Efficacy of bergamot: From anti‐inflammatory and anti‐oxidative mechanisms to clinical applications as preventive agent for cardiovascular morbidity, skin diseases, and mood alterations

Abstract We summarize the effects of bergamot (extract, juice, essential oil, and polyphenolic fraction) on cardiovascular, bone, inflammatory, skin diseases, mood alteration, anxiety, pain, and stress. This review included a total of 31 studies (20 studies on humans with 1709 subjects and 11 in animals (rats and mice)). In humans, bergamot‐derived extract (BE) exerts positive effects on hyperlipidemia with an oral dose from 150 mg to 1000 mg/day of flavonoids administered from 30 to 180 days, demonstrating an effect on body weight and in modulating total cholesterol, triglycerides, LDL, and HDL. Studies in animals confirm promising data on glucose control (500/1000 mg/day of BE with a treatment lasting 30 days) are available in rats. In animals models, bergamot essential oil (BEO, 10 mg/kg or 20 mg/kg daily for 20 weeks) increases bone volume, decreases psoriatic plaques, increases skin collagen content, and promotes hair growth. Bergamot juice (20 mg/kg) is promising in terms of pro‐inflammatory cytokine reduction. In humans, aromatherapy (from 15 to 30 min) does not appear to be useful in order to reduce stress, anxiety, and nausea, compared to placebo. Compared to baseline, BE topical application and BEO aromatherapy reduce blood diastolic and systolic pressure and could have a significant effect on improving mental conditions.

Literature suggests that bergamot plays an important role on different areas of interest as nervous system, cardiovascular health, inflammation, diabetes bone metabolism, and skin. Preliminary results show that BEO extract may reduce cardiovascular disease (Lopez, Mathers, Ezzati, Jamison, & Murray, 2006;Nelson, 2013), anxiety, stress, improvement of the cognitive function, and improvement of the sleep (Dyer et al., 2016;Saiyudthong & Marsden, 2011).
The purpose of this review is to summarize the previously published clinical studies in animals and in humans where the efficacy has been evaluated in terms of dosage and timing of administration of bergamot with regard to the nervous system, cardiometabolic markers, diabetes, inflammation, bone, and skin.

| Anti-inflammatory and anti-oxidative mechanisms of bergamot derivatives
The anti-inflammatory potential of BJ has never been evaluated after the 2011. In the study of Risitano et al., 2014 has been demonstrated that the flavonoid fraction is able to reduce protein levels of pro-inflammatory cytokines (Risitano et al., 2014).
The in vitro anti-inflammatory activity of flavonoid fraction from bergamot juice, suggesting the activation of SIRT1 and demonstrate the inhibitory effects of BJe on LPS-induced increases in mRNA transcripts and protein levels of pro-inflammatory cytokines such as IL-8 gene expression. (Borgatti et al., 2011;Xie, Zhang, & Zhang, 2013). The antioxidant activity of BJe was focused on the cytoprotective ability of BJe against oxidants, such as hydrogen peroxide (H 2 O 2 ) and (Fe 2 SO 4 ) 3 , that cause oxidative cell damage (Ferlazzo et al., 2015). Trombetta et al. (2010) evaluated the antioxidant/antiinflammatory activity of two alcoholic flavonoid-rich extracts from bergamot peel on human vessel endothelial cells (HUVECs) exposed to the pleiotropic inflammatory cytokine TNFα, a model of vascular oxidative stress, and they showed that both extracts prevented the oxidative stress induced by TNFα, modulated the activation of redoxsensitive transcription factors NF-κB, thus increasing the cell survival.

| Lipid-lowering and cardiovascular risk
This lipid-lowering effect was associated with significant reductions in biomarkers used to detect vascular oxidative damage (such as malondialdehyde, oxyLDL receptor LOX-1, and protein kinase B (PKB)), suggesting a multi-action improved potential for bergamot in patients taking statins (Gliozzi et al., 2013). Furthermore, its lipid and glycemic effects may result in a reduction of CV risk. Additionally, bergamot protects against free radical damage in the body, including the vascular endothelium, an important determinant of CV health; yet, bergamot initiates adenosine monophosphate (AMP)-activated PK (AMPK), a central regulator of energy, and thus is involved in glucose and fatty acid metabolism.

| Reducing the sensation of hunger
Bergamot contains polysaccharides and a fibrous-woody fraction that can be used in food integrators and in dietary products in order to reduce the sensation of hunger (Giannetti, Mariani, Testani, & D'Aiuto, 2010).

| Mechanism of naringin in improving the overall insulin sensitivity and glucose tolerance
Few studies have assessed the effects of bergamot on glycemic parameters, and the current positive results are not to be underestimated. Together with the finding that naringin improved overall insulin sensitivity and glucose tolerance (Mandalari et al., 2007).

| Mechanism on synaptic transmission
Microdialysis studies demonstrate that, for systemic administration, BEO increases extracellular aspartate, glycine, and taurine in the hippocampus of freely moving rats via a Ca 2+ -dependent mechanism; in fact, in experiments carried out with a cerebrospinal fluid devoid of Ca 2+ , the latter effect is suppressed suggesting that the phytocomplex interferes with the exocytotic release of amino acid neurotransmitters (Morrone et al., 2007). BEO stimulates the release of endogenous glutamate as well as of [ 3 H]D-aspartate from pre-loaded synaptosomes.
Intriguingly enough, under these experimental conditions, microdialysis experiments show that BEO does not affect basal amino acid levels in the frontoparietal cortex (penumbra region), whereas it significantly reduces excitatory amino acid, namely glutamate, efflux typically enhanced in this brain region soon after occlusion of the middle cerebral artery (Amantea et al., 2009). Bergamot oil and its major active components, namely limonene,   linalyl acetate, and linalool, have demonstrated anti-inflammatory, immunomodulatory, and wound healing activities under different conditions. Cilantro oil and its major active component, linalool, have also been reported to possess anti-inflammatory and wound healing properties. Of note, a literature search revealed no published studies of spikenard or its major active components in human cells or their anti-inflammatory and wound healing activities (Han, Beaumont, & Stevens, 2017;Han, Gibson, Eggett, & Parker, 2017).

| Mechanism on wound healing activities
F I G U R E 1 Flow chart of the study

| MATERIAL S AND ME THODS
The present systematic review was performed according to the steps by Egger, Davey-Smith, and Altman (2008) as follows: (1) Configuration of a working group: Three operators were skilled in the effects of bergamot in health, of whom one was acting as a methodological operator and two were participating as clinical operators; (2) Formulation of the revision question on the basis of considerations made in the abstract: "the state of the art on evidence regarding bergamot effects on animals and humans studies"; (

Type of study level of evidence Compound
In vivo

| RE SULTS: EFFEC TS OF B ERG AMOT ON CLINI C AL APPLI C ATI ON: FROM ANIMAL TO HUMAN
This systematic review included a total of 31 studies. In particular, we have considered 20 studies on humans with 1709 subjects.
Eleven studies in animals were considered, in particular for both rats and mice. Figure 1 represents the flowchart of the study selection process.

| Effects of bergamot on cardiovascular diseases
Concerning the effects of bergamot (BEO, BPF, BE, BJ, aromatherapy), the literature research based on the keywords "bergamot" and ["cardiovascular" or "cholesterol" or "hypertension" or "hdl" or "tryglicerides" or "ldl" or "weight"] retrieved 20 articles.
After the screening process, 15 papers were selected for full-text revision. After applying the inclusion and exclusion criteria, seven studies were excluded and eight studies both on humans (6) with 448 subjects and in animals (2) were selected for the present systematic review. Decrease noted in total cholesterol, LDL cholesterol, and apolipoprotein B (p = ns). A post hoc analysis of eight subjects with HbA1c > 5.4 and HOMA-IR score > 2 or elevated triglycerides revealed in addition a decrease in triglycerides, and plasminogen activator inhibitor type 1 (PAI-1). (p < 0.05)
BPF, in association with CAF diet, reduces total cholesterol (p < 0.05), triglycerides (p < 0.01), LDL (p < 0.05) and weight gain (p < 0.05), compared to CAF diet only. No significant reduction of these parameters was observed in SC + BPF, compared to SC. Table 1 shows human studies and the effects of bergamot on cardiovascular markers. In particular, improvements in hypercholesterolemia, triglyceridemia, and weight management are shown.
A recent 2017 study performed by Bruno (Bruno, Pandolfo, Crucitti, Maisano, et al., 2017), the treatment produced a statistically significant reduction of body weight (p = 0.004), in addition to the body mass index decrease (p = <0.05). In the second study, (daily dose of 500 mg/day for 60 days) the treatment did not show significant result (Bruno, Pandolfo, Crucitti, Cacciola, Santoro, et al., 2017).
In 2016, (Babish et al. (2016) showed that a 250 mg dose of bergamot fruit extract (BFE) and 110 mg of a blend of nine other phytoextracts showed a decrease in total cholesterol, LDL, and apolipoprotein B (p < 0.05). A post hoc analysis showed other significant effects only in eight subjects with HbA1c > 5.4 and HOMA-IR score >2 or elevated triglycerides: reduction in triglycerides and plasminogen activator inhibitor type 1 (PAI-1) (p < 0.05).
In two recent Italian clinical trials performed for 30 days, BPF at a daily dose of 500 mg or 1000 mg resulted in reducing total cholesterol, LDL, and triglycerides, and increasing HDL, compared to baseline (p < 0.0001). Furthermore, it showed a dose-dependent difference after 500 mg and 1000 mg only for HDL (p < 0.05) (Mollace et al., 2011).
In a similar study by Gliozzi, an oral administration of 1000 mg daily of BPF significantly reduced total cholesterol, LDL, and triglycerides, while enhancing HDL levels, compared to placebo group (p < 0.05) (Gliozzi et al., 2013).

| Effects on animal models
In literature, there are poor results regarding the effect of bergamot on cardiovascular diseases in animals. A study was conducted on rats using BPF (10 or 20 mg/kg/daily for 30 days), reporting a reduction in total cholesterol, LDL, and triglycerides, enhancing fecal sterols excretion, compared to controls (p < 0.05) (Bruno, Pandolfo, Crucitti, Cedro, Zoccali, et al., 2017). While it has no effect in association with SC (p = ns) administration in association with a standard chow (SC) regime did not produce the same effect (p = ns) (Parafati et al., 2015).

| Effects of bergamot on diabetes
Concerning the effects of BE, BPF, or BJ, the literature search was based on the keywords "bergamot" and ["DIABETES" OR "GLUCOSE" OR "GLYCEMIA"] and the examination retrieved five articles.
After screening, four papers were selected for full-text revision.
After applying the inclusion and exclusion criteria, two studies were excluded and two studies were selected for the present systematic review.
In Table 2, we have included studies on the effects of bergamot on diabetes for a total of two studies divided, respectively, into (1) on humans (237 subjects) and (1) on animals.

| Effects on humans
The study by (Mollace et al. (2011) on 237 subjects was focused on blood glucose levels. After a daily supplementation of 500 mg for 30 days, BPF resulted in a significant reduction on blood glucose levels compared to baseline (p < 0.0001). (Parafati et al. (2015) on rats showed that BPF reduces significantly blood glucose levels only in association with CAF diet, compared to CAF diet only (p < 0.05), while it has no effect in association with SC diet, compared to SC alone (p = ns).

| Effects of bergamot on nervous system
Concerning the effects of bergamot, the literature search was based on the keywords "bergamot" and ["NERVOUS SYSTEM" OR "STRESS" OR "ANXIETY"]. The search retrieved 30 articles.
After screening, 20 papers were selected for full-text revision.
After applying the inclusion and exclusion criteria, four studies were excluded and 16 studies were selected for the present systematic review. Table 3 includes studies on the effects of bergamot on the nervous system, for a total of 16 studies divided, respectively, into (11) on humans (831 subjects) and (5) on animals.

| Effects on humans
Literature shows several studies in humans regarding the inhalation of bergamot with controversial results on the nervous system and mood.
In particular, 15 min of BEO vapor inhalation by healthy females was tested in comparison with water vapor inhalation or rest (control). Promising results showed that salivary cortisol (CS) after inhalation was significantly different in the three groups (p = 0.003) and CS levels were lower compared to control group in both BEO vapor and water vapor group (p = 0.004 and p = 0.0049), but CS in BEO vapor group was not significantly lower than in respect of CS in water vapor group (Watanabe et al., 2015).
Two other randomized controlled trials showed similar results.
( Wiebe (1998) tested preoperative anxiety in six women with a 10 min aromatherapy based on a mixture of essential oils (vetivert, bergamot, and geranium) in comparison with a placebo group, which inhaled a hair conditioner. In this study, anxiety was reduced after exposure to both treatment and placebo, with no significant differences between groups. (Graham et al. (2003) showed that in patients subjected to radiotherapy and the effects of essential oils (lavender, bergamot, and cedarwood), there were no significant differences in HADS (hospital anxiety and depression scale) or SPHERE (somatic and psychological health report) scores between the randomly assigned groups.
However, HADS scores were significantly lower at treatment completion in the non-fragrant placebo group (carrier oil only), compared with either of the fragrant arms (p = 0.04).
Effects of bergamot on the nervous system were also evaluated in terms of blood pressure, heart variability, and pulse rate. In particular, (Liu, Lin, & Chang (2013)

Main results
Hongratanaworakit ( A recent study by Han, Gibson, et al. (2017) evaluated the effects of bergamot on mental health through the use of approved tests: BEO aromatherapy effects on mental health and well-being in comparison with distilled water aromatherapy (15 min) in 57 subjects.
Bergamot was also tested in terms of sleep improving using bergamot/sandalwood or frankincense/mandarin/lavender essential oil

| Effects of bergamot on bone
Concerning the effects of bergamot extracts on bone, the literature search was based on the keywords "bergamot" and ["BONE" or "osteoporosis" or "osteoclasts" or "osteoblasts" or "osteopenia"].
Literature retrieved two articles. After screening, one paper was selected for full-text revision. After applying the inclusion and exclusion criteria, only one study was selected for the present systematic review. Table 4 shows the study description with main results.

| Effects in animals
The oral administration of Bergapten (BEO) at 10 or 20 mg/kg daily for 20 weeks was able to significantly increase bone volume/trabecular volume ratio (BV/TV), trabecular thickness, and trabecular number (p < 0.01 for 10 mg/kg and p < 0.001 for 20 mg/kg) (Li et al., 2016).

| Effects of bergamot on inflammation
Concerning the effects of bergamot extract on inflammation, the literature search was based on the keywords "bergamot" and ["inflammation" or "ESR" or "CRP" or "citokine"].
Literature retrieved five articles. After screening, four papers were selected for full-text revision. After applying our inclusion and exclusion criteria, two studies were excluded and two studies (in vivo) were selected for the present systematic review. Table 5 shows results of the studies selected process.

| Effects of bergamot on skin
Concerning the effects of bergamot extract, the literature search was based on the keywords "bergamot" and ["PSORIASIS" OR ""OR "UVB" OR "SKIN"].
Literature review retrieved five articles. After screening, four papers were selected for full-text revision. After applying the inclusion and exclusion criteria, two studies were excluded and two studies were selected for the present systematic review.
In Table 6, studies on the effects of bergamot on skin were included, for a total of two studies divided, respectively, into 1 on humans (193 subjects) and 1 in vivo.

| Effects in humans
The clinical study on subjects with psoriasis that received a treatment with UVB + BEO applied on the psoriatic plaques 30 min before the procedures, three times weekly, showed a significant reduction of Psoriasis Area and Severity Index (PASI) compared to baseline (p < 0.001) and a reduction in the number of procedures compared to UVB treatment only (p < 0.05) (Valkova, 2007).

| Effects in animals
As regards animal studies, the topical application of bergamot extract for 42 days increased the activity of superoxide dismutase (p < 0.05) and the collagen content (p < 0.001), and decreased the content of malondialdehyde (p < 0.05) in the skin of mice. It also promoted hair growth significantly (p < 0.001) (Shao, 2003). In particular: as regards the effects on hyperlipidemia, this review summarizes that:

| D ISCUSS I ON
Treatment should involve at least an oral dose of 150 mg/day of flavonoids (Bergamot-derived extract) for 6 months (Toth et al., 2015) or an oral dose of bergamot polyphenolic fraction (BPF) from 500 to 1000 mg/day for 30/60 days (Bruno, Pandolfo, Crucitti, Cacciola, et al., 2017;Gliozzi et al., 2013;Mollace et al., 2011;Toth et al., 2015) for a reduction of body weight or decrease in total cholesterol, triglycerides, LDL and an increase of HDL. Studies in animals confirm these encouraging results, but only in animals kept on a high fat diet.
The effects of bergamot on cardiovascular outcomes were assessed in eight studies (four with level of evidence 3; one with level of evidence 1; one with level of evidence 2, and two with level of evidence 6) with a total of 448 subjects enrolled. This resume suggests that there is not enough one study of level 1 to state that there is wide scientific evidence of effectiveness of bergamot on reduction of total cholesterol, LDL cholesterol, and triglycerides.
As regards the effects on diabetes, this review summarizes that: Only one study in literature found a significant effect of 500/1000 mg/day of BPF on reducing blood glucose levels in humans, with treatment lasting 30 days (Mollace et al., 2011).
Promising data on glucose control are also available in rats with oral administration of BPF with a treatment of 50 mg/kg/day, but only in concomitance with a high fat diet (Parafati et al., 2015).
This resume suggests that there are not enough two studies of level 1 to state that there is wide scientific evidence of effectiveness of bergamot on reduction of glycaemia.
As regards the effects on nervous system, this review summarizes that: Effects of bergamot on the nervous system are controversial.
Aromatherapy (from 15 to 30 min), involving BEO vapor inhalation, appears to be significantly useful in order to reduce stress (salivary cortisol) compared to rest only (Watanabe et al., 2015) or to reduce anxiety compared to water vapor inhalation (Ni et al., 2013).
Other studies report that a 10 min aromatherapy based on bergamot and other essential oils does not significantly reduce anxiety compared to a placebo (hair conditioner) inhalation (Wiebe, 1998). Furthermore, one study in literature shows that BEO vapor aromatherapy appears to be less effective in reducing anxiety and nausea than a scented non-essential oil shampoo aromatherapy (Ndao et al., 2012). In addition, another study concluded that a 20-min inhalation of bergamot/lavender/cedarwood essential oil is less effective in reducing anxiety, compared to a non-fragrant carrier oil inhalation. (Graham et al., 2003) Accordingly to these evidences, we can affirm that BEO aromatherapy, compared to placebo group, does not appear to be useful in order to improve any aspect of mental health.
On the other hand, 10-15 min of BEO aromatherapy shows effects like reducing low-frequency power, increasing high-frequency power, and increasing heart rate variability, which are markers of a prevalence of parasympathetic activity (Chang & Shen, 2011;Liu et al., 2013). It was also observed that BEO application on skin reduces blood diastolic and systolic pressure and heart rate (Hongratanaworakit, 2011). The power of these studies is low because they compare results to baseline and not to a placebo group.
The effects of bergamot on nervous system were assessed in 16 studies (three with level of evidence 3; five with level of evidence 1; three with level of evidence 2, and five with level of evidence 6) with a total of 831 subjects. There are five studies with level of evidence 1 that underline that there is a good scientific evidence regarding effects of bergamot on nervous system. In particular, studies with level of evidence 1 agree that bergamot essential oil aromatherapy has no effect on mental health compared to other fragrant oils and, in certain situations, appears to be less effective than non-fragrant oils in reducing anxiety and nausea.
As regards the effects on bone, this review summarizes that: In animals models, the oral administration of BEO (10 mg/kg or 20 mg/kg daily for 20 weeks) plus other compounds increases bone Effects on skin and hair growth in mice.

Animal (level 6) Bergamot and boxthorn extract
In rats The skin on the back of mice was shaved topically and smeared with bergamot and boxthorn extract for 42 days Compared with control group, the extract from bergamot and boxthorn increases the activity of superoxide dismutase (p < 0.05), the content of collagen (p < 0.001) of skin and the growth of hair (p < 0.001). It decreases the content of malondialdehyde (p < 0.05) in the skin of mice volume/trabecular volume ratio (BV/TV), trabecular thickness, and trabecular number. These data must be verified in human studies (Li et al., 2016).
This resume suggests that there is not enough one study of level 6 to state that there is wide scientific evidence of effectiveness of bergamot on increase in bone mineral density.
As regards the effects on inflammation, this review summarizes that: BJ extract (20 mg/kg) for 30 days was tested by both oral and intraperitoneal administration in animals. The results are promising in terms of pro-inflammatory cytokine reduction and may represent an aid for the treatment of inflammatory bowel disease, but currently no studies in humans have been made (Impellizzeri et al., 2015(Impellizzeri et al., , 2016. This resume suggests that there is not enough one study of level 6 to state that there is wide scientific evidence of effectiveness of bergamot on humans.
As regards the effects on skin, this review summarizes that: The BEO application combined with UVB therapy on psoriatic plaques (30 min before the procedures three times weekly) reduces Psoriasis Area and Severity Index. In animals, the topical application of bergamot extract for 42 days increased skin collagen content and promoted hair growth significantly.
It seems that a topical application of Bergamot oil/extract in a certain dosage could play an active role in the skin and in promoting hair growth (Shao, 2003;Valkova, 2007). Further studies in humans are needed to confirm this effectiveness and to set a dosage.
The real effects of bergamot in humans must be better elucidated with further large clinical trials.
The key points that must be addressed for better comprehension of bergamot potentiality are the timing of supplementation, the formulation and source of the compound (juice, extract, oil, or aromatherapy etc.), and the fitting area of efficacy.
The effects of bergamot on skin were assessed in two studies (one with level of evidence 1 and one with level of evidence 6) with a total of 193 subjects. At the current state, there is a poor scientific evidence of effectiveness of bergamot on increases in the activity of superoxide dismutase and the content of collagen of skin.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

DATA S H A R I N G A N D DATA ACCE SS I B I LIT Y
All considered studies are available on PubMed and/or Google Scholar database. Text with fees was made available free from University of Pavia online library.

E TH I C A L S TATEM ENT
This study does not involve any human or animal testing.