ArticlePDF Available

Qualitative and quantitative analysis of the proautophagic activity of Citrus flavonoids from Bergamot Polyphenol Fraction


Abstract and Figures

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 flavonoids have been identified in the proautophagic fraction of the hydrolysis product of BPF (A-BPF): naringenin, hesperetin, eridictyol, 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 compounds. 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 Excel files reporting the analysis of flow cytometry data are available in the public repository.
Content may be subject to copyright.
Data Article
Qualitative and quantitative analysis of the
proautophagic activity of Citrus avonoids from
Bergamot Polyphenol Fraction
Elzbieta Janda
, Raffaele Salerno
, Concetta Martino
Antonella Lascala
, 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
article info
Article history:
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
avonoids 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 avo-
noids have been identied 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 avonoids and chemical structures of their parent com-
pounds. The second set of data shows the proautophagic activity of
BPF avonoids in a hepatic cell line HepG2 analyzed by a ow
cytometry approach. The method is based on the red to green
uorescence 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 avonoids at equal concentrations
in the presence and absence of palmitic acid and chloroquine. The
Contents lists available at ScienceDirect
journal homepage:
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:
Corresponding author at: Department of Health Sciences, Magna Graecia University, Campus Salvatore Venuta, viale
Europa, Loc. Germaneto, 88100 Catanzaro, Italy.
E-mail address: (E. Janda).
Data in Brief 19 (2018) 13271334
Excel les reporting the analysis of ow 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
Specications Table
Subject area Pharmacology and cell biology
More specic subject area Flavonoid pharmacology and autophagy
Type of data Tables, text le, graphs, dot-plots
How data was acquired liquid chromatography- high resolution mass spectrometry (LC-
HRMS) Q-ExactiveTM (Thermo Scientic) and ow 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 avonoids in
liver cells reveals the superiority of a natural polyphenol mixture over
pure avones [1].
Value of the Data
We provide output LC-HRMS parameters for naringenin, hesperetin, eriodictyol, diosmetin,
apigenin and luteolin and the list of parent avonoid glycosides found in BPF.
A fast ow 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 proportionalapproach 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 avonoid aglycones reported here, can be
used as a reference for other cell lines and compounds.
1. Data
Bergamot Polyphenol Fraction (BPF
) is a natural mixture of Citrus avonoids 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 [410]. Protective effects of BPF correlate with its ability to
stimulate autophagy in livers of rats fed cafeteria diet [9]. The proautophagic activity of BPF is
mediated by a hydrophobic fraction of hydrolysed BPF (A-BPF) containing mainly avonoid aglycones
[1].Table 1 reports the LC-HRMS output parameters for six major avonoid aglycones identied 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 avonoid 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. avonoid glycosides), shown in Fig. 1, that have been previously
identied among bergamot polyphenols [3].
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 [1]. We used this approach to
E. Janda et al. / Data in Brief 19 (2018) 132713341328
Table 1
Output parameters of LC-HRMS analysis of avonoid aglycones identied 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
m/z [MH]
Signal inten-
sity (NL) x E6
% Total
in 60 μg Parent
287.0564 287.0561 34.6 20.51 12.3 NEOERIOCITRIN, ERIODICTYOL-
261.0615 261.0612 55.9 33.4 19.9 MELITIDIN, NARINGIN
301.0722 301.0718 51.8 30.7 18.4 BRUTERIDIN, HESPERETIN-7-O-
285.0408 285.0405 3.99 2.37 1.4 LUTEOLIN-7-O-
269.0459 269.0455 7.01 4.16 2.5 RHOIFOLIN, APIGENIN-7-O-NEO-
299.0564 299.0561 15.4 9.13 5.5 DIOSMIN, NEODIOSMIN, DIOS-
E. Janda et al. / Data in Brief 19 (2018) 13271334 1329
Fig. 2. Proautophagic activities of avonoid 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
O) was
added to PA-treated cells. Red and green uorescence was recorded by ow 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-
treated controls;
po0.001 when compared to the relative PA-untreated, the same avonoid-treated
cells or
po0.01, when compared to respective PA-treated, ClQ-untreated cells. See S1 data set (Supplementary data
set S1) with row data supporting this gure.
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) 132713341330
measure the proautophagic activity of six main avonoid 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 avonoid
aglycons þ/chloroquine (ClQ) to address the autophagic ux 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 dened here as proportional analysisof 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 ow 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 le S2 contains tables with mean uores-
cence intensities values recorded in 54 independent cell samples in two independent experiments.
This Excel le also shows how the mean ATG index is calculated and normalized and performs sta-
tistical analysis of the data. Examples of ow 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 avonoids 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),
vehicle-treated cells;
po0.01 when compared to naringenin. (B) Table showing the analysis of additive effects of
six avonoid 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) 13271334 1331
2. Experimental design, materials and methods
2.1. Cell culture
GR-LC3-HepG2 cells were descried previously in Lascala et al. [1]. They were cultured in DMEM
complete medium (4.5 g/L glucose) as described for the HepG2 cells [1]. 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.
2.2. Reagents
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. [1] Mix60,orMix,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 9095% conuence) 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, ltered through a
0.45 μmlter membrane, and supplemented with FBS (10% nal 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
paralm, 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 efciency 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 nal, 150 mM stock in EtOH)
were performed 2224 h before medium change and addition of avonoid 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
[12]. Briey, 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 ow 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) 132713341332
inuence signicantly 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 identied: 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 uorescence
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 les 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. [1]. 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 [1]. 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 Scientic) mass spectrometer was operated using electrospray with
negative polarities at 35,000 resolving power (dened as FWHM at m/z200), IT 150 ms, and ACG
target¼1,000,000, in full scan analysis (mass range 140900 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 avonoids present in
60 mg/mL of A-BPF based on LCmass 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 avonoid,
which is well applicable to structurally similar compounds, according to our previous observations
[13]. For sake of simplicity, we assumed that the total quantity of six identied polyphenols corre-
sponds to 100% of A-BPF and 100% of total ion current signal intensity (TSI). SI for each avonoid was
divided by TSI and multiplied by 60 mg/mL to calculated the proportional amounts avonoids con-
tributing to A-BPF phytocomplex.
This work received a nancial 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
E. Janda et al. / Data in Brief 19 (2018) 13271334 1333
Appendix A. Supporting information
Supplementary data associated with this article can be found in the online version at https://doi.
[1] A. Lascala, C. Martino, M. Parafati, R. Salerno, M. Oliverio, D. Pellegrino, et al., Analysis of proautophagic activities of Citrus
avonoids in liver cells reveals the superiority of a natural polyphenol mixture over pure avones, J. Nutr. Biochem. (2018)
(in press).
[2] E. Janda, A. Lascala, C. Martino, S. Ragusa, S. Nucera, R. Walker, et al., Molecular mechanisms of lipid- and glucose-lowering
activities of bergamot avonoids, PharmaNutrition 4 (2016) S8S18.
[3] R. Salerno, F. Casale, C. Calandruccio, A. Procopio, Characterization of avonoids in Citrus bergamia (Bergamot) poly-
phenolic fraction by liquid chromatographyhigh resolution mass spectrometry (LC/HRMS), PharmaNutrition 4 (Supple-
ment) (2016) S1S7.
[4] J. Ehrlich, M. Gliozzi, C. Carresi, V. Musolino, E. Palma, C. Muscoli, et al., The effect of bergamot-derived polyphenolic
fraction on LDL small dense particles and non-alcoholic fatty liver disease in patients with metabolic syndrome, IJC Metab.
Endocr. 7 (Supplement 1) (2015) S7.
[5] J. Ehrlich, M. Gliozzi, E. Janda, R. Walker, F. Romeo, V. Mollace, Effect of Citrus Bergamot Polyphenol Extract on patients
with nonalcoholic fatty liver disease, Am. J Gastroenterol. 109 (2014) S152S153.
[6] M. Gliozzi, J. Maiuolo, F. Oppedisano, V. Mollace, The effect of bergamot polyphenolic fraction in patients with non
alcoholic liver steato-hepatitis and metabolic syndrome, PharmaNutrition 4 (2016) S27S31.
[7] M. Gliozzi, R. Walker, S. Muscoli, C. Vitale, S. Gratteri, C. Carresi, et al., Bergamot polyphenolic fraction enhances
rosuvastatin-induced effect on LDL-cholesterol, LOX-1 expression and protein kinase B phosphorylation in patients with
hyperlipidemia, Int. J. Cardiol. 170 (2013) 140145.
[8] V. Mollace, I. Sacco, E. Janda, C. Malara, D. Ventrice, C. Colica, et al., Hypolipemic and hypoglycaemic activity of bergamot
polyphenols: from animal models to human studies, Fitoterapia 82 (2011) 309316.
[9] M. Parafati, A. Lascala, V.M. Morittu, F. Trimboli, A. Rizzuto, E. Brunelli, et al., Bergamot polyphenol fraction prevents
nonalcoholic fatty liver disease via stimulation of lipophagy in cafeteria diet-induced rat model of metabolic syndrome,
J. Nutr. Biochem. 26 (2015) 938948.
[10] R. Walker, E. Janda, V. Mollace, The Use of Bergamot-Derived Polyphenol Fraction in Cardiometabolic Risk Prevention and
its Possible Mechanisms of Action. Polyphenols in Human Health and Disease, Elsevier Inc., San Diego (2013) 10871105 .
[11] J.H. Sheen, R. Zoncu, D. Kim, D.M. Sabatini, Defective regulation of autophagy upon leucine deprivation reveals a targetable
liability of human melanoma cells in vitro and in vivo, Cancer Cell 19 (2011) 613628.
[12] E. Janda, M. Parafati, S. Aprigliano, C. Carresi, V. Visalli, I. Sacco, et al., The antidote effect of quinone oxidoreductase
2 inhibitor against paraquat-induced toxicity in vitro and in vivo, Br. J. Pharmacol. 168 (2013) 4659.
[13] M. Oliverio, P. Costanzo, M. Nardi, C. Calandruccio, R. Salerno, A. Procopio, Tunable microwave-assisted method for the
solvent-free and catalyst-free peracetylation of natural products, Beilstein J. Org. Chem. 12 (2016) 22222233.
E. Janda et al. / Data in Brief 19 (2018) 132713341334
... HepG2 cells expressing DsRed-LC3-GFP (GR-LC3-HepG2) were generated as previously described [7,38]. Cells were seeded 3 days before treatments on 24-well plates (in triplicate) and then treated with PA or ethanol for 24 h and next day flavonoids or ethanol (vehicle) were added 6 h before harvesting (trypsinization), as previously described for flow cytometry analysis of viability [32]. ...
... We tested if there was a correlation between the NQO2 inhibitor properties of flavonoids and their ability to induce autophagy. We focused on six flavonoid aglycons, found in fruits and leaves of Citrus family plants such as bergamot [15], which are also the main compounds of Bergamot Polyphenol Fraction (BPF) ® , previously characterized for its pro-autophagic effects in hepatocytes [7,38]. Here, BPF flavonoids were compared to quercetin, a flavonol, which is known to induce autophagy in different systems [40,41]. ...
... In this assay, the ratio between red and green fluorescence (autophagy index, ATG index), measured by flow cytometry indicates the amount of lipidated LC3 (LC3-II, red) with respect to unprocessed LC3 (red and green), which correlates with an increase in autophagosome formation ( Figure 1A). ATG index measurement was performed on cells cultured in high-glucose medium and exposed for 24 h to 0.3 mM PA, to induce hepatic steatosis-like state in vitro, since in these conditions, flavonoids have been shown to stimulate a stronger autophagic response [38]. Treatment of GR-LC3-HepG2 with a low concentration of flavonoids (5 µg/mL ≈15 µM) led to a marked increase of ATG index in the cases of apigenin, luteolin, and quercetin and a tendency to enhance autophagy in case of diosmetin ( Figure 1B). ...
Full-text available
Dietary flavonoids stimulate autophagy and prevent liver dysfunction, but the upstream signaling pathways triggered by these compounds are not well understood. Certain polyphenols bind directly to NRH-quinone oxidoreductase 2 (NQO2) and inhibit its activity. NQO2 is highly expressed in the liver, where it participates in quinone metabolism, but recent evidence indicates that it may also play a role in the regulation of oxidative stress and autophagy. Here, we addressed a potential role of NQO2 in autophagy induction by flavonoids. The pro-autophagic activity of seven flavonoid aglycons correlated perfectly with their ability to inhibit NQO2 activity, and flavones such as apigenin and luteolin showed the strongest activity in all assays. The silencing of NQO2 strongly reduced flavone-induced autophagic flux, although it increased basal LC3-II levels in HepG2 cells. Both flavones induced AMP kinase (AMPK) activation, while its reduction by AMPK beta (PRKAB1) silencing inhibited flavone-induced autophagy. Interestingly, the depletion of NQO2 levels by siRNA increased the basal AMPK phosphorylation but abrogated its further increase by apigenin. Thus, NQO2 contributes to the negative regulation of AMPK activity and autophagy, while its targeting by flavones releases pro-autophagic signals. These findings imply that NQO2 works as a flavone receptor mediating autophagy and may contribute to other hepatic effects of flavonoids.
... Indeed, dietary polyphenols, a large and heterogeneous group of phytochemicals in herbs and plant-based foods, have been associated with lower risk of NAFLD [16,17]. In particular, Citrus flavonoids have been shown to exert several positive health effects on glucose and lipid metabolism in experimental models [12,16,[18][19][20][21][22] and humans [23][24][25]. ...
... Bergamot has attracted considerable attention due to its peculiar composition and high content of flavonoids, some of which are also found in other Citrus species [18,27]. BPF is a highly concentrated extract of glycosylated flavanones (naringin, neoeriocitrin and hesperidin) and flavones (diosmetin, apigenin and luteolin glycosides) from bergamot fruit juice [19,20,28]. BPF phyto-complex contains around 40% of flavonoids, but also sugars, salts and other natural compounds with a possible detoxifying activity [19,20]. ...
... BPF is a highly concentrated extract of glycosylated flavanones (naringin, neoeriocitrin and hesperidin) and flavones (diosmetin, apigenin and luteolin glycosides) from bergamot fruit juice [19,20,28]. BPF phyto-complex contains around 40% of flavonoids, but also sugars, salts and other natural compounds with a possible detoxifying activity [19,20]. A particular feature of certain flavonoid glycosides, abundant in bergamot juice and in BPF, such as bruteridin and melitidin, is the presence of covalently linked 3-hydroxy-3-methylglutaryl (HMG) moiety [28,29]. ...
Full-text available
Wrong alimentary behaviors and so-called "junk food" are a driving force for the rising incidence of non-alcoholic fatty liver disease (NAFLD) among children and adults. The "junk food" toxicity can be studied in "cafeteria" (CAF) diet animal model. Young rats exposed to CAF diet become obese and rapidly develop NAFLD. We have previously showed that bergamot (Citrus bergamia Risso et Poiteau) flavonoids, in the form of bergamot polyphenol fraction (BPF), effectively prevent CAF diet-induced NAFLD in rats. Here, we addressed if BPF can accelerate therapeutic effects of weight loss induced by a normocaloric standard chow (SC) diet. 21 rats fed with CAF diet for 16 weeks to induce NAFLD with inflammatory features (NASH) were divided into three groups. Two groups were switched to SC diet supplemented or not with BPF (CAF/SC±BPF), while one group continued with CAF diet (CAF/CAF) for 10 weeks. BPF had no effect on SC diet-induced weight loss, but it accelerated hepatic lipid droplets clearance and reduced blood triglycerides. Accordingly, BPF improved insulin sensitivity, but had little effect on leptin levels. Interestingly, the inflammatory parameters were still elevated in CAF/SC livers compared to CAF/CAF group after 10 weeks of dietary intervention, despite over 90% hepatic fat reduction. In contrast, BPF supplementation decreased hepatic inflammation by reducing interleukin 6 (Il6) mRNA expression and increasing anti-inflammatory Il10, which correlated with fewer Kupffer cells and lower inflammatory foci score in CAF/SC+BPF livers compared to CAF/SC group. These data indicate that BPF mediates a specific anti-inflammatory activity in livers recovering from NASH, while it boosts lipid-lowering and anti-diabetic effects of the dietary intervention.
... A database (Table S1 of Supplementary Materials) was built searching in the literature for the known bergamot components [9,[15][16][17][18][19][20][21][22][23][24][25]. The targeted analysis was performed by searching for all the components listed in the database on the basis of their exact mass ([M-H] − ), with a mass tolerance of 5 ppm. ...
... Most were present in both extracts, except for apigenin-7-O-rutinoside and neohesperidin-O-glucoside-O-HMG which were detected only in the leaf extract, while obacunone glucoside, limonin glucoside and obacunoic acid were detected only in BFPF. Some isomers were identified on the basis of their elution order as reported in previous studies which used RP chromatography as separation technique: for chrysoeriol/diosmetin isomers, chrysoeriol has always been reported as the first eluting isomer [18,19,21,25]; similarly, for rutinoside/neohesperidoside isomers the rutinoside is always the first eluting isomer followed by neohesperidoside [9,[15][16][17][18][19][21][22][23]25]. Figure 1 (panels a and b) shows the total ion currents (TICs) of the two extracts where the peak ions identified by a targeted approach are labelled in blue by a time-dependent progressive number. Table 2 lists the 32 ions identified with the semi-targeted approach, including 11 compounds which were definitively confirmed since they had already been identified with the targeted approach. ...
Full-text available
The aim of the study is to compare the qualitative and semi-quantitative profile of the polyphenol fraction purified from the leaf (BLPF) and fruit (BFPF) of bergamot (Citrus bergamia), and to evaluate their antioxidant and anti-inflammatory activity. The analytical qualitative profile was carried out by LC-ESI/MS using three different approaches: targeted (searching analytes already reported in bergamot extract), semi-targeted (a selective search of 3-hydroxy-3-methylglutarate [HMG] derivatives involved in the cholesterol reducing activity of BPF) and untargeted. A total number of 108 compounds were identified by using the three approaches, 100 of which are present in both the extracts thus demonstrating a good qualitative overlapping of polyphenols between the two extracts. The antioxidant activity was higher for BLPF in respect to BFPF but when normalized in respect to the polyphenol content they were almost overlapping. Both the extracts were found to dose dependently inhibit cell inflammation stimulated with IL-1α. In conclusion, the comparison of the qualitative and quantitative profile of polyphenols as well as of the antioxidant and anti-inflammatory activity of bergamot leaf and fruit well indicates that leaf is a valid source of bergamot polyphenol extraction and an even richer source of polyphenol in respect to the fruit.
... There is also an evidence indicating that BPF attenuates NAFLD and NASH in patients [23,24]. Hepatoprotective effects of BPF are associated with its ability to induce autophagy [18,22,25]. The proautophagic activity of BPF suggests that it may stimulate energy expenditure and prevent weight gain. ...
Full-text available
Bergamot flavonoids counteract dyslipidemia and hyperglycemia but fail to induce a significant weight loss. Here, we evaluated the efficacy of bergamot polyphenol extract complex (BPE-C), a novel bergamot juice-derived formulation enriched with flavonoids and pectins, on several metabolic syndrome parameters. Obese patients with atherogenic index of plasma (AIP) over 0.34 and mild hyperglycemia were recruited to a double-blind randomized trial comparing two doses of BPE-C (650 and 1300 mg daily) with placebo. Fifty-two subjects met the inclusion criteria and were assigned to three experimental groups. Fifteen subjects per group completed 90 days-trial. BPE-C reduced significantly fasting glucose by 18.1%, triglycerides by 32% and cholesterol parameters by up to 41.4%, leading to a powerful reduction of AIP (below 0.2) in the high dose group. The homeostasis model assessment of insulin resistance (HOMA-IR) and insulin levels were also reduced. Moreover, BPE-C decreased body weight by 14.8% and body mass index by 15.9% in BPE-C high group. This correlated with a significant reduction of circulating hormones balancing caloric intake, including leptin, ghrelin and upregulation of adiponectin. All effects showed a dose-dependent tendency. This study suggests that food supplements, containing full spectrum of bergamot juice components, such as BPE-C efficiently induce a combination of weight loss and insulin sensitivity effects together with a robust reduction of atherosclerosis risk.
Full-text available
Background: The peracetylation is a simple chemical modification that can be used to enhance the bioavailability of hydrophilic products and to obtain safe and stable pro-drugs. Results: A totally green, solvent-free and catalyst-free microwave (MW)-assisted method for peracetylation of natural products such as oleuropein, alpha-hederin, quercetin and rutin is presented. By simply tuning the MW heating program, polyols with chemical diverse ?OH groups or thermolabile functionalities can be peracetylated to improve the biological activity without degradation of the natural starting molecules. An evaluation of the process greenness was performed. Conclusion: The method is potentially universally applicable for green acetylation of hydrophilic biological molecules, potentially easily scalable for industrial applications, including pharmaceutical, cosmetic and food industry.
Full-text available
Bergamot (Citrus bergamia Risso et Poiteau) fruits are characterized by a particularly high content and a unique composition of flavonoids, such as neoeriocitrin, neohesperidin, naringin, melitidin and brutieridin. Bergamot juice and its concentrate, highly enriched in polyphenols-here referred to as Bergamot Polyphenol Fraction (BPF)-has been evaluated in experimental and clinical studies. Studies performed in Italy and Australia showed that BPF treatment leads to an important reduction in lipid parameters in the blood of patients with hyperlipidemia ranging from 15 up to 40% for total cholesterol and cholesterol-LDL. A striking reduction (mean 41.0±2.6%) was also observed for plasma triglyceride levels, accompanied by a significant decrease in blood glucose (22.3±1.0%) in a subgroup of patients with metabolic syndrome. Although BPF cannot be proposed as a substitute for statins in patients at high risk of cardiovascular events, it offers an excellent alternative for low-risk and for statin-intolerant patients. The robust performance of BPF in clinical practice against cardiometabolic risk factors can be explained in the light of scientific evidence showing that bergamot flavonoids influence lipid and sugar metabolism acting as 3-hydroxy-3-methlglutaryl coenzyme A (HMG-CoA) reductase inhibitors and AMP kinase (AMPK) activators.
Autophagy dysfunction has been implicated in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Natural compounds present in Bergamot Polyphenol Fraction (BPF) prevent NAFLD and induce autophagy in rat livers. Here, we employed HepG2 cells expressing DsRed-LC3-GFP, a highly sensitive model system to screen for proautophagic compounds present in BPF. BPF induced autophagy in a time- and dose-dependent fashion and the effect was amplified in cells loaded with palmitic acid. Autophagy was mediated by the hydrophobic fraction of acid-hydrolyzed BPF (A-BPF), containing six flavanone and flavone aglycones as identified by liquid chromatography- high resolution mass spectrometry (LC-HRMS). Among them naringenin, hesperitin, eriodictyol and diosmetin were weak inducers of autophagy. Apigenin showed the strongest and dose-dependent proautophagic activity at early time points (6 h). Luteolin induced a biphasic autophagic response, strong at low doses and inhibitory at higher doses. Both flavones were toxic in HepG2 cells and in differentiated human liver progenitors HepaRG upon longer treatments (24 h). In contrast, BPF and A-BPF did not show any toxicity, but induced a persistent increase in autophagic flux. A mixture of six synthetic aglycones mimicking A-BPF was sufficient to induce a similar autophagic response, but it was mildly cytotoxic. Thus, while six main BPF flavonoids fully account for its proautophagic activity, their combined effect is not sufficient to abrogate cytotoxicity of individual compounds. This suggests that a natural polyphenol phytocomplex, such as BPF is a safer and more effective strategy for the treatment of NAFLD than the use of pure flavonoids.
Bergamot (Citrus bergamia Risso et Poiteau) juice has a particularly high content and a unique composition of flavonoids. Neoeriocitrin, neohesperidin, naringin, melitidin and brutieridin represent more than 95% of Bergamot Polyphenol Fraction (BPF), while rhoifolin, diosmin, poncirin and others can be found in the remaining 5%. The brilliant performance of BPF in clinical practice against as a treatment for hyperlipidemia and moderate hyperglycemia in metabolic syndrome, awaits a plausible mechanistic explanation. Considering the overwhelming scientific evidence, it is likely that flavonoid components of BPF are responsible for majority of pharmacological effects. Here, we will review the scientific evidence showing that flavonoids, in particular citrus flavonoids present in bergamot fruits, influence lipid and sugar metabolism at the molecular level. Anti-diabetic and dyslipidemia-correcting effects of bergamot polyphenols may be explained by their ability to activate AMP kinase (AMPK), which is a central regulator of glucose and fatty acids metabolism and inhibit cAMP phosphodiesterases (PDE), involved in regulation of lipolysis in adipocytes and liver. Importantly, certain polyphenols can act as 3-hydroxy-3-methlglutaryl coenzyme A (HMG-CoA) reductase inhibitors, thereby mimicking statins action. In addition, flavonoids bind and act as natural inhibitors of quinone oxidoreductase 2 (QR2/NQO2) and other enzymes with potential roles in metabolic regulation. Finally, pleiotropic and possible synergistic effects may account for enhanced nutraceutical effects of natural flavonoid mixtures, such as BPF as compared to purified flavonoids.
The goal of the present study was the development of a UHPLC-HRMS/MS method for the identification of flavonoids present in BPF (bergamot polyphenolic fraction), a particular product derived from Bergamot juice. BPF contains over 40% flavonoids and the remaining 60% are carbohydrates, fatty acids, pectins and other compounds as well as maltodextrins which are added to allow exsiccation. Trademark and patent No. 0001380456 by Herbal and Antioxidant Derivatives S.R.L. The mass spectrometry data, obtained with an Orbitrap spectrometer, reveal the presence of principal flavonoids neoeriocitrin, naringin, neohesperidin but also of all HMG-family like bruteridin, melitidin with other HMG species and flavonoids 6,8-di-C-glicosides too. The method permits to reveal all species in high resolution full scan and to confirm their identities by HRMSMS (ddMS2, data dependent MSMS) in the same event.
Statins are the most commonly prescribed drugs to reduce cardiometabolic risk. Besides the well-known efficacy of such compounds in both preventing and treating cardiometabolic disorders, some patients experience statin-induced side effects. We hypothesize that the use of natural bergamot-derived polyphenols may allow patients undergoing statin treatment to reduce effective doses while achieving target lipid values. The aim of the present study is to investigate the occurrence of an enhanced effect of bergamot-derived polyphenolic fraction (BPF) on rosuvastatin-induced hypolipidemic and vasoprotective response in patients with mixed hyperlipidemia. A prospective, open-label, parallel group, placebo-controlled study on 77 patients with elevated serum LDL-C and triglycerides was designed. Patients were randomly assigned to a control group receiving placebo (n=15), two groups receiving orally administered rosuvastatin (10 and 20mg/daily for 30days; n=16 for each group), a group receiving BPF alone orally (1000mg/daily for 30days; n=15) and a group receiving BPF (1000mg/daily given orally) plus rosuvastatin (10mg/daily for 30days; n=15). Both doses of rosuvastatin and BPF reduced total cholesterol, LDL-C, the LDL-C/HDL-C ratio and urinary mevalonate in hyperlipidemic patients, compared to control group. The cholesterol lowering effect was accompanied by reductions of malondialdehyde, oxyLDL receptor LOX-1 and phosphoPKB, which are all biomarkers of oxidative vascular damage, in peripheral polymorphonuclear cells. Addition of BPF to rosuvastatin significantly enhanced rosuvastatin-induced effect on serum lipemic profile compared to rosuvastatin alone. This lipid-lowering effect was associated with significant reductions of biomarkers used for detecting oxidative vascular damage, suggesting a multi-action enhanced potential for BPF in patients on statin therapy.