Anti-Inflammatory Activity of Citrus Bergamia Derivatives

molecules

Review Anti-Inﬂammatory Activity of Citrus bergamia Derivatives: Where Do We Stand?

Nadia Ferlazzo 1, Santa Cirmi 1, Gioacchino Calapai 2, Elvira Ventura-Spagnolo 3, Sebastiano Gangemi 4,5 and Michele Navarra 1,*

1 Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Messina I-98168, Italy; [email protected] (N.F.); [email protected] (S.C.) 2 Department of Biomedical and Dental Sciences and Morphofunctional Imaging, University of Messina, Messina I-98125, Italy; [email protected] 3 Department of Biotechnology and Legal Medicine, University of Palermo, Palermo I-90127, Italy; [email protected] 4 Department of Clinical and Experimental Medicine, University of Messina, Messina I-98125, Italy; [email protected] 5 Institute of Applied Sciences and Intelligent Systems (ISASI), National Research Council (CNR), Pozzuoli I-80078, Italy * Correspondence: [email protected]; Tel.: +39-090-6766-431; Fax: +39-090-6766-474

Academic Editor: Derek J. McPhee Received: 15 July 2016; Accepted: 12 September 2016; Published: 23 September 2016

Abstract: Inflammatory diseases affect a large portion of the worldwide population, and chronic inflammation is a major risk factor for several dangerous pathologies. To limit the side effects of both synthetic and biological anti-inflammatory drugs, the use of herbal medicines, nutraceuticals and food supplements has increased tremendously as alternative and/or complementary medicine to treat several pathologies, including inflammation. During the last decades, the biological properties of Citrus bergamia (bergamot) derivatives have obtained important scientific achievements, and it has been suggested their use in a context of a multitarget pharmacological strategy. Here, we present an overview of the anti-inflammatory properties of bergamot extracts that could represent the scientific basis for develop novel and alternative strategies to improve health status and attenuate inflammatory conditions.

Keywords: bergamot; Citrus bergamia; inﬂammation; antioxidant activity; ﬂavonoids; natural products; complementary and alternative medicines

1. Introduction Inflammation is a complex biological reaction induced by the disruption of the tissue homeostasis, occurring in response to the presence of a biological, chemical, or physical agent in the body [1]. The classical key features of inflammation are redness, warmth, swelling, and pain. Inflammation can be acute or chronic, depending on the type of stimulus and the effectiveness of the inflammatory process resolution. Acute inflammation begins quickly and lasts a few hours or a few days. It is characterized by the exudation of fluid and plasma proteins as well as leukocyte emigration (mainly neutrophils). When the immune system successfully eliminates damaging agents in acute inflammation, the reaction disappears, but if the response fails to remove them, a chronic phase occurs. Inflammation cascades can lead to the development of diseases such as chronic asthma, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, and psoriasis. Chronic inflammation is associated with the presence of lymphocytes and macrophages, vascular proliferation, fibrosis, and tissue destruction. Experimental and clinical studies together with epidemiological observations have identified the chronic infections

Molecules 2016, 21, 1273; doi:10.3390/molecules21101273 www.mdpi.com/journal/molecules Molecules 2016, 21, 1273 2 of 11 and inflammation as major risk factors for various types of cancer. It has been estimated that the underlying infections and inflammatory reactions are linked to 15%–20% of all cancer deaths [2]. The inflammatory response is characterized by coordinate activation of various signaling pathways that regulate expression of both pro- and anti-inflammatory mediators in cells of inflamed tissue as well as leukocytes recruited from the blood. The nuclear transcription factor κB (NF-κB) is the master regulator of the inflammatory response, that drives the activation of genes associated with the transcription of inflammatory mediators, such as interleukins, tumor necrosis factor-α (TNF-α) and prostaglandins (PGs), as well as inflammatory enzymes, like inducible nitric oxide synthase (iNOS) and cyclooxygenases (COXs). Several classes of medicines, including corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs) and biologics drugs are used to treat the inflammatory disorders. However, they possess several side effects and the biologics ones are expensive to be used. To limit these drawbacks of both synthetic and biologic drugs, over the past three decades, the use of herbal medicines, nutraceuticals and food supplements has increased greatly as an alternative and/or complementary medicine to treat several pathologies, including inflammation [3]. Indeed, although natural products are not devoid of risk, generally they are safer than both synthetic and biologic drugs. Nowadays, about 80% of people around the world use natural products for the prevention and treatment of many diseases, mainly for their relative safety, efficacy and low cost as well as the compliance by patients. Plants have been the basis of many traditional medicines throughout the world for thousands of years, and continue to provide mankind with new remedies [4]. In this field, natural products offer great hope in the identification of bioactive molecules useful for the treatment of inflammatory diseases, as it happened for aspirin, the first discovered NSAID, which is still one of the bestselling drugs in the world. Polyphenols, such as flavonoids, lignans, phloroglucinols, quinones, stilbenes, phenylpropanoids, and diarylheptanoids, are a very important category of natural compounds able to modulate inflammatory pathways. Among them, luteolin [5,6], quercetin [7], wogonin [8,9], apigenin [6,8], fisetin [6] and baicalein [6] showed anti-inflammatory activity in vitro and in animal models. A growing volume of evidence supports the hypothesis that the use of a phytocomplex, such as an extract, can have a greater preventive and therapeutic success than a single biomolecule, because of both additive and synergistic effects of their individual constituents [10–13]. Moreover, all phytochemicals present in whole extracts may simultaneously modulate different targets of action in human cells, leading to a mixture of pharmacological effects contributing together to improve patient’s health [14,15]. Hence, we and other researchers have started investigating the anti-inflammatory action of a number of phytocomplexes in a context of a multitarget pharmacological strategy. In this review, we present an overview of the anti-inflammatory properties of bergamot extracts that could be the scientific basis for the development of novel and alternative strategies aimed to prevent and/or treat inflammatory conditions.

2. Oxidative Stress and Inflammation Inflammation and oxidative stress are closely related pathophysiological events that are strongly linked with one another. One of them may appear before or after the other, but when one emerges the other one is most likely to follow and then both of them take part in the pathogenesis of many disorders. Nowadays, it is clear that the prolonged low-grade inflammatory process plays a central role in the pathogenesis of many chronic diseases [16]. On the other hand, epidemiological and experimental studies strongly suggest a contribution of oxidative stress in many human diseases [16]. Just as the inflammatory process can induce oxidative stress, the latter can cause inflammation through activation of multiple pathways [17,18]. The result is that both inflammation and oxidative stress are associated with a number of chronic diseases, including diabetes, hypertension, cardiovascular diseases, neurodegenerative diseases, alcoholic liver disease, chronic kidney disease, cancer, and aging [19–22]. Under pathological inflammatory conditions there may be exaggerated generation of reactive species (RS) that can diffuse out of the cells, inducing localized oxidative stress and tissue injury [23]. Molecules 2016, 21, 1273 3 of 11

Moreover, the activated phagocytic cells produce large amounts of radical oxygen and reactive nitrogen species (ROS and RNS, respectively). Once generated, they can further generate other RS, leading to extensively damage. At the onset of inflammation, the infection or tissue damage is sensed by the pattern recognition receptors like toll-like receptors (TLR), NOD-like receptors (NLR), and the receptor for advanced glycation end products (RAGE). The stimulation of these receptors upon binding with specific molecules leads to the activation of transcription factors such as nuclear factor-κB (NF-κB) and activating protein-1 (AP-1), that in turn induce pro-inflammatory gene expression, exert antimicrobial functions and recruit additional immune cells [24,25]. It has been demonstrated that RS (i.e., hydrogen peroxide) can induce inflammation through activation of these same transcription factors [26–28]. Over the last decades, researchers have investigated on the effectiveness of antioxidants in preventing diseases like cardiovascular diseases, cancer, diabetic complications, Alzheimer’s disease, and so forth [29]. Most of the results come from in vitro studies, and clinical researches have produced controversial data, although many epidemiological data suggest that antioxidants may have a beneficial effect in many chronic diseases. However, great care should be taken in the choice of antioxidant agents and their dosage, as well as, it is most important to quantify the redox and inflammatory status to make appropriate interpretation of the findings.

3. Bergamot Derivatives Known as “bergamot”, Citrus bergamia Risso et Poiteau is a small plant belonging to the Rutaceae family, growing spontaneously in the southern coast of Calabria region (Italy), where the particular microclimate is ideal for its cultivation [30]. Bergamot fruit is used especially for the extraction of its essential oil (BEO) from the peel (by cold pressing), while the bergamot juice (BJ), derived squeezing the endocarp of the fruits, is considered a byproduct of the BEO’s production. Finally, the scraps of bergamot fruit after both BEO extraction and BJ juicing is named “bergamot pastazzo” and is used as animal feed. BEO is typically used in the cosmetic industry, being found in the composition of many fragrances, body lotions, soaps and so on. It is also used by the food industries (for flavoring tea, beverages and typical Calabrian pastries), by the pharmaceutical industries (to absorb the unpleasant smell of medicinal products and for its antiseptic and antibacterial properties [31]) and in aromatherapy [30]. Over the last decade, some researchers have started to investigate the biological properties of bergamot derivatives, obtaining important scientific achievements. For example, BEO has been evaluated for its potential neuroprotective [32] and antiproliferative effects [11,33], while BJ has been suggested as antioxidant [34,35], anti-inflammatory [36–39], anticancer [15,40–42] and hypolipidemic [43,44] drug. The chemical composition of the main Citrus bergamia derivatives, BEO and BJ, has been studied mostly by high performance liquid chromatography–mass spectrometry (HPLC-MS), gas chromatography–mass spectrometry (GC-MS) and gas chromatography–flame ionization detection (GC-FID) techniques, and has been reported in a number of papers [41,45–48]. BEO is composed of both volatile and non-volatile fractions that account for 93%–96% and 4%–7% of the total, respectively. The non-volatile components are responsible for phototoxic reactions at certain concentrations, due to the presence of furanocoumarins (or furocoumarins), especially bergapten [30]. The main constituents of BEO are terpens and furanocoumarins, among which limonene, linalyl acetate (terpens) and bergamottin (furanocoumarins) are the most abundant molecules. Other compounds present in fewer amounts are the terpenes linalool and γ-terpinene, the coumarins 5-geranyloxy-7-methoxycoumarin and citropten, as well as the furanocoumarin bergapten [30]. Their chemical structures are presented in Table1. BJ is characterized by the presence of high amount of flavonoids among which naringin, neohesperidin, neoeriocitrin and eriocitrin are the most abundant, although melitidin, hesperetin and naringenin are also present [36,41,46]. The major flavonoids in BJ and their molecular structures are shown in Table2. MoleculesMolecules20162016,, 21Molecules21,, 1273 1273 2016, 21, 1273 4 of 11 4 of4 11 of 11 Molecules2016, 21Molecules, 1273 2016, 21, 1273 4 of 11 4 of 11 Molecules2016, 21Molecules, 1273 2016, 21, 1273 4 of 11 4 of 11 MoleculesMolecules2016, 201621, 1273, 21 , 1273 4 of 114 of 11 reviewMolecules [51].2016 ,Moleculesreview The21, 1273 anti-inflammatory 2016[51]., 21The, 1273 anti-inflammatory properties of properties some BJ flavonoids of some BJ as flavonoids pure compounds as pure compoundshave been4 of 11 have 4been of 11 review [51]. reviewThe anti-inflammatory [51]. The anti-inflammatory properties of properties some BJ flavonoids of some BJ as flavonoids pure compounds as pure compoundshave been have been reviewextensively [51]. investigatedextensivelyreviewThe anti-inflammatory [51].Table investigated Theand 1. The anti-inflammatorywell main establishe properties and constituents welld inestablishe of propertiesseveral some of BEO BJ dexperimental inflavonoids andof several some their BJ experimental chemical as modelsflavonoids pure compounds structures.[52–54]. asmodels pure compounds[52–54].have been have been reviewMoleculesextensivelyreviewreview [51].2016Molecules [51]., [51].21The investigatedreviewextensively, 1273The2016 anti-inflammatoryThe anti-inflammatory, 21[51]. anti-inflammatory, 1273 investigated Theand anti-inflammatorywell establishe properties andproperties properties welld inestablishe of ofpropertiesseveral somesome of some dexperimentalBJ in offlavonoids several BJsome flavonoids BJ experimental modelsflavonoids asas pure pure as [52–54]. compoundspure compounds asmodels purecompounds compounds[52–54]. have have4 of been 11 have been have4 been of 11 been extensively investigatedextensively investigated and well establishe and welld inestablishe several dexperimental in several experimental models [52–54]. models [52–54]. extensivelyextensively investigated investigated and well establishe and welld establishe in severald experimental in several experimental models [52–54]. models [52–54]. extensivelyextensively investigated investigatedTable and 1. The welland mainTable wellestablishe constituents 1. establisheThe maind in of constituents severald BEO in several and experimental their of experimentalBEO chemical and their modelsstructures. chemical models [52–54]. structures. [52–54]. review [51].review The [51]. anti-inflammatory TableThe anti-inflammatory 1. The mainTable properties constituents 1. The mainproperties of of constituentssome BEOBEO ofBJand someflavonoids their of BEO BJchemical flavonoids and as theirpure structures. chemical compoundsas pure structures. compounds have been have been Table 1. The mainTable constituents 1. The main of constituents BEO and their of BEO chemical and their structures. chemical structures. extensivelyextensively investigatedCoumarins investigatedTable and and1. wellThe FuranocoumarinsTable mainand establishe well 1.constituents The establishe dmain in several constituentsBEOof BEOd in experimental andseveral theirofBEO BEO experimental chemical and models their structures. chemical [52–54].models Terpenes structures. [52–54]. TableTable 1. The 1. main The main constituents constituents ofBEO BEO of BEOand their andBEO theirchemical chemical structures. structures. Coumarins andCoumarins Furanocoumarins and FuranocoumarinsBEO BEO Terpenes Terpenes NameCoumarins andCoumarins MolecularFuranocoumarins and Structure FuranocoumarinsBEO BEO Name Terpenes Terpenes Molecular Structure NameTable Coumarins 1.Name TheTable andmain Coumarins1. Furanocoumarins The Molecularconstituents main and constituentsstructure Furanocoumarins of Molecular BEO andBEO of structure BEOtheir andchemical Nametheir chemicalstructures. Terpenes Molecular Name structures. structure TerpenesMolecular structure Name CoumarinsName andCoumarins Furanocoumarins Molecular and structure Furanocoumarins Molecular structure Name Terpenes Molecular Name structure Terpenes Molecular structure Name CoumarinsCoumarinsName and Furanocoumarins and Molecular Furanocoumarins structure Molecular structure Name Terpenes Molecular Name Terpenes structure Molecular structure Name Name Molecular structure Molecular structure Name Molecular Name structure Molecular structure NameName Molecular Molecular structureBEO structureBEO Name Name Molecular Molecular structure structure CoumarinsCoumarins and Furanocoumarins and Furanocoumarins Terpenes Terpenes Name Name Molecular Molecularstructure structure Name Name Molecular Molecularstructure structure BergamottinBergamottin Bergamottin LimoneneLimonene Limonene Bergamottin Bergamottin Limonene Limonene Bergamottin Bergamottin Limonene Limonene Bergamottin Bergamottin Limonene Limonene BergamottinBergamottin LimoneneLimonene

BergamottinBergamottin Limonene Limonene

Bergapten Bergapten Linalool Linalool BergaptenBergapten Bergapten LinaloolLinalool Linalool Bergapten Bergapten Linalool Linalool Bergapten Bergapten Linalool Linalool BergaptenBergapten LinaloolLinalool

Bergapten Bergapten Linalool Linalool

Citropten Citropten γ-Terpinene γ-Terpinene Citropten Citropten γ-Terpinene γ-Terpinene CitroptenCitropten Citropten γ-Terpineneγ-Terpinene γ-Terpinene Citropten Citropten γ-Terpinene γ-Terpinene

CitroptenCitropten γ -Terpineneγ-Terpinene Citropten Citropten γ-Terpinene γ-Terpinene

5-Geranyloxy-75-Geranyloxy-7 5-Geranyloxy-75-Geranyloxy-7 Linalyl acetate Linalyl acetate -methoxycoumarin5-Geranyloxy-7-methoxycoumarin5-Geranyloxy-7 Linalyl acetate Linalyl acetate 5-Geranyloxy-7--methoxycoumarin5-Geranyloxy-7-methoxycoumarin5-Geranyloxy-7 Linalyl acetate Linalyl acetate -methoxycoumarin-methoxycoumarin Linalyl acetateLinalyl acetate -methoxycoumarin-methoxycoumarin Linalyl acetate methoxycoumarin 5-Geranyloxy-75-Geranyloxy-7 5-Geranyloxy-75-Geranyloxy-7 LinalylLinalyl acetate acetate -methoxycoumarin-methoxycoumarin Linalyl acetateLinalyl acetate -methoxycoumarin-methoxycoumarin Table 2. The majorTable flavonoids2. The major in flavonoidsBJ and their in molecular BJ and their structures. molecular structures. Table 2. The majorTable flavonoids2. The major in flavonoidsBJ and their in molecular BJ and their structures. molecular structures. Table 2. The majorTable flavonoids2. The major in flavonoidsBJ and their in molecular BJ and their structures. molecular structures. Table 2. TheTable major 2. flavonoids The BJmajor Flavonoids in flavonoids BJ andBJ their Flavonoids in BJmolecular and their structures. molecular structures. Table 2. The major ﬂavonoidsBJ Flavonoids in BJBJ and Flavonoids their molecular structures. Name Name Molecular structure MolecularBJ Flavonoids structureBJ Flavonoids Name Name Molecular structure Molecular structure Name Name Molecular structure MolecularBJ FlavonoidsstructureBJ Flavonoids Name Name Molecular structure Molecular structure Name NameTable Table 2.2. Molecular The TableThe 2. major major The 2. Thestructure majorflavonoids flavonoids Molecular major flavonoids flavonoids in structurein BJ BJ and inand intheirBJ BJtheir and andmolecular Name theirmolecular their molecular molecular structures. structures. Name structures. Molecularstructures. structure Molecular structure Name Name Molecular structure Molecular BJstructure Flavonoids Name Name Molecular structure Molecular structure BJBJ Flavonoids FlavonoidsBJBJ Flavonoids Flavonoids Name Molecular Structure Name Molecular Structure NameNameName Name Molecular Molecular Molecular Molecular structure structure structure structure Name Name Name Name Molecular Molecular Molecular Molecular structure structure structure Naringin Naringin Hesperetin Hesperetin Naringin Naringin Hesperetin Hesperetin Naringin Naringin Hesperetin Hesperetin Naringin Naringin Hesperetin Hesperetin

NaringinNaringin Naringin HesperetinHesperetinHesperetin NaringinNaringin HesperetinHesperetin

NeoeriocitrinNeoeriocitrin NeohesperidinNeohesperidin NeoeriocitrinNeoeriocitrin NeohesperidinNeohesperidin Neoeriocitrin Neoeriocitrin NeohesperidinNeohesperidin NeoeriocitrinNeoeriocitrin NeohesperidinNeohesperidin

NeoeriocitrinNeoeriocitrin NeohesperidinNeohesperidin NeoeriocitrinNeoeriocitrinNeoeriocitrin NeohesperidinNeohesperidinNeohesperidin

Molecules 2016, 21, 1273 5 of 11

Table 2. Cont.

BJ Flavonoids Molecules2016Molecules, 21, 12732016 , 21, 1273 5 of 11 5 of 11 MoleculesMoleculesName2016,2016 21, 1273, 21, 1273 Molecular Structure Name Molecular Structure5 of 115 of 11

NarirutinNarirutinNarirutin EriocitrinEriocitrinEriocitrin NarirutinNarirutin EriocitrinEriocitrin

MelitidinMelitidin BrutieridinBrutieridin MelitidinMelitidinMelitidin BrutieridinBrutieridinBrutieridin

3. Antioxidant3. Antioxidant Properties Properties of Bergamot of Bergamot Derivatives Derivatives 3. Antioxidant3. Antioxidant Properties Properties of Bergamot of Bergamot Derivatives Derivatives AntioxidantA numberAntioxidant compounds of researchers compounds play have an play important documented an important protec thetive capabilityprotec roletive in of rolethe molecules preventionin the prevention present of numerous in bothof numerous BEO and AntioxidantAntioxidant compounds compounds play play an importantan important protec protectivetive role role in thein theprevention prevention of numerousof numerous diseases.BJ todiseases. counteractAlthough Although the inflammation antioxidant the antioxidant activity via modulation activityof BJ has of been ofBJ severalhas previously been pathways. previously addressed For addressed example,[55], recently [55], the recently we pharmacological better we better diseases.diseases. Although Although the antioxidantthe antioxidant activity activity of BJ of has BJ hasbeen been previously previously addressed addressed [55], [55], recently recently we betterwe better characterizedfunctionscharacterized the of chemical coumarins, the chemical composition including composition of a their flavonoid- of anti-inflammatory a flavonoid-rich extractrich fromextract activity, BJ from (BJe), BJ were and (BJe then reviewed), and investigated then byinvestigated Wu et al. characterizedcharacterized the chemicalthe chemical composition composition of a offlavonoid- a flavonoid-richrich extract extract from from BJ (BJe BJ (BJe), and), and then then investigated investigated its antioxidantin 2009its antioxidant [49 properties], and properties very in both recently inabiotic both by andabiotic Srikrishna cell-based and cell-based et assays al. [50 [34,35].],assays while [34,35].First the we anti-inflammatory First tested we the tested antioxidant the antioxidant profile and its antioxidantits antioxidant properties properties in both in both abiotic abiotic and and cell-based cell-based assays assays [34,35]. [34,35]. First First we testedwe tested the theantioxidant antioxidant activitythe structure-activityactivityof BJe using of BJe a usingrange relationship aof range tests of(Folin-Cioca tests of terpenes (Folin-Ciocalteu, was Reducinglteu, discussed Reducing Power, by Power, SouzaDPPH andDPPH ORAC coworkers and assays)ORAC in aassays)that systematic that activityactivity of BJe of BJeusing using a range a range of tests of tests (Folin-Cioca (Folin-Ciocalteu,lteu, Reducing Reducing Power, Power, DPPH DPPH and and ORAC ORAC assays) assays) that that highlightedreviewhighlighted [51its]. great The its anti-inflammatoryantioxidant great antioxidant and radical and properties radicalscavenging ofscavenging some properties BJ flavonoidsproperties related relatedto as the pure highto compounds the total high phenol total have phenol been highlightedhighlighted its greatits great antioxidant antioxidant and and radical radical scavenging scavenging properties properties related related to the to thehigh high total total phenol phenol contentextensively content[34]. Then, [34]. investigated we Then, focused we andfocused on the well cytoprotectiveon established the cytoprotectivein ability several ofability BJe experimental against of BJe againstoxidants, models oxidants, such [52 as–54 suchhydrogen]. as hydrogen contentcontent [34]. [34]. Then, Then, we focusedwe focused on theon cytoprotectivethe cytoprotective ability ability of BJe of BJeagainst against oxidants, oxidants, such such as hydrogen as hydrogen peroxideperoxide (H2O2) and(H2O (Fe2) and2SO4 )(Fe3, that2SO 4cause)3, that oxidative cause oxidative cell damage cell damage [34,35]. [We34,35]. found We that found BJe that prevented BJe prevented peroxideperoxide (H2 O(H2)2 Oand2) and (Fe2 (FeSO42)SO3, that4)3, that cause cause oxidative oxidative cell celldamage damage [34,35]. [34,35]. We Wefound found that that BJe BJeprevented prevented either4. AntioxidantHeither2O2 or H 2(FeO22 SO Propertiesor 4)(Fe3-induced2SO4)3 of-induced Bergamotoxidative oxidative damage Derivatives damage on the onA549 the cells A549 by cells different by different mechanisms mechanisms eithereither H2O H2 2orO2 (Feor 2(FeSO42)SO3-induced4)3-induced oxidative oxidative damage damage on theon theA549 A549 cells cells by differentby different mechanisms mechanisms dependingdepending from the from stressor the stressoragent. Indeed, agent. Indeed,the extract the (25extract or 50 (25 µg/mL) or 50 µg/mL)reduced reduced ROS generation ROS generation and and dependingdependingAntioxidant from from the compounds thestressor stressor agent. play agent. Indeed, an importantIndeed, the theextract protective extract (25 or(25 role50 or µg/mL) 50 in theµg/mL) preventionreduced reduced ROS of ROS numerousgeneration generation diseases.and and counteractedcounteracted the membrane the membrane lipid peroxidation, lipid peroxidation, improved improved mitochondrial mitochondrial functionality functionality and prevented and prevented Althoughcounteractedcounteracted the the antioxidant membranethe membrane lipid activity lipid peroxidation, peroxidation, of BJ has improved been improved previously mitochondrial mitochondrial addressed functionality functionality [55], recentlyand and prevented prevented we better DNA-oxidativeDNA-oxidative damage damage in A549 in cells A549 incubated cells incubated with H 2withO2 or H (Fe2O2 SOor 4(Fe)3. We2SO 4further)3. We furtherdescribed described the the characterizedDNA-oxidativeDNA-oxidative the damage chemical damage in compositionA549 in A549 cells cells incubated of incubated a flavonoid-rich with with H2O H2 extract2orO 2 (Feor 2(FeSO from42)SO3. BJWe4)3 (BJe),. Wefurther further and described then described investigated the the chelatingchelating property property of BJe in of iron-exposed BJe in iron-exposed cells, thus cells, blocking thus blocking upstream upstream redox activity redox activity as well asas wellits as its itschelating antioxidantchelating property property properties of BJe of BJein in iron-exposed bothin iron-exposed abiotic andcells, cells, cell-based thus thus blocking blocking assays upstream [ 34upstream,35]. redox First redox weactivity testedactivity as the well asantioxidant well as its as its capabilitycapability to induce to induceantioxidant antioxidant catalase. catalase. Overall, Overall, our results our resultsshow that show BJ ethat exerts BJe itsexerts antioxidant its antioxidant activitycapabilitycapability of to BJe induce to using induce antioxidant a rangeantioxidant of catalase. tests catalase. (Folin-Ciocalteu, Overall, Overall, our ourresults Reducing results show show Power,that that BJe DPPH BJexertse exerts andits antioxidantits ORAC antioxidant assays) propertiesproperties through through different different complementary complementary via: scavenging via: scavenging free radicals, free radicals, metal ion metal chelation ion chelation and and propertiesproperties through through different different complementary complementary via: via:scavenging scavenging free free radicals, radicals, metal metal ion ionchelation chelation and and boostthat ofboost highlightedcellular of cellular antioxidant its an greattioxidant defense. antioxidant defense. and radical scavenging properties related to the high total boostboost of cellular of cellular antioxidant antioxidant defense. defense. phenolTrombetta contentTrombetta et al. [34 [56]].et Then, al.evaluated [56] we evaluated focused the antioxidant/anti-inflammatory onthe the antioxidant/anti-inflammatory cytoprotective ability activity of BJe activity againstof two oxidants,ofalcoholic two alcoholic such as TrombettaTrombetta et al.et [56]al. [56] evaluated evaluated the theantioxidant/anti-inflammatory antioxidant/anti-inflammatory activity activity of twoof two alcoholic alcoholic flavonoid-richhydrogenflavonoid-rich peroxideextracts extracts from (H2 Obergamot 2from) and bergamot (Fe peel2SO on4 ) peelhuman3, that on causehumanvessel oxidativeendothelial vessel endothelial cell cells damage (HUVECs) cells [(HUVECs)34,35 exposed]. We exposed foundto that to flavonoid-richflavonoid-rich extracts extracts from from bergamot bergamot peel peel on humanon human vessel vessel endothelial endothelial cells cells (HUVECs) (HUVECs) exposed exposed to to the BJepleiotropicthe prevented pleiotropic inflammatory either inflammatory H2 Ocytokine2 or (Fe cytokine TNF-2SO4α)3, -inducedTNF-a modelα, a ofmodel oxidative vascular of vascular oxidative damage oxidative onstress. the As A549stress. hallmarks cells As hallmarks by of different of the thepleiotropic pleiotropic inflammatory inflammatory cytokine cytokine TNF- TNF-α, aα model, a model of vascular of vascular oxidative oxidative stress. stress. As hallmarksAs hallmarks of of oxidativemechanismsoxidative damage dependingdamage in TNF- inα -exposedTNF- fromα the-exposed HUVECs, stressor HUVECs, agent. the Authors Indeed, the Authors monito the extract redmonito cell (25red viability, or cell 50 µviability,g/mL) intracellular reducedintracellular ROS oxidativeoxidative damage damage in TNF-in TNF-α-exposedα-exposed HUVECs, HUVECs, the theAuthors Authors monito monitored redcell cellviability, viability, intracellular intracellular levelsgeneration oflevels MDA/HNE, of and MDA/HNE, counteracted GSH and GSH GSSG, the and membrane andGSSG, SOD and activity. lipid SOD peroxidation, activity. They showed They improved showed that both that mitochondrial extracts both extracts prevented functionality prevented levelslevels of MDA/HNE, of MDA/HNE, GSH GSH and and GSSG, GSSG, and and SOD SOD activity. activity. They They showed showed that that both both extracts extracts prevented prevented the andoxidativethe prevented oxidative stress DNA-oxidative induced stress induced by TNF- damagebyα , TNF-modulated inα, A549modulated the cells activationincubated the activation of withredox-sensitive of H 2redox-sensitiveO2 or (Fe transcription2SO4 )transcription3. We further the theoxidative oxidative stress stress induced induced by TNF-by TNF-α, modulatedα, modulated the theactivation activation of redox-sensitiveof redox-sensitive transcription transcription factorsdescribed NF-factorsκB, the NF-thus chelatingκ B,increasing thus increasing property the cell thesurvival. of BJecell insurvival. iron-exposed cells, thus blocking upstream redox activity factorsfactors NF- NF-κB, κthusB, thus increasing increasing the cellthe cellsurvival. survival. as well as its capability to induce antioxidant catalase. Overall, our results show that BJe exerts 4. Anti-Inflammatory4. Anti-Inflammatory Activity Activity of Bergamot of Bergamot Derivatives Derivatives its4. Anti-Inflammatory antioxidant4. Anti-Inflammatory properties Activity Activity through of Bergamot of different Bergamot Derivatives complementary Derivatives via: scavenging free radicals, metal ion chelationThe anti-inflammatoryThe and anti-inflammatory boost of cellular activity antioxidantactivityof bergamot of bergamot defense. derivatives derivatives has been has demonstrated been demonstrated in both inin bothvitro in vitro TheThe anti-inflammatory anti-inflammatory activity activity of bergamot of bergamot derivatives derivatives has hasbeen been demonstrated demonstrated in both in both in vitro in vitro and in vivoandTrombetta instudies. vivo studies. etKaraca al. [ 56 Karacaet] al. evaluated [57] et al.investigat [57] the investigat antioxidant/anti-inflammatoryed for theed foranti-inflammatory the anti-inflammatory activity activity activity of BEO of of twousing BEO alcoholic using andand in vivo in vivo studies. studies. Karaca Karaca et al. et [57]al. [57] investigat investigated fored thefor theanti-inflammatory anti-inflammatory activity activity of BEO of BEO using using carrageenan-inducedflavonoid-richcarrageenan-induced extracts rat paw from rat oedema paw bergamot oedema test. The peel test. auth on Theors human auth foundors vessel thatfound reduction endothelial that reduction in the cells inflammation,in (HUVECs) the inflammation, exposed carrageenan-inducedcarrageenan-induced rat pawrat paw oedema oedema test. test. The The auth authors orsfound found that that reduction reduction in the in theinflammation, inflammation, measuredto themeasured pleiotropicas reduction as reduction inflammatory of paw ofvolume, paw cytokine volume, was 95.70% TNF- wasα 95.70%with, a model indomethacin with of indomethacin vascular used oxidative as used standard as stress. standard control, As hallmarks control, measuredmeasured as reductionas reduction of paw of paw volume, volume, was was 95.70% 95.70% with with indomethacin indomethacin used used as standardas standard control, control, 27.56% 27.56%with 0.025 with mL/kg 0.025 BEO,mL/kg 30.77% BEO, 30.77%with 0.05 with mL/kg 0.05 BEOmL/kg and BEO 63.39% and 63.39%with 0.10 with mL/kg 0.10 BEOmL/kg (ED BEO50 (ED50 27.56%27.56% with with 0.025 0.025 mL/kg mL/kg BEO, BEO, 30.77% 30.77% with with 0.05 0.05 mL/kg mL/kg BEO BEO and and 63.39% 63.39% with with 0.10 0.10 mL/kg mL/kg BEO BEO (ED (ED50 50 value ofvalue 0.079 of mL/kg). 0.079 mL/kg). Borgatti Borgatti et al. [58] et haveal. [58] analyzed have analyzed the effects the ofeffects a bergamot of a bergamot epicarp epicarp extract andextract and valuevalue of 0.079 of 0.079 mL/kg). mL/kg). Borgatti Borgatti et al. et [58] al. [58] have have analyzed analyzed the effectsthe effects of a ofbergamot a bergamot epicarp epicarp extract extract and and the isolatedthe isolated cumarins cumarins on the productionon the production of IL-8 (mRNAof IL-8 (mRNA levels and levels protein and proteinrelease) release) in cystic in fibrosis cystic fibrosis the theisolated isolated cumarins cumarins on theon theproduction production of IL-8 of IL-8 (mRNA (mRNA levels levels and and protein protein release) release) in cystic in cystic fibrosis fibrosis

Molecules 2016, 21, 1273 6 of 11 of oxidative damage in TNF-α-exposed HUVECs, the Authors monitored cell viability, intracellular levels of MDA/HNE, GSH and GSSG, and SOD activity. They showed that both extracts prevented the oxidative stress induced by TNF-α, modulated the activation of redox-sensitive transcription factors NF-κB, thus increasing the cell survival.

5. Anti-Inflammatory Activity of Bergamot Derivatives The anti-inflammatory activity of bergamot derivatives has been demonstrated in both in vitro and in vivo studies. Karaca et al. [57] investigated for the anti-inflammatory activity of BEO using carrageenan-induced rat paw oedema test. The authors found that reduction in the inflammation, measured as reduction of paw volume, was 95.70% with indomethacin used as standard control, 27.56% with 0.025 mL/kg BEO, 30.77% with 0.05 mL/kg BEO and 63.39% with 0.10 mL/kg BEO (ED50 value of 0.079 mL/kg). Borgatti et al. [58] have analyzed the effects of a bergamot epicarp extract and the isolated cumarins on the production of IL-8 (mRNA levels and protein release) in cystic fibrosis IB3-1 and CuFi-1 cells treated with TNF-α or heat-inactivated Pseudomonas aeruginosa. Particularly, they showed as the most active molecules were bergapten and citropten, typically present in BEO. Other researchers studied the potential anti-inflammatory activity of the polyphenolic fraction of BJ on normal human NCTC 2544 keratinocytes exposed to interferon-gamma (IFN-γ) or histamine (H) [59]. The juice was obtained from peeled fruits by industrial pressing and squeezing. The juice was filtered and loaded on column absorbing polyphenols. Then, polyphenolic fraction was eluted by hydroalcoholic acid solution (60:40 v/v) and dried by spray-drying. IFN-γ is an essential cytokine in amplifying inflammatory reactions, which stimulates the synthesis of chemokines, that in turn attract inflammatory cells and induces the expression of inducible intercellular adhesion molecule-1 (ICAM-1). Moreover, the over-secretion of inflammatory cytokines triggers an abnormal accumulation of extracellular matrix components such as glycosaminoglycans (GAGs) [60,61]. Keratinocytes are the major constituents of the epidermis, and in inflamed skin they up-regulate various pro-inflammatory activators, such as PGE2, NO, ROS and cytokines [62]. They are actively engaged in skin inflammatory responses by attracting leukocytes and modulating their functions. The assays performed on keratinocytes demonstrated that the bergamot extract significantly reduces ICAM-1, NO, ROS and GAG production in cells exposed to IFN-γ and H in concentration-dependent manner [59]. Moreover, these Authors observed similar results when they employed neoeriocitrin, naringin or neohesperidin, the most abundant flavonoids of the BJ. Of note, the antioxidant/anti-inflammatory effect of the wool extract was superior than that of each flavonoid used alone [59]. Nisticò and coworkers [63] evaluated the effect of a 38% bergamot polyphenolic fraction (BPF) on UVB-induced photoageing by examining inflammatory cytokine expression, telomere length/telomerase alterations and cellular viability in human immortalized HaCaT keratinocytes subjected to ultraviolet (UV) radiation. They clearly showed as the phytocomplex protected HaCaT cells against UVB-induced oxidative stress, reducing IL-1β mRNA expression and restores telomere length and activity. Experiments performed in our research lab demonstrated that BJe is able to significantly reduce the LPS-pro-inflammatory action in THP-1 cells [36]. Indeed, we demonstrated that the extract inhibited both gene expression and secretion of LPS-induced pro-inflammatory cytokines (IL-6, IL-1β, TNF-α). Then, given the reported key role of NF-κB in inflammatory response induced by various stimuli, we also investigated its role in THP-1 cells exposed to LPS, showing that BJe was able to inhibit this important transcription factor. It is known that polyphenols could influence cellular function by acting as activators of SIRT1, a nuclear histone deacetylase involved in the inhibition of NF-κB signaling. Therefore, in a subset of experiments we also investigated the capability of BJe to modulate SIRT1, evidencing that the extract activated SIRT1, that in turn reduced the LPS-enhanced acetylation of p65 in THP-1 cells [36]. In the light of these observations, we wondered whether BJe may be beneficial against neuroinflammatory processes, such as those observed in Alzheimer’s disease, where the chronic Molecules 2016, 21, 1273 7 of 11 activated glial cells amplify the neurotoxicity by secreting pro-inflammatory mediators like cytokines. Hallmarks of chronic inflamed tissues are the presence of an increased number of monocytes, as well as monocyte-derived tissue macrophages, that can be referred to microglial cells in the central nervous system (CNS) [64]. To this aim we investigated the effectiveness of BJe in THP-1 cells exposed to amyloid-beta (Aβ), an experimental model of circulating monocytes/macrophages that resemble neuroinflammatory condition [38]. Exposure of THP-1 cells to Aβ significantly induced the expression and secretion of IL-6 and IL-1β in THP-1 cells, and increased the phosphorylation of ERK 1/2 as well as p46 and p54 members of JNK family. These effects were reduced by the pre-treatment with BJe, confirming its anti-inflammatory potential. Some results show that increased cytokine expression may be due to different transcription factors, such as AP-1. Notably, in this research we observed that the increased AP-1 DNA binding activity in Aβ-treated cells was reduced by the pretreatment with BJe [38]. The anti-inflammatory activity of BJe has also been demonstrated in animal models. In an in vivo model of inflammatory bowel disease, we have shown that BJe attenuated the inflammation by dinitrobenzene sulfonic acid (DNBS) [37]. The BJe-treated mice were more resistant to DNBS-induced colitis and showed less macroscopic and histological signs of inflammatory process. Moreover, the expression of important inflammatory mediators, such as TNF-α and IL-1β, as well as the neutrophil infiltration, were reduced in the colon tissues of DNBS-injected mice as consequence of BJe treatment. We also reported that BJe reduced the increase of p-JNK expression, and inhibited the NF-κB activation in the colon of DNBS-treated animals. In addition, we found reduced expression of adhesion molecules (ICAM-1 and P-selectin), nitrotyrosine, poly (ADP ribose) synthetase (PAR) and apoptosis related proteins in DNBS-injected mice treated with BJe [37]. The beneficial effects of BJe have been also demonstrated in ileum inflammation triggered by intestinal ischemia/reperfusion (I/R) injury in mice [39]. The experimental results confirmed that BJe was able to reduce histological damage, cytokines production (TNF-α and IL-1β), ICAM-1 and P-selectin expression, myeloperoxidase (MPO) activity and oxidative stress (malondialdehyde, MDA; manganese superoxide dismutase, mnSOD and inducible nitric oxide synthase, iNOS) by a mechanism involved both NF-κB and MAP kinases (JNK and p38) pathways. The studies on the anti-inflammatory effect of bergamot derivatives collected in this review are summarized in Table3.

Table 3. Principal characteristics of studies on anti-inﬂammatory effect of bergamot derivatives.

Compound Study Model Determinations References Carrageenan-induced Karaca et al.; BEO Paw volume inflammation 2007 [57] Extract from Endotelial oxidative MDA, 4-HNE, GSSG, Trombetta et al.; bergamot peel stress/inflammation GSH/GSSG, SOD 2010 [56] Extract from Borgatti et al.; Cystic fibrosis IL-8 bergamot epicarp 2011 [58] Extract from Graziano et al.; bergamot juice and Skin inflammation ICAM-1, iNOS, NO, ROS, GAG 2012 [59] flavonoids Extract from Risitano et al.; Activated monocytes IL-1β, IL-6, TNF-α, NF-κB, SIRT-1 bergamot juice 2014 [36] IL-1β, TNF-α, MPO, nitrotyrosine, Extract from Impellizzeri et al.; Colitis p-JNK, ICAM-1, P-selectin PAR, bergamot juice 2014 [37] NF-κB, BAX, BCL-2 Extract from ROS, lipid peroxidation, Ferlazzo et al.; Lung oxidative stress bergamot juice mitochondrial potential, DNA damage 2015 [34] Extract from Nisticò et al.; Skin photoaging IL-1β, telomerase activity bergamot juice 2015 [63] Molecules 2016, 21, 1273 8 of 11

Table 3. Cont.

Compound Study Model Determinations References Extract from Currò et al.; Neuroinﬂammation IL-1β, IL-6, ERK1/2, p-JNK, AP-1 bergamot juice 2016 [38] Extract from ROS, lipid peroxidation, mitohcondrial Ferlazzo et al.; Lung oxidative stress bergamot juice potential, DNA damage, catalase 2016 [35] histological alterations, IL-1β, TNF-α, Extract from Intestinal Impellizzeri et al.; MDA, MnSOD, ICAM-1, P-selectin, bergamot juice ischemia/reperfusion injury 2016 [39] NF-κB, iNOS, p-p38, p-JNK

6. Conclusions Despite the remarkable medical advances, inflammation remains a serious health problem. Recently, interest in natural product has led both researchers and the wellness industry to explore natural sources for alternative and complementary medicine to prevent and/or treat inflammatory diseases. Several studies on the beneficial effects of bergamot derivatives in the inflammation field have been published, increasing the attention towards this Citrus species as a potential source of natural antioxidant/anti-inflammatory remedies. This is the first review that collects and critically reports the literature on this topic. It may represent a scientific basis for the development of novel strategies to improve health status and attenuate inflammatory conditions.

Acknowledgments: This review has been written within the framework of the “MEPRA” (PO FESR Sicilia 2007/2013, Linea d’Intervento 4.1.1.1, CUP G73F11000050004) and “ABSIB” (PSR Calabria 2007/2013 misura 124) projects to MN. Author Contributions: N.F. wrote the paper; S.C. and E.V.S. collected the literature; S.G. and G.C. revised the paper; M.N. conceived the study and assisted in writing the paper. Conﬂicts of Interest: The authors declare no conﬂict of interests.

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