Qualitative and quantitative analysis of the
proautophagic activity of Citrus ﬂavonoids from
Bergamot Polyphenol Fraction
, Raffaele Salerno
, Concetta Martino
, Daniele La Russa
, Manuela Oliverio
Department of Health Sciences, Magna Graecia University, Campus Germaneto, Catanzaro, Italy
Interregional Research Center for Food Safety and Health, Catanzaro, Italy
Department of Ecology, University of Calabria, Rende, Cosenza, Italy
Received 20 April 2018
Received in revised form
15 May 2018
Accepted 28 May 2018
Available online 31 May 2018
Bergamot Polyphenol Fraction (BPF
) is a natural mixture of Citrus
ﬂavonoids extracted from processed bergamot fruits. It has been
shown to counteract cardiovascular risk factors and to prevent
liver steatosis in rats and patients. Hepatic effects of BPF correlate
with its ability to stimulate liver autophagy. Six aglyconic ﬂavo-
noids have been identiﬁed in the proautophagic fraction of the
hydrolysis product of BPF (A-BPF): naringenin, hesperetin, eri-
dictyol, diosmetin, apigenin and luteolin. We report here the
output parameters of high resolution mass spectrometry analysis
of these ﬂavonoids and chemical structures of their parent com-
pounds. The second set of data shows the proautophagic activity of
BPF ﬂavonoids in a hepatic cell line HepG2 analyzed by a ﬂow
cytometry approach. The method is based on the red to green
ﬂuorescence intensity ratio analysis of DsRed -LC3- GFP, which is
stably expressed in HepG2 cells. Proportional analysis of ATG
indexes allowed us to address a relative contribution of individual
compounds to the proautophagic activity of the A-BPF mixture and
evaluate if the effect was additive. Qualitative analysis of ATG
indexes compared the effects of ﬂavonoids at equal concentrations
in the presence and absence of palmitic acid and chloroquine. The
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/dib
Data in Brief
2352-3409/&2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license
DOI of original article: https://doi.org/10.1016/j.jnutbio.2018.04.005
Corresponding author at: Department of Health Sciences, Magna Graecia University, Campus “Salvatore Venuta”, viale
Europa, Loc. Germaneto, 88100 Catanzaro, Italy.
E-mail address: firstname.lastname@example.org (E. Janda).
Data in Brief 19 (2018) 1327–1334
Excel ﬁles reporting the analysis of ﬂow cytometry data are
available in the public repository.
&2018 The Authors. Published by Elsevier Inc. This is an open
access article under the CC BY license
Subject area Pharmacology and cell biology
More speciﬁc subject area Flavonoid pharmacology and autophagy
Type of data Tables, text ﬁle, graphs, dot-plots
How data was acquired liquid chromatography- high resolution mass spectrometry (LC-
HRMS) Q-ExactiveTM (Thermo Scientiﬁc) and ﬂow cytometry (FACS
Canto II, BD Biosciences)
Data format Raw and analyzed
Experimental factors Flavonoid aglycons and their mixtures, treatments for 6 h, chlor-
oquine 2 h, palmitic acid added for 22 h and then withdrawn
Experimental features GR-LC3-HepG2 cells (HepG2 cells expressing DsRed -LC3- GFP)
Data source location Campus Germaneto, Catanzaro, Italy
Related research article Lascala et al. Analysis of proautophagic activities of Citrus ﬂavonoids in
liver cells reveals the superiority of a natural polyphenol mixture over
pure ﬂavones .https://doi.org/10.1016/j.jnutbio.2018.04.005
Value of the Data
We provide output LC-HRMS parameters for naringenin, hesperetin, eriodictyol, diosmetin,
apigenin and luteolin and the list of parent ﬂavonoid glycosides found in BPF.
A fast ﬂow cytometry method to analyze autophagy is illustrated in a detailed manner and sup-
ported by row data, so it can be easily reproduced by other researchers.
We describe here a “proportional”approach to the analysis of the proautophagic activities in a mix
of compounds that can be applied to other mixtures.
The autophagy index (ATG index) data for six typical ﬂavonoid aglycones reported here, can be
used as a reference for other cell lines and compounds.
Bergamot Polyphenol Fraction (BPF
) is a natural mixture of Citrus ﬂavonoids extracted from
processed bergamot fruits [2,3]. It has been shown to counteract cardiovascular risk factors and to
prevent liver steatosis in rats and patients [4–10]. Protective effects of BPF correlate with its ability to
stimulate autophagy in livers of rats fed cafeteria diet . The proautophagic activity of BPF is
mediated by a hydrophobic fraction of hydrolysed BPF (A-BPF) containing mainly ﬂavonoid aglycones
.Table 1 reports the LC-HRMS output parameters for six major ﬂavonoid aglycones identiﬁed in
A-BPF, such as retention times (RT), theoretical and measured mass to charge ratio (m/z), signal
intensity as well as a calculated relative and absolute abundance of each ﬂavonoid in the mix (see
material and methods). The aglycones listed in Table 1 (column A) originate from several known and
unknown parent compounds (i.e. ﬂavonoid glycosides), shown in Fig. 1, that have been previously
identiﬁed among bergamot polyphenols .
The exposure of hepatocytes to palmitic acid (PA) causes an accumulation of intracellular lipid
droplets and models non-alcoholic fatty liver disease (NAFLD) in vitro . We used this approach to
E. Janda et al. / Data in Brief 19 (2018) 1327–13341328
Output parameters of LC-HRMS analysis of ﬂavonoid aglycones identiﬁed in A-BPF and their expected parent compounds. Column G shows a theoretical quantitative representation of
aglycones in A-BPF, while the H column the amount (in μg) of aglycones in 60 μg of A-BPF, as calculated based on data in G.
Name RT (MIN) Molecular
sity (NL) x E6
in 60 μg Parent
ERIODICTYOL 21.09 C
287.0564 287.0561 34.6 20.51 12.3 NEOERIOCITRIN, ERIODICTYOL-
NARINGENIN 23.61 C
261.0615 261.0612 55.9 33.4 19.9 MELITIDIN, NARINGIN
HESPERETIN 24.38 C
301.0722 301.0718 51.8 30.7 18.4 BRUTERIDIN, HESPERETIN-7-O-
LUTEOLIN 24.46 C
285.0408 285.0405 3.99 2.37 1.4 LUTEOLIN-7-O-
APIGENIN 26.47 C
269.0459 269.0455 7.01 4.16 2.5 RHOIFOLIN, APIGENIN-7-O-NEO-
DIOSMETIN 26.70 C
299.0564 299.0561 15.4 9.13 5.5 DIOSMIN, NEODIOSMIN, DIOS-
E. Janda et al. / Data in Brief 19 (2018) 1327–1334 1329
Fig. 2. Proautophagic activities of ﬂavonoid aglycones in hepatic cells with high lipid content. ClQ co-treatments further
increase ATG index. (A) GR-LC3-HepG2 cells were treated with PA 0,3mM or vehicle (ethanol, EtOH) for 22 h. Subsequently, the
medium was exchanged and aglycones (30 μg/mL) were added for 6 h. 2 h before analysis ClQ (50 μM) or vehicle (H
added to PA-treated cells. Red and green ﬂuorescence was recorded by ﬂow cytometry. The graph shows the mean ATG index
þ/SEM, from four independent experiments performed each time with triplicate independent samples. Naringenin (Nari),
hesperetin (Hesp) and eriodictyol (Erio), followed by diosmetin(Dios), apigenin (Apig) and luteolin (Lute). Red/green ratio data
are normalized to controls. Statistical analysis: student T-test; *po0.05, **po0.01, ***po0.001, when compared to vehicle-
po0.001 when compared to the relative PA-untreated, the same ﬂavonoid-treated
po0.01, when compared to respective PA-treated, ClQ-untreated cells. See S1 data set (Supplementary data
set S1) with row data supporting this ﬁgure.
Fig. 1. Structures of parent compounds of 6 main aglycones present in A-BPF and their sugar moieties: O-Glu, O-glucoside;
C-glucose, C-glucoside; O-Neo, O-Neohesperidoside; O-Neo-HMG, 3-hydroxy-3-methyl-glutaryl-neohesperidoside.
E. Janda et al. / Data in Brief 19 (2018) 1327–13341330
measure the proautophagic activity of six main ﬂavonoid aglycones present in A-BPF in the presence
and absence of lipotoxic stress (Fig. 2). This was done by the qualitative analysis, i.e. equal con-
centrations of aglycones were used to induce autophagy in HepG2 cells expressing DsRed -LC3- GFP,
which turns red when autophagy is induced or LC3-II accumulates [1,11] . GR-LC3-HepG2 cells were
treated with PA (0.3 mM) to cause intracellular lipid overload, and 22 h later exposed to ﬂavonoid
aglycons þ/chloroquine (ClQ) to address the autophagic ﬂux modulation. These data were then
compared with ATG index (red/green ratio) induced in the absence of PA by six polyphenols in
independent experiments performed otherwise under identical conditions (Fig. 2 and Supplementary
data set S1).
Next, we addressed the quantitative contribution of each aglycone present in A-BPF to the
proautophagic activity of A-BPF, which we deﬁned here as “proportional analysis”of proautophagic
activity as opposed to “qualitative analysis”, shown in Fig. 2, where equal doses of compounds are
compared for their activity. We calculated the proportional amounts of six aglycones, as described in
Experimental design. Then we treated GR-LC3-HepG2 cells with calculated amounts of standards and
analyzed autophagy by ﬂow cytometry 6 h post-treatment. In the same experiment, we also mea-
sured the ATG index induced by the mix of all six compounds, used at proportional concentrations to
reconstitute 60 μg/mL of A-BPF (Mix60) (Table 1). The ATG index data for six tested aglycones and
their Mix are reported in Fig. 3A. The data in Fig. 3A are supported by Supplementary data set S2
presenting the row data used for this analysis. The Excel ﬁle S2 contains tables with mean ﬂuores-
cence intensities values recorded in 54 independent cell samples in two independent experiments.
This Excel ﬁle also shows how the mean ATG index is calculated and normalized and performs sta-
tistical analysis of the data. Examples of ﬂow cytometry dot-plots with raw data and relevant gates
used for analysis are also attached to Supplemetary data set S2.
Next, we tried to evaluate if six polyphenols contributed in additive or synergistic fashion to the
overall autophagic activity of BPF. The data in Fig. 3B are a numeric representation of data presented
in Fig. 3A and they show that the sum of mean ATG index increases caused by individual compounds
is higher than the ATG index induced by the Mix of aglycones. These data would rather suggest
additive as well competitive effects, rather than synergistic effects of tested ﬂavonoids on autophagy.
Fig. 3. Proportional analysis of proautophagic activities of aglycones present in A-BPF. (A) GR-LC3-HepG2 cells were treated for
6 h with the doses of pure aglycones corresponding to those present in 60μg A-BPF (reported as numbers of μg/mL after
compound abbreviation) or with the mix of these compounds (Mix60) and analysed for ATG index in six independent samples
þ/SEM. Statistical analysis: two-tailed, unpaired T-test; *po0.05, **po0.01, ***po0.001 when compared to control (CTRL),
po0.01 when compared to naringenin. (B) Table showing the analysis of additive effects of
six ﬂavonoid aglycones on ATG index. See Supplementary data set S2 (Multimedia Component 3) with row data supporting this
E. Janda et al. / Data in Brief 19 (2018) 1327–1334 1331
2. Experimental design, materials and methods
2.1. Cell culture
GR-LC3-HepG2 cells were descried previously in Lascala et al. . They were cultured in DMEM
complete medium (4.5 g/L glucose) as described for the HepG2 cells . For experiments, the cells
were seeded at the density 4 10
and the last medium change was performed 24 h before the
end of the experiment.
Naringenin, hesperetin, eriodictyol, diosmetin, apigenin and luteolin were purchased from
Extrasynthese (Geney Cedex, France) as Z99% pure (HPLC-grade) powders, which were solubilized in
EtOH 100% to stock solutions 5 mg/mL, except for diosmetin and apigenin diluted as 2 mg/mL stock
due to their low solubility. BPF
was provided by Herbal and Antioxidant Derivatives srl (H&AD srl),
Bianco, Italy. A-BPF was prepared by acid hydrolysis of BPF and isolation of hydrophobic phases from
crude hydrolysate, according to the procedure described in Lascala et al.  “Mix60”,or“Mix”,was
prepared by mixing ethanol solutions of six aglycones (as above) in natural proportions as found in
A-BPF, according to the data presented in Table 1, column G and H. ChlQ (25 mM stock in PBS), PA (0.1
to 1 M stock in EtOH) were from Sigma-Aldrich. These compounds were kept in aliquots at 80 °Cor
20 °C (shorter storage) and thawed shortly before each treatment.
2.3. Production of retroviruses coding for DsRed-LC3-GFP
To produce GR-LC3-HepG2 cells stably expressing DsRed-LC3-GFP reporter, recombinant retro-
viruses coding were generated and used to infect HepG2 cells. To produce viral stocks, HEK 293 Tcells
(one 100 mm plate at 90–95% conﬂuence) were transfected with pUMVC (10 μg), pCMV-VSV- G (4 μg)
and pQCXI-Puro-DsRed-LC3-GFP (10 μg) from Addgene (Cambridge, MA, USA), using Lipofectamine
2000 (Life Tech., Invitrogen, 11668027) according to manufacturer's instructions. After an overnight
incubation, cell medium was replaced with 5 mL of RPMI medium (Life Tech., Invitrogen, 11875093),
supplemented with 2% FBS. 48 h post-transfection, cell supernatant was collected, ﬁltered through a
0.45 μmﬁlter membrane, and supplemented with FBS (10% ﬁnal concentration). Prior to viral
transduction, HepG2 cells were seeded at a concentration of 4 10
/well in six-well plates, and 5 mL
of viral supernatant was collected and used to infect cells by spinoculation in 6-well plate sealed with
paraﬁlm, at 720 g, T¼32 °C, in presence of polybrene (8 μg/mL, Sigma-Aldrich, 107689) for 50 min.
After 4 h of incubation at 37 °C, 5% CO
, cells were washed with PBS and switched to standard
medium. At 48 h post-infection, puromycin (2 μg/mL, Sigma-Aldrich, P8833) was added for 8 days.
The transduction efﬁciency was evaluated by FACS analysis as the EGFP-positive cell fraction. To
maintain high expression of DsRed-LC3-GFP the cells were cultivated in DMEM complete supple-
mented with puromycin (1 μg/mL). For experiments cells were plated without puromycin.
2.4. Flow cytometry analysis of autophagy
GR-LC3-HepG2 cells were seeded on 24-well plates and cultured as described in Cell culture sec-
tion above. After 3 days pre-treatments with PA (Sigma-Aldrich, 0,3 mM ﬁnal, 150 mM stock in EtOH)
were performed 22–24 h before medium change and addition of ﬂavonoid aglycones or other sub-
stances 6 h before cell harvesting. ClQ (50 μM in H2O) was added 2 h before cell harvesting. For the
treatments all wells were treated with the same volume of EtOH (usually 3 μL), DMSO and water,
which were used as vehicles. Each treatment series were performed in triplicate, but at different
times. 6 h later the cells were washed once in PBS and collected by trypsinization as described before
. Brieﬂy, pelleted cells were resuspended in 0.45 mL PBS containing 1% FBS and 0.1 mM EDTA
(Sigma-Aldrich, E5134). To exclude dead cells in analysis they were treated with 75 μl of trypan blue
(TB) solution (0.008% in PBS), added 60 s before ﬂow cytometry recording. This was not necessary for
experiments with low cell mortality (below 5% in all samples). However, the addition of TB did not
E. Janda et al. / Data in Brief 19 (2018) 1327–13341332
inﬂuence signiﬁcantly the ATG index. Cells were acquired in 502 nm (FITC, green), 556 nm (PE, red)
and 655 (PerCP-Cy5, blue) channels by FACSCanto II (BD Biosciences, Erenbodgem, Belgium). Popu-
lations of interest were identiﬁed: single cell population (or P1), viable single cells (or P6) and GFP
and Ds-Red/GFP positive population (indicated as Q2). See row data sets S1 and S2. Mean ﬂuorescence
intensity (MFI) for red and green channels was determined in the populations of interest by BD
FACSDiva software. The autophagy index (ATG index) was calculated as the ratio of red to green
channel MFI in triplicate samples for each experimental point for a populations of interest (P6 or Q2).
The data were normalized to the mean ATG index of three control samples. For further details see
examples of ATG index analysis in Excel ﬁles provided as in Supplementary data sets S1 and S2.
2.5. A-BPF preparation and analysis
A-BPF was obtained from BPF
by acid hydrolysis, as reported in Lascala et al. . Subsequently, 2
mg of A-BPF were used to prepare a sample for LC-HRMS analysis according to the procedures
described in the companion paper . BPF
is a kind gift of the owner of the BPF trademark, Herbal
and Antioxidant Derivatives S.r.l. (H&AD S.r.l.), Bianco (RC), Italy.
2.6. Mass spectrometry
Q-ExactiveTM (Thermo Scientiﬁc) mass spectrometer was operated using electrospray with
negative polarities at 35,000 resolving power (deﬁned as FWHM at m/z200), IT 150 ms, and ACG
target¼1,000,000, in full scan analysis (mass range 140–900 amu). Source conditions were: spray
voltage 2.9 KV, sheath gas: 30, arbitrary units, Auxiliary gas: 10, probe heater temperature: 280 °C;
capillary temperature: 320 °C; S-Lens RF Level: 50. The instrument was calibrated by Thermo cali-
bration solutions prior to the beginning the analysis.
2.7. Proportional analysis of activity of individual compounds present A-BPF phytocomplex
To perform a proportional analysis, we estimated the amounts of six major ﬂavonoids present in
60 mg/mL of A-BPF based on LC–mass spectrometry data described in Table 1. To this end, we assumed
that the ion current signal intensity (SI) is proportional to the relative quantity of each ﬂavonoid,
which is well applicable to structurally similar compounds, according to our previous observations
. For sake of simplicity, we assumed that the total quantity of six identiﬁed polyphenols corre-
sponds to 100% of A-BPF and 100% of total ion current signal intensity (TSI). SI for each ﬂavonoid was
divided by TSI and multiplied by 60 mg/mL to calculated the proportional amounts ﬂavonoids con-
tributing to A-BPF phytocomplex.
This work received a ﬁnancial support from H&AD S.r.l., Bianco (RC), Italy (BPF and liver genes
2017) and from Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR, grant FFARB 2017).
The authors wish to thank Antonio Pisano, Experimental and Clinical Medicine Department, UMG,
Catanzaro, for helpful hints and suggestions and Daniele Vumbaca from Department of Health
Sciences, UMG, Catanzaro, Italy for excellent technical assistance.
Transparency document. Supporting information
Transparency data associated with this article can be found in the online version at https://doi.org/
E. Janda et al. / Data in Brief 19 (2018) 1327–1334 1333
Appendix A. Supporting information
Supplementary data associated with this article can be found in the online version at https://doi.
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