vol.170 issue 2 iss n.0167-5271
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INTERNATIONAL JOURNAL OF CARDIOLOGY
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ANDREW I.C. COATS
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Bergamot polyphenolic fraction enhances rosuvastatin-induced effect on
LDL-cholesterol, LOX-1 expression and protein kinase B phosphorylation in
patients with hyperlipidemia
Micaela Gliozzi a,1, RossWalker a,1, Saverio Muscoli b,1, Cristiana Vitale c,1, Santo Gratteri a,1, Cristina Carresi a,1,
Vincenzo Musolino a,1, Vanessa Russo a,1, Elzbieta Janda a,1, Salvatore Ragusa a,1, Antonio Aloe a,1,
Ernesto Palma a,1, Carolina Muscoli a,1, Franco Romeo b,1, Vincenzo Mollace a,c,⁎,1
a Research Centre for Food Safety & Health (IRC-FSH), University “Magna Graecia”, Catanzaro, Italy
b Department of Medicine, Chair of Cardiology, University of Rome Tor Vergata, Rome, Italy
c IRCCS San Raffaele, Rome, Italy
a r t i c l e i n f o a b s t r a c t
Article history:
Received 2 April 2013
Received in revised form 3 August 2013
Accepted 30 August 2013
Available online 8 September 2013
Keywords:
Hyperlipidemia
Cardiometabolic risk
Bergamot polyphenolic fraction
Rosuvastatin
Background: Statins are the most commonly prescribed drugs to reduce cardiometabolic risk. Besides the wellknown
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 polyphenolsmay
allowpatients 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 patientswithmixed hyperlipidemia.
Methods: 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 20 mg/daily for 30 days; n = 16 for
each group), a group receiving BPF alone orally (1000 mg/daily for 30 days; n = 15) and a group receiving
BPF (1000 mg/daily given orally) plus rosuvastatin (10 mg/daily for 30 days; n = 15).
Results: 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.
Conclusions: Addition of BPF to rosuvastatin significantly enhanced rosuvastatin-induced effect on serumlipemic
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.
© 2013 Published by Elsevier Ireland Ltd.
1. Introduction
Dyslipidemia (or hypercholesterolemia) is an important risk factor
for the development of atherosclerosis and coronary artery disease, as
shown in several lipid intervention trials and prospective studies
[1]. Increased concentrations of low-density lipoprotein cholesterol
(LDL-C), total blood cholesterol (TC) and triglycerides (TG) comprise
the main pathogenic risk profile. Conditions of insulin resistance
such as impaired glucose tolerance or “prediabetes” are also associated
with a high risk of cardiovascular disease (CVD) and are often
accompanied by low levels of high-density lipoprotein cholesterol
(HDL-C) [2]. The majority of therapeutic protocols rely on drugs
that belong to the “statin” family. Statins inhibit the activity of 3-
hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, which catalyzes
the rate-limiting step in mevalonate biosynthesis, a key intermediate
in cholesterol metabolism. A meta-analysis of placebo-controlled
“standard dose” statin trials shows a reduction in cardiovascular mortality
averaging 20% and a decrease in major cardiovascular events by
approximately 25% [3]. Treatment with high-dose statins was shown
to reduce the morbidity by 36%, and a reduction of cardiovascular
International Journal of Cardiology 170 (2013) 140–145
⁎ Corresponding author at: Faculty of Pharmacy, Department of Health Sciences,
University “Magna Graecia”, Complesso Ninì Barbieri, Roccelletta di Borgia, 88100
Catanzaro, Italy. Tel.:+39 0961 369 4128,+39 0961 369 5732; fax:+39 0961 369 4237.
E-mail address: mollace@unicz.it (V. Mollace).
1 Each author takes responsibility for all aspects of the reliability and freedomfrombias
of the data presented and their discussed interpretation.
0167-5273/$ – see front matter © 2013 Published by Elsevier Ireland Ltd.
http://dx.doi.org/10.1016/j.ijcard.2013.08.125
Contents lists available at ScienceDirect
International Journal of Cardiology
journal homepage: www.elsevier .com/locate/ijcard
Author's personal copy
events up to 40% [4]. Despite the significant clinical benefits provided
by statins, many patients, in particular those with diabetes
or metabolic syndrome, do not achieve their recommended lowdensity
and high-density lipoprotein (LDL-C, HDL-C) cholesterol
target goals with statins alone [5]. Moreover, statins have been reported
to cause dose-related side effects, the more serious including
liver disease or severe myopathy, in up to 22% of patients
eligible for this therapeutic approach [6,7]. This limits the use of
statins and suggests the need for alternative and/or supplementary
therapeutic approaches.
Experimental and epidemiological evidence suggests that dietary
polyphenols, in particular flavonoids, may play a role in ameliorating
atherosclerosis, due to a pleiotropic anti-oxidative and
anti-inflammatory effect proposed as underlying mechanisms
[8–11]. In support of this hypothesis, it has been shown that certain
plant polyphenols, such as nobiletin and poncirin, inhibit monocyte
activation in vitro [9,12–17], and the enhancing effects of
such components may play a central role in this beneficial effect.
This observation provides the rationale for the optimal use of
appropriate, evidence-based phytochemicals as an adjunct to
treat hyperlipidemia with the aim of preventing cardiovascular
events.
Bergamot (Citrus bergamia) is an endemic plant growing in the
Calabrian region of Southern Italy with a unique profile of flavonoid
and glycosides present in its juice and albedo, such as neoeriocitrin,
neohesperidin, naringin, rutin, neodesmin, rhoifolin and poncirin.
Bergamot differs from other citrus fruits, not only for the composition,
but also for the particularly high content of flavonoids [18,19]. Some
of these flavonoids, such as naringin (which is also present in
grapefruit), have already been shown to be active in animal models
of atherosclerosis [20,21], while neoeriocitrin and rutin have been
found to exhibit a strong capacity to inhibit LDL oxidation [22]. Importantly,
bergamot juice is rich in 3-hydroxy-3-methylglutaryl
neohesperidosides of hesperetin (brutieridine) and naringenin
(melitidine) with an ability to inhibit HMG-CoA reductase [23].
These compounds are likely to contribute to the substantial
hypolipidemic effects of bergamot juice and to the vasoprotective
effects of bergamot oil derivatives in rats [24,25]. However, no clinical
study has been performed to date to evaluate the fruit's potential
dyslipidemia-correcting properties.
Recently, we found that bergamot-derived polyphenolic fraction
(BPF), given orally both in animal models of diet-induced hyperlipidemia
and in patients suffering from metabolic syndrome, produces a
significant and sustained reduction of serum cholesterol, triglycerides
and glycaemia [26]. This effect was accompanied by a significant improvement
in vascular reactivity in patients with both hyperlipidemia
and elevated serum glucose, thus suggesting a potential protective
role for the use of BPF in patients with metabolic syndrome and
cardiometabolic risk.
Apart from these results, no evidence has been collected to date, on
themechanism of action of bergamot-derived extract and on its potential
benefit when used in comparison with statins in patients with metabolic
syndrome.
To address these issues, we conducted a prospective, openlabel,
parallel group, placebo-controlled study on patients with elevated
serum LDL-C and triglycerides, to investigate the effect of
BPF in modulating serum lipidemic profile in patients suffering
from mixed hyperlipidemia, either untreated or treated with
rosuvastatin. The effect of rosuvastatin with BPF on the expression
of biomarkers of vascular oxidative stress and circulating cell viability
was also studied in these patients. In particular, we examined
the accumulation of malondialdehyde (MDA), a marker of lipid peroxidation,
the expression of LOX-1 receptor for oxidized LDL and protein
kinase B (PKB) phosphorylation in peripheral mononuclear cells
(PMC) in patients taking BPF alone or in combination with rosuvastatin
treatment.
2. Materials and methods
2.1. Plant material
C. bergamia Risso & Poiteau fruits were collected from plants located in a range of
90 km from Bianco to Reggio Calabria, Italy.
2.2. Preparation of BPF
Bergamot juice was obtained from peeled-off fruits by squeezing. The juice was oil
fraction-depleted by stripping, clarified by ultra-filtration and loaded on to a suitable polystyrene
resin column able to absorb polyphenol compounds ofmolecular weight between
300 and 600 Da (Mitsubishi). Polyphenol fractions were eluted by a 1 mMKOH solution.
The basic eluate was incubated at a rocking platform to reduce the fumocumarin content.
The shaking time was adjusted proportionally to the amount of fumocumarin contaminants.
Next, the phytocomplex derived fromthe defurocoumarinization process was neutralized
by filtration on cationic resin at acidic pH. Finally itwas vacuum dried andminced
to the desired particle size to obtain BPF powder. BPF powder was analyzed for flavonoid,
furocumarin and other polyphenol content. In addition, all toxicological analyses were
performed, including heavy metal, pesticide, phthalate and sinephrine content
which revealed the absence of known toxic compounds at significant levels (data
not shown). Standardmicrobiological test showed that the final BPF was free of mycotoxins
and contaminating bacteria.
Finally, 500 mg aliquots of the BPF powder supplementedwith 50 mg of ascorbic acid
as antioxidantwere encapsulated into suitable gelatin capsules by a semi-automatic gelatin
encapsulation device employing an authorized pharmaceutical manufacturer (Plants,
Messina, Italy). All procedures have been performed according to Good Manufacture Practice
(GMP) headlines of European Legislation.
The main flavonoids identified in BPF were neoeriocitrin (370 ppm), naringin
(520 ppm), and neohesperidin (310 ppm). Tablets containing 1000 mg of maltodestrin
supplemented with 50 mg ascorbic acid were used as placebo.
2.3. Patient selection and study design
A prospective, open-label, parallel group, placebo-controlled studywas designed.
We enrolled 77 patients suffering from mixed hypercholesterolemia (LDL-C levels of
N160 mg/dl, and triglycerides levels N225 mg/dl) from a primary care setting at the
Department of Cardiology of the IRCCS San Raffaele– Rome and at the Atherosclerosis
Prevention Ambulatory centre at the University of Rome “Tor Vergata” facility. Age,
gender, and body mass index were matched among all subjects. Patients were randomly
assigned to five groups, all following a low fat diet: a control group receiving
placebo (n = 15), two groups receiving oral rosuvastatin (10 and 20 mg/daily for
30 days; n = 16 for each group), a group receiving BPF alone orally (1000 mg/daily
for 30 days; n = 15) and a group receiving BPF (1000 mg/daily given orally) plus
rosuvastatin (10 mg/daily for 30 days; n = 15). In all groups, we determined the efficacy
of each treatment by monitoring serum lipid profiles and urinary mevalonic
acid concentration (expressing inhibition of HMG-CoA reductase).
We excluded patients with overt liver disease, chronic renal failure, hypothyroidism,
myopathy, uncontrolled diabetes, severe hypertension, stroke, acute coronary events
within the preceding 30 days, coronary revascularization within the preceding 3 months,
or alcohol abuse. No patient was taking hormone replacement therapy or antioxidant vitamin
supplements during the 2 months preceding our study.
All the patients were given placebo, rosuvastatin, BPF alone or BPF plus rosuvastatin
before meals daily during a 30 day treatment period. A research nurse counted pills at
the end of treatment tomonitor compliance. The patients were seen at leastweekly during
the study. To minimize side effects, we measured serum asparate aminotransferase, alanine
aminotransferase, creatine kinase, blood urea nitrogen, creatinine and blood cell
counts before and after therapy. Seven (7) patients taking placebo and 16 patients taking
BPF and/or rosuvastatin were smokers. Two (2) patients taking placebo, and 13 patients
taking BPF alone and/or rosuvastatin were also taking ACE inhibitors, angiotensin AT1 receptor
blockers or beta adrenergic blockers to control blood pressure. No additional medications
including aspirin or nonsteroidal anti-inflammatory drugs were allowed during
the study period to avoid confounding effects of other drugs. Anti-hypertensive drugs
were withheld for ≥48 h before starting the study. Blood samples for assessing lipid profile
and 24 h urinary collectionwere performed at day 0 and after 30 day treatment in placebo,
rosuvastatin and rosuvastatin/BPF-treated patients. At the same time, blood samples
were collected for assessing baseline values for blood cell counts, transaminases, creatinine
blood levels and PMN separation and culturing.
All patients participating in the study signed an informed consent according to the
European Legislation and the protocol was previously submitted and approved by the Regional
Ethical Committee. In addition, the study protocol was performed according to the
ethical guidelines of the 1975 Declaration of Helsinki, as reflected in a priori approval by
the institution's human research committee.
2.4. Serum lipids
Total cholesterol, LDL-C, HDL-C and triglycerides in serum samples of patients were
determined colorimetrically and enzymatically using commercial assay kits (BioSystems
S.A., Barcelona, Spain).
M. Gliozzi et al. / International Journal of Cardiology 170 (2013) 140–145 141
Author's personal copy
2.5. Urinary mevalonic acid detection
Twenty-four-hour urine samples were collected from each patient before and after
treatment with placebo, supplement and/or drugs. Total volume was recorded, and aliquots
were frozen at −20 °C. Frozen urine samples were thawed and centrifuged at
1200 g for 30 min to remove solids. Urinary mevalonic acid (MVA) concentrations were
determined by means of a modification of the radioenzymatic isotope-dilution method
of Popjak (26). The intra-subject coefficient of variation was evaluated; 90% of all values
were found to lie within 35% of a single 24-h determination, whereas for two determinations,
the variation was 25%. The intra-assay coefficient of variation in each run was less
than 5%, whereas the inter-assay variation, based on the repeated analysis of the same
standard over a 2-year period, was 3.9% (25). The completeness of urine samples was determined
on the basis of subjects' records and their careful questioning; if these were
deemed incomplete, a repeated collection was obtained.
2.6. Mononuclear cell isolation
Twenty milliliter of heparinised blood was diluted with the same volume of PBS and
carefully layered over 6 ml of Ficoll-Paque Plus (Amersham Biosciences, USA). The mixturewas
centrifuged at 400 ×g for 40 min at 18–20 °C. The interface containing peripheral
blood mononuclear cells (PMNs) was collected and washed two times with PBS. This
preparation contained approximately 25% monocytes and 75% lymphocytes. Further purification
of the monocytes from the PMNs fraction was accomplished by allowing the
monocytes to adhere to plastic. Using 25 cm2 culture dishes, PMNs were cultured for
45 min in RPMI 1640medium (with glutamax-I, Dutch modification, Gibco BRL) containing
10% pooled human serum. The nonadhering lymphocytes were removed by washing
the culture dishes with PBS, after which the adherentmonocytes were harvested. The reagents
used for the isolation of monocytes contained no detectable endotoxins, as determined
by the E-Toxate assay (Sigma, St. Louis, USA).
2.7. Cell extracts and SDS-PAGE Western blotting
In order to study phosphor-PKB expression, PMNswere lysed with ice-cold lysis buffer
(20 mM Tris–HCl, pH 7.5; 150 mM NaCl; 2 mM EDTA; 2 mM EGTA; 1% Triton X-100;
1 nM okadaic acid, protease inhibitor cocktail - Sigma; phosphatase inhibitor cocktail —
Calbiochem), centrifuged at 10,000 ×g for 15 min at +4 °C. Alternatively, for LOX-1 detection,
PMNs were lysed with a different ice-cold lysis buffer (50 mM Tris pH 8,
150 mM NaCl, 1% Nonidet P-40; 2 mM Pefablock, 1 microM ml−1 Aprotinin, 10 microM
ml−1 Leupepeptin, 2 microM ml−1 Pepstatin A, trypsin inhibitor), centrifuged at
13,000 ×g for 30 min at+4 °C. Protein concentrations in supernatants were determined
by standard Bradford assay.
Lysates from each treatment (30 micrograms/lane) were resolved by 10% SDS–polyacrylamide
gel electrophoresis and transferred to nitrocellulosemembranes. After incubation
in blocking solution (4% dry nonfat milk, Sigma–Aldrich, St. Louis, MO), membranes
were incubated with anti-LOX-1 (1:10,000; gift from Professor T. Sawamura, Osaka,
Japan); anti-phospho-PKB (Ser 473) (1:2000; Cell Signaling Technology, USA). Membraneswere
rinsed and then incubatedwith anti-mouse/anti-rabbit IgG antibody (GE
Healthcare, Buckinghamshire, England) for 1 h at room temperature. The specific
complex was detected by an enhanced chemiluminescence detection system (GE
Healthcare, Buckinghamshire, England) and relative intensities of protein bands
were analyzed by MSF-300G scanner (Microtek International, Inc., Hsinchu,
Taiwan). Membranes were stripped with restore western blot stripping reagent following
the manufacturer's instructions (Pierce Chemical, Rockford, IL) and then
blocked (1 h at room temperature) with blocking solution and incubated with
mouse monoclonal anti-alpha-actin (1:1000 dilution; Sigma) or anti-PKB (1:2000;
Cell Signaling Technology, USA). After rinsing, the membranes were incubated with
antimouse horseradish peroxidase-conjugated secondary antibody (1:20,000 dilution;
GE Healthcare, Buckinghamshire, England) and the specific complex was detected
by the enhanced chemiluminescence detection system (GE Healthcare,
Buckinghamshire, England). The relative intensities of protein bands were analyzed
by MSF-300G scanner.
2.8. Malondialdehyde determinations
Malondialdehyde (MDA), a reliable biomarker used for studying lipid peroxidation,
was measured in PMNs of hyperlipemic patients either treated or not with BPF,
rosuvastatin or their combination. Levels of MDA were measured in PMNs isolated from
blood samples collected at the day 0 and 30 of treatment. Briefly, cells were homogenized
in potassium chloride (1.15%) and frozen in liquid nitrogen. Chloroform (2 ml) was then
added to each homogenate and then spun for 30 min.
The organic layer of the sample was removed and dried under nitrogen gas and reconstituted
with 100 μl of saline. MDA generation was evaluated by the assay of
thiobarbituric acid (TBA)-reacting compounds. The addition of a solution of 20 μl of sodium
dodecyl sulphate (SDS; 8.1%), 150 μl of 20% acetic acid solution (pH3.5), 150 μl l of
0.8% TBA and 400 μl l of distilled water, produced a chromogenic product which was
extracted in n-butanol and pyridine. Then, the organic layer was removed andMDA levels
read at 532 nm and expressed as nmol MDA/g prot.
2.9. Statistical analyses
Bartlett's testwas performed on each set of data to ensure that variance of the setswas
homogenous. In case of homogenous set of data, ANOVA was performed to determine the
treatment effects, and Dunnett's test was employed as appropriate. In case of heterogenous
data, F test was carried out to determine which pairs of groups were heterogenous.
This was followed by Student's t tests.
3. Results
3.1. The effect of BPF and/or plus rosuvastatin on serum lipids of patients
with mixed hyperlipidemia
Oral administration of BPF (1000 mg/daily for 30 consecutive days;
n = 15) in patients suffering from mixed hyperlipidemia significantly
reduced total cholesterol, LDL-C, triglyceride and enhanced HDL-C
levels (Table 1). No change in serum total cholesterol, LDL-C, HDL-C
and triglycerides was found in the group of patients receiving placebo
compared to basal lipidemic profile (Table 1).
Neither symptoms nor hematochemical signs of toxicitywere found
in any patients undergoing BPF and/or placebo administration (not
shown). In 2 patients treated with BPF/daily, a moderate gastric pyrosis
was observed. However, none of the patients taking BPF interrupted the
treatment.
Administration of rosuvastatin (10 and 20 mg/daily; n = 16 for
each group; Table 1) produced a significant (P b 0.05) reduction of
total cholesterol and LDL-C compared to placebo. Minimal and insignificant
changes in triglycerides occurred after treating patients with
rosuvastatin at both doses daily. Furthermore, an elevation of HDL-C
was found.
Association of BPF (1000 mg/daily) with the lower dose of
rosuvastatin (10 mg) for 30 consecutive days produced a significant
enhancement of the hypolipidemic effect of rosuvastatin compared
with the effect of rosuvastatin alone (Table 1). Indeed, the lower dose
of rosuvastatin (10 mg/daily) when given in combination with a single
oral dose of BPF (1000 mg/daily) produced a significant decrease on
total cholesterol and LDL-C serum levels in hyperlipidemic patients
when compared to rosuvastatin alone, an effect which was nearly the
same than the one produced by 20 mg of rosuvastatin. In addition, triglycerides
were reduced by 36 ± 5% and HDL-C was increased by
37 ± 2%, an effect which was significantly higher while compared to
the use of rosuvastatin alone.
3.2. The effect of BPF and/or rosuvastatin on urinary mevalonate (MVA)
Twenty-four-hour urinary MVA excretion in hyperlipidemic patients
undergoing BPF (1000 mg/daily for 30 days), rosuvastatin (10
and 20 mg/daily for 30 days) or combination of BPF (1000 mg) plus
rosuvastatin was significantly decreased (P N0.05) in all groups compared
to placebo (Fig. 1). Interestingly, the group of patients receiving
both BPF and the low concentration of rosuvastatin, showed that BPF
produced a supplementary urinary excretion of MVA compared to
Table 1
Data on lipidemic serum profile of 77 patients undergoing 30 consecutive day treatment
with a daily dose of placebo, rosuvastatin (10 or 20 mg), BPF 1000 mg and BPF
1000 mg + rosuvastatin 10 mg, respectively. Data are expressed in mg/dl.
Total cholesterol LDL-C HDL-C Triglycerides
Basal levels 278 ± 4 191 ± 3 38 ± 2 238 ± 5
Placebo 275 ± 4 190 ± 2 38 ± 3 235 ± 5
Rosuvastatin 10 mg 195 ± 3⁎ 115 ± 4⁎ 42 ± 3⁎ 200 ± 4⁎
Rosuvastatin 20 mg 174 ± 4⁎ 87 ± 3⁎ 48 ± 3⁎ 202 ± 5⁎
BPF 1000 mg 191 ± 5⁎ 113 ± 4⁎ 45 ± 4⁎ 165 ± 3⁎
Rosuvastatin 10 mg + BPF
1000 mg
172 ± 3⁎ 90 ± 4⁎ 52 ± 4⁎ 152 ± 5⁎
⁎ P b 0.05 vs placebo.
142 M. Gliozzi et al. / International Journal of Cardiology 170 (2013) 140–145
Author's personal copy
rosuvastatin alone, confirming that BPF contributed to inhibiting HMGCoA
reductase.
3.3. The effect of BPF and/or rosuvastatin on MDA, phosphoPKB and LOX-1
levels in peripheral PMNs of hyperlipemic patients
Peripheral PMNswere isolated fromthe peripheral blood of each patient
entering the study and re-suspended into Krebs buffer. The levels
ofMDA (amarker of lipid peroxidation) and the expression of LOX-1 receptor
for oxyLDL were detected in peripheral PMNs before and at the
end of the treatment with BPF and rosuvastatin given alone or in
combination.
The basal levels ofMDA and the expression of LOX-1 have been previously
found elevated in patients with hyperlipidemia compared to a
group of normolipidemic patients (n = 15; not shown). Administration
of BPF (1000 mg/daily for 30 days), rosuvastatin (10 and 20 mg/daily
for 30 days) or a combination of both (1000 mg plus 10 mg, respectively)
significantly decreased both MDA levels and LOX-1 expression in
PMNs of patients with mixed hyperlipidemia (Figs. 2 and 3). In particular,
the effect of BPF produced a further enhancement of rosuvastatin
antioxidant effect (as shown by decreasedMDA levels) and a significant
increment of rosuvastatin-induced activity in LOX-1 expression. Both
effects significantly correlated with vasoprotective properties of statins
in patients with cardiometabolic risk. The effect of BPF in combination
with rosuvastatin also resulted in an enhanced expression of PKB levels
in PMNs of hyperlipemic patients (Fig. 4). In particular,we found an enhanced
phosphorylation of PKB, an effect which accounts for a better
vasoprotection against factors supporting smoothmuscle cell proliferation
and vascular damage.
4. Discussion
Our data show that bergamot extract rich in polyphenols (BPF),
given orally for 30 consecutive days allows the reduction of daily dose
of rosuvastatin while achieving target lipid values in patients with
mixed dyslipidemia (elevated serumLDL-Cwith hypertriglyceridemia).
This effectwas characterized by a reduction of both total cholesterol and
LDL-C and amplified the increase of HDL-C produced by rosuvastatin
alone. This enhancement of statin-induced effects suggests a potential
benefit in the reduction of cardiometabolic risk. Furthermore, the reduction
of serumcholesterol in patients taking both BPF and rosuvastatin is
accompanied by a significant reduction in triglyceride levels, an effect
which has not been found with rosuvastatin alone, thus suggesting an
additive role of BPF in statin-induced hypolipidemic response.
These data are in accordance with previous evidence showing that
bergamot derivatives possess antilipidemic and vasoprotective effects
both in ratmodels of hyperlipidemia and vascular disease and in hyperlipidemic
patients [24–26]. In addition, there is a significant additive
0
0,5
1
1,5
2
2,5
3
PLACEBO ROSUVASTATIN ROSUVASTATIN BPF BPF +
ROSUVASTATIN
urinary mevalonic acid excretion
(μmol/day)
10 mg 20 mg 1000 mg 10 mg
Fig. 1. The graph showsmean values for 24 h urinary mevalonic acid excretion in patients
at 0 time (black columns) and after 30 days (grey columns) of oral treatment with placebo,
BPF (1000 mg/daily), rosuvastatin (10 and 20 mg/daily) and rosuvastatin (10 mg/
daily plus BPF 1000 mg/daily). Error bars show the standard deviation (S.D.). * - indicates
a statistically significant change compared to the placebo group at P b0.05.
0
10
20
30
40
50
60
70
80
90
PLACEBO ROSUVASTATIN ROSUVASTATIN BPF BPF +
ROSUVASTATIN
MDA nmol g-1
10 mg 20 mg 1000 mg 10 mg
Fig. 2. The graph showsmean values for malondialdehyde (lipid peroxidation) in PMNs of
patients after 30 days of oral treatment with placebo, BPF (1000 mg/daily), rosuvastatin
(10 and 20 mg/daily) and rosuvastatin (10 mg/daily plus BPF 1000 mg/daily). Error bars
showthe standard deviation (S.D.). * - indicates a statistically significant change compared
to the placebo group at P b0.05.
Fig. 3. Representative western blotting analysis showing the expression of oxyLDL receptor
LOX-1 in peripheral PMNs of patients with mixed hyperlipidemia patients after
30 days of oral treatment with placebo, BPF (1000 mg/daily), rosuvastatin (10 and
20 mg/daily) and rosuvastatin (10 mg/daily plus BPF 1000 mg/daily).
Fig. 4. Representative western blotting analysis showing the state of phosphorylation of
PKB (phosphoPKB) in peripheral PMNs of patientswithmixed hyperlipidemia patients either
untreated (CTRL) as well as after 30 days of oral treatment with placebo, BPF
(1000 mg/daily), rosuvastatin (10 and 20 mg/daily) and rosuvastatin (10 mg/daily plus
BPF 1000 mg/daily).
M. Gliozzi et al. / International Journal of Cardiology 170 (2013) 140–145 143
Author's personal copy
effect of BPF with rosuvastatin (compared with either alone), an effect
consistent with the further reduction seen in urinaryMVA as detected
in patients after treatment with both BPF and lower doses of
rosuvastatin.
Beyond the clear antilipemic/hypoglycemic response which follows
30 day treatment with BPF in patients, the mechanism of action of this
bergamot derivative remains to be elucidated. Previous data showed
that non-nutritive constituents of Citrus family fruits, such as pectin
and flavonoids found in peel extracts, cause lowering of serum and/or
tissue cholesterol levels by modulating hepatic HMG-CoA levels, possibly
by binding bile acids and increasing the turnover rate of blood and
liver cholesterol [27–31]. Since BPF showed enhancement in the excretion
of fecal sterols in rats [26], such a mechanism may contribute to
both the hypolipidemic and hypoglycemic response of bergamot derivatives.
However, a major contribution to the hypolipidemic response
found in patients undergoing BPF treatment seems to be related to the
modulatory properties in the flavonone glycoside component of the
bergamot juice extract, in particular naringin and neo-hesperidin.
Indeed, evidence exists that dietary hesperetin reduces hepatic TG
accumulation associated with a reduced activity of TG synthetic enzyme,
PAP [32]. In addition, in vitro studies suggest that hesperetin
may, in part, reduce apoB levels [33]. Togetherwith an enhanced expression
of the LDL receptor, these mechanisms may explain, at least
in part, the hypocholesterolemic properties of the citrus flavonoids
[34] and of BPF, as found in our experiments.
Furthermore, naringenin has been shown to act at multiple levels in
regulating lipid metabolism in patients [35]. Moreover, naringin was
found to reduce both VLDL-derived and endogenously synthesized
fatty acids, preventing muscle triglyceride accumulation and, finally,
improving overall insulin sensitivity and glucose tolerance [36].
The effect of both naringin and hesperidin seems also to involve direct
inhibition of the HMG-CoA reductase enzyme system [37]. This is
confirmed by our data in which MVA, the end product of HMG-CoA
reductase activity, was reduced by treatment with BPF. Furthermore,
ACAT activities as a result of naringin supplementation could account
for the decrease of fecal neutral sterols [37]. In addition, BPF seems to
contain high concentration of buteridine and melitidine [38], two
flavonone derivatives shown to selectively inhibit HMG-CoA reductase,
and this may well explain the potency of BPF in reducing cholesterol
levels [23].
Our data also showan additive vaso-protective effect of BPFwhen
given associated with rosuvastatin, an effect probably due to the antioxidant
properties of bergamot polyphenols. This is shown by the
enhanced reduction of both LOX-1 and phosphorylation of PKB in
PMNs of patients undergoing rosuvastatin treatment in combination
with BPF and by simultaneous reduction ofMDA, a biomarker of lipid
peroxidation. This confirms previous studies showing that BPF given
in hyperlipemic patients, improves reactive vasodilatation [26]. Furthermore,
we recently showed that expression of LOX-1 receptor is
associated with smooth cell proliferation in injured blood vessel, an
effect counteracted by bergamot extract [25] as well as by exogenous
antioxidants [39]. Finally, BPF contributed to the statin-induced increase
of PKB phosphorylation. This may be relevant due to the evidence
that PKB is a member of the second-messenger family that
regulates the sub-family of protein kinases implicated in signaling
downstream of tyrosine kinases and PI3-kinase.When phopshorylated,
PKB leads to vasoprotection, an effect which is confirmed by evidence
that statins are known to produce protection against vascular atherogenic
injury also via PKB phosphorylation in response to mitogens and
survival factors [40]. Thus, exogenous supply of natural antioxidants,
such as bergamot-derived polyphenols, highlighted the modulation of
peripheral biomarkers of cardiometabolic risk induced by rosuvastatin
showing an additive vasoprotective potential for this natural extract
in patients with hyperlipemia.
In conclusion, our data show that BPF enhances the effect of
rosuvastatin in normalizing the serum lipemic profile. Clinically, this
may allow a reduction in daily rosuvastatin doses for maintaining patients
to target levels of cholesterol. In addition, BPF contributes to the
reduction in oxidative processes found in hyperlipemic patients, thus
suggesting a potential vasoprotective benefit of using such natural polyphenols
in hyperlipemic patients undergoing statin treatment.
Acknowledgments
This paper has been supported by PON MIUR a3_00359. We would
like to thank Prof. James Ehlrich (University of Denver, Colorado, USA)
for the contribution in discussing data and revising the manuscript.
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Bergamot polyphenolic fraction enhances rosuvastatin-induced effect on
LDL-cholesterol, LOX-1 expression and protein kinase B phosphorylation in
patients with hyperlipidemia
Micaela Gliozzi a,1, RossWalker a,1, Saverio Muscoli b,1, Cristiana Vitale c,1, Santo Gratteri a,1, Cristina Carresi a,1,
Vincenzo Musolino a,1, Vanessa Russo a,1, Elzbieta Janda a,1, Salvatore Ragusa a,1, Antonio Aloe a,1,
Ernesto Palma a,1, Carolina Muscoli a,1, Franco Romeo b,1, Vincenzo Mollace a,c,⁎,1
a Research Centre for Food Safety & Health (IRC-FSH), University “Magna Graecia”, Catanzaro, Italy
b Department of Medicine, Chair of Cardiology, University of Rome Tor Vergata, Rome, Italy
c IRCCS San Raffaele, Rome, Italy
a r t i c l e i n f o a b s t r a c t
Article history:
Received 2 April 2013
Received in revised form 3 August 2013
Accepted 30 August 2013
Available online 8 September 2013
Keywords:
Hyperlipidemia
Cardiometabolic risk
Bergamot polyphenolic fraction
Rosuvastatin
Background: Statins are the most commonly prescribed drugs to reduce cardiometabolic risk. Besides the wellknown
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 polyphenolsmay
allowpatients 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 patientswithmixed hyperlipidemia.
Methods: 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 20 mg/daily for 30 days; n = 16 for
each group), a group receiving BPF alone orally (1000 mg/daily for 30 days; n = 15) and a group receiving
BPF (1000 mg/daily given orally) plus rosuvastatin (10 mg/daily for 30 days; n = 15).
Results: 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.
Conclusions: Addition of BPF to rosuvastatin significantly enhanced rosuvastatin-induced effect on serumlipemic
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.
© 2013 Published by Elsevier Ireland Ltd.
1. Introduction
Dyslipidemia (or hypercholesterolemia) is an important risk factor
for the development of atherosclerosis and coronary artery disease, as
shown in several lipid intervention trials and prospective studies
[1]. Increased concentrations of low-density lipoprotein cholesterol
(LDL-C), total blood cholesterol (TC) and triglycerides (TG) comprise
the main pathogenic risk profile. Conditions of insulin resistance
such as impaired glucose tolerance or “prediabetes” are also associated
with a high risk of cardiovascular disease (CVD) and are often
accompanied by low levels of high-density lipoprotein cholesterol
(HDL-C) [2]. The majority of therapeutic protocols rely on drugs
that belong to the “statin” family. Statins inhibit the activity of 3-
hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, which catalyzes
the rate-limiting step in mevalonate biosynthesis, a key intermediate
in cholesterol metabolism. A meta-analysis of placebo-controlled
“standard dose” statin trials shows a reduction in cardiovascular mortality
averaging 20% and a decrease in major cardiovascular events by
approximately 25% [3]. Treatment with high-dose statins was shown
to reduce the morbidity by 36%, and a reduction of cardiovascular
International Journal of Cardiology 170 (2013) 140–145
⁎ Corresponding author at: Faculty of Pharmacy, Department of Health Sciences,
University “Magna Graecia”, Complesso Ninì Barbieri, Roccelletta di Borgia, 88100
Catanzaro, Italy. Tel.:+39 0961 369 4128,+39 0961 369 5732; fax:+39 0961 369 4237.
E-mail address: mollace@unicz.it (V. Mollace).
1 Each author takes responsibility for all aspects of the reliability and freedomfrombias
of the data presented and their discussed interpretation.
0167-5273/$ – see front matter © 2013 Published by Elsevier Ireland Ltd.
http://dx.doi.org/10.1016/j.ijcard.2013.08.125
Contents lists available at ScienceDirect
International Journal of Cardiology
journal homepage: www.elsevier .com/locate/ijcard
Author's personal copy
events up to 40% [4]. Despite the significant clinical benefits provided
by statins, many patients, in particular those with diabetes
or metabolic syndrome, do not achieve their recommended lowdensity
and high-density lipoprotein (LDL-C, HDL-C) cholesterol
target goals with statins alone [5]. Moreover, statins have been reported
to cause dose-related side effects, the more serious including
liver disease or severe myopathy, in up to 22% of patients
eligible for this therapeutic approach [6,7]. This limits the use of
statins and suggests the need for alternative and/or supplementary
therapeutic approaches.
Experimental and epidemiological evidence suggests that dietary
polyphenols, in particular flavonoids, may play a role in ameliorating
atherosclerosis, due to a pleiotropic anti-oxidative and
anti-inflammatory effect proposed as underlying mechanisms
[8–11]. In support of this hypothesis, it has been shown that certain
plant polyphenols, such as nobiletin and poncirin, inhibit monocyte
activation in vitro [9,12–17], and the enhancing effects of
such components may play a central role in this beneficial effect.
This observation provides the rationale for the optimal use of
appropriate, evidence-based phytochemicals as an adjunct to
treat hyperlipidemia with the aim of preventing cardiovascular
events.
Bergamot (Citrus bergamia) is an endemic plant growing in the
Calabrian region of Southern Italy with a unique profile of flavonoid
and glycosides present in its juice and albedo, such as neoeriocitrin,
neohesperidin, naringin, rutin, neodesmin, rhoifolin and poncirin.
Bergamot differs from other citrus fruits, not only for the composition,
but also for the particularly high content of flavonoids [18,19]. Some
of these flavonoids, such as naringin (which is also present in
grapefruit), have already been shown to be active in animal models
of atherosclerosis [20,21], while neoeriocitrin and rutin have been
found to exhibit a strong capacity to inhibit LDL oxidation [22]. Importantly,
bergamot juice is rich in 3-hydroxy-3-methylglutaryl
neohesperidosides of hesperetin (brutieridine) and naringenin
(melitidine) with an ability to inhibit HMG-CoA reductase [23].
These compounds are likely to contribute to the substantial
hypolipidemic effects of bergamot juice and to the vasoprotective
effects of bergamot oil derivatives in rats [24,25]. However, no clinical
study has been performed to date to evaluate the fruit's potential
dyslipidemia-correcting properties.
Recently, we found that bergamot-derived polyphenolic fraction
(BPF), given orally both in animal models of diet-induced hyperlipidemia
and in patients suffering from metabolic syndrome, produces a
significant and sustained reduction of serum cholesterol, triglycerides
and glycaemia [26]. This effect was accompanied by a significant improvement
in vascular reactivity in patients with both hyperlipidemia
and elevated serum glucose, thus suggesting a potential protective
role for the use of BPF in patients with metabolic syndrome and
cardiometabolic risk.
Apart from these results, no evidence has been collected to date, on
themechanism of action of bergamot-derived extract and on its potential
benefit when used in comparison with statins in patients with metabolic
syndrome.
To address these issues, we conducted a prospective, openlabel,
parallel group, placebo-controlled study on patients with elevated
serum LDL-C and triglycerides, to investigate the effect of
BPF in modulating serum lipidemic profile in patients suffering
from mixed hyperlipidemia, either untreated or treated with
rosuvastatin. The effect of rosuvastatin with BPF on the expression
of biomarkers of vascular oxidative stress and circulating cell viability
was also studied in these patients. In particular, we examined
the accumulation of malondialdehyde (MDA), a marker of lipid peroxidation,
the expression of LOX-1 receptor for oxidized LDL and protein
kinase B (PKB) phosphorylation in peripheral mononuclear cells
(PMC) in patients taking BPF alone or in combination with rosuvastatin
treatment.
2. Materials and methods
2.1. Plant material
C. bergamia Risso & Poiteau fruits were collected from plants located in a range of
90 km from Bianco to Reggio Calabria, Italy.
2.2. Preparation of BPF
Bergamot juice was obtained from peeled-off fruits by squeezing. The juice was oil
fraction-depleted by stripping, clarified by ultra-filtration and loaded on to a suitable polystyrene
resin column able to absorb polyphenol compounds ofmolecular weight between
300 and 600 Da (Mitsubishi). Polyphenol fractions were eluted by a 1 mMKOH solution.
The basic eluate was incubated at a rocking platform to reduce the fumocumarin content.
The shaking time was adjusted proportionally to the amount of fumocumarin contaminants.
Next, the phytocomplex derived fromthe defurocoumarinization process was neutralized
by filtration on cationic resin at acidic pH. Finally itwas vacuum dried andminced
to the desired particle size to obtain BPF powder. BPF powder was analyzed for flavonoid,
furocumarin and other polyphenol content. In addition, all toxicological analyses were
performed, including heavy metal, pesticide, phthalate and sinephrine content
which revealed the absence of known toxic compounds at significant levels (data
not shown). Standardmicrobiological test showed that the final BPF was free of mycotoxins
and contaminating bacteria.
Finally, 500 mg aliquots of the BPF powder supplementedwith 50 mg of ascorbic acid
as antioxidantwere encapsulated into suitable gelatin capsules by a semi-automatic gelatin
encapsulation device employing an authorized pharmaceutical manufacturer (Plants,
Messina, Italy). All procedures have been performed according to Good Manufacture Practice
(GMP) headlines of European Legislation.
The main flavonoids identified in BPF were neoeriocitrin (370 ppm), naringin
(520 ppm), and neohesperidin (310 ppm). Tablets containing 1000 mg of maltodestrin
supplemented with 50 mg ascorbic acid were used as placebo.
2.3. Patient selection and study design
A prospective, open-label, parallel group, placebo-controlled studywas designed.
We enrolled 77 patients suffering from mixed hypercholesterolemia (LDL-C levels of
N160 mg/dl, and triglycerides levels N225 mg/dl) from a primary care setting at the
Department of Cardiology of the IRCCS San Raffaele– Rome and at the Atherosclerosis
Prevention Ambulatory centre at the University of Rome “Tor Vergata” facility. Age,
gender, and body mass index were matched among all subjects. Patients were randomly
assigned to five groups, all following a low fat diet: a control group receiving
placebo (n = 15), two groups receiving oral rosuvastatin (10 and 20 mg/daily for
30 days; n = 16 for each group), a group receiving BPF alone orally (1000 mg/daily
for 30 days; n = 15) and a group receiving BPF (1000 mg/daily given orally) plus
rosuvastatin (10 mg/daily for 30 days; n = 15). In all groups, we determined the efficacy
of each treatment by monitoring serum lipid profiles and urinary mevalonic
acid concentration (expressing inhibition of HMG-CoA reductase).
We excluded patients with overt liver disease, chronic renal failure, hypothyroidism,
myopathy, uncontrolled diabetes, severe hypertension, stroke, acute coronary events
within the preceding 30 days, coronary revascularization within the preceding 3 months,
or alcohol abuse. No patient was taking hormone replacement therapy or antioxidant vitamin
supplements during the 2 months preceding our study.
All the patients were given placebo, rosuvastatin, BPF alone or BPF plus rosuvastatin
before meals daily during a 30 day treatment period. A research nurse counted pills at
the end of treatment tomonitor compliance. The patients were seen at leastweekly during
the study. To minimize side effects, we measured serum asparate aminotransferase, alanine
aminotransferase, creatine kinase, blood urea nitrogen, creatinine and blood cell
counts before and after therapy. Seven (7) patients taking placebo and 16 patients taking
BPF and/or rosuvastatin were smokers. Two (2) patients taking placebo, and 13 patients
taking BPF alone and/or rosuvastatin were also taking ACE inhibitors, angiotensin AT1 receptor
blockers or beta adrenergic blockers to control blood pressure. No additional medications
including aspirin or nonsteroidal anti-inflammatory drugs were allowed during
the study period to avoid confounding effects of other drugs. Anti-hypertensive drugs
were withheld for ≥48 h before starting the study. Blood samples for assessing lipid profile
and 24 h urinary collectionwere performed at day 0 and after 30 day treatment in placebo,
rosuvastatin and rosuvastatin/BPF-treated patients. At the same time, blood samples
were collected for assessing baseline values for blood cell counts, transaminases, creatinine
blood levels and PMN separation and culturing.
All patients participating in the study signed an informed consent according to the
European Legislation and the protocol was previously submitted and approved by the Regional
Ethical Committee. In addition, the study protocol was performed according to the
ethical guidelines of the 1975 Declaration of Helsinki, as reflected in a priori approval by
the institution's human research committee.
2.4. Serum lipids
Total cholesterol, LDL-C, HDL-C and triglycerides in serum samples of patients were
determined colorimetrically and enzymatically using commercial assay kits (BioSystems
S.A., Barcelona, Spain).
M. Gliozzi et al. / International Journal of Cardiology 170 (2013) 140–145 141
Author's personal copy
2.5. Urinary mevalonic acid detection
Twenty-four-hour urine samples were collected from each patient before and after
treatment with placebo, supplement and/or drugs. Total volume was recorded, and aliquots
were frozen at −20 °C. Frozen urine samples were thawed and centrifuged at
1200 g for 30 min to remove solids. Urinary mevalonic acid (MVA) concentrations were
determined by means of a modification of the radioenzymatic isotope-dilution method
of Popjak (26). The intra-subject coefficient of variation was evaluated; 90% of all values
were found to lie within 35% of a single 24-h determination, whereas for two determinations,
the variation was 25%. The intra-assay coefficient of variation in each run was less
than 5%, whereas the inter-assay variation, based on the repeated analysis of the same
standard over a 2-year period, was 3.9% (25). The completeness of urine samples was determined
on the basis of subjects' records and their careful questioning; if these were
deemed incomplete, a repeated collection was obtained.
2.6. Mononuclear cell isolation
Twenty milliliter of heparinised blood was diluted with the same volume of PBS and
carefully layered over 6 ml of Ficoll-Paque Plus (Amersham Biosciences, USA). The mixturewas
centrifuged at 400 ×g for 40 min at 18–20 °C. The interface containing peripheral
blood mononuclear cells (PMNs) was collected and washed two times with PBS. This
preparation contained approximately 25% monocytes and 75% lymphocytes. Further purification
of the monocytes from the PMNs fraction was accomplished by allowing the
monocytes to adhere to plastic. Using 25 cm2 culture dishes, PMNs were cultured for
45 min in RPMI 1640medium (with glutamax-I, Dutch modification, Gibco BRL) containing
10% pooled human serum. The nonadhering lymphocytes were removed by washing
the culture dishes with PBS, after which the adherentmonocytes were harvested. The reagents
used for the isolation of monocytes contained no detectable endotoxins, as determined
by the E-Toxate assay (Sigma, St. Louis, USA).
2.7. Cell extracts and SDS-PAGE Western blotting
In order to study phosphor-PKB expression, PMNswere lysed with ice-cold lysis buffer
(20 mM Tris–HCl, pH 7.5; 150 mM NaCl; 2 mM EDTA; 2 mM EGTA; 1% Triton X-100;
1 nM okadaic acid, protease inhibitor cocktail - Sigma; phosphatase inhibitor cocktail —
Calbiochem), centrifuged at 10,000 ×g for 15 min at +4 °C. Alternatively, for LOX-1 detection,
PMNs were lysed with a different ice-cold lysis buffer (50 mM Tris pH 8,
150 mM NaCl, 1% Nonidet P-40; 2 mM Pefablock, 1 microM ml−1 Aprotinin, 10 microM
ml−1 Leupepeptin, 2 microM ml−1 Pepstatin A, trypsin inhibitor), centrifuged at
13,000 ×g for 30 min at+4 °C. Protein concentrations in supernatants were determined
by standard Bradford assay.
Lysates from each treatment (30 micrograms/lane) were resolved by 10% SDS–polyacrylamide
gel electrophoresis and transferred to nitrocellulosemembranes. After incubation
in blocking solution (4% dry nonfat milk, Sigma–Aldrich, St. Louis, MO), membranes
were incubated with anti-LOX-1 (1:10,000; gift from Professor T. Sawamura, Osaka,
Japan); anti-phospho-PKB (Ser 473) (1:2000; Cell Signaling Technology, USA). Membraneswere
rinsed and then incubatedwith anti-mouse/anti-rabbit IgG antibody (GE
Healthcare, Buckinghamshire, England) for 1 h at room temperature. The specific
complex was detected by an enhanced chemiluminescence detection system (GE
Healthcare, Buckinghamshire, England) and relative intensities of protein bands
were analyzed by MSF-300G scanner (Microtek International, Inc., Hsinchu,
Taiwan). Membranes were stripped with restore western blot stripping reagent following
the manufacturer's instructions (Pierce Chemical, Rockford, IL) and then
blocked (1 h at room temperature) with blocking solution and incubated with
mouse monoclonal anti-alpha-actin (1:1000 dilution; Sigma) or anti-PKB (1:2000;
Cell Signaling Technology, USA). After rinsing, the membranes were incubated with
antimouse horseradish peroxidase-conjugated secondary antibody (1:20,000 dilution;
GE Healthcare, Buckinghamshire, England) and the specific complex was detected
by the enhanced chemiluminescence detection system (GE Healthcare,
Buckinghamshire, England). The relative intensities of protein bands were analyzed
by MSF-300G scanner.
2.8. Malondialdehyde determinations
Malondialdehyde (MDA), a reliable biomarker used for studying lipid peroxidation,
was measured in PMNs of hyperlipemic patients either treated or not with BPF,
rosuvastatin or their combination. Levels of MDA were measured in PMNs isolated from
blood samples collected at the day 0 and 30 of treatment. Briefly, cells were homogenized
in potassium chloride (1.15%) and frozen in liquid nitrogen. Chloroform (2 ml) was then
added to each homogenate and then spun for 30 min.
The organic layer of the sample was removed and dried under nitrogen gas and reconstituted
with 100 μl of saline. MDA generation was evaluated by the assay of
thiobarbituric acid (TBA)-reacting compounds. The addition of a solution of 20 μl of sodium
dodecyl sulphate (SDS; 8.1%), 150 μl of 20% acetic acid solution (pH3.5), 150 μl l of
0.8% TBA and 400 μl l of distilled water, produced a chromogenic product which was
extracted in n-butanol and pyridine. Then, the organic layer was removed andMDA levels
read at 532 nm and expressed as nmol MDA/g prot.
2.9. Statistical analyses
Bartlett's testwas performed on each set of data to ensure that variance of the setswas
homogenous. In case of homogenous set of data, ANOVA was performed to determine the
treatment effects, and Dunnett's test was employed as appropriate. In case of heterogenous
data, F test was carried out to determine which pairs of groups were heterogenous.
This was followed by Student's t tests.
3. Results
3.1. The effect of BPF and/or plus rosuvastatin on serum lipids of patients
with mixed hyperlipidemia
Oral administration of BPF (1000 mg/daily for 30 consecutive days;
n = 15) in patients suffering from mixed hyperlipidemia significantly
reduced total cholesterol, LDL-C, triglyceride and enhanced HDL-C
levels (Table 1). No change in serum total cholesterol, LDL-C, HDL-C
and triglycerides was found in the group of patients receiving placebo
compared to basal lipidemic profile (Table 1).
Neither symptoms nor hematochemical signs of toxicitywere found
in any patients undergoing BPF and/or placebo administration (not
shown). In 2 patients treated with BPF/daily, a moderate gastric pyrosis
was observed. However, none of the patients taking BPF interrupted the
treatment.
Administration of rosuvastatin (10 and 20 mg/daily; n = 16 for
each group; Table 1) produced a significant (P b 0.05) reduction of
total cholesterol and LDL-C compared to placebo. Minimal and insignificant
changes in triglycerides occurred after treating patients with
rosuvastatin at both doses daily. Furthermore, an elevation of HDL-C
was found.
Association of BPF (1000 mg/daily) with the lower dose of
rosuvastatin (10 mg) for 30 consecutive days produced a significant
enhancement of the hypolipidemic effect of rosuvastatin compared
with the effect of rosuvastatin alone (Table 1). Indeed, the lower dose
of rosuvastatin (10 mg/daily) when given in combination with a single
oral dose of BPF (1000 mg/daily) produced a significant decrease on
total cholesterol and LDL-C serum levels in hyperlipidemic patients
when compared to rosuvastatin alone, an effect which was nearly the
same than the one produced by 20 mg of rosuvastatin. In addition, triglycerides
were reduced by 36 ± 5% and HDL-C was increased by
37 ± 2%, an effect which was significantly higher while compared to
the use of rosuvastatin alone.
3.2. The effect of BPF and/or rosuvastatin on urinary mevalonate (MVA)
Twenty-four-hour urinary MVA excretion in hyperlipidemic patients
undergoing BPF (1000 mg/daily for 30 days), rosuvastatin (10
and 20 mg/daily for 30 days) or combination of BPF (1000 mg) plus
rosuvastatin was significantly decreased (P N0.05) in all groups compared
to placebo (Fig. 1). Interestingly, the group of patients receiving
both BPF and the low concentration of rosuvastatin, showed that BPF
produced a supplementary urinary excretion of MVA compared to
Table 1
Data on lipidemic serum profile of 77 patients undergoing 30 consecutive day treatment
with a daily dose of placebo, rosuvastatin (10 or 20 mg), BPF 1000 mg and BPF
1000 mg + rosuvastatin 10 mg, respectively. Data are expressed in mg/dl.
Total cholesterol LDL-C HDL-C Triglycerides
Basal levels 278 ± 4 191 ± 3 38 ± 2 238 ± 5
Placebo 275 ± 4 190 ± 2 38 ± 3 235 ± 5
Rosuvastatin 10 mg 195 ± 3⁎ 115 ± 4⁎ 42 ± 3⁎ 200 ± 4⁎
Rosuvastatin 20 mg 174 ± 4⁎ 87 ± 3⁎ 48 ± 3⁎ 202 ± 5⁎
BPF 1000 mg 191 ± 5⁎ 113 ± 4⁎ 45 ± 4⁎ 165 ± 3⁎
Rosuvastatin 10 mg + BPF
1000 mg
172 ± 3⁎ 90 ± 4⁎ 52 ± 4⁎ 152 ± 5⁎
⁎ P b 0.05 vs placebo.
142 M. Gliozzi et al. / International Journal of Cardiology 170 (2013) 140–145
Author's personal copy
rosuvastatin alone, confirming that BPF contributed to inhibiting HMGCoA
reductase.
3.3. The effect of BPF and/or rosuvastatin on MDA, phosphoPKB and LOX-1
levels in peripheral PMNs of hyperlipemic patients
Peripheral PMNswere isolated fromthe peripheral blood of each patient
entering the study and re-suspended into Krebs buffer. The levels
ofMDA (amarker of lipid peroxidation) and the expression of LOX-1 receptor
for oxyLDL were detected in peripheral PMNs before and at the
end of the treatment with BPF and rosuvastatin given alone or in
combination.
The basal levels ofMDA and the expression of LOX-1 have been previously
found elevated in patients with hyperlipidemia compared to a
group of normolipidemic patients (n = 15; not shown). Administration
of BPF (1000 mg/daily for 30 days), rosuvastatin (10 and 20 mg/daily
for 30 days) or a combination of both (1000 mg plus 10 mg, respectively)
significantly decreased both MDA levels and LOX-1 expression in
PMNs of patients with mixed hyperlipidemia (Figs. 2 and 3). In particular,
the effect of BPF produced a further enhancement of rosuvastatin
antioxidant effect (as shown by decreasedMDA levels) and a significant
increment of rosuvastatin-induced activity in LOX-1 expression. Both
effects significantly correlated with vasoprotective properties of statins
in patients with cardiometabolic risk. The effect of BPF in combination
with rosuvastatin also resulted in an enhanced expression of PKB levels
in PMNs of hyperlipemic patients (Fig. 4). In particular,we found an enhanced
phosphorylation of PKB, an effect which accounts for a better
vasoprotection against factors supporting smoothmuscle cell proliferation
and vascular damage.
4. Discussion
Our data show that bergamot extract rich in polyphenols (BPF),
given orally for 30 consecutive days allows the reduction of daily dose
of rosuvastatin while achieving target lipid values in patients with
mixed dyslipidemia (elevated serumLDL-Cwith hypertriglyceridemia).
This effectwas characterized by a reduction of both total cholesterol and
LDL-C and amplified the increase of HDL-C produced by rosuvastatin
alone. This enhancement of statin-induced effects suggests a potential
benefit in the reduction of cardiometabolic risk. Furthermore, the reduction
of serumcholesterol in patients taking both BPF and rosuvastatin is
accompanied by a significant reduction in triglyceride levels, an effect
which has not been found with rosuvastatin alone, thus suggesting an
additive role of BPF in statin-induced hypolipidemic response.
These data are in accordance with previous evidence showing that
bergamot derivatives possess antilipidemic and vasoprotective effects
both in ratmodels of hyperlipidemia and vascular disease and in hyperlipidemic
patients [24–26]. In addition, there is a significant additive
0
0,5
1
1,5
2
2,5
3
PLACEBO ROSUVASTATIN ROSUVASTATIN BPF BPF +
ROSUVASTATIN
urinary mevalonic acid excretion
(μmol/day)
10 mg 20 mg 1000 mg 10 mg
Fig. 1. The graph showsmean values for 24 h urinary mevalonic acid excretion in patients
at 0 time (black columns) and after 30 days (grey columns) of oral treatment with placebo,
BPF (1000 mg/daily), rosuvastatin (10 and 20 mg/daily) and rosuvastatin (10 mg/
daily plus BPF 1000 mg/daily). Error bars show the standard deviation (S.D.). * - indicates
a statistically significant change compared to the placebo group at P b0.05.
0
10
20
30
40
50
60
70
80
90
PLACEBO ROSUVASTATIN ROSUVASTATIN BPF BPF +
ROSUVASTATIN
MDA nmol g-1
10 mg 20 mg 1000 mg 10 mg
Fig. 2. The graph showsmean values for malondialdehyde (lipid peroxidation) in PMNs of
patients after 30 days of oral treatment with placebo, BPF (1000 mg/daily), rosuvastatin
(10 and 20 mg/daily) and rosuvastatin (10 mg/daily plus BPF 1000 mg/daily). Error bars
showthe standard deviation (S.D.). * - indicates a statistically significant change compared
to the placebo group at P b0.05.
Fig. 3. Representative western blotting analysis showing the expression of oxyLDL receptor
LOX-1 in peripheral PMNs of patients with mixed hyperlipidemia patients after
30 days of oral treatment with placebo, BPF (1000 mg/daily), rosuvastatin (10 and
20 mg/daily) and rosuvastatin (10 mg/daily plus BPF 1000 mg/daily).
Fig. 4. Representative western blotting analysis showing the state of phosphorylation of
PKB (phosphoPKB) in peripheral PMNs of patientswithmixed hyperlipidemia patients either
untreated (CTRL) as well as after 30 days of oral treatment with placebo, BPF
(1000 mg/daily), rosuvastatin (10 and 20 mg/daily) and rosuvastatin (10 mg/daily plus
BPF 1000 mg/daily).
M. Gliozzi et al. / International Journal of Cardiology 170 (2013) 140–145 143
Author's personal copy
effect of BPF with rosuvastatin (compared with either alone), an effect
consistent with the further reduction seen in urinaryMVA as detected
in patients after treatment with both BPF and lower doses of
rosuvastatin.
Beyond the clear antilipemic/hypoglycemic response which follows
30 day treatment with BPF in patients, the mechanism of action of this
bergamot derivative remains to be elucidated. Previous data showed
that non-nutritive constituents of Citrus family fruits, such as pectin
and flavonoids found in peel extracts, cause lowering of serum and/or
tissue cholesterol levels by modulating hepatic HMG-CoA levels, possibly
by binding bile acids and increasing the turnover rate of blood and
liver cholesterol [27–31]. Since BPF showed enhancement in the excretion
of fecal sterols in rats [26], such a mechanism may contribute to
both the hypolipidemic and hypoglycemic response of bergamot derivatives.
However, a major contribution to the hypolipidemic response
found in patients undergoing BPF treatment seems to be related to the
modulatory properties in the flavonone glycoside component of the
bergamot juice extract, in particular naringin and neo-hesperidin.
Indeed, evidence exists that dietary hesperetin reduces hepatic TG
accumulation associated with a reduced activity of TG synthetic enzyme,
PAP [32]. In addition, in vitro studies suggest that hesperetin
may, in part, reduce apoB levels [33]. Togetherwith an enhanced expression
of the LDL receptor, these mechanisms may explain, at least
in part, the hypocholesterolemic properties of the citrus flavonoids
[34] and of BPF, as found in our experiments.
Furthermore, naringenin has been shown to act at multiple levels in
regulating lipid metabolism in patients [35]. Moreover, naringin was
found to reduce both VLDL-derived and endogenously synthesized
fatty acids, preventing muscle triglyceride accumulation and, finally,
improving overall insulin sensitivity and glucose tolerance [36].
The effect of both naringin and hesperidin seems also to involve direct
inhibition of the HMG-CoA reductase enzyme system [37]. This is
confirmed by our data in which MVA, the end product of HMG-CoA
reductase activity, was reduced by treatment with BPF. Furthermore,
ACAT activities as a result of naringin supplementation could account
for the decrease of fecal neutral sterols [37]. In addition, BPF seems to
contain high concentration of buteridine and melitidine [38], two
flavonone derivatives shown to selectively inhibit HMG-CoA reductase,
and this may well explain the potency of BPF in reducing cholesterol
levels [23].
Our data also showan additive vaso-protective effect of BPFwhen
given associated with rosuvastatin, an effect probably due to the antioxidant
properties of bergamot polyphenols. This is shown by the
enhanced reduction of both LOX-1 and phosphorylation of PKB in
PMNs of patients undergoing rosuvastatin treatment in combination
with BPF and by simultaneous reduction ofMDA, a biomarker of lipid
peroxidation. This confirms previous studies showing that BPF given
in hyperlipemic patients, improves reactive vasodilatation [26]. Furthermore,
we recently showed that expression of LOX-1 receptor is
associated with smooth cell proliferation in injured blood vessel, an
effect counteracted by bergamot extract [25] as well as by exogenous
antioxidants [39]. Finally, BPF contributed to the statin-induced increase
of PKB phosphorylation. This may be relevant due to the evidence
that PKB is a member of the second-messenger family that
regulates the sub-family of protein kinases implicated in signaling
downstream of tyrosine kinases and PI3-kinase.When phopshorylated,
PKB leads to vasoprotection, an effect which is confirmed by evidence
that statins are known to produce protection against vascular atherogenic
injury also via PKB phosphorylation in response to mitogens and
survival factors [40]. Thus, exogenous supply of natural antioxidants,
such as bergamot-derived polyphenols, highlighted the modulation of
peripheral biomarkers of cardiometabolic risk induced by rosuvastatin
showing an additive vasoprotective potential for this natural extract
in patients with hyperlipemia.
In conclusion, our data show that BPF enhances the effect of
rosuvastatin in normalizing the serum lipemic profile. Clinically, this
may allow a reduction in daily rosuvastatin doses for maintaining patients
to target levels of cholesterol. In addition, BPF contributes to the
reduction in oxidative processes found in hyperlipemic patients, thus
suggesting a potential vasoprotective benefit of using such natural polyphenols
in hyperlipemic patients undergoing statin treatment.
Acknowledgments
This paper has been supported by PON MIUR a3_00359. We would
like to thank Prof. James Ehlrich (University of Denver, Colorado, USA)
for the contribution in discussing data and revising the manuscript.
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- zafferano
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Re: Open conversazione
by zafferano » Mon Feb 01, 2016 5:37 am
1
1.Introduction
Hyperlipidemia or hypercholesterolemia are important
risk factors for the development of atherosclerosis and
coronary artery disease [1, 2]. Main pathogenic blood
parameters are increased concentrations of cholesterol bound
to Low-density Lipoprotein (cLDL), total blood cholesterol
(totChol) and triglycerides (TG). Conditions of insulin
resistance such as impaired glucose tolerance or
"prediabetes" are also characterized by a high risk of
cardiovascular diseases (CVD) [3]. Majority of therapeutic
protocols rely on drugs that belong to statin family. Statins
inhibit the activity
of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase,
which catalyzes the rate-limiting step in mevalonate
biosynthesis, a key intermediate in cholesterol metabolism.
This is associated to a
decrease in totChol and a switch from cLDL to High-density
Lipoprotein (cHDL) fraction. Despite the significant clinical
benefits provided by statins [1], many patients, in particular
those with metabolic syndrome, do not achieve their
recommended low-density and high-density lipoprotein
(LDL, HDL) cholesterol target goals with statins [3].
Moreover, the use of statins is forbidden in more than 40% of
patients eligible for this therapeutic approach, mostly for the
occurrence of side effects including myalgia, myopathy or
liver disease and rhabdomyolysis in more severe cases [4, 5].
This limits the use of statins and suggest the need of
alternative therapeutic approaches. Experimental and
epidemiological studies suggest that dietary polyphenols, in
particular flavonoids, may play a role in ameliorating
atherosclerosis and pleiotropic anti-oxidative and
nflammatory effects have been proposed as an underlying
mechanism [6-8]. Yet these compounds when analysed
separately show rather weak cholesterol-lowering effects in
animal experiments [9]. In contrast, some natural
compositions of plant polyphenols seem to possess a good
hypolipemic activity both in animal models and in human
studies, suggesting that synergistic effects of individual
compounds may play a central role in the beneficial effects
[6, 8, 9]. This observation provides the rationale for the
identification of optimally synergizing elements and their
plant sources with the best ability to prevent hyperlipidemia
and cardiovascular complications.
Bergamot (Citrus Bergamia) is an endemic plant of Calabrian
region in Southern Italy with an unique profile of flavonoid
and flavonoid glycosides present in its juice and albedo, such
as neoeriocitrin, neohesperidin, naringin, rutin, neodesmin,
rhoifolin and poncirin. Bergamot differs from other Citrus
fruits not only because of the composition of its flavonoids,
but also because of their particularly high content [10, 11].
Among them naringin, present also in grapefruit, have
already been reported to be active in animal models of
atherosclerosis [12], while neoeriocitrin and rutin have been
shown to inhibit LDL oxidation [13]. Importantly, bergamot
juice (BJ) is rich in neohesperidosides of hesperetin and
naringenin, such as melitidine and brutieridine. These
flavonoids possess 3-hydroxy-3-methylglutaryl moiety with a
structural similarity to the natural substrate of HMG-CoA
reductase and are likely to exhibit statin-like proprieties [14].
Experimental evidence obtained in animal models of dietinduced
hypercholesterolemia and renal damage [15, 16] as
well as in the rat model of mechanical stress-induced vascular
2
injury [17] supports the hypolipemic and vasoprotective effects of bergamot constituents. However, the therapeutic potential of bergamot has never been investigated in human studies, even though the traditional use of BJ in Calabrian region suggested since long time its potential beneficial use in counteracting atherosclerosis.
The present study has been carried out to verify, in a rat model of diet-induced hyperlipemia, the effect of bergamot-derived polyphenolic fraction (BPF) on totChol, cLDL, cHDL, TG and blood glucose. This effect was also investigated by givin orally BPF for 30 consecutive days in 237 patients suffering from isolated or mixed hyperlipemia either associated or not with hyperglycaemia. In
patients we have also studied the possible contribution of HMG-CoA reductase inhibition by BPF to its hypolipemic activity and this effect was also compared with changes of reactive vasodilation to verify the potential beneficial effect of BPF in modulating the imbalanced endothelial reactivity.
2. Materials and methods
2.1 Plant Material. C. bergamia Risso & Poiteau fruits were collected from plantations located between Reggio Calabria and Bianco, in 90 Km long costal area in South Italy.
2.2 Preparation of BPF. BJ was obtained from peeled-off fruits by industrial pressing and squeezing. The juice was oil fraction-depleted by stripping, clarified by ultra-filtration and loaded on a suitable polystyrene resin columns absorbing polyphenol compounds of MW between 300 to 600 Da (Mitsubishi). Polyphenol fractions were eluted by a mild KOH solution. Next the fitocomplex was neutralized by filtration on cationic resin at acidic pH. Finally it was vacuum dried and minced to the desired particle size to obtain BPF powder. BPF powder was analysed by HPLC for flavonoid and other polyphenol content. In addition, toxicological analysis were performed including heavy metal, pesticide, phthalate and sinephrine content which revealed the absence of known toxic compounds (data not shown). Standard microbiological tests detected no mycotoxins and bacteria. Finally, 500 mg aliquots of the BPF powder supplemented with 50 mg of ascorbic acid as antioxidant were encapsulated with a semi-automatic gelatin encapsulation device by an authorized manufacturer (Plants, Messina, Italy). Tablets containing 500 mg of maltodextrin supplemented with 50 mg ascorbic acid were prepared for placebo studies. All procedures have been performed according to the European Community Guidelines concerning dietary supplements.
2.3 High Pressure Liquid Chromatography (HPLC) analysis was performed on Fast AGILENT 1200 HPLC system, equipped with DAD detector and ZORBAX Eclipse XDB - C18 column, 50 mm. 2 l of the sample (BPF diluted in 50% ethanol and filtered with 0.2 m filter) was injected on two solvent gradient of water and acetonitrile. Different gradients were used for the determination of flavonoid content or possible fumocumarin contaminats. The flow-rate was 3 ml/min and the column was maintained at 35 ºC. The detector was monitored at 280 nm. Flavonoid and furocumarin pure standards were purchased from Sigma-Aldrich. Brutieridin and melitidin were identified according to Di Donna [14]. . The estimated concentration of 5 main flavonoids was: neoeriocitrin (77700 ppm), naringin (63011 ppm), neohesperidin (72056 ppm), melitidin (15606 ppm) and brutieridin (33202 ppm).
2.4 Animal studies. Male Wistar rats (Harlan, Italy), weighing 180–200 g, were used for the experiments. The animals were kept under stable and controlled conditions (temperature, 22º C; humidity, 60%) with water ad libitum. Animal care was in accordance with Italian regulations on protection of animals used for experimental and other
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scientific purposes (D.M. 116192), as well as with the EC regulations (Off. J. Eur. Communities 1986, L 358).
The effect of BPF on tChol, cLDL, cHDL, tryglycerides and glucose were evaluated in Wistar rats fed a hypercholesterolemic diet composed of a standard diet (Harlan), supplemented with cholesterol 2% (Sigma-Aldrich, pur. 95%), 0.2% cholic acid (min. 98%, Sigma) and 4.8% palm oil . The rats were divided into four groups of 10 animals each:
• Group I (normolipidemic controls) was kept on a standard diet (Harlan) for 30 days.
• Group II (hyperlipidemic controls) received the hypercholesterolemic diet for 30 days.
• Group III and IV received the hypercholesterolemic diet for 30 days; from the 1st to the 30th day, each rat was administered by gavage with BPF (10 and 20 mg/Kg/rat daily, same route).
During the experiment, animals were weighed weekly, and 24 h food consumption was recorded daily. On day 29, rats were individually housed in metabolic cages. At the end of the study, the animals were fasted overnight; blood samples were collected from the penile vein of the rats and serum was separated and stored at -20°C until analyzed. The analysis of totChol, cLDL and cHDL was performed as described below.
Fecal Neutral Sterols and Bile Acids Determination. Neutral sterols and bile acids in the fecal samples of rats fed a hypercholesterolemic diet either untreated or treated with 20 mg/Kg of BPF for 30 consecutive days were extracted according to the method described previously (15). Briefly, feces were freeze–dried for 48 h, minced into fine powder, solubilized in KOH solution and saponified by autoclaving at 120 °C for 1 h; after the addition of NaCl solution, neutral sterols were extracted several times with ethyl ether as described in (24). The upper phases were pooled, evaporated with a rotary evaporator, and dried under nitrogen. The concentration of fecal neutral sterols was determined by gas chromatographic (GC) analysis. Before the GC-analysis, all samples were diluted 1:10 v/v in n-hexane and R-cholestanol was added as internal standard.
2.5 Human studies. We used a randomized, double-blind, placebo-controlled study design that was approved by the Regional Ethical Committee. All the patients participating to the study signed an informed consensus according to the European Legislation. The randomisation scheme was generated by a computerised procedure. Neither the investigators nor the patients knew the randomisation code, block size or the results of the blood lipid concentrations until after the statistical analysis. Furthermore, the statistical analyses were conducted before breaking the randomisation code.
2.5.1 Intervention and procedures
In the screening visit, conducted 321 days before randomisation, all subjects received standardised dietary advice in order to reduce the variability of their baseline lipid values. It was attempted to keep the calorie intake as constant as possible by advising the subjects to reduce their morning and evening meal by approximately the same amount of calories as they received through the study nutrients. There was, however, no detailed diary recording of calorie intake in any of the study phases. Age, gender, and body mass index were matched among all subjects. We recruited 237 patients suffering from hypercholesterolemia from a primary care setting at the Department of Cardiology at University of Rome “Tor Vergata” and at the Vascular Medicine and Atherosclerosis Unit, Cardiology, Villa Salus Medical Center, Marinella di Bruzzano, Italy. Patients enrolled into the study were divided into three groups: group A, 104 subjects with isolated hypercholesterolemia, HC (cLDL levels ≥ 130 mg/dl); group B, 42 patients with hyperlipidemia (hypercholesterolemia and hypertryglyceridemia, HC/HT) and group C, 59 patients with mixed hyperlipidemia and glycemia over 110 mg/dl, HC/HT/HG.
Each group was divided into three subgroups. The first received an oral dose of BPF (500 mg/day; A1, B1 and C1), the second received 1000 mg/day of BPF (A2, B2 and C2) and the third received placebo (APL, BPL, and CPL).
The last group “D” or “post-statin” comprised 32 patients who stopped simvastatin therapy due to muscular pain and a significant elevation of serum creatine-phospho-kinase (CPK). Post-statin patients received 1500 mg/day of BPF daily after a washout period of 60 days and were asked to observe a 1600 Kcal/day diet.
We excluded patients with overt liver disease, chronic renal failure, hypothyroidism, myopathy, uncontrolled diabetes, severe hypertension, stroke, acute coronary events within the preceding 30 days, coronary revascularization within the preceding 3 months, or alcohol abuse. None of the patients took hormone replacement therapy or antioxidant or vitamin supplements during the 2 months preceding our study.
All the 237 patients were given placebo or BPF daily (before meal) for consecutive 30 days treatment period. The patients were seen every 7 days during the study and the compliance was monitored. To monitor possibile side effects, we measured serum asparate aminotransferase, alanine aminotransferase, creatine kinase, blood urea nitrogen and creatinine and blood cell counts before and after therapy. 18 placebo patients and 24 patients taking BPF took beta adrenergic blockers or calcium channel blockers to control blood pressure. These drugs were withheld for ≥48 h before starting the BPF treatment. No additional medications including aspirin or nonsteroidal anti-inflammatory drugs were allowed during the study period to avoid confounding effects.
2.6 Urinary mevalonic acid detection. Twenty-four-hour urine samples were collected from each patient before and after treatment with BPF or placebo. Total volume was recorded, and aliquots were frozen at -20°C. Urinary mevalonic acid (MVA) concentrations were determined by a modified radioenzymatic isotope-dilution method of Popjak [18].
2.7 Endothelial function. Brachial arterial blood pressure was measured with a mercury sphygmomanometer after patients sat rested for 10 min or longer. The mean value of 3 measurements was calculated. Endothelial function was measured from brachial artery flow-mediated vasodilatation with B-mode ultrasound imaging of the brachial artery and by assessing the increase in artery diameter during reactive hyperemia.
2.8 Statystical analysis
In case of homogenous set of data ANOVA was performed to determine the treatment effects, and Dunnett's test was employed as appropriate. In case of heterogenous data, F test was carried out to determine which pairs of groups are heterogenous. This was followed by Cochran's or Student's t tests, as appropriate. The analysis was performed by Statistical analysis add-in component of Microsoft Excel 2007.
3. Results
To assess the nutraceutical proprieties of bergamot flavonoids we concentrated bergamot juice in a form of powder, highly
4
enriched in polyphenols. This was achieved by a standardized procedure of partial purification of bergamot polyphenol fraction (BPF) on a polystyrene resin column. The BPF preparation used in all studies contained 26 - 28 % of 5 main flavonoids. 6 -6,15 g of BPF correspond to 1000 ml bergamot juice in terms of flavonoid content. The content of 5 main flavonoids in a standardized BPF, according to HPLC analysis was as follows: neoeriocitrin (7,7 %±0,4%), naringin (6,3%±0,33%), neohesperidin (7,2% ± 0,35%), melitidin (1,56% ± 0,11%) and brutieridin (3,32% ± 0.17%).
3.1 Animal studies
In animals fed an hypercholesterolemic diet for 30 consecutive days, an elevation of tChol, cLDL and tryglicerides was found compared to baseline values (n= 10; Fig. 1). Administration of BPF (10 and 20 mg/Kg/daily; n=10 for each dose) for 30 days in diet-induced hypercholesterolemic rats produced significant reduction in tChol, cLDL and tryglycerides, an effect accompanied by moderate elevation of cHDL (Fig. 1A). No significant difference in weekly mean body weight, in BPF treatment, versus hyperlipidemic controls was found; moreover, no reduction in 24 h food consumption was observed (data not shown).
Figure 1. Bergamot Polyphenol Fraction (BPF; 10 and 20 mg/Kg/day) reduces total cholesterol (tChol), LDL cholesterol (cLDL) and tryglicerides (TG) and enhances fecal sterols excretion in Wistar rats fed a hyperlipemic diet. A) BPF was administrated daily by gavage (10 and 20 mg/Kg/daily; n=10 for each dose) for 30 days in diet-induced hypercholesterolemic rats. At the day 30 the blood was drawn from penile vein and tChol, cLDL, cHDL and tryglycerides were analysed by standard methods. Statistical analysis was performed as described in material and methods. § - indicates statistically significant change compared to control rats kept on high-cholesterol diet for P < 0.05, * - indicates a statistically significant change compared to normolipemic rats for P < 0.05. B) The animal feces were collected from control (CONTROL), hyperlipemic (HYPERCHOL) and BPF treated hyperlipemic animals (BPF) (20 mg/Kg/day) on the day 30 of the treatment, and sterols and bile acids were extracted with ethyl ether and analysed by gas chromatography as described in material and methods. Statistical analysis was performed as described in material and methods. § - indicates a statistically significant change compared to control rats kept on high-cholesterol diet for p < 0.05.
Moreover, BPF was subjected also to acute (5000 mg/kg) and subchronic animal toxicity studies according to OECD guidelines at 50, 200, 1000 mg/kg per day doses (n=5 for each dose). All performed tests have shown lack of any evident animal toxicity including hematochemical parameters, behavioral tests, histopathological evaluation of liver, kidney and brain tissues (data available as on-line supplementary material, Table S2 and S3).
3.1.1 Fecal Neutral Sterols and Bile Acids.
Fecal output of total bile acids and neutral sterols was found to be enhanced significantly, in the BPF-treated group compared to the
hyperlipemic group (Fig. 1B), suggesting that bergamot extract enhances the epato-biliary turnover and cholesterol consumption as previously suggested for bergamot juice (15).
3.2 Studies in patients
3.2.1 Effect of BPF on serum lipids and glucose in patients.
Treatment with BPF (500, 1000 mg/day) for 30 consecutive days in patients suffering from isolated HC, (group A), mixed hyperlipidemia (HC/HT, group B) and metabolic syndrome (group C) led to a strong reduction in totChol, cLDL and a significant increase in cHDL in majority of subjects. No significant changes in the mean cholesterol parameters were recorded after 30 days maltodextrin treatment for all placebo groups (Table 1).
The significant reduction was also observed for triglycerides levels in patients with HT (Table 2). In particular, group C, comprising 59 metabolic syndrome patients, suffering from HC/HT/HG, responded very well to BPF therapy. The initial mean values such as 278 mg/ml in totChol, 188 mg/ml in cLDL and 267 mg/ml in TG, dropped to 199, 126 and 158 mg/ml, respectively (Fig. 2) after the treatment with high BPF dose. The reduction in cLDL was accompanied by a dose-dependent elevation in cHDL in all patients. In the 10% subjects with the best response the effect on cHDL was very striking -64.6% (Table 1). In addition, metabolic syndrome patients presented a highly significant (P<0.0001) reduction in blood glucose levels (mean value of -18.9% under 500 mg BPF treatment (C1 group) and -22.4% in C2 group). No changes in glucose levels were recorded after 30 days in the placebo group (Fig.2, Table 2). These data suggest that BPF induces a complex effects on metabolic
regulation. As reported in Table 1 the maximum effect for all cholesterol parameters was seen in patients taking 1000 mg BPF daily, but differences between 500 mg and 1000 mg dosage were statistically significant only for cHDL. In addition, we evaluated the efficacy of BPF depending on the metabolic disorder, but the differences were not statistically significant (P>0.05) (Table 1). This suggest that BPF consumption corrects common mechanisms of cholesterol metabolism similarly deregulated in all 3 groups of patients.
3.2.2 BPF as post-statin treatment
To test the responsiveness of the patients in whom the use of statins was forbidden for the appearance of side effects, we recruited 32 patients suffering from statin toxicity. These patients stopped taking statins for 2 months and than they were asked to take 3 capsules of BPF daily (1500 mg). This treatment proved to be very efficient (Fig. 3). 30/32 patients responded well and after 30 days a mean -25% reduction in tChol and -27.6% in cLDL was observed (Table 1), without re-appearance of side effects. This indicates that BPF could
be considered as an alternative treatment in patients with a relevant intolerance to statins.
Neither symptoms nor hematochemical signs of toxicity were found in all patients undergoing the BPF treatment. In 6 patients treated daily with 500 mg and in 11 patients taking 1000 mg of BPF a moderate gastric pyrosis was observed. However, none of the patients taking BPF interrupted the treatment.
5
Table 1. Percent variations in totChol, cLDL, cHDL (%Δ) in patients subjected to the 30 day BPF treatment
1 Mean changes in blood parameters for each group or subgroup of patients were calculated by adding the changes recorded for individual patients and dividing them by the number of patients (Nr P). b For division of all recruited patients in groups and subgroups see section 2.4.1 c No response – Number of patients that show a smaller than 5% reduction in totChol, cLDL or a lower than 5% increase in cHDL after 30 days treatment with BPF. Note that in some cases bigger than 5% changes were recorded in the placebo treated patients. d Best 10% - mean value in the subgroup of the best 10% responders. * Statistically significant difference compared to 500 mg BPF dose.
Figure 2. Bergamot Polyphenol Fraction (BPF) reduces total cholesterol (tChol), LDL-cholesterol (cLDL), tryglicerides (Tryg.) and blood glucose (Bglucose) levels in patients with metabolic syndrome (mixed hyperlipidemia and associated hyperglycemia = HC/HT/HG, group C).The graphs show mean values for indicated blood parameters from 59 patients diagnosed with metabolic syndrome before the BPF treatment with 500 mg/day (group C1, n=20) or 1000 mg/day (group C2, n=19) or placebo (group CPL, n=20). The indicated patients’ blood parameters expressed in mg/dl were analysed on the day 0 (Before) and day 31 (After) of the treatment. Error bars show the standard deviation (S.D.). * - indicates a statistically significant change compared to the placebo group at P <0.0001; # - indicates a statistically significant change between C1 and C2 subgroups at P < 0.05.
Figure 3. Response to BPF in the statin intolerant group of patients (group D). 32 patients who were obliged to stop simavastatin therapy due to its side effects were given 1500 mg/ml BPF daily 60 days after the statin withdrawal. The indicated patients’ blood parameters were recorded in mg/dl (axis Y) before and after the BPF treatment (30 days). Statistical analysis was performed as described in material and methods; * - indicates a statistically significant change compared to the blood parameters before the treatment at P< 0.0001.
.2.3 MVA urinary concentrations in patients
Twenty-four-hour urinary MVA excretion in patients undergoing BPF treatment (500-1000 mg/ day for 30 days) decreased (P<0.05), from a baseline value of 2.12 + 0.32 to 1.56 + 0.28 and 1.34 + 0.26 mol/day, respectively. On discontinuation of drug therapy, urinary MVA levels increased to 1.82 + 0.31 and 1.65 + 0.44 mol/day in the first week, with no further increase seen at 4 weeks (1.88 + 0.26 mol/day). No changes were seen in the group of patients who received placebo over 30 day treatment (1.98 + 0.30 mol).
Patients
groupb
Sub-groupb
Nr
P
BPF dose
daily
(mg)
totChol
cLDL
cHDL
% Δ ± SEM
No resp.c
Best 10%d
% Δ ± SEM
No resp.
Best 10%
% Δ ± SEM
No resp.
Best 10%
A A1 35 500 -20.7 ± 1.9 6 -34.6 -23.0 ± 1.9 4 - 37.2 25.9 ± 2.3 0 50.0 A2 37 1000 -30.9 ± 1.5 2 -40.0 -38.6 ± 1.5 0 -49.1 39.0 ± 2.8 * 0 68.6
APL
32
0
-0.4 ± 0.4
32
-
-1.7 ± 0.5
27
-
0.5 ± 1.1
22
-
B B1 14 500 -21.9 ± 1.8 1 -28.3 -25.3 ± 2.0 0 -34.6 17.3 ± 1.4 0 26.7 B2 14 1000 -27.7 ± 3.4 2 -41.5 -33.4 ± 3.9 2 -43.6 35.8 ± 4.2 * 0 66.7
BPL
14
0
-0.5 ± 0.5
14
-
-0.5 ± 0.7
13
-
-1.3 ± 1.8
11
-
C C1 20 500 -24.7 ± 2.6 2 -41.7 -26.8 ± 3.6 1 -53.6 16.5 ± 1.6 0 42.9 C2 19 1000 -28.1 ± 2.6 1 -41.1 -33.2 ± 3.0 1 -47.0 29.6 ± 1.8 * 0 64.6
CPL
20
0
0.5 ± 0.5
20
-
-0.9 ± 1.4
18
-
2.9 ± 2.0
15
-
D - 32 1500 -25.0 ± 1.6 2 -39.8 -27.6 ± 0.5 0 -32.4 23.8 ± 1.7 0 41.1
A+B
+C - 69 500 -21.8 ± 1.4 9 -37.8 -24.1 ± 1.5 5 -45.0 22.3 ± 1.3 0 -48.6 - 70 1000 -29.4 ± 1.3 5 -40.6 -36.0 ± 1.4 * 3 -47.9 40.1 ± 1.9 * 0 -66.4 - 66 0 -0.1± 0.3 66 - -1.1 ± 0.5 58 - 1.2 ± 0.9 48 -
6
Table 2. Percent variations in TG and blood glucose in patients suffering from HC/HT
and HC/HT/HG subjected to the 30 day BPF treatment
1 Mean changes in blood parameters for each group or subgroup of patients were calculated as for Table 1.
b, c, d for legend see the legend to the Table 1.
3.2.4 Reactive vasodilatation.
Before starting the BPF treatment, flow-mediated vasodilation was found reduced in patients suffering from isolated HC or mixed HC/HT (group A and B, C), being the latter effect more pronounced in the subgroup of patients with moderate elevation of serum glucose (group C) (Fig. 4). After 30 days of BPF treatment with (5000 and 1000 mg/daily for 30 consecutive days), flow-mediated vasodilation increased significantly, whereas no changes have been observed in patients receiving placebo (Fig. 4). This suggests that BPF is able to improve the impaired endothelium-mediated vasodilation in hyperlipidemic patients with or without hyperglycemia.
Figure 4. The effect of BPF (500 and 1000 mg/day) on reactive vasodilation in patients suffering from isolated hypercholesterolemia (HC) or mixed hyperlipidemia (HC/HT) and associated hyperglycemia (HC/HT/HG). After 30 days of treatment with placebo or BPF (500 or 1000 mg/day), endothelial function was assessed in 3 groups of patients (14 to 30 patients per group). Flow-mediated vasodilation was measured from brachial artery diameter during reactive hyperemia. * indicates a statistically significant change compared to the respective placebo group of patients at P < 0.05; # indicates statistically significant changes compared to the 500 mg BPF group at P < 0.01; + indicates statistically significant changes compared to HC group at P < 0.01.
4. Discussion
Based on the data presented in this study, the BPF treatment leads to a significant reduction of coronary artery disease risk and other cardiovascular complications according to the headlines of National Cholesterol Education Programme ( NCEP – ATP III ) in a rat model of diet-induced hyperlipemia. This effect was also confirmed in human studies carried out in patients with pure or mixed hyperlipemia either ot not associated with hyperglycaemia (metabolic syndrome).Scientific evidence obtained with Citrus flavonoids and other non nutritive constituents of Citrus fruits, provides some mechanistic explanations for the beneficial effects of bergamot juice. Previous data showed that Citrus peel extracts, rich in pectins and flavonoids, cause lowering of cholesterol levels by modulating hepatic HMG-CoA levels, possibly by binding bile acids and increasing the turnover rate of blood and liver cholesterol [19-21]. Since BJ was showed to enhance the excretion of fecal sterols in rats [15], such a mechanism may contribute to its hypolipemic and hypoglycemic effect found in patients under BPF treatment. Special contribution to the hypolipemic response seems to be related to the modulatory properties of flavonone glycoside components of BPF, in particular naringin and neohesperidin. Indeed, evidence exists that dietary hesperetin reduces hepatic TG accumulation and this is associated with the reduced activity of TG synthetic enzymes, such as phosphatidate phosphohydrolase [22]. In addition, in vitro studies suggest that naringenin and hesperetin decrease the availability of lipids for assembly of apoB-containing lipoproteins, an effect mediated by reduced activities of of acyl CoA:cholesterol acyltransferases (ACAT) [23].Importantly, BPF is rich in buteridine and melitidine, which are 3-hydroxy-3-methylglutaryl derivatives of hesperetin and naringenin, respectively. Given the structural similarity to HMG-CoA reductase substrate, these compounds have been proposed to possess the statin-like proprieties, by selective inhibition of HMG-CoA reductase [14]. In addition, the classical glycoside derivative of naringenin, which is naringin has been shown to inhibit hepatic HMG-CoA reductase [24]. Therefore it is likely that melitidine and brutieridine in concert with naringin and other flavonone glycosides might be responsible for the striking potency of BPF in reducing cholesterol levels. The direct action of BPF on HMG-CoA reductase activity is suggested by a significant mevalonate reduction in the urine of patients under BPF treatment in our study. The oxidative stress and the inflammatory processes in the endothelium have been shown to reduce reactive NO-dependent vasodilation. Well documented antioxidant and anti-inflammatory mechanisms
Patients
Group b
Sub-group
Nr P
BPF dose
daily
(mg)
Triglycerides
Glucose
% Δ ± SEM
No responsec
Best 10%d
% Δ ± SEM
No response
Best 10%
B B1 14 500 -28.2 ± 3.9 2 -46.8 - - -34.6 B2 14 1000 -37.9 ± 3.3 1 -47.9 - - -43.6
BPL
14
0
0.1 ± 0.5
14
-
-
-
-
C C1 20 500 -32.7 ± 2.5 0 -41.6 -18.9 ± 1.2 0 -27.1 C2 19 1000 -41.0 ± 2.6 1 -49.6 -22.4 ± 1.0 0 -32.2
CPL
20
0
0.0 ± 0.6
19
-
-0.5 ± 0.7
20
-
7
regulated by Citrus flavonoids, such as increasing superoxide dismutase and catalase activities and protecting the plasma vitamin E (25) may well attenutate overproduction of oxygen reactive species in the vascular wall thereby restoring the imbalanced endothelial function, as we observed in our patients by studying reactive vasodilation (Fig. 4). Another potential benefit from BPF treatment is related to its hypoglycemic activity (Fig.2). Among few mechanistic studies on hypoglycemic effects of flavonoids, it has been shown that naringenin, similarly to other polyphenols significantly increased AMP kinase (AMPK) activity and glucose uptake in muscle cells and liver [26, 27]. The hypoglycaemic activity of naringenin in vivo can be more complex and may depend on multilevel effects on lipid metabolism that lead to increased insulin sensitivity and glucose tolerance as shown in animal models of metabolic syndrome [28].
On the basis of our data, BPF oral supplements contribute to lowering plasma cholesterol and lipids in a rat model of diet-induced hyperlipemia and in patients, in a range of potency comparable with low dose statins. Thus BPF offers a safe alternative for patients suffering from statins toxicity. In addition, the possibility to reduce blood glucose by 15%-25% suggest a phytotherapeutic approach to control the prediabetic states in patients with metabolic syndrome.
Aknowledgments
We thank Domenico Malara, Malara s.r.l., Reggio Calabria, Italy and Franco Nava, Piacenza, Italy for developing BPF purification procedure. We are grateful to Artur Janda, Jagellonian University, Cracow, Poland, for help with the statistical analysis and Capua s.r.l, Reggio Calabria, Italy, for technical support.
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[17] Mollace, V., Ragusa, S., Sacco, I., Muscoli, C., Sculco, F., Visalli, V., et al., The protective effect of bergamot oil extract on lecitine-like oxyLDL receptor-1 expression in balloon injury-related neointima formation, J Cardiovasc Pharmacol Ther. 13 (2008) 120-9.
[18] Pappu, A.S., Illingworth, D.R., Contrasting effects of lovastatin and cholestyramine on low-density lipoprotein cholesterol and 24-hour urinary mevalonate excretion in patients with heterozygous familial hypercholesterolemia, J Lab Clin Med. 114 (1989) 554-62.
[19] Bok, S.H., Lee, S.H., Park, Y.B., Bae, K.H., Son, K.H., Jeong, T.S., et al., Plasma and hepatic cholesterol and hepatic activities of 3-hydroxy-3-methyl-glutaryl-CoA reductase and acyl CoA: cholesterol transferase are lower in rats fed citrus peel extract or a mixture of citrus bioflavonoids, J Nutr. 129 (1999) 1182-5.
[20] Marounek, M., Volek, Z., Synytsya, A., Copikova, J., Effect of pectin and amidated pectin on cholesterol homeostasis and cecal metabolism in rats fed a high-cholesterol diet, Physiol Res. 56 (2007) 433-42.
[21] Terpstra, A.H., Lapre, J.A., de Vries, H.T., Beynen, A.C., Dietary pectin with high viscosity lowers plasma and liver cholesterol concentration and plasma cholesteryl ester transfer protein activity in hamsters, J Nutr. 128 (1998) 1944-9.
[22] Cha, J.Y., Cho, Y.S., Kim, I., Anno, T., Rahman, S.M., Yanagita, T., Effect of hesperetin, a citrus flavonoid, on the liver triacylglycerol content and phosphatidate phosphohydrolase activity in orotic acid-fed rats, Plant Foods Hum Nutr. 56 (2001) 349-58.
[23] Wilcox, L.J., Borradaile, N.M., de Dreu, L.E., Huff, M.W., Secretion of hepatocyte apoB is inhibited by the flavonoids, naringenin and hesperetin, via reduced activity and expression of ACAT2 and MTP, J Lipid Res. 42 (2001) 725-34.
[24] Kim, H.J., Oh, G.T., Park, Y.B., Lee, M.K., Seo, H.J., Choi, M.S., Naringin alters the cholesterol biosynthesis and
8
antioxidant enzyme activities in LDL receptor-knockout mice under cholesterol fed condition, Life Sci. 74 (2004) 1621-34.
[25] Jeon, S.M., Bok, S.H., Jang, M.K., Lee, M.K., Nam, K.T., Park, Y.B., et al., Antioxidative activity of naringin and lovastatin in high cholesterol-fed rabbits, Life Sci. 69 (2001) 2855-66.
[26] Hwang, J.T., Kwon, D.Y., Yoon, S.H., AMP-activated protein kinase: a potential target for the diseases prevention by natural occurring polyphenols, N Biotechnol. 26 (2009) 17-22.
[27] Zygmunt, K., Faubert, B., Macneil, J., Tsiani, E., Naringenin, a citrus flavonoid, increases muscle cell glucose uptake via AMPK, Biochem Biophys Res Commun.
[28] Mulvihill, E.E., Allister, E.M., Sutherland, B.G., Telford, D.E., Sawyez, C.G., Edwards, J.Y., et al., Naringenin prevents dyslipidemia, apolipoprotein B overproduction, and hyperinsulinemia in LDL receptor-null mice with diet-induced insulin resistance, Diabetes. 58 (2009) 2198-210.
1.Introduction
Hyperlipidemia or hypercholesterolemia are important
risk factors for the development of atherosclerosis and
coronary artery disease [1, 2]. Main pathogenic blood
parameters are increased concentrations of cholesterol bound
to Low-density Lipoprotein (cLDL), total blood cholesterol
(totChol) and triglycerides (TG). Conditions of insulin
resistance such as impaired glucose tolerance or
"prediabetes" are also characterized by a high risk of
cardiovascular diseases (CVD) [3]. Majority of therapeutic
protocols rely on drugs that belong to statin family. Statins
inhibit the activity
of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase,
which catalyzes the rate-limiting step in mevalonate
biosynthesis, a key intermediate in cholesterol metabolism.
This is associated to a
decrease in totChol and a switch from cLDL to High-density
Lipoprotein (cHDL) fraction. Despite the significant clinical
benefits provided by statins [1], many patients, in particular
those with metabolic syndrome, do not achieve their
recommended low-density and high-density lipoprotein
(LDL, HDL) cholesterol target goals with statins [3].
Moreover, the use of statins is forbidden in more than 40% of
patients eligible for this therapeutic approach, mostly for the
occurrence of side effects including myalgia, myopathy or
liver disease and rhabdomyolysis in more severe cases [4, 5].
This limits the use of statins and suggest the need of
alternative therapeutic approaches. Experimental and
epidemiological studies suggest that dietary polyphenols, in
particular flavonoids, may play a role in ameliorating
atherosclerosis and pleiotropic anti-oxidative and
nflammatory effects have been proposed as an underlying
mechanism [6-8]. Yet these compounds when analysed
separately show rather weak cholesterol-lowering effects in
animal experiments [9]. In contrast, some natural
compositions of plant polyphenols seem to possess a good
hypolipemic activity both in animal models and in human
studies, suggesting that synergistic effects of individual
compounds may play a central role in the beneficial effects
[6, 8, 9]. This observation provides the rationale for the
identification of optimally synergizing elements and their
plant sources with the best ability to prevent hyperlipidemia
and cardiovascular complications.
Bergamot (Citrus Bergamia) is an endemic plant of Calabrian
region in Southern Italy with an unique profile of flavonoid
and flavonoid glycosides present in its juice and albedo, such
as neoeriocitrin, neohesperidin, naringin, rutin, neodesmin,
rhoifolin and poncirin. Bergamot differs from other Citrus
fruits not only because of the composition of its flavonoids,
but also because of their particularly high content [10, 11].
Among them naringin, present also in grapefruit, have
already been reported to be active in animal models of
atherosclerosis [12], while neoeriocitrin and rutin have been
shown to inhibit LDL oxidation [13]. Importantly, bergamot
juice (BJ) is rich in neohesperidosides of hesperetin and
naringenin, such as melitidine and brutieridine. These
flavonoids possess 3-hydroxy-3-methylglutaryl moiety with a
structural similarity to the natural substrate of HMG-CoA
reductase and are likely to exhibit statin-like proprieties [14].
Experimental evidence obtained in animal models of dietinduced
hypercholesterolemia and renal damage [15, 16] as
well as in the rat model of mechanical stress-induced vascular
2
injury [17] supports the hypolipemic and vasoprotective effects of bergamot constituents. However, the therapeutic potential of bergamot has never been investigated in human studies, even though the traditional use of BJ in Calabrian region suggested since long time its potential beneficial use in counteracting atherosclerosis.
The present study has been carried out to verify, in a rat model of diet-induced hyperlipemia, the effect of bergamot-derived polyphenolic fraction (BPF) on totChol, cLDL, cHDL, TG and blood glucose. This effect was also investigated by givin orally BPF for 30 consecutive days in 237 patients suffering from isolated or mixed hyperlipemia either associated or not with hyperglycaemia. In
patients we have also studied the possible contribution of HMG-CoA reductase inhibition by BPF to its hypolipemic activity and this effect was also compared with changes of reactive vasodilation to verify the potential beneficial effect of BPF in modulating the imbalanced endothelial reactivity.
2. Materials and methods
2.1 Plant Material. C. bergamia Risso & Poiteau fruits were collected from plantations located between Reggio Calabria and Bianco, in 90 Km long costal area in South Italy.
2.2 Preparation of BPF. BJ was obtained from peeled-off fruits by industrial pressing and squeezing. The juice was oil fraction-depleted by stripping, clarified by ultra-filtration and loaded on a suitable polystyrene resin columns absorbing polyphenol compounds of MW between 300 to 600 Da (Mitsubishi). Polyphenol fractions were eluted by a mild KOH solution. Next the fitocomplex was neutralized by filtration on cationic resin at acidic pH. Finally it was vacuum dried and minced to the desired particle size to obtain BPF powder. BPF powder was analysed by HPLC for flavonoid and other polyphenol content. In addition, toxicological analysis were performed including heavy metal, pesticide, phthalate and sinephrine content which revealed the absence of known toxic compounds (data not shown). Standard microbiological tests detected no mycotoxins and bacteria. Finally, 500 mg aliquots of the BPF powder supplemented with 50 mg of ascorbic acid as antioxidant were encapsulated with a semi-automatic gelatin encapsulation device by an authorized manufacturer (Plants, Messina, Italy). Tablets containing 500 mg of maltodextrin supplemented with 50 mg ascorbic acid were prepared for placebo studies. All procedures have been performed according to the European Community Guidelines concerning dietary supplements.
2.3 High Pressure Liquid Chromatography (HPLC) analysis was performed on Fast AGILENT 1200 HPLC system, equipped with DAD detector and ZORBAX Eclipse XDB - C18 column, 50 mm. 2 l of the sample (BPF diluted in 50% ethanol and filtered with 0.2 m filter) was injected on two solvent gradient of water and acetonitrile. Different gradients were used for the determination of flavonoid content or possible fumocumarin contaminats. The flow-rate was 3 ml/min and the column was maintained at 35 ºC. The detector was monitored at 280 nm. Flavonoid and furocumarin pure standards were purchased from Sigma-Aldrich. Brutieridin and melitidin were identified according to Di Donna [14]. . The estimated concentration of 5 main flavonoids was: neoeriocitrin (77700 ppm), naringin (63011 ppm), neohesperidin (72056 ppm), melitidin (15606 ppm) and brutieridin (33202 ppm).
2.4 Animal studies. Male Wistar rats (Harlan, Italy), weighing 180–200 g, were used for the experiments. The animals were kept under stable and controlled conditions (temperature, 22º C; humidity, 60%) with water ad libitum. Animal care was in accordance with Italian regulations on protection of animals used for experimental and other
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scientific purposes (D.M. 116192), as well as with the EC regulations (Off. J. Eur. Communities 1986, L 358).
The effect of BPF on tChol, cLDL, cHDL, tryglycerides and glucose were evaluated in Wistar rats fed a hypercholesterolemic diet composed of a standard diet (Harlan), supplemented with cholesterol 2% (Sigma-Aldrich, pur. 95%), 0.2% cholic acid (min. 98%, Sigma) and 4.8% palm oil . The rats were divided into four groups of 10 animals each:
• Group I (normolipidemic controls) was kept on a standard diet (Harlan) for 30 days.
• Group II (hyperlipidemic controls) received the hypercholesterolemic diet for 30 days.
• Group III and IV received the hypercholesterolemic diet for 30 days; from the 1st to the 30th day, each rat was administered by gavage with BPF (10 and 20 mg/Kg/rat daily, same route).
During the experiment, animals were weighed weekly, and 24 h food consumption was recorded daily. On day 29, rats were individually housed in metabolic cages. At the end of the study, the animals were fasted overnight; blood samples were collected from the penile vein of the rats and serum was separated and stored at -20°C until analyzed. The analysis of totChol, cLDL and cHDL was performed as described below.
Fecal Neutral Sterols and Bile Acids Determination. Neutral sterols and bile acids in the fecal samples of rats fed a hypercholesterolemic diet either untreated or treated with 20 mg/Kg of BPF for 30 consecutive days were extracted according to the method described previously (15). Briefly, feces were freeze–dried for 48 h, minced into fine powder, solubilized in KOH solution and saponified by autoclaving at 120 °C for 1 h; after the addition of NaCl solution, neutral sterols were extracted several times with ethyl ether as described in (24). The upper phases were pooled, evaporated with a rotary evaporator, and dried under nitrogen. The concentration of fecal neutral sterols was determined by gas chromatographic (GC) analysis. Before the GC-analysis, all samples were diluted 1:10 v/v in n-hexane and R-cholestanol was added as internal standard.
2.5 Human studies. We used a randomized, double-blind, placebo-controlled study design that was approved by the Regional Ethical Committee. All the patients participating to the study signed an informed consensus according to the European Legislation. The randomisation scheme was generated by a computerised procedure. Neither the investigators nor the patients knew the randomisation code, block size or the results of the blood lipid concentrations until after the statistical analysis. Furthermore, the statistical analyses were conducted before breaking the randomisation code.
2.5.1 Intervention and procedures
In the screening visit, conducted 321 days before randomisation, all subjects received standardised dietary advice in order to reduce the variability of their baseline lipid values. It was attempted to keep the calorie intake as constant as possible by advising the subjects to reduce their morning and evening meal by approximately the same amount of calories as they received through the study nutrients. There was, however, no detailed diary recording of calorie intake in any of the study phases. Age, gender, and body mass index were matched among all subjects. We recruited 237 patients suffering from hypercholesterolemia from a primary care setting at the Department of Cardiology at University of Rome “Tor Vergata” and at the Vascular Medicine and Atherosclerosis Unit, Cardiology, Villa Salus Medical Center, Marinella di Bruzzano, Italy. Patients enrolled into the study were divided into three groups: group A, 104 subjects with isolated hypercholesterolemia, HC (cLDL levels ≥ 130 mg/dl); group B, 42 patients with hyperlipidemia (hypercholesterolemia and hypertryglyceridemia, HC/HT) and group C, 59 patients with mixed hyperlipidemia and glycemia over 110 mg/dl, HC/HT/HG.
Each group was divided into three subgroups. The first received an oral dose of BPF (500 mg/day; A1, B1 and C1), the second received 1000 mg/day of BPF (A2, B2 and C2) and the third received placebo (APL, BPL, and CPL).
The last group “D” or “post-statin” comprised 32 patients who stopped simvastatin therapy due to muscular pain and a significant elevation of serum creatine-phospho-kinase (CPK). Post-statin patients received 1500 mg/day of BPF daily after a washout period of 60 days and were asked to observe a 1600 Kcal/day diet.
We excluded patients with overt liver disease, chronic renal failure, hypothyroidism, myopathy, uncontrolled diabetes, severe hypertension, stroke, acute coronary events within the preceding 30 days, coronary revascularization within the preceding 3 months, or alcohol abuse. None of the patients took hormone replacement therapy or antioxidant or vitamin supplements during the 2 months preceding our study.
All the 237 patients were given placebo or BPF daily (before meal) for consecutive 30 days treatment period. The patients were seen every 7 days during the study and the compliance was monitored. To monitor possibile side effects, we measured serum asparate aminotransferase, alanine aminotransferase, creatine kinase, blood urea nitrogen and creatinine and blood cell counts before and after therapy. 18 placebo patients and 24 patients taking BPF took beta adrenergic blockers or calcium channel blockers to control blood pressure. These drugs were withheld for ≥48 h before starting the BPF treatment. No additional medications including aspirin or nonsteroidal anti-inflammatory drugs were allowed during the study period to avoid confounding effects.
2.6 Urinary mevalonic acid detection. Twenty-four-hour urine samples were collected from each patient before and after treatment with BPF or placebo. Total volume was recorded, and aliquots were frozen at -20°C. Urinary mevalonic acid (MVA) concentrations were determined by a modified radioenzymatic isotope-dilution method of Popjak [18].
2.7 Endothelial function. Brachial arterial blood pressure was measured with a mercury sphygmomanometer after patients sat rested for 10 min or longer. The mean value of 3 measurements was calculated. Endothelial function was measured from brachial artery flow-mediated vasodilatation with B-mode ultrasound imaging of the brachial artery and by assessing the increase in artery diameter during reactive hyperemia.
2.8 Statystical analysis
In case of homogenous set of data ANOVA was performed to determine the treatment effects, and Dunnett's test was employed as appropriate. In case of heterogenous data, F test was carried out to determine which pairs of groups are heterogenous. This was followed by Cochran's or Student's t tests, as appropriate. The analysis was performed by Statistical analysis add-in component of Microsoft Excel 2007.
3. Results
To assess the nutraceutical proprieties of bergamot flavonoids we concentrated bergamot juice in a form of powder, highly
4
enriched in polyphenols. This was achieved by a standardized procedure of partial purification of bergamot polyphenol fraction (BPF) on a polystyrene resin column. The BPF preparation used in all studies contained 26 - 28 % of 5 main flavonoids. 6 -6,15 g of BPF correspond to 1000 ml bergamot juice in terms of flavonoid content. The content of 5 main flavonoids in a standardized BPF, according to HPLC analysis was as follows: neoeriocitrin (7,7 %±0,4%), naringin (6,3%±0,33%), neohesperidin (7,2% ± 0,35%), melitidin (1,56% ± 0,11%) and brutieridin (3,32% ± 0.17%).
3.1 Animal studies
In animals fed an hypercholesterolemic diet for 30 consecutive days, an elevation of tChol, cLDL and tryglicerides was found compared to baseline values (n= 10; Fig. 1). Administration of BPF (10 and 20 mg/Kg/daily; n=10 for each dose) for 30 days in diet-induced hypercholesterolemic rats produced significant reduction in tChol, cLDL and tryglycerides, an effect accompanied by moderate elevation of cHDL (Fig. 1A). No significant difference in weekly mean body weight, in BPF treatment, versus hyperlipidemic controls was found; moreover, no reduction in 24 h food consumption was observed (data not shown).
Figure 1. Bergamot Polyphenol Fraction (BPF; 10 and 20 mg/Kg/day) reduces total cholesterol (tChol), LDL cholesterol (cLDL) and tryglicerides (TG) and enhances fecal sterols excretion in Wistar rats fed a hyperlipemic diet. A) BPF was administrated daily by gavage (10 and 20 mg/Kg/daily; n=10 for each dose) for 30 days in diet-induced hypercholesterolemic rats. At the day 30 the blood was drawn from penile vein and tChol, cLDL, cHDL and tryglycerides were analysed by standard methods. Statistical analysis was performed as described in material and methods. § - indicates statistically significant change compared to control rats kept on high-cholesterol diet for P < 0.05, * - indicates a statistically significant change compared to normolipemic rats for P < 0.05. B) The animal feces were collected from control (CONTROL), hyperlipemic (HYPERCHOL) and BPF treated hyperlipemic animals (BPF) (20 mg/Kg/day) on the day 30 of the treatment, and sterols and bile acids were extracted with ethyl ether and analysed by gas chromatography as described in material and methods. Statistical analysis was performed as described in material and methods. § - indicates a statistically significant change compared to control rats kept on high-cholesterol diet for p < 0.05.
Moreover, BPF was subjected also to acute (5000 mg/kg) and subchronic animal toxicity studies according to OECD guidelines at 50, 200, 1000 mg/kg per day doses (n=5 for each dose). All performed tests have shown lack of any evident animal toxicity including hematochemical parameters, behavioral tests, histopathological evaluation of liver, kidney and brain tissues (data available as on-line supplementary material, Table S2 and S3).
3.1.1 Fecal Neutral Sterols and Bile Acids.
Fecal output of total bile acids and neutral sterols was found to be enhanced significantly, in the BPF-treated group compared to the
hyperlipemic group (Fig. 1B), suggesting that bergamot extract enhances the epato-biliary turnover and cholesterol consumption as previously suggested for bergamot juice (15).
3.2 Studies in patients
3.2.1 Effect of BPF on serum lipids and glucose in patients.
Treatment with BPF (500, 1000 mg/day) for 30 consecutive days in patients suffering from isolated HC, (group A), mixed hyperlipidemia (HC/HT, group B) and metabolic syndrome (group C) led to a strong reduction in totChol, cLDL and a significant increase in cHDL in majority of subjects. No significant changes in the mean cholesterol parameters were recorded after 30 days maltodextrin treatment for all placebo groups (Table 1).
The significant reduction was also observed for triglycerides levels in patients with HT (Table 2). In particular, group C, comprising 59 metabolic syndrome patients, suffering from HC/HT/HG, responded very well to BPF therapy. The initial mean values such as 278 mg/ml in totChol, 188 mg/ml in cLDL and 267 mg/ml in TG, dropped to 199, 126 and 158 mg/ml, respectively (Fig. 2) after the treatment with high BPF dose. The reduction in cLDL was accompanied by a dose-dependent elevation in cHDL in all patients. In the 10% subjects with the best response the effect on cHDL was very striking -64.6% (Table 1). In addition, metabolic syndrome patients presented a highly significant (P<0.0001) reduction in blood glucose levels (mean value of -18.9% under 500 mg BPF treatment (C1 group) and -22.4% in C2 group). No changes in glucose levels were recorded after 30 days in the placebo group (Fig.2, Table 2). These data suggest that BPF induces a complex effects on metabolic
regulation. As reported in Table 1 the maximum effect for all cholesterol parameters was seen in patients taking 1000 mg BPF daily, but differences between 500 mg and 1000 mg dosage were statistically significant only for cHDL. In addition, we evaluated the efficacy of BPF depending on the metabolic disorder, but the differences were not statistically significant (P>0.05) (Table 1). This suggest that BPF consumption corrects common mechanisms of cholesterol metabolism similarly deregulated in all 3 groups of patients.
3.2.2 BPF as post-statin treatment
To test the responsiveness of the patients in whom the use of statins was forbidden for the appearance of side effects, we recruited 32 patients suffering from statin toxicity. These patients stopped taking statins for 2 months and than they were asked to take 3 capsules of BPF daily (1500 mg). This treatment proved to be very efficient (Fig. 3). 30/32 patients responded well and after 30 days a mean -25% reduction in tChol and -27.6% in cLDL was observed (Table 1), without re-appearance of side effects. This indicates that BPF could
be considered as an alternative treatment in patients with a relevant intolerance to statins.
Neither symptoms nor hematochemical signs of toxicity were found in all patients undergoing the BPF treatment. In 6 patients treated daily with 500 mg and in 11 patients taking 1000 mg of BPF a moderate gastric pyrosis was observed. However, none of the patients taking BPF interrupted the treatment.
5
Table 1. Percent variations in totChol, cLDL, cHDL (%Δ) in patients subjected to the 30 day BPF treatment
1 Mean changes in blood parameters for each group or subgroup of patients were calculated by adding the changes recorded for individual patients and dividing them by the number of patients (Nr P). b For division of all recruited patients in groups and subgroups see section 2.4.1 c No response – Number of patients that show a smaller than 5% reduction in totChol, cLDL or a lower than 5% increase in cHDL after 30 days treatment with BPF. Note that in some cases bigger than 5% changes were recorded in the placebo treated patients. d Best 10% - mean value in the subgroup of the best 10% responders. * Statistically significant difference compared to 500 mg BPF dose.
Figure 2. Bergamot Polyphenol Fraction (BPF) reduces total cholesterol (tChol), LDL-cholesterol (cLDL), tryglicerides (Tryg.) and blood glucose (Bglucose) levels in patients with metabolic syndrome (mixed hyperlipidemia and associated hyperglycemia = HC/HT/HG, group C).The graphs show mean values for indicated blood parameters from 59 patients diagnosed with metabolic syndrome before the BPF treatment with 500 mg/day (group C1, n=20) or 1000 mg/day (group C2, n=19) or placebo (group CPL, n=20). The indicated patients’ blood parameters expressed in mg/dl were analysed on the day 0 (Before) and day 31 (After) of the treatment. Error bars show the standard deviation (S.D.). * - indicates a statistically significant change compared to the placebo group at P <0.0001; # - indicates a statistically significant change between C1 and C2 subgroups at P < 0.05.
Figure 3. Response to BPF in the statin intolerant group of patients (group D). 32 patients who were obliged to stop simavastatin therapy due to its side effects were given 1500 mg/ml BPF daily 60 days after the statin withdrawal. The indicated patients’ blood parameters were recorded in mg/dl (axis Y) before and after the BPF treatment (30 days). Statistical analysis was performed as described in material and methods; * - indicates a statistically significant change compared to the blood parameters before the treatment at P< 0.0001.
.2.3 MVA urinary concentrations in patients
Twenty-four-hour urinary MVA excretion in patients undergoing BPF treatment (500-1000 mg/ day for 30 days) decreased (P<0.05), from a baseline value of 2.12 + 0.32 to 1.56 + 0.28 and 1.34 + 0.26 mol/day, respectively. On discontinuation of drug therapy, urinary MVA levels increased to 1.82 + 0.31 and 1.65 + 0.44 mol/day in the first week, with no further increase seen at 4 weeks (1.88 + 0.26 mol/day). No changes were seen in the group of patients who received placebo over 30 day treatment (1.98 + 0.30 mol).
Patients
groupb
Sub-groupb
Nr
P
BPF dose
daily
(mg)
totChol
cLDL
cHDL
% Δ ± SEM
No resp.c
Best 10%d
% Δ ± SEM
No resp.
Best 10%
% Δ ± SEM
No resp.
Best 10%
A A1 35 500 -20.7 ± 1.9 6 -34.6 -23.0 ± 1.9 4 - 37.2 25.9 ± 2.3 0 50.0 A2 37 1000 -30.9 ± 1.5 2 -40.0 -38.6 ± 1.5 0 -49.1 39.0 ± 2.8 * 0 68.6
APL
32
0
-0.4 ± 0.4
32
-
-1.7 ± 0.5
27
-
0.5 ± 1.1
22
-
B B1 14 500 -21.9 ± 1.8 1 -28.3 -25.3 ± 2.0 0 -34.6 17.3 ± 1.4 0 26.7 B2 14 1000 -27.7 ± 3.4 2 -41.5 -33.4 ± 3.9 2 -43.6 35.8 ± 4.2 * 0 66.7
BPL
14
0
-0.5 ± 0.5
14
-
-0.5 ± 0.7
13
-
-1.3 ± 1.8
11
-
C C1 20 500 -24.7 ± 2.6 2 -41.7 -26.8 ± 3.6 1 -53.6 16.5 ± 1.6 0 42.9 C2 19 1000 -28.1 ± 2.6 1 -41.1 -33.2 ± 3.0 1 -47.0 29.6 ± 1.8 * 0 64.6
CPL
20
0
0.5 ± 0.5
20
-
-0.9 ± 1.4
18
-
2.9 ± 2.0
15
-
D - 32 1500 -25.0 ± 1.6 2 -39.8 -27.6 ± 0.5 0 -32.4 23.8 ± 1.7 0 41.1
A+B
+C - 69 500 -21.8 ± 1.4 9 -37.8 -24.1 ± 1.5 5 -45.0 22.3 ± 1.3 0 -48.6 - 70 1000 -29.4 ± 1.3 5 -40.6 -36.0 ± 1.4 * 3 -47.9 40.1 ± 1.9 * 0 -66.4 - 66 0 -0.1± 0.3 66 - -1.1 ± 0.5 58 - 1.2 ± 0.9 48 -
6
Table 2. Percent variations in TG and blood glucose in patients suffering from HC/HT
and HC/HT/HG subjected to the 30 day BPF treatment
1 Mean changes in blood parameters for each group or subgroup of patients were calculated as for Table 1.
b, c, d for legend see the legend to the Table 1.
3.2.4 Reactive vasodilatation.
Before starting the BPF treatment, flow-mediated vasodilation was found reduced in patients suffering from isolated HC or mixed HC/HT (group A and B, C), being the latter effect more pronounced in the subgroup of patients with moderate elevation of serum glucose (group C) (Fig. 4). After 30 days of BPF treatment with (5000 and 1000 mg/daily for 30 consecutive days), flow-mediated vasodilation increased significantly, whereas no changes have been observed in patients receiving placebo (Fig. 4). This suggests that BPF is able to improve the impaired endothelium-mediated vasodilation in hyperlipidemic patients with or without hyperglycemia.
Figure 4. The effect of BPF (500 and 1000 mg/day) on reactive vasodilation in patients suffering from isolated hypercholesterolemia (HC) or mixed hyperlipidemia (HC/HT) and associated hyperglycemia (HC/HT/HG). After 30 days of treatment with placebo or BPF (500 or 1000 mg/day), endothelial function was assessed in 3 groups of patients (14 to 30 patients per group). Flow-mediated vasodilation was measured from brachial artery diameter during reactive hyperemia. * indicates a statistically significant change compared to the respective placebo group of patients at P < 0.05; # indicates statistically significant changes compared to the 500 mg BPF group at P < 0.01; + indicates statistically significant changes compared to HC group at P < 0.01.
4. Discussion
Based on the data presented in this study, the BPF treatment leads to a significant reduction of coronary artery disease risk and other cardiovascular complications according to the headlines of National Cholesterol Education Programme ( NCEP – ATP III ) in a rat model of diet-induced hyperlipemia. This effect was also confirmed in human studies carried out in patients with pure or mixed hyperlipemia either ot not associated with hyperglycaemia (metabolic syndrome).Scientific evidence obtained with Citrus flavonoids and other non nutritive constituents of Citrus fruits, provides some mechanistic explanations for the beneficial effects of bergamot juice. Previous data showed that Citrus peel extracts, rich in pectins and flavonoids, cause lowering of cholesterol levels by modulating hepatic HMG-CoA levels, possibly by binding bile acids and increasing the turnover rate of blood and liver cholesterol [19-21]. Since BJ was showed to enhance the excretion of fecal sterols in rats [15], such a mechanism may contribute to its hypolipemic and hypoglycemic effect found in patients under BPF treatment. Special contribution to the hypolipemic response seems to be related to the modulatory properties of flavonone glycoside components of BPF, in particular naringin and neohesperidin. Indeed, evidence exists that dietary hesperetin reduces hepatic TG accumulation and this is associated with the reduced activity of TG synthetic enzymes, such as phosphatidate phosphohydrolase [22]. In addition, in vitro studies suggest that naringenin and hesperetin decrease the availability of lipids for assembly of apoB-containing lipoproteins, an effect mediated by reduced activities of of acyl CoA:cholesterol acyltransferases (ACAT) [23].Importantly, BPF is rich in buteridine and melitidine, which are 3-hydroxy-3-methylglutaryl derivatives of hesperetin and naringenin, respectively. Given the structural similarity to HMG-CoA reductase substrate, these compounds have been proposed to possess the statin-like proprieties, by selective inhibition of HMG-CoA reductase [14]. In addition, the classical glycoside derivative of naringenin, which is naringin has been shown to inhibit hepatic HMG-CoA reductase [24]. Therefore it is likely that melitidine and brutieridine in concert with naringin and other flavonone glycosides might be responsible for the striking potency of BPF in reducing cholesterol levels. The direct action of BPF on HMG-CoA reductase activity is suggested by a significant mevalonate reduction in the urine of patients under BPF treatment in our study. The oxidative stress and the inflammatory processes in the endothelium have been shown to reduce reactive NO-dependent vasodilation. Well documented antioxidant and anti-inflammatory mechanisms
Patients
Group b
Sub-group
Nr P
BPF dose
daily
(mg)
Triglycerides
Glucose
% Δ ± SEM
No responsec
Best 10%d
% Δ ± SEM
No response
Best 10%
B B1 14 500 -28.2 ± 3.9 2 -46.8 - - -34.6 B2 14 1000 -37.9 ± 3.3 1 -47.9 - - -43.6
BPL
14
0
0.1 ± 0.5
14
-
-
-
-
C C1 20 500 -32.7 ± 2.5 0 -41.6 -18.9 ± 1.2 0 -27.1 C2 19 1000 -41.0 ± 2.6 1 -49.6 -22.4 ± 1.0 0 -32.2
CPL
20
0
0.0 ± 0.6
19
-
-0.5 ± 0.7
20
-
7
regulated by Citrus flavonoids, such as increasing superoxide dismutase and catalase activities and protecting the plasma vitamin E (25) may well attenutate overproduction of oxygen reactive species in the vascular wall thereby restoring the imbalanced endothelial function, as we observed in our patients by studying reactive vasodilation (Fig. 4). Another potential benefit from BPF treatment is related to its hypoglycemic activity (Fig.2). Among few mechanistic studies on hypoglycemic effects of flavonoids, it has been shown that naringenin, similarly to other polyphenols significantly increased AMP kinase (AMPK) activity and glucose uptake in muscle cells and liver [26, 27]. The hypoglycaemic activity of naringenin in vivo can be more complex and may depend on multilevel effects on lipid metabolism that lead to increased insulin sensitivity and glucose tolerance as shown in animal models of metabolic syndrome [28].
On the basis of our data, BPF oral supplements contribute to lowering plasma cholesterol and lipids in a rat model of diet-induced hyperlipemia and in patients, in a range of potency comparable with low dose statins. Thus BPF offers a safe alternative for patients suffering from statins toxicity. In addition, the possibility to reduce blood glucose by 15%-25% suggest a phytotherapeutic approach to control the prediabetic states in patients with metabolic syndrome.
Aknowledgments
We thank Domenico Malara, Malara s.r.l., Reggio Calabria, Italy and Franco Nava, Piacenza, Italy for developing BPF purification procedure. We are grateful to Artur Janda, Jagellonian University, Cracow, Poland, for help with the statistical analysis and Capua s.r.l, Reggio Calabria, Italy, for technical support.
References
[1] Baigent, C., Keech, A., Kearney, P.M., Blackwell, L., Buck, G., Pollicino, C., et al., Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins, Lancet. 366 (2005) 1267-78.
[2] Gielen, S., Sandri, M., Schuler, G., Teupser, D., Risk factor management: antiatherogenic therapies, Eur J Cardiovasc Prev Rehabil. 16 Suppl 2 (2009) S29-36.
[3] Jones, P.H., Expert perspective: reducing cardiovascular risk in metabolic syndrome and type 2 diabetes mellitus beyond low-density lipoprotein cholesterol lowering, Am J Cardiol. 102 (2008) 41L-7L.
[4] Alsheikh-Ali, A.A., Karas, R.H., The relationship of statins to rhabdomyolysis, malignancy, and hepatic toxicity: evidence from clinical trials, Curr Atheroscler Rep. 11 (2009) 100-4.
[5] Joy, T.R., Hegele, R.A., Narrative review: statin-related myopathy, Ann Intern Med. 150 (2009) 858-68.
[6] Devaraj, S., Jialal, I., Vega-Lopez, S., Plant sterol-fortified orange juice effectively lowers cholesterol levels in mildly hypercholesterolemic healthy individuals, Arterioscler Thromb Vasc Biol. 24 (2004) e25-8.
[7] Dohadwala, M.M., Vita, J.A., Grapes and cardiovascular disease, J Nutr. 139 (2009) 1788S-93S.
[8] Gorinstein, S., Caspi, A., Libman, I., Lerner, H.T., Huang, D., Leontowicz, H., et al., Red grapefruit positively influences serum triglyceride level in patients suffering from coronary atherosclerosis: studies in vitro and in humans, J Agric Food Chem. 54 (2006) 1887-92.
[9] Kurowska, E.M., Manthey, J.A., Hypolipidemic effects and absorption of citrus polymethoxylated flavones in hamsters with diet-induced hypercholesterolemia, J Agric Food Chem. 52 (2004) 2879-86.
[10] Dugo, P., Presti, M.L., Ohman, M., Fazio, A., Dugo, G., Mondello, L., Determination of flavonoids in citrus juices by micro-HPLC-ESI/MS, J Sep Sci. 28 (2005) 1149-56.
[11] Nogata, Y., Sakamoto, K., Shiratsuchi, H., Ishii, T., Yano, M., Ohta, H., Flavonoid composition of fruit tissues of citrus species, Biosci Biotechnol Biochem. 70 (2006) 178-92.
[12] Choe, S.C., Kim, H.S., Jeong, T.S., Bok, S.H., Park, Y.B., Naringin has an antiatherogenic effect with the inhibition of intercellular adhesion molecule-1 in hypercholesterolemic rabbits, J Cardiovasc Pharmacol. 38 (2001) 947-55.
[13] Yu, J., Wang, L., Walzem, R.L., Miller, E.G., Pike, L.M., Patil, B.S., Antioxidant activity of citrus limonoids, flavonoids, and coumarins, J Agric Food Chem. 53 (2005) 2009-14.
[14] Di Donna, L., De Luca, G., Mazzotti, F., Napoli, A., Salerno, R., Taverna, D., et al., Statin-like principles of bergamot fruit (Citrus bergamia): isolation of 3-hydroxymethylglutaryl flavonoid glycosides, J Nat Prod. 72 (2009) 1352-4.
[15] Miceli, N., Mondello, M.R., Monforte, M.T., Sdrafkakis, V., Dugo, P., Crupi, M.L., et al., Hypolipidemic effects of Citrus bergamia Risso et Poiteau juice in rats fed a hypercholesterolemic diet, J Agric Food Chem. 55 (2007) 10671-7.
[16] Trovato, A., Taviano, M.F., Pergolizzi, S., Campolo, L., De Pasquale, R., Miceli, N., Citrus bergamia risso & poiteau juice protects against renal injury of diet-induced hypercholesterolemia in rats, Phytother Res. 24 (2010) 514-9.
[17] Mollace, V., Ragusa, S., Sacco, I., Muscoli, C., Sculco, F., Visalli, V., et al., The protective effect of bergamot oil extract on lecitine-like oxyLDL receptor-1 expression in balloon injury-related neointima formation, J Cardiovasc Pharmacol Ther. 13 (2008) 120-9.
[18] Pappu, A.S., Illingworth, D.R., Contrasting effects of lovastatin and cholestyramine on low-density lipoprotein cholesterol and 24-hour urinary mevalonate excretion in patients with heterozygous familial hypercholesterolemia, J Lab Clin Med. 114 (1989) 554-62.
[19] Bok, S.H., Lee, S.H., Park, Y.B., Bae, K.H., Son, K.H., Jeong, T.S., et al., Plasma and hepatic cholesterol and hepatic activities of 3-hydroxy-3-methyl-glutaryl-CoA reductase and acyl CoA: cholesterol transferase are lower in rats fed citrus peel extract or a mixture of citrus bioflavonoids, J Nutr. 129 (1999) 1182-5.
[20] Marounek, M., Volek, Z., Synytsya, A., Copikova, J., Effect of pectin and amidated pectin on cholesterol homeostasis and cecal metabolism in rats fed a high-cholesterol diet, Physiol Res. 56 (2007) 433-42.
[21] Terpstra, A.H., Lapre, J.A., de Vries, H.T., Beynen, A.C., Dietary pectin with high viscosity lowers plasma and liver cholesterol concentration and plasma cholesteryl ester transfer protein activity in hamsters, J Nutr. 128 (1998) 1944-9.
[22] Cha, J.Y., Cho, Y.S., Kim, I., Anno, T., Rahman, S.M., Yanagita, T., Effect of hesperetin, a citrus flavonoid, on the liver triacylglycerol content and phosphatidate phosphohydrolase activity in orotic acid-fed rats, Plant Foods Hum Nutr. 56 (2001) 349-58.
[23] Wilcox, L.J., Borradaile, N.M., de Dreu, L.E., Huff, M.W., Secretion of hepatocyte apoB is inhibited by the flavonoids, naringenin and hesperetin, via reduced activity and expression of ACAT2 and MTP, J Lipid Res. 42 (2001) 725-34.
[24] Kim, H.J., Oh, G.T., Park, Y.B., Lee, M.K., Seo, H.J., Choi, M.S., Naringin alters the cholesterol biosynthesis and
8
antioxidant enzyme activities in LDL receptor-knockout mice under cholesterol fed condition, Life Sci. 74 (2004) 1621-34.
[25] Jeon, S.M., Bok, S.H., Jang, M.K., Lee, M.K., Nam, K.T., Park, Y.B., et al., Antioxidative activity of naringin and lovastatin in high cholesterol-fed rabbits, Life Sci. 69 (2001) 2855-66.
[26] Hwang, J.T., Kwon, D.Y., Yoon, S.H., AMP-activated protein kinase: a potential target for the diseases prevention by natural occurring polyphenols, N Biotechnol. 26 (2009) 17-22.
[27] Zygmunt, K., Faubert, B., Macneil, J., Tsiani, E., Naringenin, a citrus flavonoid, increases muscle cell glucose uptake via AMPK, Biochem Biophys Res Commun.
[28] Mulvihill, E.E., Allister, E.M., Sutherland, B.G., Telford, D.E., Sawyez, C.G., Edwards, J.Y., et al., Naringenin prevents dyslipidemia, apolipoprotein B overproduction, and hyperinsulinemia in LDL receptor-null mice with diet-induced insulin resistance, Diabetes. 58 (2009) 2198-210.
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Re: Open conversazione
by zafferano » Mon Feb 01, 2016 5:40 am
Title :
Topic r
Option:
Protection against doxorubicine-induced cardiomyopathy by
bergamot polyphenols through myocyte survival and cardiac stem cell
activation
O3.O2 - Stem cells and cell therapy
Young Investigator Award (YIA)
C. Carresil, I. Aquilal, F. Marinol, V. Musolinol, C, Correalel, M. Gliozzil, GM. Ellison2, B.
Nad,al-Ginard2, D. Torellal, V. Mollacel - (1) Magna Graecia University of Catanzaro,
Catanzaro, Italy (2) King's College London, London, United Kingdom
Purpose: The clinical use of Doxorubicin (DOX) has the serious drawback ot
cardiotoxicity, which over time causes a cardiomyopathy leading to heart failure. The
molecular pathogenesis of anthracycline cardiotoxicity remains highly controversial.
Recently, it has been suggested that resident endogenous cardiac stemlprogenitor cell
(eCSC) depletion contributes to DOX-induced cardiomyopathy. Dietary polyphenols
play a beneficial cardiovascular protecting role due to their pleiotropic antioxidative/
inffammatory effects. Thus, we have investigated whether a mixture of
flavonoids extracted from Bergamot (Citrus Bergamia, àD endemic plant from
Sogthern ltaly), the bergamot-derived polyphenolic fraction (BPF), could attenuate
rnyocyte damage and improve myocyte regeneration through eCSC activation in DOXinduced
cardiomyopathy. Methods: We first assessed BPF's effects on DOX-induced
damage in CSCs in vitro. For in vivo studies, Wistar male rats (n=36) were randomly
assigned to receive intra-peritoneal injection of saline (that served as controls, CTRL,
n=6), BPF (20mg/kg dailV, n=10), DOX (6 doses of 2,5m9/Kg from day 1to day 14,
n=10), and DOX+BPF (n=10). To track new cardiac cell formation all animals were
implanted with subcutaneous micro-pumps to systemically release BrdU over 21 days
when the rats were sacrificed. Results: BPF significantly reduces reactive oxygen
species (ROS) accumulation and apoptotic death in CSCs in vitro. Intriguingly, BPF
enhanced CSC specification into beating cardiomyocytes in vitro. Importantly, BPF in
vivo treatment was able to prevent DOX-Induced LV impairment. Echocardiography
inraging demonstrated that DOX+BPF group had a significantly improved ejection
fraction, fractional shortening and myocardial strain when compared to DOX-treated
rats. DOX caused significant myocyte apoptosis with reactive myocyte hypertrophy
when compared to CTRL. c-kit+ eCSC number was not significantly higher in DOX
compared to CTRL. Only rare BrdU labelled cardiac cell nuclei but no BrdU+ myocytes
were identlfied in DOX-induced cardiomyopathy, showing a lack of myocardial
regeneration. On the contrary, BPF significantly reduced myocyte loss and myocyte
hypertrophy by DOX. This improvement was associated with an increased number of
activated BrdU+ eCSCs and differentiating newly-formed BrdU+ cardiomyocytes.
Conclusions: BPF reduces DOX-induced cardiotoxicity decreasing myocyte loss and
enhancing CSC activation and cardiomyocyte replacement. These data suggest that a
BPF-supptemented diet could have beneficial effects in attenuating cardiotoxicity in
patients requiring anthracycline chemiotherapy.
Topic r
Option:
Protection against doxorubicine-induced cardiomyopathy by
bergamot polyphenols through myocyte survival and cardiac stem cell
activation
O3.O2 - Stem cells and cell therapy
Young Investigator Award (YIA)
C. Carresil, I. Aquilal, F. Marinol, V. Musolinol, C, Correalel, M. Gliozzil, GM. Ellison2, B.
Nad,al-Ginard2, D. Torellal, V. Mollacel - (1) Magna Graecia University of Catanzaro,
Catanzaro, Italy (2) King's College London, London, United Kingdom
Purpose: The clinical use of Doxorubicin (DOX) has the serious drawback ot
cardiotoxicity, which over time causes a cardiomyopathy leading to heart failure. The
molecular pathogenesis of anthracycline cardiotoxicity remains highly controversial.
Recently, it has been suggested that resident endogenous cardiac stemlprogenitor cell
(eCSC) depletion contributes to DOX-induced cardiomyopathy. Dietary polyphenols
play a beneficial cardiovascular protecting role due to their pleiotropic antioxidative/
inffammatory effects. Thus, we have investigated whether a mixture of
flavonoids extracted from Bergamot (Citrus Bergamia, àD endemic plant from
Sogthern ltaly), the bergamot-derived polyphenolic fraction (BPF), could attenuate
rnyocyte damage and improve myocyte regeneration through eCSC activation in DOXinduced
cardiomyopathy. Methods: We first assessed BPF's effects on DOX-induced
damage in CSCs in vitro. For in vivo studies, Wistar male rats (n=36) were randomly
assigned to receive intra-peritoneal injection of saline (that served as controls, CTRL,
n=6), BPF (20mg/kg dailV, n=10), DOX (6 doses of 2,5m9/Kg from day 1to day 14,
n=10), and DOX+BPF (n=10). To track new cardiac cell formation all animals were
implanted with subcutaneous micro-pumps to systemically release BrdU over 21 days
when the rats were sacrificed. Results: BPF significantly reduces reactive oxygen
species (ROS) accumulation and apoptotic death in CSCs in vitro. Intriguingly, BPF
enhanced CSC specification into beating cardiomyocytes in vitro. Importantly, BPF in
vivo treatment was able to prevent DOX-Induced LV impairment. Echocardiography
inraging demonstrated that DOX+BPF group had a significantly improved ejection
fraction, fractional shortening and myocardial strain when compared to DOX-treated
rats. DOX caused significant myocyte apoptosis with reactive myocyte hypertrophy
when compared to CTRL. c-kit+ eCSC number was not significantly higher in DOX
compared to CTRL. Only rare BrdU labelled cardiac cell nuclei but no BrdU+ myocytes
were identlfied in DOX-induced cardiomyopathy, showing a lack of myocardial
regeneration. On the contrary, BPF significantly reduced myocyte loss and myocyte
hypertrophy by DOX. This improvement was associated with an increased number of
activated BrdU+ eCSCs and differentiating newly-formed BrdU+ cardiomyocytes.
Conclusions: BPF reduces DOX-induced cardiotoxicity decreasing myocyte loss and
enhancing CSC activation and cardiomyocyte replacement. These data suggest that a
BPF-supptemented diet could have beneficial effects in attenuating cardiotoxicity in
patients requiring anthracycline chemiotherapy.
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Re: Open conversazione
by zafferano » Mon Feb 01, 2016 5:45 am
The Use of Bergamot-derived Polyphenol
Fraction in Cardiometabolic Risk Prevention
and its Possible Mechanisms of Action
Ross Walker*, Elzbieta Janda† and Vincenzo Mollace‡
*Consultant Cardiologist, Sydney Adventist Hospital, Sydney, Australia †Department of Health Sciences, University
“Magna Graecia,” Campus Salvatore Venuta, Germaneto, Catanzaro, Italy ‡Department of Health Sciences, University
“Magna Graecia” of Catanzaro, Complesso Nini’ Barbieri, Roccelletta di Borgia, Catanzaro, Italy
s0010
1. INTRODUCTION
p0010 Hypercholesterolemia (increased cholesterol levels),
hyperlipidemia (increased triglycerides (TG) and cholesterol)
and hyperglycemia (blood glucose over
100 mg/dL) are important risk factors for the development
of atherosclerosis and coronary artery disease.1,2
Increased concentrations of total blood cholesterol
(tChol, $200 mg/dL) as well as high ratio of cholesterol
bound to low-density lipoprotein (cLDL) with
respect to the cholesterol bound to high-density lipoprotein
(cHDL) are considered to be the main pathogenic
blood parameters. However, conditions of
insulin resistance such as impaired glucose tolerance
or prediabetes are also characterized by a high risk of
atherosclerotic vascular diseases (AVD).3 The presence
of all cardiometabolic risk factors that occur in the metabolic
syndrome particularly accelerate atherosclerotic
changes, and are treated with polypharmacy, as discussed
in Section 3.1.
p0015 Experimental and epidemiological evidence suggests
that dietary polyphenols, such as flavonoids,
may prevent atherosclerosis by counteracting its risk
factors.46 Accordingly, better clinical results by
increasing the dosage and quality of consumed flavonoids
should be expected. This idea has been exploited
in the case of bergamot juice flavonoids and proved to
work in clinical practice.
p0020 Bergamot (Citrus bergamia Risso et Poiteau) belongs
to the family Rutaceae, genus Citrus, and is an endemic
plant from the Calabrian region in Italy. The plant is
very sensitive to the pedoclimatic conditions of the soil
and it grows almost exclusively in the narrow coastal
area from Reggio Calabria to Locri, in the most southern
part of the Italian Peninsula (Figure 84.1).
Bergamot juice was traditionally recognized by the
local population of Calabria, as a remedy for “fatty
arteries” and heart problems. The medicinal use of bergamot
derivatives, forgotten for decades, is now being
rediscovered. This started from a demonstration in animal
models of hypolipemic properties of bergamot
juice.7 Next, Mollace and co-workers8 addressed the
efficacy of a concentrated mixture of bergamot flavonoids,
so-called Bergamot Polyphenol Fraction (BPF),
in experimental and clinical settings. These authors
showed, using animal models and through human
studies, that BPF is effective in combating several
symptoms of metabolic syndrome such as increased
tChol, cLDL and TG, increased blood glucose levels
and endothelial dysfunction in a group of metabolic
syndrome patients. The effects on mean cholesterol
parameters were comparable with a moderate dose of
simavastatin (20 mg daily), including a marked
increase in cHDL levels. In addition, 1000 mg BPF
taken for 30 days reduced moderate hyperglycemia by
more than 20%.8
The brilliant performance of BPF on MS and dyslipi- p0025
demia is astonishing and needs a mechanistic explanation.
Unfortunately, so far there is very limited
experimental evidence proving the modulation of one
1085
Polyphenols in Human Health and Disease.
DOI: http://dx.doi.org/10.1016/B978-0-12-398456-2.00084-0 © 2014 Elsevier Inc. All rights reserved.
Watson 978-0-12-398456-2 00084
Fraction in Cardiometabolic Risk Prevention
and its Possible Mechanisms of Action
Ross Walker*, Elzbieta Janda† and Vincenzo Mollace‡
*Consultant Cardiologist, Sydney Adventist Hospital, Sydney, Australia †Department of Health Sciences, University
“Magna Graecia,” Campus Salvatore Venuta, Germaneto, Catanzaro, Italy ‡Department of Health Sciences, University
“Magna Graecia” of Catanzaro, Complesso Nini’ Barbieri, Roccelletta di Borgia, Catanzaro, Italy
s0010
1. INTRODUCTION
p0010 Hypercholesterolemia (increased cholesterol levels),
hyperlipidemia (increased triglycerides (TG) and cholesterol)
and hyperglycemia (blood glucose over
100 mg/dL) are important risk factors for the development
of atherosclerosis and coronary artery disease.1,2
Increased concentrations of total blood cholesterol
(tChol, $200 mg/dL) as well as high ratio of cholesterol
bound to low-density lipoprotein (cLDL) with
respect to the cholesterol bound to high-density lipoprotein
(cHDL) are considered to be the main pathogenic
blood parameters. However, conditions of
insulin resistance such as impaired glucose tolerance
or prediabetes are also characterized by a high risk of
atherosclerotic vascular diseases (AVD).3 The presence
of all cardiometabolic risk factors that occur in the metabolic
syndrome particularly accelerate atherosclerotic
changes, and are treated with polypharmacy, as discussed
in Section 3.1.
p0015 Experimental and epidemiological evidence suggests
that dietary polyphenols, such as flavonoids,
may prevent atherosclerosis by counteracting its risk
factors.46 Accordingly, better clinical results by
increasing the dosage and quality of consumed flavonoids
should be expected. This idea has been exploited
in the case of bergamot juice flavonoids and proved to
work in clinical practice.
p0020 Bergamot (Citrus bergamia Risso et Poiteau) belongs
to the family Rutaceae, genus Citrus, and is an endemic
plant from the Calabrian region in Italy. The plant is
very sensitive to the pedoclimatic conditions of the soil
and it grows almost exclusively in the narrow coastal
area from Reggio Calabria to Locri, in the most southern
part of the Italian Peninsula (Figure 84.1).
Bergamot juice was traditionally recognized by the
local population of Calabria, as a remedy for “fatty
arteries” and heart problems. The medicinal use of bergamot
derivatives, forgotten for decades, is now being
rediscovered. This started from a demonstration in animal
models of hypolipemic properties of bergamot
juice.7 Next, Mollace and co-workers8 addressed the
efficacy of a concentrated mixture of bergamot flavonoids,
so-called Bergamot Polyphenol Fraction (BPF),
in experimental and clinical settings. These authors
showed, using animal models and through human
studies, that BPF is effective in combating several
symptoms of metabolic syndrome such as increased
tChol, cLDL and TG, increased blood glucose levels
and endothelial dysfunction in a group of metabolic
syndrome patients. The effects on mean cholesterol
parameters were comparable with a moderate dose of
simavastatin (20 mg daily), including a marked
increase in cHDL levels. In addition, 1000 mg BPF
taken for 30 days reduced moderate hyperglycemia by
more than 20%.8
The brilliant performance of BPF on MS and dyslipi- p0025
demia is astonishing and needs a mechanistic explanation.
Unfortunately, so far there is very limited
experimental evidence proving the modulation of one
1085
Polyphenols in Human Health and Disease.
DOI: http://dx.doi.org/10.1016/B978-0-12-398456-2.00084-0 © 2014 Elsevier Inc. All rights reserved.
Watson 978-0-12-398456-2 00084
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Re: Open conversazione
by zafferano » Mon Feb 01, 2016 5:46 am
or another metabolic pathway by BPF itself. However,
as discussed in Section 5, the vast scientific literature
suggests that certain individual flavonoids present in
BPF are implicated in the regulation of several metabolic
enzymes, expressed in the liver, blood and endothelial
cells. In addition, natural polyphenols show less
specific, antioxidant properties, that depend on the
free radical scavenging ability of hydroxyl groups
linked to carbon aromatic rings.911 Together with
antioxidant properties, dietary flavonoids and their
metabolites may modulate basic signal transduction
pathways of every cell leading to anti-proliferative,
anti-aging and immune responses as well as other beneficial
effects for human health, as discussed in other
chapters of this volume and in the vast literature to be
found on the subject.9,10 Finally, besides intracellular,
molecular effects, flavonoids, in cooperation with pectins,
can work at the level of intestine and liver to stimulate
fat excretion and reduce fat absorption, which
augments the direct activity on enzymes, involved in the
regulation of carbohydrate and lipid metabolism.7,12,13
p0030 In this chapter, we will discuss chemical and pharmacological
properties of flavonoids, present in bergamot
fruits (Section 2). Section 3 will examine the experimental
evidence, along with clinical and observational studies
demonstrating efficacy of BPF in combating
cardiometabolic risk factors. The use of BPF, its advantages
and limitations will be discussed in the context of
conventional statin therapy (Section 4). Section 5 will
focus on possible molecular mechanisms of action of
bergamot polyphenols that could account for the therapeutic
effects of BPF.
2. CHEMICAL AND FUNCTIONAL s0015
CHARACTERIZATIONS OF BERGAMOT
FLAVONOIDS
Bergamot juice is particularly rich in flavanone- p0035
O-glycosides (see Table 84.1) and is characterized by a
unique profile of flavonoids, showing partial similarity
to Citrus x myrtifolia Raf. (chinotto)14 and Citrus aurantium
L.15 It contains relatively rare neoeriocitrin and
neohesperidin and, as recently discovered, rare brutieridin
and melitidin bearing 3-hydroxy-
3-methylglutaric acid (HMG) moiety16 (Figure 84.2, see
also Plate 13). High-throughput analysis of flavonoid
content in 45 citrus species and cultivars, reported by
Nogata et al.,17 shows that the amount of the flavonoids
per volume unit of juice, or per mass unit of
f0010 FIGURE 84.1 Mature bergamot fruits of Femminello cultivar (left) and the geographical distribution of bergamot cultivars in Calabria.
Bergamot plantations are restricted to a narrow costal area in the most south part of Italian peninsula, between Reggio Calabria and Locri.
This is due to high sensitivity to pedoclimatic conditions of bergamot trees.
1086 84. THE USE OF BERGAMOT-DERIVED POLYPHENOL FRACTION IN CARDIOMETABOLIC RISK PREVENTION
8. CARDIAC HEALTH AND POLYPHENOLS
Watson 978-0-12-398456-2 00084
as discussed in Section 5, the vast scientific literature
suggests that certain individual flavonoids present in
BPF are implicated in the regulation of several metabolic
enzymes, expressed in the liver, blood and endothelial
cells. In addition, natural polyphenols show less
specific, antioxidant properties, that depend on the
free radical scavenging ability of hydroxyl groups
linked to carbon aromatic rings.911 Together with
antioxidant properties, dietary flavonoids and their
metabolites may modulate basic signal transduction
pathways of every cell leading to anti-proliferative,
anti-aging and immune responses as well as other beneficial
effects for human health, as discussed in other
chapters of this volume and in the vast literature to be
found on the subject.9,10 Finally, besides intracellular,
molecular effects, flavonoids, in cooperation with pectins,
can work at the level of intestine and liver to stimulate
fat excretion and reduce fat absorption, which
augments the direct activity on enzymes, involved in the
regulation of carbohydrate and lipid metabolism.7,12,13
p0030 In this chapter, we will discuss chemical and pharmacological
properties of flavonoids, present in bergamot
fruits (Section 2). Section 3 will examine the experimental
evidence, along with clinical and observational studies
demonstrating efficacy of BPF in combating
cardiometabolic risk factors. The use of BPF, its advantages
and limitations will be discussed in the context of
conventional statin therapy (Section 4). Section 5 will
focus on possible molecular mechanisms of action of
bergamot polyphenols that could account for the therapeutic
effects of BPF.
2. CHEMICAL AND FUNCTIONAL s0015
CHARACTERIZATIONS OF BERGAMOT
FLAVONOIDS
Bergamot juice is particularly rich in flavanone- p0035
O-glycosides (see Table 84.1) and is characterized by a
unique profile of flavonoids, showing partial similarity
to Citrus x myrtifolia Raf. (chinotto)14 and Citrus aurantium
L.15 It contains relatively rare neoeriocitrin and
neohesperidin and, as recently discovered, rare brutieridin
and melitidin bearing 3-hydroxy-
3-methylglutaric acid (HMG) moiety16 (Figure 84.2, see
also Plate 13). High-throughput analysis of flavonoid
content in 45 citrus species and cultivars, reported by
Nogata et al.,17 shows that the amount of the flavonoids
per volume unit of juice, or per mass unit of
f0010 FIGURE 84.1 Mature bergamot fruits of Femminello cultivar (left) and the geographical distribution of bergamot cultivars in Calabria.
Bergamot plantations are restricted to a narrow costal area in the most south part of Italian peninsula, between Reggio Calabria and Locri.
This is due to high sensitivity to pedoclimatic conditions of bergamot trees.
1086 84. THE USE OF BERGAMOT-DERIVED POLYPHENOL FRACTION IN CARDIOMETABOLIC RISK PREVENTION
8. CARDIAC HEALTH AND POLYPHENOLS
Watson 978-0-12-398456-2 00084
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Re: Open conversazione
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t0010 TABLE 84.1 Flavonoid Content in Bergamot Fruits (Juice and Albedo).
Compound
Content Position on the
Ranking List
for Flavonoid
Content* Remarks
Juice
mg/L
Albedo
mg/kg
MAJOR FLAVANONES O-GLYCOSIDES
Naringin 248b, 523a 670a 3a
Neohesperidin 295b, 763a 686a 1a
Neoeriocitrin 296b, 293a 435a 1a
Bruteridin 230c 250300d 1e Several times higher than in C. aurantium
and C. mirtifolia.
Melitidin 133c 150200d 1e Several times higher than in C. aurantium
and C. mirtifolia.
Poncirin (7-O-neohesperidoside
of isosakuranetin)
64f, 1870 (?)a 777a 1a Big difference, because of comparing
hand-squeezed juicea with industrial juiceb.
Eriocitrin 13.415.6b 13.4a 6.3a Relatively low content compared
to other Citrus fruits
Narirutin 17.7a 13.9a Relatively low content compared
to other Citrus fruits
MAJOR FLAVONES
Rhoifolin 53.268.1b 39.9a 21a 1a,f Industrial juicef
Neodiosmin 19.027.1b 48.1a 13.8a 1f2a Industrial juicef
Rutin 28.3a 0a 3a
Vicenin-2 (Apigenin 6,8-di-C-glucoside) 58.363.2b,f 1f Industrial juicef
Diosmetin 6,8-di-C-glucoside
(Lucenin-2 40-methyl ether)
37.949.7b 2f Industrial juice, second conc. after Citrus limonf
Chrysoeriol 7-O-neohesperoside
40 glucoside
11.613.2b 1f Industrial juicef
Stellarin-2 (Chrysoeriol 6,
8-di-C-glucoside)
5.87.3b
Lucenin-2 (Luteolin 6,8-di-C-glucoside) 6.47.5b
Isovitexin (Apigenin 6-C-glucoside) 4.55.3b
Scoparin (Chrysoeriol 8-C-glucoside) 7.27.9b
Diosmetin 8-C-glucoside (Orientin
40-methyl ether)
7.68.8b
*Position in the classification of Citrus juices according the content of a specific flavonoid, based on indicated references.
aNogata et al.17
bGattuso et al.18
cUnpublished results, obtained in samples of industrial juice, courtesy of D. Malara, Reggio Calabria, Italy.
dDi Donna et al.16
eBarreca et al.14
fGattuso
Compound
Content Position on the
Ranking List
for Flavonoid
Content* Remarks
Juice
mg/L
Albedo
mg/kg
MAJOR FLAVANONES O-GLYCOSIDES
Naringin 248b, 523a 670a 3a
Neohesperidin 295b, 763a 686a 1a
Neoeriocitrin 296b, 293a 435a 1a
Bruteridin 230c 250300d 1e Several times higher than in C. aurantium
and C. mirtifolia.
Melitidin 133c 150200d 1e Several times higher than in C. aurantium
and C. mirtifolia.
Poncirin (7-O-neohesperidoside
of isosakuranetin)
64f, 1870 (?)a 777a 1a Big difference, because of comparing
hand-squeezed juicea with industrial juiceb.
Eriocitrin 13.415.6b 13.4a 6.3a Relatively low content compared
to other Citrus fruits
Narirutin 17.7a 13.9a Relatively low content compared
to other Citrus fruits
MAJOR FLAVONES
Rhoifolin 53.268.1b 39.9a 21a 1a,f Industrial juicef
Neodiosmin 19.027.1b 48.1a 13.8a 1f2a Industrial juicef
Rutin 28.3a 0a 3a
Vicenin-2 (Apigenin 6,8-di-C-glucoside) 58.363.2b,f 1f Industrial juicef
Diosmetin 6,8-di-C-glucoside
(Lucenin-2 40-methyl ether)
37.949.7b 2f Industrial juice, second conc. after Citrus limonf
Chrysoeriol 7-O-neohesperoside
40 glucoside
11.613.2b 1f Industrial juicef
Stellarin-2 (Chrysoeriol 6,
8-di-C-glucoside)
5.87.3b
Lucenin-2 (Luteolin 6,8-di-C-glucoside) 6.47.5b
Isovitexin (Apigenin 6-C-glucoside) 4.55.3b
Scoparin (Chrysoeriol 8-C-glucoside) 7.27.9b
Diosmetin 8-C-glucoside (Orientin
40-methyl ether)
7.68.8b
*Position in the classification of Citrus juices according the content of a specific flavonoid, based on indicated references.
aNogata et al.17
bGattuso et al.18
cUnpublished results, obtained in samples of industrial juice, courtesy of D. Malara, Reggio Calabria, Italy.
dDi Donna et al.16
eBarreca et al.14
fGattuso
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Re: Open conversazione
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solid parts of the fruit, is absolutely the highest in bergamot
compared to other Citrus species. These observations
were then confirmed for industrial bergamot
juice obtained by fruit crashing.18 A lower content of
flavonoids is present in manually squeezed bergamot
juice.19,20 This discrepancy can be explained by
differences in processing and different distribution of
flavonoids between the vacuole juice, albedo (the inner
white part of the peel), flavedo (external peel rich in
aromatic oils), and inner fibers of the fruits. Indeed,
the majority of flavonoids are several times more
abundant in albedo than in the juice,17 and industrial
pressing causes a release of albedo compounds. This
processing enriches the total flavonoid content of the
juice to more than 1600 mg/L in the industrial juices
from the initial 373 up to 512 mg/L, present in the
“hand-squeezed” juices of two different Bergamot cultivars
Fantastico and Feminello, respectively.19 Thus, the
industrial bergamot juice likely shows the highest concentration
of polyphenols among non-concentrated
natural juices.
p0040 Bergamot fruits (juice, albedo, flavedo, and fibers)
show the highest concentrations of flavanones: neoeriocitrin,
neohesperidin, naringin, poncirin, melitidin
and brutieridin, and the highest content of certain flavones:
rhoifolin and neodiosmin among 45 different
Citrus species17 (see Table 84.1). A high content was
confirmed for rhoifolin and neodiosmin by another
independent study that also identified other highly
abundant flavones in bergamot juice: vicenin-2, diosmetin
di-C-glucoside and chryseriol 7-O-neohesperoside
and lower amounts of other flavonoids: lucenin-2,
stellarin-2, isovitexin, scoparin, diosmetin 8-C-glucoside,
chrysoeriol 7-O-neohesperoside-40-O-glycoside
and others.18 Another peculiar feature of bergamot
juice is a high content of unsaturated fatty acids,
accounting for 80% of total acids, such as oleic acid,
linoleic acid and palmitic acid.21
p0045 As a medicinal food, bergamot juice is not always
available and is subjected to fast deterioration. In the
search of a more stable and possibly more powerful
pharmaceutical surrogate of bergamot juice, a method
to concentrate and exsiccate bergamot juice was developed.
According to this method, bergamot juice is concentrated
by a preparative size exclusion
chromatography based on polystyrene gel filtration.8
Polyphenols and other macromolecules of the molecular
size over 300 Da are retained on the column, eluted
with acids and exsiccated with other clarified juice
components to give rise to a flavonoid-enriched bergamot
powder or BPF.8 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
(D. Malara, personal communication). In contrast to
BPF, bergamot juice derivatives obtained by spry-drier
method rich maximum 1% polyphenols concentration
(D. Malara, personal communication). The main polyphenol
components of BPF are flavonoids and their
composition basically mirrors the bergamot juice polyphenol
profile (see Figure 84.2, and Plate 13), with the
only difference that flavonoids are over 200 times
more concentrated in BPF. Flavanones glycosides
account for 95% of total flavonoids present in BPF
(and in bergamot juice), while flavones can be found
in the remaining 5% (Table 84.1 and D. Malara and
C. Malara, unpublished observations).
Currently, there are no published bioavailability and p0050
pharmacokinetic studies for BPF. However, absorption,
metabolism and excretion parameters have been
described for several individual flavonoids present in
bergamot juice. It is well known that flavonoid glycosides
are hydrolyzed to aglycones by baterial flora of
the gut. Gut microflora hydrolysis is thought to favor
flavonoid glycoside bioavailability.22 When sugar unit
is removed, the resulting aglycone can be absorbed
more readily.23 Indeed, flavonoid aglycones of diosmin,
hesperidin and naringin, diffuse usually easily through
the plasma membrane of Caco-2 cells, in contrast to the
respective glycosides. Moreover, naringin, hesperidin,
rutin and poncirin are hydrolyzed to their respective
aglycones by human intestinal microflora, and the
resulting aglycones are absorbed better.24 However, the
bioavilability of flavonoids is low and it is estimated
that only 10% of total consumed polyphenols are
absorbed, although these numbers vary between species
and individuals.23 When inside the enterocytes part
of aglycones are subjected to intestinal metabolism,
such as glucuronidation and sulfation. Phase II metabolism
is the main route of metabolism for polyphenols.
Conjugation of free phenolic groups via glucuronidation
and/or sulfation will increase their polarity and
water solubility, enabling their elimination from the
body.23 Generally, citrus and other flavonoids have a
short life-time and metabolites are thought to lose their
biological activity. Nevertheless, recent data suggest
that sulfonated or methylated and much less glucuronidated
metabolites of resveratrol maintain full or partial
activity of the parent compound. In addition, sulfonation
has been shown to increase the activity for some
molecular targets of resveratrol.25
Therefore although not properly investigated, it is p0055
likely aglycones of bergamot flavonoids and their
metabolites contribute to the modulation of the intracellular
targets and the subsequent biological response.
3. PHARMACOLOGICAL EFFECTS OF s0020
BERGAMOT POLYPHENOL FRACTION
ON CARDIOVASCULAR RISK FACTORS
3.1 Metabolic Syndrome and Cardiovascular s0025
Risk Factors
AVD is the leading cause of death in developed p0060
countries.26 Coronary artery disease is the commonest
form of cardiovascular disease accounting for around a
third of all-cause mortality in people over the age of 35.
AVD is caused by atherosclerosis, which is defined as
the deposition of lipids, inflammatory cells and mediators
in the subendothelial layer of blood vessels,
eventually leading to thickening and hardening of
3. PHARMACOLOGICAL EFFECTS OF BERGAMOT POLYPHENOL FRACTION ON CARDIOVASCULAR RISK FACTORS 1089
8. CARDIAC HEALTH AND POLYPHENOLS
Watson 978-0-12-398456-2 00084
medium- to large-sized arteries. Atherosclerosis has a
very long pre-symptomatic phase and can begin even
in-utero in predisposed individuals. The presymptomatic
phase of atherosclerosis averages between
4060 years, although clinical presentation seldom
occurs before this time frame and commonly thereafter.
The presentation, thus depends on the rates of atherosclerotic
deposition in the blood vessel wall, often lasting
decades, while common clinical manifestation of
AVD, such as acute myocardial infarction,
unstable angina, stroke or sudden cardiac death, usually
occur following the rupture of a large plaque in the
blood vessel wall.
p0065 It is well-established that the abundance of fat and
sugar in diets found in industrialized western society is
the most important risk factor for developing AVD.
Indeed, as many of the developing nations adopt more
affluent life styles, an increasing rates of cardiovascular
disease is seen.27 This is because the diet influences blood
parameters that may become AVD risk factors if out of
established limits. For example, the average cholesterol
levels in malnourished regions (for example, rural China)
are between 7797 mg/dL (22.5 mmol/L).
Atherosclerosis is a rarity in these regions.28 Once food
becomes available and in plentiful supply, the cholesterol
level rises above 3 mmol/L and it is above this level that
atherosclerosis occurs. In fact, in the majority of cases,
people with lifelong cholesterol levels below 4 mmol/L
are rarely troubled by significant complications of
atherosclerosis.28
p0070 Excessive fat and sugar consumption, linked to
genetic predisposition and poor lifestyle behaviors
may lead to the development of a pathological state,
called metabolic syndrome (MS). MS is very common
and 1030% of individuals in industrialized countries
suffer from this condition. MS is defined as a cluster of
cardiovascular risk factors, including atherogenic dyslipidemia,
insulin resistance or glucose intolerance, visceral
obesity, hypertension and endothelial
dysfunction.
p0075 There are four basic components of MS:
o0010 1. Hyperglycemia (fasting plasma glucose $110 mg/dl
(6.1 mmol/L))
o0015 2. Hypertension (systolic blood pressure .130 or
diastolic blood pressure .85 mmHg)
o0020 3. Dyslipidemia
o0025 a) Increased cholesterol .200 mg/dL (5 mmol/L)
o0030 b) Increased Triglyceride .150 mg/dL (1.5 mmol/L)
o0035 c) Reduced cHDL ,55 mg/dL, (1.3 mmol/L)
o0040 4. Abdominal obesity
o0045 a) Males .95 cm
o0050 b) Females .80 cm
p0125 The presence of all components of MS is associated
with a particularly increased risk of accelerated
atherosclerosis and cardiovascular events.29 However,
the probability of developing AVD (cardiovascular
risk) is also increased in individuals with isolated metabolic
dysfunctions, such as increased cholesterol
levels (hypercholesterolemia) or increased level of triglycerides
(hypertriglyceridemia) and in patients with
type-1 and -2 diabetes as well as in patients with glucose
intolerance and hypertension.
Pharmacological treatment of any cardiometabolic p0130
risk factors is clearly a preventive medicinal approach,
since it slows down the process of atherosclerosis and
reduces the probability of cardiovascular events. For
this reason, it would be preferable that such a treatment
is based on diet and natural drugs, and starts as
early as possible in adult age; and not on conventional
and aggressive polypharmacy started when MS or
even ADV is well-advanced.
The experimental and clinical evidence will be pre- p0135
sented here, showing that BPF—a concentrate of natural
bergamot polyphenols—is a suitable treatment for
MS and other metabolic pathologies associated with an
increased cardiovascular risk of atherosclerosis and
life-threatening cardiovascular events.
3.2 Hypolipidemic Effects s0030
The first evidence of the cholesterol-lowering prop- p0140
erties of bergamot polyphenols comes from experimental
studies on rats fed a high-cholesterol diet.7 In this
laboratory model of diet-induced hypocholesterolemia,
rats on a 2% cholesterol and 20% oil diet developed
extremely high levels of total blood cholesterol (tChol,
700 mg/dL). The animals treated daily with 1 ml bergamot
juice for 28 days showed a marked reduction in
total blood cholesterol (tChol) by (29.3%) , triglicerides
(46.2%) and cLDL (51.7%). These results were confirmed
in an independent work by Mollace et al.,8
addressing the efficacy of the bergamot polyphenol
mixture, BPF. Another important pharmacological
finding observed by Miceli et al.7 was evident reduction
of lipid steatosis in the liver, defined as a decrease
in the number and size of hepatic lipid droplets and a
reduction in inflammatory changes due to fat accumulation
in the liver. In addition, the anatomical analysis
of arteries indicated some protective effect of bergamot
juice against mild endothelial thickening. Next
Mollace’s group confirmed these observations and
demonstrated a prevention of accelerated stenosis with
the use of Bergamot extract. In this model of stenosis/
atherosclerosis, the balloon injury is induced to the
carotid artery in laboratory rats.30 Bergamot extract (in
this case a non-volatile part of bergamot oil) uniformly
prevented rapid thickening and inflammatory changes
in the arteries subjected to balloon injury.
1090 84. THE USE OF BERGAMOT-DERIVED POLYPHENOL FRACTION IN CARDIOMETABOLIC RISK PREVENTION
8. CARDIAC HEALTH AND POLYPHENOLS
Watson 978-0-12-398456-2 00084
medium- to large-sized arteries. Atherosclerosis has a
very long pre-symptomatic phase and can begin even
in-utero in predisposed individuals. The presymptomatic
phase of atherosclerosis averages between
4060 years, although clinical presentation seldom
occurs before this time frame and commonly thereafter.
The presentation, thus depends on the rates of atherosclerotic
deposition in the blood vessel wall, often lasting
decades, while common clinical manifestation of
AVD, such as acute myocardial infarction,
unstable angina, stroke or sudden cardiac death, usually
occur following the rupture of a large plaque in the
blood vessel wall.
p0065 It is well-established that the abundance of fat and
sugar in diets found in industrialized western society is
the most important risk factor for developing AVD.
Indeed, as many of the developing nations adopt more
affluent life styles, an increasing rates of cardiovascular
disease is seen.27 This is because the diet influences blood
parameters that may become AVD risk factors if out of
established limits. For example, the average cholesterol
levels in malnourished regions (for example, rural China)
are between 7797 mg/dL (22.5 mmol/L).
Atherosclerosis is a rarity in these regions.28 Once food
becomes available and in plentiful supply, the cholesterol
level rises above 3 mmol/L and it is above this level that
atherosclerosis occurs. In fact, in the majority of cases,
people with lifelong cholesterol levels below 4 mmol/L
are rarely troubled by significant complications of
atherosclerosis.28
p0070 Excessive fat and sugar consumption, linked to
genetic predisposition and poor lifestyle behaviors
may lead to the development of a pathological state,
called metabolic syndrome (MS). MS is very common
and 1030% of individuals in industrialized countries
suffer from this condition. MS is defined as a cluster of
cardiovascular risk factors, including atherogenic dyslipidemia,
insulin resistance or glucose intolerance, visceral
obesity, hypertension and endothelial
dysfunction.
p0075 There are four basic components of MS:
o0010 1. Hyperglycemia (fasting plasma glucose $110 mg/dl
(6.1 mmol/L))
o0015 2. Hypertension (systolic blood pressure .130 or
diastolic blood pressure .85 mmHg)
o0020 3. Dyslipidemia
o0025 a) Increased cholesterol .200 mg/dL (5 mmol/L)
o0030 b) Increased Triglyceride .150 mg/dL (1.5 mmol/L)
o0035 c) Reduced cHDL ,55 mg/dL, (1.3 mmol/L)
o0040 4. Abdominal obesity
o0045 a) Males .95 cm
o0050 b) Females .80 cm
p0125 The presence of all components of MS is associated
with a particularly increased risk of accelerated
atherosclerosis and cardiovascular events.29 However,
the probability of developing AVD (cardiovascular
risk) is also increased in individuals with isolated metabolic
dysfunctions, such as increased cholesterol
levels (hypercholesterolemia) or increased level of triglycerides
(hypertriglyceridemia) and in patients with
type-1 and -2 diabetes as well as in patients with glucose
intolerance and hypertension.
Pharmacological treatment of any cardiometabolic p0130
risk factors is clearly a preventive medicinal approach,
since it slows down the process of atherosclerosis and
reduces the probability of cardiovascular events. For
this reason, it would be preferable that such a treatment
is based on diet and natural drugs, and starts as
early as possible in adult age; and not on conventional
and aggressive polypharmacy started when MS or
even ADV is well-advanced.
The experimental and clinical evidence will be pre- p0135
sented here, showing that BPF—a concentrate of natural
bergamot polyphenols—is a suitable treatment for
MS and other metabolic pathologies associated with an
increased cardiovascular risk of atherosclerosis and
life-threatening cardiovascular events.
3.2 Hypolipidemic Effects medium- to large-sized arteries. Atherosclerosis has a
very long pre-symptomatic phase and can begin even
in-utero in predisposed individuals. The presymptomatic
phase of atherosclerosis averages between
4060 years, although clinical presentation seldom
occurs before this time frame and commonly thereafter.
The presentation, thus depends on the rates of atherosclerotic
deposition in the blood vessel wall, often lasting
decades, while common clinical manifestation of
AVD, such as acute myocardial infarction,
unstable angina, stroke or sudden cardiac death, usually
occur following the rupture of a large plaque in the
blood vessel wall.
p0065 It is well-established that the abundance of fat and
sugar in diets found in industrialized western society is
the most important risk factor for developing AVD.
Indeed, as many of the developing nations adopt more
affluent life styles, an increasing rates of cardiovascular
disease is seen.27 This is because the diet influences blood
parameters that may become AVD risk factors if out of
established limits. For example, the average cholesterol
levels in malnourished regions (for example, rural China)
are between 7797 mg/dL (22.5 mmol/L).
Atherosclerosis is a rarity in these regions.28 Once food
becomes available and in plentiful supply, the cholesterol
level rises above 3 mmol/L and it is above this level that
atherosclerosis occurs. In fact, in the majority of cases,
people with lifelong cholesterol levels below 4 mmol/L
are rarely troubled by significant complications of
atherosclerosis.28
p0070 Excessive fat and sugar consumption, linked to
genetic predisposition and poor lifestyle behaviors
may lead to the development of a pathological state,
called metabolic syndrome (MS). MS is very common
and 1030% of individuals in industrialized countries
suffer from this condition. MS is defined as a cluster of
cardiovascular risk factors, including atherogenic dyslipidemia,
insulin resistance or glucose intolerance, visceral
obesity, hypertension and endothelial
dysfunction.
p0075 There are four basic components of MS:
o0010 1. Hyperglycemia (fasting plasma glucose $110 mg/dl
(6.1 mmol/L))
o0015 2. Hypertension (systolic blood pressure .130 or
diastolic blood pressure .85 mmHg)
o0020 3. Dyslipidemia
o0025 a) Increased cholesterol .200 mg/dL (5 mmol/L)
o0030 b) Increased Triglyceride .150 mg/dL (1.5 mmol/L)
o0035 c) Reduced cHDL ,55 mg/dL, (1.3 mmol/L)
o0040 4. Abdominal obesity
o0045 a) Males .95 cm
o0050 b) Females .80 cm
p0125 The presence of all components of MS is associated
with a particularly increased risk of accelerated
atherosclerosis and cardiovascular events.29 However,
the probability of developing AVD (cardiovascular
risk) is also increased in individuals with isolated metabolic
dysfunctions, such as increased cholesterol
levels (hypercholesterolemia) or increased level of triglycerides
(hypertriglyceridemia) and in patients with
type-1 and -2 diabetes as well as in patients with glucose
intolerance and hypertension.
Pharmacological treatment of any cardiometabolic p0130
risk factors is clearly a preventive medicinal approach,
since it slows down the process of atherosclerosis and
reduces the probability of cardiovascular events. For
this reason, it would be preferable that such a treatment
is based on diet and natural drugs, and starts as
early as possible in adult age; and not on conventional
and aggressive polypharmacy started when MS or
even ADV is well-advanced.
The experimental and clinical evidence will be pre- p0135
sented here, showing that BPF—a concentrate of natural
bergamot polyphenols—is a suitable treatment for
MS and other metabolic pathologies associated with an
increased cardiovascular risk of atherosclerosis and
life-threatening cardiovascular events.
3.2 Hypolipidemic Effects s0030
The first evidence of the cholesterol-lowering prop- p0140
erties of bergamot polyphenols comes from experimental
studies on rats fed a high-cholesterol diet.7 In this
laboratory model of diet-induced hypocholesterolemia,
rats on a 2% cholesterol and 20% oil diet developed
extremely high levels of total blood cholesterol (tChol,
700 mg/dL). The animals treated daily with 1 ml bergamot
juice for 28 days showed a marked reduction in
total blood cholesterol (tChol) by (29.3%) , triglicerides
(46.2%) and cLDL (51.7%). These results were confirmed
in an independent work by Mollace et al.,8
addressing the efficacy of the bergamot polyphenol
mixture, BPF. Another important pharmacological
finding observed by Miceli et al.7 was evident reduction
of lipid steatosis in the liver, defined as a decrease
in the number and size of hepatic lipid droplets and a
reduction in inflammatory changes due to fat accumulation
in the liver. In addition, the anatomical analysis
of arteries indicated some protective effect of bergamot
juice against mild endothelial thickening. Next
Mollace’s group confirmed these observations and
demonstrated a prevention of accelerated stenosis with
the use of Bergamot extract. In this model of stenosis/
atherosclerosis, the balloon injury is induced to the
carotid artery in laboratory rats.30 Bergamot extract (in
this case a non-volatile part of bergamot oil) uniformly
prevented rapid thickening and inflammatory changes
in the arteries subjected to balloon injury.
1090 84. THE USE OF BERGAMOT-DERIVED POLYPHENOL FRACTION IN CARDIOMETABOLIC RISK PREVENTION
8. CARDIAC HEALTH AND POLYPHENOLS
Watson 978-0-12-398456-2 00084
The final evidence of hypolipemic effects of bergamot
polyphenols comes from an extensive placebocontrolled
human clinical trial.8 This study recruited
237 patients who had been divided into three groups:
hypercholesterolemic (HC, increased tChol, cLDL, low
cHDL), mixed dyslipidemic (HC and increased TG),
and metabolic syndrome patients (HC, HT and
increased blood glucose HG). The patients received 500
or 1000 mg BPF daily. After 30 days, 90% of the patients
in all three groups responded with a significant reduction
in lipid blood parameters ranging from 5 up to
45% decrease (Table 84.2). In particular, more than a
45% decrease for cLDL was achieved in 10% patients.
The most striking reduction (mean 41.062.6%) was
observed for plasma triglyceride levels. Excellent mean
reduction for other blood parameters was achieved:
228.1% for totChol and 233.2% for cLDL and 130.3%
for cHDL in a group of MS patients (Table 84.2 and
Figure 84.3). Patients with isolated HC and HC1HT
responded in a similar way to treatment with BPF
AU:1 (Table 84.3, groups A and B vs C). Placebo-treated
patients showed a maximum 5% reduction in
cholesterol parameters and triglycerides. Less striking,
but significant results were obtained in a small group of
statin-intolerant patients (group D), who started BPF
therapy after a considerable “wash-out period” (4
weeks) to reduce the rebound effect that takes place
after statin withdrawal (Table 84.2, group D).
In order to verify pharmacological properties of p0150
BPF, an independent observational study has been performed
in Australia on over 600 patients, with MS or
hyperlipidemia. All patients were subjected to 1300 mg
BPF therapy. Although, with all treatments, there was
a group of non-responders, when all patients were
considered, there was (on average) a 29% reduction in
cholesterol, a 36% reduction in cLDL, up to a 40%
reduction in triglycerides and up to a 40% increase in
HDL levels (R. Walker, unpublished observations).
3.3 Hypoglycemic Effects and Improvement of s0035
Endothelial Function
The clinical study by Mollace et al. p0155 8 showed other
important pharmacological properties of BPF. Indeed,
t0015 TABLE 84.2 A summary of the Results from Clinical Trial Testing of the Efficacy of BPF Treatment (30 days) Against tChol, cLDL,
cHDL in Patients with Hypercholesterolemia (HC, Group A), Mixed Hyperlipidemia (HC/HT, Group B), MS (HC/HT/HG, Group C) and
Patients Intolerant to Statins (Group D).
BPF
Dose
Daily
(mg)
tChol cLDL cHDL
Patients
Group
Sub-
Groupa
Nr
Pb
% Δ6
SEMc
No
Resp.d
Best
10%e
% Δ6
SEM
No
Resp.
Best
10%
% Δ6
SEM
No
Resp.
Best
10%
A A1 35 500 220.761.9 6 234.6 223.061.9 4 237.2 25.962.3 0 50.0
A2 37 1000 230.961.5 2 240.0 238.661.5 0 249.1 39.062.8* 0 68.6
APL 32 0 20.460.4 32 2 21.760.5 27 2 0.561.1 22 2
B B1 14 500 221.961.8 1 228.3 225.362.0 0 234.6 17.361.4 0 26.7
B2 14 1000 227.763.4 2 241.5 233.463.9 2 243.6 35.864.2* 0 66.7
BPL 14 0 20.560.5 14 2 20.560.7 13 2 21.361.8 11 2
C C1 20 500 224.762.6 2 241.7 226.863.6 1 253.6 16.561.6 0 42.9
C2 19 1000 228.162.6 1 241.1 233.263.0 1 247.0 29.661.8* 0 64.6
CPL 20 0 0.560.5 20 2 20.961.4 18 2 2.962.0 15 2
D 2 32 1500 225.061.6 2 239.8 227.660.5 0 232.4 23.861.7 0 41.1
A1B1C 2 69 500 221.861.4 9 237.8 224.161.5 5 245.0 22.361.3 0 248.6
2 70 1000 229.461.3 5 240.6 236.061.4* 3 247.9 40.161.9* 0 266.4
2 66 0 20.16 0.3 66 21.160.5 58 2 1.260.9 48 2
aFor division of all recruited patients in groups and subgroups, see Section 2.3.1.
bNr P, number of patients recruited for each treatment group (AD) and subgroup: 1 (low dose BPF), 2 (high dose BPF) or PL (placebo).
cMean changes in blood parameters for each group or subgroup of patients were calculated by adding the changes recorded for individual patients and dividing them by the
number of patients (Nr P).
dNo response Number of patients that show a smaller than 5% reduction in totChol, cLDL or a lower than 5% increase in cHDL after 30 days treatment with BPF. Note that
in some cases bigger than 5% changes were recorded in the placebo treated patients.
eBest 10%, mean value in the subgroup of the best 10% responders.
*Statistically significant difference compared to 500 mg BPF dose.
The table was prepared according to the data published in Mollace et al.8 Copyright Elsevier Inc.
3. PHARMACOLOGICAL EFFECTS OF BERGAMOT POLYPHENOL FRACTION ON CARDIOVASCULAR RISK FACTORS 1091
8. CARDIAC HEALTH AND POLYPHENOLS
Watson 978
the hypolipemic effect in patients with MS was accompanied
by a significant reduction in blood glucose
levels. In particular, the mean reduction in glucose was
218.9% and 222.4% in patients taking 500 and
1000 mg BPF daily, respectively8 (Table 84.2 and
Figure 84.3). This observation was unexpected and
clearly suggests that the pharmacological effects of
BPF go beyond statin-like activity. This interesting
metformin-like property of BPF was partially confirmed
in the Australian observational study.
p0160 The patients with elevated cardiometabolic risk factors
usually suffer from endothelial stiffness, i.e., the
reduction of flow-mediated vasodilatation. This problem
is particularly pronounced in MS patients with
elevated blood glucose. Indeed, the study by Mollace
et al.8 showed that patients with mixed
hyperlipidemia and hyperglycemia (HC/HT/HG)
with lowest flow-mediated vasodilatation before the
treatment, showed a particular B50% improvement
of endothelial function after 30 days of 1000 mg BPF
therapy. Significant improvement was also observed
in other groups patients (see Figure 84.4).
s0040
4. STATIN THERAPY AND BPF
4.1 Advantages and Disadvantages of s0045
Conventional Therapy Based on Statins
The most commonly prescribed drug across the p0165
world against hypercholesterolemia is Atorvastatin,
which belongs to statin family of cholesterol-lowering
f0020 FIGURE 84.3 Reduction of total cholesterol (tCho), LDL cholesterol (cLDL), triglycerides (TG) and glucose levels and increase of HDLcholesterol
(cHDL) in the blood of patients with metabolic syndrome. A group of 59 metabolic syndrome patients was divided in two
groups. Twenty-nine patients were asked to take 500 mg BPF once or twice a day while 30 patients took equivalent amounts of placebo for 30
days. Note the significant reduction in all blood parameters accompanied by the increase in “good cholesterol” cHDL. The graphs show the
mean values for indicated blood parameters from the relative treatment group. Group C1, n520, 500 mg/day, group C2, n519, 1000 mg/
day, group CPL, n520, placebo. The indicated patients’ blood parameters, all expressed in mg/dLl, were analyzed on day 0 (before) and day
30 (after) of treatment. Error bars show the standard deviation (SD). *Indicates a statistically significant change compared to the placebo group
at p ,0.0001; #Indicates a statistically significant change between C1 and C2 subgroups at p ,0.05. From Mollace et al.8
t0020 TABLE 84.3 The Percent Variations in TG and Blood Glucose in Patients Suffering from HC/HTand HC/HT/HG Subjected to the 30
Day BPF Treatment.
BPF Dose Daily
(mg)
Triglycerides Glucose
Patients
Group
Subgroup
Nr
Pa
%Δ6
SEMb
No
Responsec
Best
10%d %Δ6SEM
No
Response
Best
10%
B B1 14 500 228.263.9 2 246.8 234.6
B2 14 1000 237.963.3 1 247.9 243.6
BPL 14 0 0.1 to 0.5 14
C C1 20 500 232.762.5 0 241.6 218.961.2 0 227.1
C2 19 1000 241.062.6 1 249.6 222.461.0 0 232.2
CPL 20 0 0.060.6 19 20.560.7 20
aNogata et al.17
bGattuso et al.18
cUnpublished results, obtained in samples of industrial juice, courtesy of D. Malara, Reggio Calabria, Italy.
dDi Donna et al.16
Prepared according to the data published in Mollace et al.8 Copyright Elsevier.
Treatment groups or subgroups of patients are indicated B-C and . . .1,. . .2, . . .PL according to codes described in the title of Table 84.1.
1092 84. THE USE OF BERGAMOT-DERIVED POLYPHENOL FRACTION IN CARDIOMETABOLIC RISK PREVENTION
8. CARDIAC HEALTH AND POLYPHENOLS
Watson 978-0-12-398456-2 00084
drugs. Statins have been one of the greatest advances
in cardiology over the past 50 years.
p0170 The advantages of statin therapy are:
u0010 • Proven clinical efficiency. Depending on dose, statins
reduce LDL anywhere between 3060%, based on
the particular statin and dose. Typically the
reduction in cardiovascular events is somewhere
2030%.31 The recent Jupiter trial suggested a 50%
reduction in cardiac events with a moderate dose of
Rosuvastatin (20 mg) but this was in an
intermediate risk group with an overall low number
of events, albeit statistically significant according to
the Jupiter study.32
u0015 • Tolerability and compliance. Most patients tolerate low
to moderate doses, which only needs to be taken as
a daily dose. However, a number of studies have
clearly shown that compliance is poor with only
50% of patients still taking their medications twelve
months after the initial prescription.33
u0020 • Pleiotropic effects. Not only do statins have powerful
total cholesterol and LDL lowering effects, there is
also good evidence to support non-lipid benefits
such as anti-inflammatory, antioxidant and antiplatelet
effects. Statins have also been shown to
reduce plaque size and burden.34
p0190 There are, however, disadvantages to the long-term
use of statins. Although the medical profession has
had a “long-term love affair” with statins, there has
been much disquiet amongst the complementary medical
world and the general public. Several of the
patients attribute their many side effects, both subtle
and not so subtle, to their statin therapy. Statins are
well-described as contributing to myalgia and other
muscle related issues, including an elevated CPK.
Although the clinical trials suggests the incidence of
muscle problems is only 12%, in real world clinical
practice this occurs in around 1020% of patients.35
Twenty percent of patients taking (especially fat soluble)
statins experience neuro-cognitive symptoms.
Liver abnormalities have been described and abnormal
liver function is commonly seen.35
For a number of years, cancer concerns have been p0195
raised in regard to chronic statin therapy but there has
been no consistent data to prove or deny this relationship.
36 There is no doubt that statins are effective
agents with a central role in the management of AVD,
but it is, in fact, this powerful, metabolic regulatory
effect that also raises some concerns.
Some people who take statins, often commencing in p0200
their fourth or fifth decade of life, may potentially be
maintained on these agents for anywhere between 20
to 50 years, based on their longevity.
Cholesterol is a vital component of cell membrane p0205
structure and function, along with a key component of
steroid, bile salts and vitamin D metabolism. To date,
there have been no data regarding the potential deleterious
effects of prolonged statin therapy, the majority
of clinical trials running for less than 10 years. In fact,
four studies over the past 2 years have suggested a
50% increased risk for diabetes in patients ingesting
statins long-term.37 Finally, recent studies have raised
doubts as to the benefits of statin use in primary prevention.
A recent meta-analysis of 65,229 patients
reviewing 11 studies demonstrated no statistically significant
reduction in all-cause mortality.38
A recent Cochrane review of 14 trials, involving p0210
34,272 patients showed no evidence of improved quality
of life or cost effectiveness in primary prevention
patients.39
Thus, in the authors’ opinion, there needs to be a p0215
different approach to the management of hypercholesterolemia
and associated lipid disorders. The evidence
strongly suggests the routine use of statins as part of
the management of all patients with established vascular
disease or with strong evidence of a significant atherosclerotic
load on appropriate non-evasive screening
tools (e.g., coronary calcium scoring, carotid intimal—
medical thickness measurements, etc.). To date, however,
there are no scientific data supporting the use of
statins for isolated lipid abnormalities, in the absence
of other significant cardiac risk factors, especially
when, for example, a coronary calcium score places
the individual either at 0 or in a low percentile risk
range (e.g., ,50th percentile for score and age). In
these individuals, the current data suggests no benefit
and possibly harm from long-term exposure, such as
compared to other Citrus species. These observations
were then confirmed for industrial bergamot
juice obtained by fruit crashing.18 A lower content of
flavonoids is present in manually squeezed bergamot
juice.19,20 This discrepancy can be explained by
differences in processing and different distribution of
flavonoids between the vacuole juice, albedo (the inner
white part of the peel), flavedo (external peel rich in
aromatic oils), and inner fibers of the fruits. Indeed,
the majority of flavonoids are several times more
abundant in albedo than in the juice,17 and industrial
pressing causes a release of albedo compounds. This
processing enriches the total flavonoid content of the
juice to more than 1600 mg/L in the industrial juices
from the initial 373 up to 512 mg/L, present in the
“hand-squeezed” juices of two different Bergamot cultivars
Fantastico and Feminello, respectively.19 Thus, the
industrial bergamot juice likely shows the highest concentration
of polyphenols among non-concentrated
natural juices.
p0040 Bergamot fruits (juice, albedo, flavedo, and fibers)
show the highest concentrations of flavanones: neoeriocitrin,
neohesperidin, naringin, poncirin, melitidin
and brutieridin, and the highest content of certain flavones:
rhoifolin and neodiosmin among 45 different
Citrus species17 (see Table 84.1). A high content was
confirmed for rhoifolin and neodiosmin by another
independent study that also identified other highly
abundant flavones in bergamot juice: vicenin-2, diosmetin
di-C-glucoside and chryseriol 7-O-neohesperoside
and lower amounts of other flavonoids: lucenin-2,
stellarin-2, isovitexin, scoparin, diosmetin 8-C-glucoside,
chrysoeriol 7-O-neohesperoside-40-O-glycoside
and others.18 Another peculiar feature of bergamot
juice is a high content of unsaturated fatty acids,
accounting for 80% of total acids, such as oleic acid,
linoleic acid and palmitic acid.21
p0045 As a medicinal food, bergamot juice is not always
available and is subjected to fast deterioration. In the
search of a more stable and possibly more powerful
pharmaceutical surrogate of bergamot juice, a method
to concentrate and exsiccate bergamot juice was developed.
According to this method, bergamot juice is concentrated
by a preparative size exclusion
chromatography based on polystyrene gel filtration.8
Polyphenols and other macromolecules of the molecular
size over 300 Da are retained on the column, eluted
with acids and exsiccated with other clarified juice
components to give rise to a flavonoid-enriched bergamot
powder or BPF.8 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
(D. Malara, personal communication). In contrast to
BPF, bergamot juice derivatives obtained by spry-drier
method rich maximum 1% polyphenols concentration
(D. Malara, personal communication). The main polyphenol
components of BPF are flavonoids and their
composition basically mirrors the bergamot juice polyphenol
profile (see Figure 84.2, and Plate 13), with the
only difference that flavonoids are over 200 times
more concentrated in BPF. Flavanones glycosides
account for 95% of total flavonoids present in BPF
(and in bergamot juice), while flavones can be found
in the remaining 5% (Table 84.1 and D. Malara and
C. Malara, unpublished observations).
Currently, there are no published bioavailability and p0050
pharmacokinetic studies for BPF. However, absorption,
metabolism and excretion parameters have been
described for several individual flavonoids present in
bergamot juice. It is well known that flavonoid glycosides
are hydrolyzed to aglycones by baterial flora of
the gut. Gut microflora hydrolysis is thought to favor
flavonoid glycoside bioavailability.22 When sugar unit
is removed, the resulting aglycone can be absorbed
more readily.23 Indeed, flavonoid aglycones of diosmin,
hesperidin and naringin, diffuse usually easily through
the plasma membrane of Caco-2 cells, in contrast to the
respective glycosides. Moreover, naringin, hesperidin,
rutin and poncirin are hydrolyzed to their respective
aglycones by human intestinal microflora, and the
resulting aglycones are absorbed better.24 However, the
bioavilability of flavonoids is low and it is estimated
that only 10% of total consumed polyphenols are
absorbed, although these numbers vary between species
and individuals.23 When inside the enterocytes part
of aglycones are subjected to intestinal metabolism,
such as glucuronidation and sulfation. Phase II metabolism
is the main route of metabolism for polyphenols.
Conjugation of free phenolic groups via glucuronidation
and/or sulfation will increase their polarity and
water solubility, enabling their elimination from the
body.23 Generally, citrus and other flavonoids have a
short life-time and metabolites are thought to lose their
biological activity. Nevertheless, recent data suggest
that sulfonated or methylated and much less glucuronidated
metabolites of resveratrol maintain full or partial
activity of the parent compound. In addition, sulfonation
has been shown to increase the activity for some
molecular targets of resveratrol.25
Therefore although not properly investigated, it is p0055
likely aglycones of bergamot flavonoids and their
metabolites contribute to the modulation of the intracellular
targets and the subsequent biological response.
3. PHARMACOLOGICAL EFFECTS OF s0020
BERGAMOT POLYPHENOL FRACTION
ON CARDIOVASCULAR RISK FACTORS
3.1 Metabolic Syndrome and Cardiovascular s0025
Risk Factors
AVD is the leading cause of death in developed p0060
countries.26 Coronary artery disease is the commonest
form of cardiovascular disease accounting for around a
third of all-cause mortality in people over the age of 35.
AVD is caused by atherosclerosis, which is defined as
the deposition of lipids, inflammatory cells and mediators
in the subendothelial layer of blood vessels,
eventually leading to thickening and hardening of
3. PHARMACOLOGICAL EFFECTS OF BERGAMOT POLYPHENOL FRACTION ON CARDIOVASCULAR RISK FACTORS 1089
8. CARDIAC HEALTH AND POLYPHENOLS
Watson 978-0-12-398456-2 00084
medium- to large-sized arteries. Atherosclerosis has a
very long pre-symptomatic phase and can begin even
in-utero in predisposed individuals. The presymptomatic
phase of atherosclerosis averages between
4060 years, although clinical presentation seldom
occurs before this time frame and commonly thereafter.
The presentation, thus depends on the rates of atherosclerotic
deposition in the blood vessel wall, often lasting
decades, while common clinical manifestation of
AVD, such as acute myocardial infarction,
unstable angina, stroke or sudden cardiac death, usually
occur following the rupture of a large plaque in the
blood vessel wall.
p0065 It is well-established that the abundance of fat and
sugar in diets found in industrialized western society is
the most important risk factor for developing AVD.
Indeed, as many of the developing nations adopt more
affluent life styles, an increasing rates of cardiovascular
disease is seen.27 This is because the diet influences blood
parameters that may become AVD risk factors if out of
established limits. For example, the average cholesterol
levels in malnourished regions (for example, rural China)
are between 7797 mg/dL (22.5 mmol/L).
Atherosclerosis is a rarity in these regions.28 Once food
becomes available and in plentiful supply, the cholesterol
level rises above 3 mmol/L and it is above this level that
atherosclerosis occurs. In fact, in the majority of cases,
people with lifelong cholesterol levels below 4 mmol/L
are rarely troubled by significant complications of
atherosclerosis.28
p0070 Excessive fat and sugar consumption, linked to
genetic predisposition and poor lifestyle behaviors
may lead to the development of a pathological state,
called metabolic syndrome (MS). MS is very common
and 1030% of individuals in industrialized countries
suffer from this condition. MS is defined as a cluster of
cardiovascular risk factors, including atherogenic dyslipidemia,
insulin resistance or glucose intolerance, visceral
obesity, hypertension and endothelial
dysfunction.
p0075 There are four basic components of MS:
o0010 1. Hyperglycemia (fasting plasma glucose $110 mg/dl
(6.1 mmol/L))
o0015 2. Hypertension (systolic blood pressure .130 or
diastolic blood pressure .85 mmHg)
o0020 3. Dyslipidemia
o0025 a) Increased cholesterol .200 mg/dL (5 mmol/L)
o0030 b) Increased Triglyceride .150 mg/dL (1.5 mmol/L)
o0035 c) Reduced cHDL ,55 mg/dL, (1.3 mmol/L)
o0040 4. Abdominal obesity
o0045 a) Males .95 cm
o0050 b) Females .80 cm
p0125 The presence of all components of MS is associated
with a particularly increased risk of accelerated
atherosclerosis and cardiovascular events.29 However,
the probability of developing AVD (cardiovascular
risk) is also increased in individuals with isolated metabolic
dysfunctions, such as increased cholesterol
levels (hypercholesterolemia) or increased level of triglycerides
(hypertriglyceridemia) and in patients with
type-1 and -2 diabetes as well as in patients with glucose
intolerance and hypertension.
Pharmacological treatment of any cardiometabolic p0130
risk factors is clearly a preventive medicinal approach,
since it slows down the process of atherosclerosis and
reduces the probability of cardiovascular events. For
this reason, it would be preferable that such a treatment
is based on diet and natural drugs, and starts as
early as possible in adult age; and not on conventional
and aggressive polypharmacy started when MS or
even ADV is well-advanced.
The experimental and clinical evidence will be pre- p0135
sented here, showing that BPF—a concentrate of natural
bergamot polyphenols—is a suitable treatment for
MS and other metabolic pathologies associated with an
increased cardiovascular risk of atherosclerosis and
life-threatening cardiovascular events.
3.2 Hypolipidemic Effects s0030
The first evidence of the cholesterol-lowering prop- p0140
erties of bergamot polyphenols comes from experimental
studies on rats fed a high-cholesterol diet.7 In this
laboratory model of diet-induced hypocholesterolemia,
rats on a 2% cholesterol and 20% oil diet developed
extremely high levels of total blood cholesterol (tChol,
700 mg/dL). The animals treated daily with 1 ml bergamot
juice for 28 days showed a marked reduction in
total blood cholesterol (tChol) by (29.3%) , triglicerides
(46.2%) and cLDL (51.7%). These results were confirmed
in an independent work by Mollace et al.,8
addressing the efficacy of the bergamot polyphenol
mixture, BPF. Another important pharmacological
finding observed by Miceli et al.7 was evident reduction
of lipid steatosis in the liver, defined as a decrease
in the number and size of hepatic lipid droplets and a
reduction in inflammatory changes due to fat accumulation
in the liver. In addition, the anatomical analysis
of arteries indicated some protective effect of bergamot
juice against mild endothelial thickening. Next
Mollace’s group confirmed these observations and
demonstrated a prevention of accelerated stenosis with
the use of Bergamot extract. In this model of stenosis/
atherosclerosis, the balloon injury is induced to the
carotid artery in laboratory rats.30 Bergamot extract (in
this case a non-volatile part of bergamot oil) uniformly
prevented rapid thickening and inflammatory changes
in the arteries subjected to balloon injury.
1090 84. THE USE OF BERGAMOT-DERIVED POLYPHENOL FRACTION IN CARDIOMETABOLIC RISK PREVENTION
8. CARDIAC HEALTH AND POLYPHENOLS
Watson 978-0-12-398456-2 00084
medium- to large-sized arteries. Atherosclerosis has a
very long pre-symptomatic phase and can begin even
in-utero in predisposed individuals. The presymptomatic
phase of atherosclerosis averages between
4060 years, although clinical presentation seldom
occurs before this time frame and commonly thereafter.
The presentation, thus depends on the rates of atherosclerotic
deposition in the blood vessel wall, often lasting
decades, while common clinical manifestation of
AVD, such as acute myocardial infarction,
unstable angina, stroke or sudden cardiac death, usually
occur following the rupture of a large plaque in the
blood vessel wall.
p0065 It is well-established that the abundance of fat and
sugar in diets found in industrialized western society is
the most important risk factor for developing AVD.
Indeed, as many of the developing nations adopt more
affluent life styles, an increasing rates of cardiovascular
disease is seen.27 This is because the diet influences blood
parameters that may become AVD risk factors if out of
established limits. For example, the average cholesterol
levels in malnourished regions (for example, rural China)
are between 7797 mg/dL (22.5 mmol/L).
Atherosclerosis is a rarity in these regions.28 Once food
becomes available and in plentiful supply, the cholesterol
level rises above 3 mmol/L and it is above this level that
atherosclerosis occurs. In fact, in the majority of cases,
people with lifelong cholesterol levels below 4 mmol/L
are rarely troubled by significant complications of
atherosclerosis.28
p0070 Excessive fat and sugar consumption, linked to
genetic predisposition and poor lifestyle behaviors
may lead to the development of a pathological state,
called metabolic syndrome (MS). MS is very common
and 1030% of individuals in industrialized countries
suffer from this condition. MS is defined as a cluster of
cardiovascular risk factors, including atherogenic dyslipidemia,
insulin resistance or glucose intolerance, visceral
obesity, hypertension and endothelial
dysfunction.
p0075 There are four basic components of MS:
o0010 1. Hyperglycemia (fasting plasma glucose $110 mg/dl
(6.1 mmol/L))
o0015 2. Hypertension (systolic blood pressure .130 or
diastolic blood pressure .85 mmHg)
o0020 3. Dyslipidemia
o0025 a) Increased cholesterol .200 mg/dL (5 mmol/L)
o0030 b) Increased Triglyceride .150 mg/dL (1.5 mmol/L)
o0035 c) Reduced cHDL ,55 mg/dL, (1.3 mmol/L)
o0040 4. Abdominal obesity
o0045 a) Males .95 cm
o0050 b) Females .80 cm
p0125 The presence of all components of MS is associated
with a particularly increased risk of accelerated
atherosclerosis and cardiovascular events.29 However,
the probability of developing AVD (cardiovascular
risk) is also increased in individuals with isolated metabolic
dysfunctions, such as increased cholesterol
levels (hypercholesterolemia) or increased level of triglycerides
(hypertriglyceridemia) and in patients with
type-1 and -2 diabetes as well as in patients with glucose
intolerance and hypertension.
Pharmacological treatment of any cardiometabolic p0130
risk factors is clearly a preventive medicinal approach,
since it slows down the process of atherosclerosis and
reduces the probability of cardiovascular events. For
this reason, it would be preferable that such a treatment
is based on diet and natural drugs, and starts as
early as possible in adult age; and not on conventional
and aggressive polypharmacy started when MS or
even ADV is well-advanced.
The experimental and clinical evidence will be pre- p0135
sented here, showing that BPF—a concentrate of natural
bergamot polyphenols—is a suitable treatment for
MS and other metabolic pathologies associated with an
increased cardiovascular risk of atherosclerosis and
life-threatening cardiovascular events.
3.2 Hypolipidemic Effects medium- to large-sized arteries. Atherosclerosis has a
very long pre-symptomatic phase and can begin even
in-utero in predisposed individuals. The presymptomatic
phase of atherosclerosis averages between
4060 years, although clinical presentation seldom
occurs before this time frame and commonly thereafter.
The presentation, thus depends on the rates of atherosclerotic
deposition in the blood vessel wall, often lasting
decades, while common clinical manifestation of
AVD, such as acute myocardial infarction,
unstable angina, stroke or sudden cardiac death, usually
occur following the rupture of a large plaque in the
blood vessel wall.
p0065 It is well-established that the abundance of fat and
sugar in diets found in industrialized western society is
the most important risk factor for developing AVD.
Indeed, as many of the developing nations adopt more
affluent life styles, an increasing rates of cardiovascular
disease is seen.27 This is because the diet influences blood
parameters that may become AVD risk factors if out of
established limits. For example, the average cholesterol
levels in malnourished regions (for example, rural China)
are between 7797 mg/dL (22.5 mmol/L).
Atherosclerosis is a rarity in these regions.28 Once food
becomes available and in plentiful supply, the cholesterol
level rises above 3 mmol/L and it is above this level that
atherosclerosis occurs. In fact, in the majority of cases,
people with lifelong cholesterol levels below 4 mmol/L
are rarely troubled by significant complications of
atherosclerosis.28
p0070 Excessive fat and sugar consumption, linked to
genetic predisposition and poor lifestyle behaviors
may lead to the development of a pathological state,
called metabolic syndrome (MS). MS is very common
and 1030% of individuals in industrialized countries
suffer from this condition. MS is defined as a cluster of
cardiovascular risk factors, including atherogenic dyslipidemia,
insulin resistance or glucose intolerance, visceral
obesity, hypertension and endothelial
dysfunction.
p0075 There are four basic components of MS:
o0010 1. Hyperglycemia (fasting plasma glucose $110 mg/dl
(6.1 mmol/L))
o0015 2. Hypertension (systolic blood pressure .130 or
diastolic blood pressure .85 mmHg)
o0020 3. Dyslipidemia
o0025 a) Increased cholesterol .200 mg/dL (5 mmol/L)
o0030 b) Increased Triglyceride .150 mg/dL (1.5 mmol/L)
o0035 c) Reduced cHDL ,55 mg/dL, (1.3 mmol/L)
o0040 4. Abdominal obesity
o0045 a) Males .95 cm
o0050 b) Females .80 cm
p0125 The presence of all components of MS is associated
with a particularly increased risk of accelerated
atherosclerosis and cardiovascular events.29 However,
the probability of developing AVD (cardiovascular
risk) is also increased in individuals with isolated metabolic
dysfunctions, such as increased cholesterol
levels (hypercholesterolemia) or increased level of triglycerides
(hypertriglyceridemia) and in patients with
type-1 and -2 diabetes as well as in patients with glucose
intolerance and hypertension.
Pharmacological treatment of any cardiometabolic p0130
risk factors is clearly a preventive medicinal approach,
since it slows down the process of atherosclerosis and
reduces the probability of cardiovascular events. For
this reason, it would be preferable that such a treatment
is based on diet and natural drugs, and starts as
early as possible in adult age; and not on conventional
and aggressive polypharmacy started when MS or
even ADV is well-advanced.
The experimental and clinical evidence will be pre- p0135
sented here, showing that BPF—a concentrate of natural
bergamot polyphenols—is a suitable treatment for
MS and other metabolic pathologies associated with an
increased cardiovascular risk of atherosclerosis and
life-threatening cardiovascular events.
3.2 Hypolipidemic Effects s0030
The first evidence of the cholesterol-lowering prop- p0140
erties of bergamot polyphenols comes from experimental
studies on rats fed a high-cholesterol diet.7 In this
laboratory model of diet-induced hypocholesterolemia,
rats on a 2% cholesterol and 20% oil diet developed
extremely high levels of total blood cholesterol (tChol,
700 mg/dL). The animals treated daily with 1 ml bergamot
juice for 28 days showed a marked reduction in
total blood cholesterol (tChol) by (29.3%) , triglicerides
(46.2%) and cLDL (51.7%). These results were confirmed
in an independent work by Mollace et al.,8
addressing the efficacy of the bergamot polyphenol
mixture, BPF. Another important pharmacological
finding observed by Miceli et al.7 was evident reduction
of lipid steatosis in the liver, defined as a decrease
in the number and size of hepatic lipid droplets and a
reduction in inflammatory changes due to fat accumulation
in the liver. In addition, the anatomical analysis
of arteries indicated some protective effect of bergamot
juice against mild endothelial thickening. Next
Mollace’s group confirmed these observations and
demonstrated a prevention of accelerated stenosis with
the use of Bergamot extract. In this model of stenosis/
atherosclerosis, the balloon injury is induced to the
carotid artery in laboratory rats.30 Bergamot extract (in
this case a non-volatile part of bergamot oil) uniformly
prevented rapid thickening and inflammatory changes
in the arteries subjected to balloon injury.
1090 84. THE USE OF BERGAMOT-DERIVED POLYPHENOL FRACTION IN CARDIOMETABOLIC RISK PREVENTION
8. CARDIAC HEALTH AND POLYPHENOLS
Watson 978-0-12-398456-2 00084
The final evidence of hypolipemic effects of bergamot
polyphenols comes from an extensive placebocontrolled
human clinical trial.8 This study recruited
237 patients who had been divided into three groups:
hypercholesterolemic (HC, increased tChol, cLDL, low
cHDL), mixed dyslipidemic (HC and increased TG),
and metabolic syndrome patients (HC, HT and
increased blood glucose HG). The patients received 500
or 1000 mg BPF daily. After 30 days, 90% of the patients
in all three groups responded with a significant reduction
in lipid blood parameters ranging from 5 up to
45% decrease (Table 84.2). In particular, more than a
45% decrease for cLDL was achieved in 10% patients.
The most striking reduction (mean 41.062.6%) was
observed for plasma triglyceride levels. Excellent mean
reduction for other blood parameters was achieved:
228.1% for totChol and 233.2% for cLDL and 130.3%
for cHDL in a group of MS patients (Table 84.2 and
Figure 84.3). Patients with isolated HC and HC1HT
responded in a similar way to treatment with BPF
AU:1 (Table 84.3, groups A and B vs C). Placebo-treated
patients showed a maximum 5% reduction in
cholesterol parameters and triglycerides. Less striking,
but significant results were obtained in a small group of
statin-intolerant patients (group D), who started BPF
therapy after a considerable “wash-out period” (4
weeks) to reduce the rebound effect that takes place
after statin withdrawal (Table 84.2, group D).
In order to verify pharmacological properties of p0150
BPF, an independent observational study has been performed
in Australia on over 600 patients, with MS or
hyperlipidemia. All patients were subjected to 1300 mg
BPF therapy. Although, with all treatments, there was
a group of non-responders, when all patients were
considered, there was (on average) a 29% reduction in
cholesterol, a 36% reduction in cLDL, up to a 40%
reduction in triglycerides and up to a 40% increase in
HDL levels (R. Walker, unpublished observations).
3.3 Hypoglycemic Effects and Improvement of s0035
Endothelial Function
The clinical study by Mollace et al. p0155 8 showed other
important pharmacological properties of BPF. Indeed,
t0015 TABLE 84.2 A summary of the Results from Clinical Trial Testing of the Efficacy of BPF Treatment (30 days) Against tChol, cLDL,
cHDL in Patients with Hypercholesterolemia (HC, Group A), Mixed Hyperlipidemia (HC/HT, Group B), MS (HC/HT/HG, Group C) and
Patients Intolerant to Statins (Group D).
BPF
Dose
Daily
(mg)
tChol cLDL cHDL
Patients
Group
Sub-
Groupa
Nr
Pb
% Δ6
SEMc
No
Resp.d
Best
10%e
% Δ6
SEM
No
Resp.
Best
10%
% Δ6
SEM
No
Resp.
Best
10%
A A1 35 500 220.761.9 6 234.6 223.061.9 4 237.2 25.962.3 0 50.0
A2 37 1000 230.961.5 2 240.0 238.661.5 0 249.1 39.062.8* 0 68.6
APL 32 0 20.460.4 32 2 21.760.5 27 2 0.561.1 22 2
B B1 14 500 221.961.8 1 228.3 225.362.0 0 234.6 17.361.4 0 26.7
B2 14 1000 227.763.4 2 241.5 233.463.9 2 243.6 35.864.2* 0 66.7
BPL 14 0 20.560.5 14 2 20.560.7 13 2 21.361.8 11 2
C C1 20 500 224.762.6 2 241.7 226.863.6 1 253.6 16.561.6 0 42.9
C2 19 1000 228.162.6 1 241.1 233.263.0 1 247.0 29.661.8* 0 64.6
CPL 20 0 0.560.5 20 2 20.961.4 18 2 2.962.0 15 2
D 2 32 1500 225.061.6 2 239.8 227.660.5 0 232.4 23.861.7 0 41.1
A1B1C 2 69 500 221.861.4 9 237.8 224.161.5 5 245.0 22.361.3 0 248.6
2 70 1000 229.461.3 5 240.6 236.061.4* 3 247.9 40.161.9* 0 266.4
2 66 0 20.16 0.3 66 21.160.5 58 2 1.260.9 48 2
aFor division of all recruited patients in groups and subgroups, see Section 2.3.1.
bNr P, number of patients recruited for each treatment group (AD) and subgroup: 1 (low dose BPF), 2 (high dose BPF) or PL (placebo).
cMean changes in blood parameters for each group or subgroup of patients were calculated by adding the changes recorded for individual patients and dividing them by the
number of patients (Nr P).
dNo response Number of patients that show a smaller than 5% reduction in totChol, cLDL or a lower than 5% increase in cHDL after 30 days treatment with BPF. Note that
in some cases bigger than 5% changes were recorded in the placebo treated patients.
eBest 10%, mean value in the subgroup of the best 10% responders.
*Statistically significant difference compared to 500 mg BPF dose.
The table was prepared according to the data published in Mollace et al.8 Copyright Elsevier Inc.
3. PHARMACOLOGICAL EFFECTS OF BERGAMOT POLYPHENOL FRACTION ON CARDIOVASCULAR RISK FACTORS 1091
8. CARDIAC HEALTH AND POLYPHENOLS
Watson 978
the hypolipemic effect in patients with MS was accompanied
by a significant reduction in blood glucose
levels. In particular, the mean reduction in glucose was
218.9% and 222.4% in patients taking 500 and
1000 mg BPF daily, respectively8 (Table 84.2 and
Figure 84.3). This observation was unexpected and
clearly suggests that the pharmacological effects of
BPF go beyond statin-like activity. This interesting
metformin-like property of BPF was partially confirmed
in the Australian observational study.
p0160 The patients with elevated cardiometabolic risk factors
usually suffer from endothelial stiffness, i.e., the
reduction of flow-mediated vasodilatation. This problem
is particularly pronounced in MS patients with
elevated blood glucose. Indeed, the study by Mollace
et al.8 showed that patients with mixed
hyperlipidemia and hyperglycemia (HC/HT/HG)
with lowest flow-mediated vasodilatation before the
treatment, showed a particular B50% improvement
of endothelial function after 30 days of 1000 mg BPF
therapy. Significant improvement was also observed
in other groups patients (see Figure 84.4).
s0040
4. STATIN THERAPY AND BPF
4.1 Advantages and Disadvantages of s0045
Conventional Therapy Based on Statins
The most commonly prescribed drug across the p0165
world against hypercholesterolemia is Atorvastatin,
which belongs to statin family of cholesterol-lowering
f0020 FIGURE 84.3 Reduction of total cholesterol (tCho), LDL cholesterol (cLDL), triglycerides (TG) and glucose levels and increase of HDLcholesterol
(cHDL) in the blood of patients with metabolic syndrome. A group of 59 metabolic syndrome patients was divided in two
groups. Twenty-nine patients were asked to take 500 mg BPF once or twice a day while 30 patients took equivalent amounts of placebo for 30
days. Note the significant reduction in all blood parameters accompanied by the increase in “good cholesterol” cHDL. The graphs show the
mean values for indicated blood parameters from the relative treatment group. Group C1, n520, 500 mg/day, group C2, n519, 1000 mg/
day, group CPL, n520, placebo. The indicated patients’ blood parameters, all expressed in mg/dLl, were analyzed on day 0 (before) and day
30 (after) of treatment. Error bars show the standard deviation (SD). *Indicates a statistically significant change compared to the placebo group
at p ,0.0001; #Indicates a statistically significant change between C1 and C2 subgroups at p ,0.05. From Mollace et al.8
t0020 TABLE 84.3 The Percent Variations in TG and Blood Glucose in Patients Suffering from HC/HTand HC/HT/HG Subjected to the 30
Day BPF Treatment.
BPF Dose Daily
(mg)
Triglycerides Glucose
Patients
Group
Subgroup
Nr
Pa
%Δ6
SEMb
No
Responsec
Best
10%d %Δ6SEM
No
Response
Best
10%
B B1 14 500 228.263.9 2 246.8 234.6
B2 14 1000 237.963.3 1 247.9 243.6
BPL 14 0 0.1 to 0.5 14
C C1 20 500 232.762.5 0 241.6 218.961.2 0 227.1
C2 19 1000 241.062.6 1 249.6 222.461.0 0 232.2
CPL 20 0 0.060.6 19 20.560.7 20
aNogata et al.17
bGattuso et al.18
cUnpublished results, obtained in samples of industrial juice, courtesy of D. Malara, Reggio Calabria, Italy.
dDi Donna et al.16
Prepared according to the data published in Mollace et al.8 Copyright Elsevier.
Treatment groups or subgroups of patients are indicated B-C and . . .1,. . .2, . . .PL according to codes described in the title of Table 84.1.
1092 84. THE USE OF BERGAMOT-DERIVED POLYPHENOL FRACTION IN CARDIOMETABOLIC RISK PREVENTION
8. CARDIAC HEALTH AND POLYPHENOLS
Watson 978-0-12-398456-2 00084
drugs. Statins have been one of the greatest advances
in cardiology over the past 50 years.
p0170 The advantages of statin therapy are:
u0010 • Proven clinical efficiency. Depending on dose, statins
reduce LDL anywhere between 3060%, based on
the particular statin and dose. Typically the
reduction in cardiovascular events is somewhere
2030%.31 The recent Jupiter trial suggested a 50%
reduction in cardiac events with a moderate dose of
Rosuvastatin (20 mg) but this was in an
intermediate risk group with an overall low number
of events, albeit statistically significant according to
the Jupiter study.32
u0015 • Tolerability and compliance. Most patients tolerate low
to moderate doses, which only needs to be taken as
a daily dose. However, a number of studies have
clearly shown that compliance is poor with only
50% of patients still taking their medications twelve
months after the initial prescription.33
u0020 • Pleiotropic effects. Not only do statins have powerful
total cholesterol and LDL lowering effects, there is
also good evidence to support non-lipid benefits
such as anti-inflammatory, antioxidant and antiplatelet
effects. Statins have also been shown to
reduce plaque size and burden.34
p0190 There are, however, disadvantages to the long-term
use of statins. Although the medical profession has
had a “long-term love affair” with statins, there has
been much disquiet amongst the complementary medical
world and the general public. Several of the
patients attribute their many side effects, both subtle
and not so subtle, to their statin therapy. Statins are
well-described as contributing to myalgia and other
muscle related issues, including an elevated CPK.
Although the clinical trials suggests the incidence of
muscle problems is only 12%, in real world clinical
practice this occurs in around 1020% of patients.35
Twenty percent of patients taking (especially fat soluble)
statins experience neuro-cognitive symptoms.
Liver abnormalities have been described and abnormal
liver function is commonly seen.35
For a number of years, cancer concerns have been p0195
raised in regard to chronic statin therapy but there has
been no consistent data to prove or deny this relationship.
36 There is no doubt that statins are effective
agents with a central role in the management of AVD,
but it is, in fact, this powerful, metabolic regulatory
effect that also raises some concerns.
Some people who take statins, often commencing in p0200
their fourth or fifth decade of life, may potentially be
maintained on these agents for anywhere between 20
to 50 years, based on their longevity.
Cholesterol is a vital component of cell membrane p0205
structure and function, along with a key component of
steroid, bile salts and vitamin D metabolism. To date,
there have been no data regarding the potential deleterious
effects of prolonged statin therapy, the majority
of clinical trials running for less than 10 years. In fact,
four studies over the past 2 years have suggested a
50% increased risk for diabetes in patients ingesting
statins long-term.37 Finally, recent studies have raised
doubts as to the benefits of statin use in primary prevention.
A recent meta-analysis of 65,229 patients
reviewing 11 studies demonstrated no statistically significant
reduction in all-cause mortality.38
A recent Cochrane review of 14 trials, involving p0210
34,272 patients showed no evidence of improved quality
of life or cost effectiveness in primary prevention
patients.39
Thus, in the authors’ opinion, there needs to be a p0215
different approach to the management of hypercholesterolemia
and associated lipid disorders. The evidence
strongly suggests the routine use of statins as part of
the management of all patients with established vascular
disease or with strong evidence of a significant atherosclerotic
load on appropriate non-evasive screening
tools (e.g., coronary calcium scoring, carotid intimal—
medical thickness measurements, etc.). To date, however,
there are no scientific data supporting the use of
statins for isolated lipid abnormalities, in the absence
of other significant cardiac risk factors, especially
when, for example, a coronary calcium score places
the individual either at 0 or in a low percentile risk
range (e.g., ,50th percentile for score and age). In
these individuals, the current data suggests no benefit
and possibly harm from long-term exposure, such as
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Re: Open conversazione
by zafferano » Mon Feb 01, 2016 5:59 am
I HAD ENOUGH OF THIS I CAN PASTE AND COPY FOR DAYS ,and I don't know how to send as email in the forum
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Re: Open conversazione
by Zoff82 » Mon Feb 01, 2016 12:50 pm
Last edited by Zoff82 on Mon Feb 01, 2016 3:45 pm, edited 5 times in total.
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Re: Open conversazione
by juventino_italiano » Mon Feb 01, 2016 1:28 pm
You hens still clucking?
Dino, let it go...he doesn't get it.
Dino, let it go...he doesn't get it.
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Re: Open conversazione
by Zoff82 » Mon Feb 01, 2016 2:03 pm
I'm truly sad the great Maro can't throw his 2 cents in.
It's a diversion from Inter getting beat badly the past 2 games. How much of a hypocrite does Man-weenie look like after his gestures yesterday? If Sarri got suspended for 2 games, Man-weenie should get the same. Of course it wouldn't have happened when Inter & Moratti after sabotaging and setting up the top teams in the league for their advantage but I'm sure he'll get punished for it. If he doesn't, it would be criminal. After all, Sarri shouldn't have been suspended. All he did was state facts by sending homosexual slurs towards Mancini. I think it was the itself that got him suspended, not the fact he made a gay reference to a gay man. It was only a matter of time for Inter's luck to run out with the way they've been playing. Teams are going to catch on and he isn't smart enough to change tactics and be more unpredictable.
It's a diversion from Inter getting beat badly the past 2 games. How much of a hypocrite does Man-weenie look like after his gestures yesterday? If Sarri got suspended for 2 games, Man-weenie should get the same. Of course it wouldn't have happened when Inter & Moratti after sabotaging and setting up the top teams in the league for their advantage but I'm sure he'll get punished for it. If he doesn't, it would be criminal. After all, Sarri shouldn't have been suspended. All he did was state facts by sending homosexual slurs towards Mancini. I think it was the itself that got him suspended, not the fact he made a gay reference to a gay man. It was only a matter of time for Inter's luck to run out with the way they've been playing. Teams are going to catch on and he isn't smart enough to change tactics and be more unpredictable.
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Re: Open conversazione
by zafferano » Mon Feb 01, 2016 11:46 pm
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Re: Open conversazione
by zafferano » Mon Feb 01, 2016 11:50 pm
. Grapefruit, contraindicated in statins, possibly has the same properties as Bergamot (my theory, not fact but based on similar studies, I do believe that's the case) Grapefruit increases the drug levels of statins via inhibition of the CYP3A4 enzyme which decrease metabolic rate, increases the serum concentration (not so much in Crestor vs Lipitor).
nothing to do with grapefruit
we do know about grapefruit
and no use discussing anymore
you mind is set
nothing to do with grapefruit
we do know about grapefruit
and no use discussing anymore
you mind is set
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Re: Open conversazione
by zafferano » Tue Feb 02, 2016 12:40 am
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Re: Open conversazione
by Bagg10 » Tue Feb 02, 2016 12:46 am
Monday February 1 2016
Napoli fans banned for Juve clash
By Football Italia staff
http://www.football-italia.net/79283/na ... juve-clash
There are reports that Napoli fans will be banned from attending the Scudetto clash with Juventus on February 13.
There are currently two points between the clubs at the summit of the Serie A table, and they are due to meet in less than two weeks at Juventus Stadium.
However, widespread reports in Italy say that visiting fans have been forbidden from attending the match.
The ban is believed to be specific to fans who live in the Naples area, but there are still many Partenopei supporters living in Turin.
Juventus fans were banned from attending the first meeting between the clubs earlier this season at the San Paolo due to security fears.
Napoli fans banned for Juve clash
By Football Italia staff
http://www.football-italia.net/79283/na ... juve-clash
There are reports that Napoli fans will be banned from attending the Scudetto clash with Juventus on February 13.
There are currently two points between the clubs at the summit of the Serie A table, and they are due to meet in less than two weeks at Juventus Stadium.
However, widespread reports in Italy say that visiting fans have been forbidden from attending the match.
The ban is believed to be specific to fans who live in the Naples area, but there are still many Partenopei supporters living in Turin.
Juventus fans were banned from attending the first meeting between the clubs earlier this season at the San Paolo due to security fears.
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Re: Open conversazione
by Zoff82 » Tue Feb 02, 2016 1:31 am
No no Briz. You can't just go off and neglect what you feel like. It's got plenty to do wit grapefruit. Very similar mechanism of action with flavonoids and polypehnols. Obviously, you should get prescriptions for Adderall at your next conference. You can't focus on a topic even after I told you what to focus on. What do you do? Pick something different, after I asked you to focus on my arguments. It isn't about my mind being set. It's about me standing my ground on facts. Facts & reasoning you apparently can't argue.If you wanna try to fact check me, fact check me on the things I've said. Other than that, take a hike with your product. I don't wanna hear about it again. You wanna talk about how bad Inter is and how gay Mancini is, feel free to post. Other than that, don't wanna talk about Bergamot. You just don't understand.
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Re: Open conversazione
by Bagg10 » Tue Feb 02, 2016 9:04 am
No more posts about Bergamet in this thread. Open a Bergamet thread in a different miscellaneous section and run wild with it. I don't want to see anymore of that shit that looks like ad spam in the Juve threads.
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Re: Open conversazione
by Zoff82 » Tue Feb 02, 2016 7:07 pm
That's exactly what it is: shit. Except for my posts...those are quality, and facts. Think Sarri got in Mancini's head about the gay comments? Apparently he's getting a divorce according to briz. I think Mancini's wife caught him in Moratti's office with his pants down.
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Re: Open conversazione
by zafferano » Wed Feb 03, 2016 2:11 am
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