGaA,
Darmstadt, Germany), according to the manufacturer’s instructions
(MCH100102 Muse Count & Viability assay kit, Merck Millipore).
For the cell cycle analysis, hGF were seeded and treated as above. The
quantification of the percentage of cells in the different cell phases was
performed using the Muse Cell Analyzer (MCH100106 Muse cell
cycle assay kit, Merck Millipore) as previously reported.49
Real-Time RT-PCR Analysis. The gene expression in hGF was
quantified by performing a real-time RT-PCR analysis. Briefly, hGF
(3500 cells/cm2) were treated in complete medium with compound 6
(1−5 μM) in the absence or in the presence of H2O2 (200 μM) or
LPS (1 μg/mL) for 72 h. The cells were collected, and the total RNA
was extracted using the Rneasy mini kit (Qiagen, Hilden, Germany)
according to the manufacturer’s instructions. The purity of the RNA
samples was determined by measuring the absorbance at 260/280 nm.
mRNA (500 ng) was retrotranscribed to single-strand cDNA using
the i-Script cDNA synthesis kit (BioRad) following the manufac-
turer’s instructions. Real-time RT-PCR reactions were performed with
Fluocycle II SYBR in the presence of 50 ng of cDNA and 200 nM
forward and reverse primers.48,50 The primers were designed to span
intron/exon boundaries to exclude genomic DNA impurity, and β-
actin was used as the housekeeping gene. p53 FOR: 5′-
CTTTGAGGTGCGTGTTTGTG-3′ REV: 5′-GTGGTT-
TCTTCTTTGGCTGG-3′ (161 bp); p21 FOR: 5′-TGCCGA-
AGTCAGTTCCTTG-3′ REV: 5′-CATGGGTTCTGACG-
GACATC-3′ (134 bp); p16INK4A (CDKN2A) FOR: 5′-
GACCCCGCCACTCTCACC-3′ REV: 5′-CCTGTAGG-
ACCTTCGGTGACTGA-3′ (318 bp); IL-6 FOR: 5′-TCCTCGA-
CGGCATCTCA-3′ REV: 5′-TTTTCACCAGGCAAGTCTCCT-3′
(165 bp); IL-8 FOR: 5′-AAGAGAGCTCTGTCTGGACC-3′ REV:
5′-GATATTCTCTTGGCCCTTGG-3′ (408 bp); IL-10 FOR: 5′-
CAAGCTGAGAACCAAGACCC-3′ REV: 5′-AAGATGTCA-
AACTCACTCATGGC-3′ (141 bp); β-actin FOR: 5′-
GCACTCTTCCAGCCTTCCTTCC-3′ REV: 5′-GAGCCGCC-
GATCCACACG-3′ (254 bp). All reactions were performed for 40
cycles using the following temperature profiles: 98 °C for 30 s; 57 °C
for 20 s; and 72 °C for 30 s. The mRNA levels were normalized with
β-actin mRNA levels, and the relative expression was calculated using
the Ct value. PCR specificity was determined by both melting curve
analysis and gel electrophoresis.
The EtOAc fraction was subjected to flash silica gel CC using an
Isolera Biotage (SNAP 340 g column, flow rate 90 mL/min), eluting
with CHCl3, followed by increasing concentrations of MeOH in
CHCl3 (between 1% and 100%), collecting fractions of 27 mL, which
were grouped by TLC into four major fractions (A2−D2). Fractions
B2 (69.0 mg) and C2 (105.6 mg) were subjected to RP-HPLC with
MeOH−H2O (5.5:4.5) to yield compound 10 (1.7 mg, tR 31 min)
from fraction B2 and compounds 11 (2.8 mg, tR 17 min) and 10 (11.4
mg, tR 37 min) from fraction C2.
Compound (1): amorphous powder; [α]2D5 −8 (c 0.1, MeOH); H
1
1
and 13C NMR data of the aglycone moiety, see Table 1; H and 13C
NMR of the sugar moieties, see Table 2; ESIMS m/z 1395 [M − H]−,
1249 [M − H − 146]−, 1071 [M − H − 162 − 162]−, 1419 [M +
Na]+, 1257 [M + Na − 162]+, 1095 [M + Na − 162 − 162]+;
HRESIMS m/z 1419.6357 [M + Na]+, 1257.5852 [M + Na − 162]+
(calcd for C65H104O32Na 1419.6403).
Compound (2): amorphous powder; [α]2D5 −10.1 (c 0.1, MeOH);
1
1H and 13C NMR data of the aglycone moiety, see Table 1; H and
13C NMR of the sugar moieties, see Table 2; ESIMS m/z 1257 [M +
Na]+, 1095 [M + Na − 162]+, 1111 [M + Na − 146]+, 933 [M + Na
− 162 − 162]+, 625 [M + Na − 162 − 162 − 146 − 162]+, 447 [M +
Na − 162 − 162 − 146 − 162 − 132 − 46]+; HRESIMS m/z
1257.5826 [M + Na]+, 1095.6554 [M + Na − 162]+ (calcd for
C59H94O27Na 1257.5875).
Compound (3): amorphous powder; [α]2D5 −21 (c 0.1, MeOH); 1H
and 13C NMR data of the aglycone moiety were superimposable to
1
those reported for 2; H and 13C NMR of the sugar moieties, see
Table 2; HRESIMS m/z 1241.5990 [M + Na]+, 1095.6798 [M + Na
− 146]+ (calcd for C59H94O26Na 1241.5926).
Compound (4): amorphous powder; [α]2D5 −7 (c 0.1, MeOH); H
1
and 13C NMR data of the aglycone moiety were superimposable to
1
those reported for 2; H and 13C NMR of the sugar moieties were
superimposable to those reported for 1; HRESIMS m/z 1419.6390
[M + Na]+, 1257.7781 [M + Na − 162]+, 1095.6188 [M + Na − 162
− 162]+ (calcd for C65H104O32Na 1419.6403).
Compound (5): amorphous powder; [α]2D5 −25 (c 0.1, MeOH); 1H
and 13C NMR, see Tables 1 and 2; ESIMS m/z 457 [M − H]−, 413
[M − H − 44]−, 295 [M − H − 162]−, 277 [M − H − 180]−, 251
[M − H − 162 − 44]−, 233 [M − H − 162 − 18 − 44]−, 189 [M −
H − 162 − 18 − 44 − 44]− 481 [M + Na]+, 463 [M + Na − 18]+,
,
437 [M + Na − 44]+; HRESIMS m/z 481.1666 [M + Na]+ (calcd for
C21H30O11Na 481.1680).
Acid Hydrolysis of Compounds 1−5. Acid hydrolysis of
compounds 1−5 was performed as reported previously.39 D-Glucose,
L-rhamnose, and L-arabinose were identified as the sugar unit in each
case by comparison with the retention times of authentic samples.
Cell Culture. Human gingival fibroblast cells were purchased from
CLS Cell Line Service GmbH (Germany), lot. number 300703-
1541SF. Cells were maintained in DMEM-F12 supplemented with
HEPES, 5% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100
mg/mL streptomycin at 37 °C in 5% CO2. Cells were used between
passage 4 and 7.
Cell Proliferation Assay. hGF cells were seeded in 96-well
microplates (1500 cells/well) in complete medium. After 24 h the
medium was changed to noncomplete medium and cells were starved
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Statistical Analysis. Data were analyzed with GraphPad Prism
program (GraphPad Software Inc., San Diego, CA, USA). All data are
represented as the mean SD. Statistical analysis was performed as
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.9b01030.
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NMR spectra of compounds 1−5 (PDF)
AUTHOR INFORMATION
Corresponding Author
■
́
M.; Jimenez-Mejías, P.; Lattanzi, E.; Marchetti, D.; Martinetto, E.;
Nunziatina De Tommasi − Dipartimento di Farmacia,
Masin, R. R.; Medagli, P.; Passalacqua, N. G.; Peccenini, S.; Pennesi,
R.; Pierini, B.; Podda, L.; Poldini, L.; Prosser, F.; Raimondo, F. M.;
Roma-Marzio, F.; Rosati, L.; Santangelo, A.; Scoppola, A.;
Scortegagna, S.; Selvaggi, A.; Selvi, F.; Soldano, A.; Stinca, A.;
Wagensommer, R. P.; Wilhalm, T.; Bartolucci, F. Plant Biosystems
2018, 152, 556−592.
̀
Universita degli Studi di Salerno, 84084 Fisciano, SA, Italy;
orcid.org/0000-0003-1707-4156; Phone: +39-089-
969754; Email: detommasi@unisa.it; Fax: +39-089-969602
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Universitari e di Perfezionamento, 56127 Pisa, Italy
Claudia Martini − Dipartimento di Farmacia and Centro
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.9b01030
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by Fondi di Ateneo
PRA_2017_30, University of Pisa, and FARB 2018, University
of Salerno. We also thank Dr. Lara Russo for her expert
technical assistance.
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