Clinical application of bergamot ( Citrus bergamia ) for reducing high cholesterol and cardiovascular disease markers

The bergamot is a citrus fruit native to southern Italy with traditional uses that include improving immune response and cardiovascular function. There are a variety of phytochemicals that have been found in the bergamot including brutieridin and melitidin as well as other flavonoids, flavones O-glucosides and C-glucosides. Multiple clinical trials have provided evidence that different forms of orally administered bergamot can reduce total cholesterol and low-density lipoprotein cholesterol. In vitro mechanistic studies have provided evidence that polyphenols from the bergamot can alter the function of AMPK and pancreatic cholesterol ester hydrolase (pCEH). The use of bergamot in multiple clinical trials has consistently shown that it is well tolerated in studies ranging from 30 days to 12 weeks. This mini-review reports on the clinical studies performed with different forms of bergamot along with their effectiveness in reducing total cholesterol and LDL cholesterol in patients with hypercholesterolemia.

Dyslipidemia is an important risk factor for the development of atherosclerosis and eventual coronary artery disease. Dyslipidemia is evidenced by increased concentrations (i.e. hyperlipidemia) of low-density lipoprotein cholesterol (LDL-C), total blood cholesterol, and triglycerides. Hyperlipidemia is often accompanied by insulin resistance including impaired glucose tolerance, or "pre-diabetes", and low levels of high-density lipoprotein cholesterol (HDL-C) [7]. The three most common pharmacological approaches to lowering hypercholesterolemia include bile acid sequestrants, statins, and inhibitors of cholesterol absorption (i.e. ezetimibe). Of these three, the statin family represents the most favored approach as evidenced by current protocols favoring statin use, annual sales of statins, and clinical studies suggesting significant reductions in cardiovascular events, morbidity, and mortality with statins [8]. The primary mechanism of statins includes the inhibition of the enzyme catalyzing the rate-limiting step in mevalonate biosynthesis. This key intermediate in cholesterol metabolism is essential for de novo cholesterol synthesis. Along with the inhibition of cholesterol synthesis, there are known dose related side effects shown in estimates as high as 22% of patients utilizing statins, including liver disease or severe myopathy [9,10]. Given the documented benefits of lowering LDL-C, triglycerides, and total cholesterol, additional dietary and phytochemical approaches should be investigated as alternative methods to reducing indices of hyperlipidemia. One such example includes the bergamot fruit that has been investigated in pre-clinical and clinical studies for improving dysplipidemia.
The tree Citrus bergamia belonging to the Rutaceae family is found in the Calabria region specifically, due to its unique climate that is suitable for its growth. Essential oils of the bergamot peel are well characterized and used extensively in products ranging from the food industry, pharmaceutical industry, and the cosmetic industry [1,11]. Previous studies have suggested that the essential oil contains up to 93-96% volatile phytochemicals that include monoterpenes (25-53%), linalool (2-20%) and linalyl acetate (15-40%). The non-volatile compounds include waxes, pigments, coumarins, and psoralens. The bergamot fruit also contains flavonoids that include neoeriocitrin, naringin and neohesperidin among many others that have been of interest for their cardiovascular benefits. In this review we will evaluate the clinical evidence for bergamot as a strategy for improving dyslipidemia.

Inhibiting oxidation of LDL particles
Oxidation of low-density lipoprotein particles is a harmful form of cholesterol that results from free radical damage. This form of oxidative damage, along with increased inflammatory events, has been associated with atherosclerosis that ultimately alters cardiovascular blood flow. Several constituents including naringin, neoeriocitrin, and rutin from the bergamot have been reported to lower the oxidation of LDL particles. Studies using naringin, neoeriocitrin and rutin reported them to have antioxidant activity in in vitro antioxidant models by beta-carotene-linoleic acid, 1,1-diphenyl-2-picryl hydrazyl (DPPH), superoxide, and hamster low-density lipoprotein (LDL) [17].
In another study, male New Zealand rabbits were fed a high cholesterol diet and divided into three groups as follows: 1) placebo (i.e. control group) 2) naringin and 3) lovastatin [18]. The results revealed that naringin significantly reduced fatty streak formation and macrophage infiltration in endothelial cells. In addition, naringin was found to be hepatoprotective while lovastatin was not found to be hepatoprotective. Naringin-inhibitedcholesterol also induced elevation of intercellular adhesion molecule-1 (ICAM-1) in endothelial cells. ICAM-1 levels have been reported to be elevated in response to normal immune function disruption in endothelial cells leading to atherosclerosis [19].
Reactive oxygen species (ROS) including superoxide (O 2 − ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals (OH − ) can directly damage cells in the cardiovascular system and induce proinflammatory events. In addition, ROS can induce the formation of peroxynitrite (ONOO − ) and is associated with neointima formation. This formation of scar tissue can also result from a balloon angioplasty procedure. An in vivo study evaluated the impact of bergamot on injured blood vessels following angioplasty in rats [20]. Pre-treatment of rats with the nonvolatile fraction of bergamot reduced free radical formation and Lectin-like oxyLDL receptor-1 (LOX-1). These results show that 14 days of consecutive administration of bergamot oil antagonized the effects of smooth muscle cell proliferation and neointima formation in the rat carotid artery following angioplasty.
Oxidized LDL leads to vasoconstriction mediated by the inflammatory thromboxane A2 [21]. Studies have suggested that glomerular injuries and hemodynamic abnormalities of the kidney may by directly caused by interaction of oxidized LDL with mesangial cells Wheeler et al., 1994. The renal protective properties of bergamot juice were tested in rats receiving a hyperlipidemic diet [22]. Bergamot juice (1 mL) was found to significantly decrease malondialdehyde (MDA) levels compared to hyperlipidemic controls (4.10 ± 0.10 nmol/mg protein and 4.78 ± 0.15 nmol/mg protein, respectively). Biochemical data also reported that histological preparations of the kidney suggests that bergamot juice prevented the development of renal damage from hypercholesterolemia.

Hypolipidemic properties of bergamot polyphenols
HMG-CoA reductase is the rate controlling enzyme in the mevalonate pathway that is responsible for cholesterol synthesis. The class of compounds known as statins are potent inhibitors of HMG-CoA by competitively binding the active site where HMG binds. This makes HMG-CoA a valuable target for reducing cholesterol levels. A study by Di Donna et al in 2009 proposed two molecules from bergamot, neohesperidin and naringin, as sharing structural similarity to statins [23]. A more recent study by Leopoldini reported through computational modelling that bergamot's statin-like molecules bind to HMG-CoA at Arg590, Ser684, Asp690, Lys692, and Lys735 residues, as well as at the nonpolar amino acids [24]. To date, there has not been any definitive in vitro validation that flavonoids from bergamot share a similar mechanism for inhibition of HMG-CoA reductase. Though no clinical trials have reported on the coenzyme Q10 levels following bergamot administration, it may be another possible benefit over statins, as statins are well known to decrease plasma levels of coenzyme Q10 [25].
A second mechanism that has been proposed with bergamot polyphenols is the activation of adenosine monophosphate-activated protein kinase (AMPK). Activation of AMPK by small molecules improves glucose homeostasis, lipid profiles, blood pressure and insulin resistance and is one of the proposed mechanisms of metformin. Naringin was found to promote phosphorylation of AMPK in the liver at threonine-172 in C57BL/6J mice receiving a high fat diet [26]. These results were further confirmed in HepG2 cells that were exposed to naringin. A study by Sui et al revealed that naringin activates AMPK altering the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9), sterol regulatory element-binding proteins (SREBPs), and low-density lipoprotein receptor (LDLR) [27]. The results of this study identified naringin as an AMPK activator in mice, leading to a downregulated expression of SREBPs and PCSK9, and an increased expression of LDLR to reduce the body weight of obese C57BL/6J mice. A statistically significant decrease in triglycerides, LDL and total cholesterol was observed. Increasing the expression of LDL receptor is beneficial for promoting the endocytosis of cholesterol rich LDL.
In vivo studies have reported that oral administration of bergamot juice can reduce blood cholesterol and improve the atherogenic index in mice. Hand pressed bergamot juice was administered to Wistar rats weighing 180 to 200 grams while being administered a high cholesterol diet [28]. diet or a high cholesterol diet. Rats receiving bergamot polyphenol fraction at 10 mg/kg by oral gavage were found to inhibit pCEH activity.
Babish (2016)-An observational, one-arm study was conducted with 11 human participants (3 male and 8 female; age 38-65 years) and evaluated a combination of 9 plant extracts that included bergamot fruit extract [32]. The extract (i.e. F105) was formulated with apple fruit extract, bergamot fruit extract, blueberry fruit concentrate, capsicum fruit, grape seed extract, grape skin extract, green tea leaf extract, mangosteen pericarp extract, olive leaf extract, and turmeric root & rhizome extract in a number of ratios beginning with Nauman and Johnson Page 5 Integr Food Nutr Metab. Author manuscript; available in PMC 2019 May 02.

Gliozzi (2013)-
A prospective, open-label, parallel group, placebo-controlled study with 77 human subjects with elevated LDL and triglycerides were administered 1) placebo (n=15) 2) rosuvastatin 10 mg (n=16) 3) rosuvastatin 20 mg (n=16) 4) bergamot polyphenol fraction (BPF) (n=15) or 5) bergamot polyphenol fraction with rosuvastatin (n=15) [33]. The total duration of the study was 30 days. Capsules containing 500 mg of bergamot polyphenol fraction with 50 mg of ascorbic acid were encapsulated for the study. The principle flavonoids in the bergamot polyphenol fraction were neoeriocitrin, naringin, and neohesperidin. Both doses of rosuvastatin and BPF reduced total cholesterol, LDL, and urinary mevalonate. The results of this study suggest a combination of rosuvastatin and BPF were safe when taken together for 30 days. Further research beyond 30 days would be needed to determine if rosuvastatin and BPF can safely continue to be taken in combination.
Mollace (2011)-A randomized, double-blind, placebo-controlled clinical trial evaluated bergamot (500 mg or 1,000 mg per day) for three months to reduce total cholesterol, reduce LDL, and increase HDL [34]. A total of 237 human subjects were enrolled in the study. Total cholesterol was reduced by 20% (500 mg of bergamot) and 30.9% (1,000 mg of bergamot). LDL was reduced by 23% (500 mg bergamot) and by 38.6% (1,000 mg of bergamot). HDL was increased by 25.9% (500 mg of bergamot) and by 39% (1,000 mg of bergamot). 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. Interestingly, this study enrolled 32 human subjects who experienced statin toxicity. Prior to their enrollment to the bergamot study, human subjects stopped taking statins for 2 months. They were then administered 1500 mg of BPF daily. After 30 days, those patients receiving BPF had changes in total cholesterol (−25%) and LDL (−27.6%) without reappearance of statin toxicity. Taken together, the results of this study suggest that BPF can reduce total cholesterol, LDL, and triglycerides.

Conclusion
The results of five different clinical trials (Table 1) using bergamot in various forms suggest the polyphenol fraction can lower LDL-C and total cholesterol. Several studies suggested that bergamot polyphenols can reduce triglycerides and increase HDL-C, however, the results were not consistent across all studies. One possible explanation for this variability (i.e. TG and HDL-C) is that bergamot preparation, extraction, and standardization varied in Nauman  several studies. Consistently in all of the clinical trials bergamot appeared to be well tolerated with studies ranging from 30 days to 6 months. There are several weaknesses in the design of several of the clinical trials that used an open label design (Table 1). However, it should be noted that each patient can serve as their own control since cholesterol was quantified prior to bergamot and at the completion of the study. Three of the studies suggested an increase in HDL by up to 4 mg/dl (Table 1). This is significant because HDL is often difficult to increase apart from lifestyle changes. Regarding the mechanism of action there are several possible mechanisms that may be responsible for improving cholesterol lab values including activation of AMPK and inhibition of pancreatic cholesterol ester hydrolase (pCEH). As of now the suggestions that bergamot inhibits HMG-CoA reductase appear to be largely based on molecular modeling and will require further studies to confirm this proposed mechanism of action. Taken together, these early clinical trials along with the mechanistic studies that have been performed suggest that bergamot can reduce total cholesterol and LDL-C through mechanisms that are distinct from current pharmaceutical approaches.