Bergamot Medical Research

Background: Statins represent the compounds mostly used worldwide in counteracting high serum cholesterol and reducing cardiometabolic risk. Beside the consistent benefit in using statins for lowering serum cholesterol, many patients undergo side effects and the use of natural compounds often represent a valid alternative for statin-intolerant patients. Recently, bergamot polyphenolic fraction (BPF) has been found to reduce significantly blood cholesterol, triglycerides and oxyLDL in animal models of hyperlipemia and in patients, thus suggesting potential benefit in using such derivative in patients in which the use of statins is controindicated. Here we report on the effect of BPF in patients suffering from high cholesterol and triglycerides either untreated or treated with rosuvastatin

Results: Rosuvastatin dose dependently reduced by 42+4 % and 56 +5%, LDL-C. Similar effect was seen in total cholesterol, non-high-density lipoprotein cholesterol (non-HDL-C) levels, the LDL-C/ HDL-C ratio and urinary mevalonate as determined at the end of treatment compared to control group. In addition, cholesterol lowering effect of rosuvastatin was accompanied by reduced LOX-1 and PhpsphoPKB in peripheral mononuclear cells, suggesting an effect on toxic oxyLDL formation. No significant changes in triglycerides was seen in rosuvastatin-treated groups. In BPF-treated group, a 31+3 % reduction of LDL-C and significant improvement on lipid serum profile was seen, including a significant reduction by 37+3 % of triglycerides.

Furthermore, in BPF–treated patients, mononuclear expression of LOX-1 and PhpsphoPKB were reduced in a way similar to the one produced by rosuvastatin, thus suggesting an antioxidant effect of BPF in serum lipoproteins. Finally, addition of BPF (1000 mg/daily) to rosuvastatin (10 mg/daily) significantly enhanced rosuvastatin effect on serum cholesterol compared to rosuvastatin alone (LDL-C reduction was 52+4%), an effect associated to significant attenuation of hypertriglyceridemia and to significant reduction of both urinary mevalonate and expression of LOX-1 and Phospho PKB in mononuclear cells, suggesting a multiaction synergistic potential for BPF in statin-taking patients.

In August 2012.

Bergamot Polyphenolic Fraction Potentiates Rosuvastatin-Induced Effect On LDL-Cholesterol, LOX-1 Expression and Protien Kinase B Phosphorilation In Patients

Mollace, R. Walker, 1S. Muscoli, 2G. Rosano, and 1F. Romeo Faculty of Pharmacy, University Magna Graecia of Catanzaro; 1 Chair of Cardiology, University of Rome Tor Vergata, Rome, Italy and 2San Raffaele IRCCS Pisana, Rome, Italy

Background: Statins represent the compounds mostly used worldwide in counteracting high serum cholesterol and reducing cardiometabolic risk. Beside the consistent benefit in using statins for lowering serum cholesterol, many patients undergo side effects and the use of natural compounds often represent a valid alternative for statin-intolerant patients. Recently, bergamot polyphenolic fraction (BPF) has been found to reduce significantly blood cholesterol, triglycerides and oxyLDL in animal models of hyperlipemia and in patients, thus suggesting potential benefit in using such derivative in patients in which the use of statins is controindicated. Here we report on the effect of BPF in patients suffering from high cholesterol and triglycerides either untreated or treated with rosuvastatin.

 Results: Rosuvastatin dose dependently reduced by 42+4 % and 56 +5%, LDL-C. Similar effect was seen in total cholesterol, non-high-density lipoprotein cholesterol (non-HDL-C) levels, the LDL-C/ HDL-C ratio and urinary mevalonate as determined at the end of treatment compared to control group. In addition, cholesterol lowering effect of rosuvastatin was accompanied by reduced LOX-1 and PhpsphoPKB in peripheral mononuclear cells, suggesting an effect on toxic oxyLDL formation. No significant changes in triglycerides was seen in rosuvastatin-treated groups. In BPF-treated group, a 31+3 % reduction of LDL-C and significant improvement on lipid serum profile was seen, including a significant reduction by 37+3 % of triglycerides. Furthermore, in BPF–treated patients, mononuclear expression of LOX-1 and PhpsphoPKB were reduced in a way similar to the one produced by rosuvastatin, thus suggesting an antioxidant effect of BPF in serum lipoproteins. Finally, addition of BPF (1000 mg/daily) to rosuvastatin (10 mg/daily) significantly enhanced rosuvastatin effect on serum cholesterol compared to rosuvastatin alone (LDL-C reduction was 52+4%), an effect associated to significant attenuation of hypertriglyceridemia and to significant reduction of both urinary mevalonate and expression of LOX-1 and Phospho PKB in mononuclear cells, suggesting a multiaction synergistic potential for BPF in statin-taking patients.

Abstract: Abstract of a study to be published by Elsevier, in a book on the role of polyphenols

The Mechanisms of Hypolipidemic and Hypoglycemic Activities of Bergamot Flavonoids

Elizbieta Janda*, Vanessa Russo, Maddalena Parafati, Salvatore Ragusa and Vincenzo Mollace – Department of Health Sciences, University Manga Graecia, Catanzaro, Italy.

Bergamot (Citrus Bergamia) juice has a particularly high content and a unique composition of flavonoids. Neoeriocitrin, neohesperidin, naringin, melitidin and brutieridin represent more than 95% of Bergamot Polyphenol Fraction (BPF), while rhoifolin, diosmin, poncirin and others can be found in the remaining 5%. The brilliant performance of BPF in clinical practice against, and as a treatment for, hyperlipidemia and moderate hyperglycemia in metabolic syndrome, awaits a plausible mechanistic explanation.

Considering the overwhelming scientific evidence, it is likely that flavonoid components of BPF are responsible for the majority of pharmacological effects. Here, we will review the scientific evidence showing that flavonoids, in particular citrus flavonoids rich in Bergamot, influence lipid and sugar metabolism at the molecular level. Anti-diabetic and dyslipidemia-correcting effects of Bergamot polyphenold may be explained by their ability to activate AMP kinase (AMPK), which is a central regulator of glucose and fatty acids metabolism, and inhibit cAMP phosphodiesterases (PDE) involved in regulation of lipolysis in adipocytes and liver.

Importantly, certain polyphenols can act as a 3-hydroxy-3-methlglutaryl coenzyme A (HMG-CoA) reductase inhibitors, thereby mimicking statins action. In addition, flavonoids bind and act as natural inhibitors of quinone oxidoreductase 2 (QR2) and other enzymes, with potential roles in metabolic regulation. Finally, pleiotropic and possible synergistic effects may account for enhanced nutraceutical effects of natural flavonoid mixtures, such as BPF as compared to purified flavonoids.

1. Introduction

Metabolic syndrome (MS) is a cluster of common cardiovascular risk factors, including, atherogenic dyslipidemia, insulin resistance or glucose intolerance, visceral obesity, hypertention and endothelial dysfunction. 10-30% of individuals in industrialized countries, including Italy, suffer from this condition. MS is associated with an increased risk of accelerated atherosclerosis and cardiovascular events [1].

Cardiovascular risk factors are also represented by different dyslipidemias, such as increased cholesterol levels (hypercholesterolemia) or increased level of tryglicerides (hypertryglicedemia), that occur separately or by diabetes. Experimental and epidemiological evidence suggest that dietary polyphenols, such as flavonoids may prevent atherosclerosis by counteracting its risk factors [2][3][4].

Accordingly, we should expect better clinical results by increasing the dosage and quality of consumed flavonoids. Indeed, as discussed in previous chapters by Mollace and Walker, a concentrated mixture of bergamot (Citrus Bergamia) flavonoids, so called Bergamot Polyphenol Fraction (BPF), shows brilliant results in clinical practice. BPF derived from bergamot juice has particularly high (40%) and unique composition of polyphenols.

Bergamot, as the endemic plant of Calabria, occupied and continues to occupy and important place in Calabrian economy as the main source for the production essential oil used in the cosmetic industry. However, the medicinal use of bergamot derivatives, forgotten for decades now is being rediscovered. For example, bergamot juice was considered by local population of Calabria, as a remedy for “fatty arteries” and heart diseases.

This inspired Mollace and co-workers to address the efficacy of bergamot juice and then its derivative BPF in experimental and clinical settings [5]. The effects on mean cholesterol parameters were comparable with a moderate dose of simavastatin (20 mg daily), including a marked increase in cHDL levels (see the chapter by Walker and Mollace). In addition, 1000 mg BPF taken for 30 days reduced moderate hyperglycemia by more than 20% [5].

The brilliant performance of BPF on MS and dyslipidemia is astonishing and needs a mechanistic explanation. Unfortunately, so far there is very limited experimental evidence proving the modulation ofone or another metabolic pathway by BPF itself. However, the vast scientific literature suggest that certain individual flavonoids present in BPF are implicated in the regulation of several metabolic enzymes, expressed in the liver, blood and endothelial wall cells. These regulation in many cases may be direct, i.e. mediated by the physical interaction of a receptor (specific enzyme) and the flavonoid compound and may lead to either inhibition or activation of the catalytic function of the enzymes.

In addition, natural polyphenols show less specific, antioxidant properties, that depend on the free radicals scavenging ability of hydroxyl groups linked to carbon aromatic rings [6][7][8]. The antioxidant features of bergamot flavonoids are separately discussed in the chapter by I. Korkina.

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 and other beneficial effects for human health, as discussed in vast literature on the subject [6][7]. Finally, 5 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 [9][10][11].

Here, we will review the scientific evidence showing that citrus flavonoids, present in bergamot juice and albedo may influence lipid and sugar metabolism at the molecular level via AMPK, PDE and other enzymes modulation. In this regard we will focus on direct molecular receptors of flavonoids, identified by crystallography and computational chemistry studies. We will also discuss a possible contribution of flavonoids to intestine and liver physiology. At the end we propose that pleiotropic and synergistic effects in natural mixtures of polyphenols defined here as “fitocomplex” may account for their superior performance in vivo compared to purified flavonoids.

Bergamot flavonoids and their metabolism

Bergamot juice is particularly rich in flavanones and flavones belonging to flavonoids group (see Fig. 1) and is characterized by a unique profile of flavonoids. It contains relatively rare neoeriocitrin and neohesperidin and, as recently discovered, rare brutieridin and melitidin. Careful analysis of flavonoid content in 42 citrus species and cultivars, reported by Nogata et al. leads to the conclusion that the amount of the flavonoids per volume unit of juice, or per mass unit of albedo (peels), is absolutely the highest in bergamot compared to other Citrus fruits.

Bergamot shows the highest concentrations of flavanones: neoeriocitrin, neohesperidin, naringin, melitidin and brutieridin, and the highest content of certain flavones: rhoifolin, neodiosmin, poncirin and rutin among all 42 different Citrusspecies . Bergamot juice can be further concentrated by a patented method, involving a preparative size exclusion chromatography based on polystyrene gel filtration and the eluate exsiccation to give rise to a polyphenol-enriched powder, BPF [5]. BPF contains over 40% flavonoids, carbohydrates, pectins, and other compounds, in contrast to bergamot juice powders obtained by spry-drier method that 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, with the only difference that flavonoids are over 200 times more concentrated in BPF. 95% of flavonoids present in BPF (and in bergamot juice) are flavanones, while flavones can be found in the remaining 5% (Fig. 1 and D. Malara and C. Malara, unpublished observations).

Up to date there are no published bioavailability and 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 hydrolysed to aglycones by bacterial flora of the gut.

Gut microflora hydrolysis is thought to favor flavonoid glycoside bioavailability. When sugar unit is removed, the resulting aglycone can be absorbed more readily [12]. 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.

However, the bioavailability 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. 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. 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.

Therefore although not properly investigated, it is likely both aglycones of citrus flavonoids and their metabolites contribute to the modulation of the intracellular targets and the subsequent biological response. In such a case, the concentration of these compounds achieved in the plasma would suggest that they elicit in vivo majority of the molecular effects discovered in vitro, even though bioavailability of citrus flavonoids is low and they have short half-life.

3. Molecular targets and antidiabetic and antilipemic effects of flavonoids

Several mechanisms have been proposed to explain anti-diabetic and anti-lipemic effects of flavonoids, but AMP kinase (AMPK) activation, discussed in the section 3.1, seems the most plausible. AMPK is a crucial regulator of glucose and fatty acids metabolism {Hwang, 2009 #23; Zygmunt, 2011 #15} in all tissues. AMPK is also an important target of metformin, a well-known anti-diabetic drug. The evidence discussed in section 3.1. suggest, that undoubtedly certain flavonoids, including naringin, present in Bergamot can activate AMPK, but it is not clear what is the exact molecular mechanism, since neither metformin nor flavonoids bind directly to AMPK.

Bergamot polyphenols can act as HMG-CoA reductase inhibitors, thus mimicking statins action. This has been first suggested for naringin and more recently for melitidin and brutieridin. Structural characteristics of latter compounds allow them to mimic the natural substrates of HMG-CoA and block the rate-limiting step in cholesterol synthesis. In section 3.2, we will critically review the scientific evidence for the inhibition of cholesterol pathway by these flavonoids.

In the section 3.3 we will discuss a direct flavonoid target identified by computational chemistry studies which is cAMP phosphodiesterase (PDE). PDE inhibition by certain citrus flavonoids has been shown to stimulate lipolysis. Computational chemistry identified also several other potential targets of flavonoids and they will be briefly discussed in section 3.5.

Crystallographic studies provide a more powerful evidence of a direct physical interaction between a compound and the target enzyme. Such studies are available for polyphenol resveratrol and quinone oxidoreductase 2 (QR2).

As discussed in section 3.6, functional studies indicate that resveratrol and other flavonoids, including those present in bergamot potently inhibit QR2 . The problem is that biological function of the direct interaction QR2-flavonoid, is still unknown, although QR2 may play a role in the metabolism of steroids, including cholesterol, as discussed later.

3.1 AMPK activation

AMP (adenosine monophosphate)-activated protein kinase (AMPK) is a master regulator of the metabolic 21 pathways involved in ATP production in mammalian cells. AMPK works a sensor of AMP/ATP levels. The 22 intracellular AMP increases, when energy status is low and binds to AMPK to allow its activation. AMPK plays a central role in regulating glucose and lipid metabolism and enery production in several different organs. To ensure the energy production, AMPK, not only facilitates glucose uptake, but it triggers several catabolic and blocks anabolic pathways.

There is overwhelming evidence that different flavonoids can activate AMPK, both in vitro and in vivo as a result of dietary supplementation with flavonoids in animal and human studies, as recently demonstrated. Citrus flavonoids (naringin, hesperdin and others) have been shown to exert potent hypolipidemic effect and amielorate atherogenic dyslipidemia in animal studies. Naringin is an example of Bergamo flavanones that can activate AMPK. Another example is rutin. Rutin is a flavon which is particularly rich in Bergamot. Recently, rutin has been shown to induce AMPK in hepatocytes and pancreatic beta cells. In hepatocytes, beside AMPK stimulation, rutin suppressed oleic acid-induced lipid accumulation.

Taken together, these results suggest that AMPK activation is a molecular target of many natural polyphenols, including naringin and rutin, rich in Bergamot and AMPK may be responsible for therapeutic effects of polyphenols against various metabolic disorders, including obesity, diabetes, hyperlipidemia and non-alcoholic fatty liver disease.

 

 

MECHANISMS UNDERLYING THE ANTI-TUMORAL EFFECTS OF CITRUS BERGAMIA JUICE

PLoS One. 2013; 8(4): e61484.
Published online 2013 April 16. doi: 10.1371/journal.pone.0061484
PMCID: PMC3628853

Simona Delle Monache[1], Patrizia Sanità[1], Elena Trapasso[2], Maria Rita Ursino[2], Paola Dugo[2], Marina Russo[2], Nadia Ferlazzo[2], Gioacchino Calapai[3], Adriano Angelucci[1], and Michele Navarra*[2][4]
Irina V. Lebedeva, Editor

 

Abstract: Based on the growing deal of data concerning the biological activity of flavonoid-rich natural products, the aim of the present study was to explore, in vitro, the potential anti-tumoral activity of Citrus Bergamia (Bergamot) juice (BJ), determining its molecular interaction with cancer cells. Here we show that BJ reduced growth rate of different cancer cell lines, with the maximal growth inhibition observed in neuroblastoma cells (SH-SY5Y) after 72 hrs. of exposure to 5% BJ. The SH-SY5Y anti-proliferative effect elicited by BJ was not due to a cytotoxic action, and it did not induce apoptosis. Instead, BJ stimulated the arrest in the G1 phase of cell cycle and determined a modification in cellular morphology, causing a marked increase of detached cells. The inhibition of adhesive capacity on different physiologic substrates and on endothelial cells monolayer were correlated with an impairment of actin filaments, a reduction in the expression of the active form of focal adhesion kinase (FAK) that, in turn, caused inhibition of cell migration. In parallel, BJ seemed to hinder the association between the neural cell adhesion molecule (NCAM) and FAK. Our data suggest a mechanisms through which BJ can inhibit important molecular pathways related to cancer-associated aggressive phenotype, and offer new suggestions for further studies on the role of BJ in cancer treatment.

Introduction: Citrus Bergamia Risso & Poiteau, a small tree belonging to the Rutaceae family, is cultivated almost exclusively along the southern coast of Calabria region (Italy), where the particular environmental conditions are favorable for its cultivation. Bergamot fruit is mostly used for the extraction of essential oil, widely used in perfume industry and recently investigated for its beneficial effects in neuroprotection [1]. Bergamot juice (BJ), instead, obtained from the endocarp of the fruit, and is considered just a secondary and discarded product. Different studies have analyzed the chemical composition of BJ [2][3][4][5] revealing its elevated content in flavonoids most of which can exert beneficial effect on human health. The most recurrent flavonoids present in BJ include flavanones and flavones.

Inhibition of carcinogenesis by flavonoids has been demonstrated both in vitro and in vivo [6]. Several underlying mechanisms have been proposed, including the suppression of cyclooxygenase-2 (COX-2) expression [7], decrease of reactive oxygen species (ROS) [8][9], modulation of oncogenic signalling pathways and down-regulation of nuclear transcription factor kappa B (NF-kB) [10][11]. The resulting effects are the arrest of proliferation and the induction of the apoptosis in cancer cells [12][13]. Moreover, several flavonoids have demonstrated radical-scavenging and anti-inflammatory activities [14][15][16]. Importantly, flavonoids do not present any significant toxicological risk and the safety margin for their therapeutic use in humans is very large [17]. In addition, other studies suggest that flavonoids could inhibit tumoral invasion and metastasis [18][19][20].

Findings in animals and investigations by using different cellular models suggest that certain flavonoids could effectively inhibit also tumour progression. In particular, naringin, one of the flavonoids present in the BJ, was able to reduce the high glucose-induced ICAM-1 expression via the p38 MAPK signalling pathway, contributing to the inhibition of monocyte adhesion to endothelial cells [19]. Moreover it has been demonstrated that flavanone and 2′-OH flavanone perturb the invasion and metastasis of lung cancer cells probably through inactivation of ERK 1/2 and p38MAPK signalling pathways [20].

Much of these effects can be attributable to the ability of flavonoids to interact with mitogenic or migratory signalling pathways but the precise underlying molecular mechanisms remain largely unclear.

The anti-tumor potential of natural products rich in flavonoids is probably underestimated and further studies elucidating their molecular interaction are warranted. In this study we demonstrate that BJ has a potential anti-tumoral capacity and that in a neuroblastoma cell model this action is realized mainly through an early impairment in cell adhesive and migratory machinery.

Bergamot polyphenols demonstrate significant lipid lowering effects, along with positive benefits in metabolic syndrome.

Mollace V, Sacco I, Janda E, Malara C, Ventrice D, Colica C, Visalli V, Muscoli S, Ragusa S, Muscoli C, Rotiroti D, Romero F, Walker R *

Conclusions: Bergamot extract (BPF) 1,000mg daily represents an effective alternative for statin-intolerant patients; adjunctive therapy to statins and effective treatment for metabolic syndrome.

REFERENCES

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Research

Mollace.V, Muscoli.S, Janda.E, Sacco.I, Ventrice.D, Iannone.M, Visalli.V, Rotiroti. D, Romeo.F
Hypolipemic and Hypoglycaemic activity of bergamot polyphenol: from animal studies to human studies. J.Fitoterapia Vol 81 Issue 8

 

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Di Donna.L, De Luca.G, Mazzotti.F, Napoli.A, Salerno.R, Taverna.D, Sindona.G. (2009)
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Yu.J, Wang.L, Walzem.R.L, Miller.E.G, Pike. L.M, Patil.B.S. (2005) Antioxidant activity of citrus Limonoids, flavonoids and courmarins. J. Agric Food Chem 53: 2009-2014

 

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Micelli.N, Mondello.M.R, Monforte.M.T, Sdrafkakis.V, Dugo.P, Crupi.M.L, Taviano.M.F, De Pasquale.R, Trovato.A. (2007)
Hypolipidemic effects of Citrus Bergamia Risso et Poiteau juice in rats fed a hypercholesterolemic diet. J. Agric Food Chem 55: 10671-10677.

 

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Mollace.V, Ragusa.S, Sacco.I, Muscoli.C, Sculco.F, Visalli.V, Palma.E, Muscoli.S, Mondello.L, et al (2008)
The protective effect of bergamot oil extract on lecitine like oxy LDL receptor-1 expression In balloon injury-related neointima formation. J Cardiovasc Pharmacol Ther 13: 120-129

 

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Li.J.M, Che.C.T, Lau.C.B, Leung.P.S, Cheng.C.H, (2006)
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PUBLISHED CLINICAL TRIALS

1. Hypolipemic and hypoglycaemic activity of bergamot polyphenols: from animal models to human studies. Fitoterapia Vol 82 Iss 3 04/2011
2. Bergamot polyphenolic fraction enhances rosuvastatin-induced effect on LDL-Cholesterol, LOX-1 expression and protein kinase B phosphorylation in patients with hyperlipidemia, I.J.C 170 (2013)
3. The effect of Beramot-Derived Polyphenolic Fraction on LDL small dense particles and Non Alcoholic Fatty Liver Disease in Patients with Metabolic Syndrome. ABC Journal 04/2014
4. Use of a novel and natural antioxidant compound (Bergamot) in the management of statine intorlerance. European Heart Journal. 06/2014
5. Mechanisms Underlying the Anti-Tumoral Effects of Citrus bergamia Juice. Plos One 2013
6. The use of Bergamot Derived Polyphenolic Fraction in Cardiometabolic Risk Prevention and possible Mechanismd of Action. Ch84 Cardiac Health Elsevier 2014
7. Bergamot polyphenol fraction prevents non alcoholic fatty liver disease via stimulation of lipophagy in cafereria diet- induced rat model of metabolic syndrome. J.Nutr.Biochem 05.30.2015
8. Telomere and Telomerase modulation by Bergamot Polyphenolic Fraction in experimental photoageing in human keratinocytes. J.Biol Regul Homeost Agents 2015
9. Citrus Beramot Improves Atherogenic Lipoprotein Particle Characteristics in Patients with Non-Alcoholic Fatty Liver Disease and Metabolic Syndrome. J.Clinical Lipidology. May-June 2015
10. The Hepatic Effects of Citrus Bergamot Polyphenolic Fraction (BPF) on Patients with Non-alcoholic Fatty Liver Disease and Metabolic Syndrome. J. Clinical Lipidology. May-June 2015.
11. Bergamot Polyphenolic Fraction counteracts Erectile Dysfunction occurring in patients suffering from type 2 Diabetes.J.Pharma Nutrition 2016.