British Journal of Nutrition

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1 British Journl of Nutrition (2013), 109, q The Authors 2012 doi: /s The ntioxidnt effet of -ryophyllene protets rt liver from ron tetrhloride-indued firosis y inhiiting hepti stellte ell tivtion Miguel Angel Cllej 1, Jose Mrí Vieites 2, Trinidd Montero-Meterdez 3,Mrí Isel Torres 4, Mrí José Fus 5, Angel Gil 5 nd Antonio Suárez 6 * 1 UGC Frmi, Hospitl Universitrio Virgen de ls Nieves, Grnd, Spin 2 Bioquími de Alimentos, Instituto de Investigiones Mrins (CSIC), Vigo, Spin 3 Biohemil Phrmology, Willim Hrvey Reserh Institute, Queen Mry University of London, London, UK 4 Deprtmento de Biologí Experimentl, Universidd de Jen, Jen, Spin 5 Deprtmento de Bioquími y Biologí Moleulr 2, Fultd de Frmi, Universidd de Grnd, Grnd, Spin 6 Instituto de Nutriión y Tenologí de los Alimentos, Centro de Investigión Biomédi, Armill, Spin (Sumitted 1 April 2011 Finl revision reeived 20 Ferury 2012 Aepted 27 Ferury 2012 First pulished online 1 My 2012) British Journl of Nutrition Astrt Plnt-sed whole foods provide thousnds of iotive metolites to the humn diet tht redue the risk of developing hroni diseses. -Cryophyllene (CAR) is ommon onstituent of the essentil oil of numerous plnts, vegetles, fruits nd mediinl hers, nd hs een used s flvouring gent sine the 1930 s. Here, we report the ntioxidnt tivity of CAR, its protetive effet on liver firosis nd its inhiitory pity on hepti stellte ell (HSC) tivtion. CAR ws tested for the inhiition of lipid peroxidtion nd s free rdil svenger. CAR hd higher inhiitory pity on lipid peroxidtion thn prouol, -humulene nd -toopherol. Also, CAR showed high svenging tivities ginst hydroxyl rdil nd superoxide nion. The tivity of 5-lipoxygense, n enzyme tht tively prtiiptes in firogenesis, ws signifintly inhiited y CAR. Cron tetrhloride-treted rts reeived CAR t 2, 20 nd 200 mg/kg. CAR signifintly improved liver struture, nd redued firosis nd the expression of Col11, Tgf1 nd Timp1 genes. Oxidtive stress ws used to estlish model of HSC tivtion with overprodution of extrellulr mtrix proteins. CAR (1 nd 10 mm) inresed ell viility nd signifintly redued the expression of firoti mrker genes. CAR, sesquiterpene present in numerous plnts nd foods, is s nturl ntioxidnt tht redues ron tetrhloride-medited liver firosis nd inhiits hepti ell tivtion. Key words: -Cryophyllene: Liver firosis: Antioxidnts: Hepti stellte ells Epidemiologil studies hve onsistently shown tht regulr onsumption of fruits nd vegetles s well s whole grins is strongly ssoited with redued risk of developing hroni diseses, suh s ner nd CVD, whih re the top two uses of deth in most industrilised ountries (1). These studies highlighted the unique hemil fetures of fruits, vegetles, spies nd grins. Plnt-sed whole foods provide thousnds of primry nd seondry metolites to the humn diet tht re sored into the ody due to their low-moleulr lipophili nture. These phytohemils re iotive non-nutrient plnt ompounds tht n e lssified into vrious fmilies, suh s lkloids, flvonoids, isoflvones, isothioyntes, orgno-sulphur ompounds, psiinoids, phytosterols, rotenoids nd terpenoids (2). Prtiulrly, terpenoids re perhps the most diverse fmily of nturl produts synthesised from plnts, serving rnge of importnt physiologil funtions. Terpenoids re the mjor voltile onstituents of essentil oils tht generte the flvour fingerprint of vegetles, fruits nd spies (3). The essentil oils of plnts, fruits nd vegetles re nturl, omplex, multiomponent systems omposed minly of terpenes in ddition to some other non-terpene omponents. Formed vi the isoprene pthwy, these ompounds re grouped into hydrorons (limonene), lohols (menthol), ldehydes (itrl), ketones (rvone), ids nd esters (gernyl ette). Sine this pthwy opertes to different extent in plnts, mny of the sme hemil onstituents re present in wide vriety of essentil oils. A wide rnge of terpenoids hs demonstrted Arevitions: ALAT, lnine minotrnsferse; ASAT, sprtte minotrnsferse; CAR, -ryophyllene; Col11, type I ollgen 1; DPPH, 1,1-diphenyl- 2-pirylhydrzyl; HSC, hepti stellte ell; LDH, ltte dehydrogense; TBARS, thiorituri id-retive sustnes; TGF-1, trnsforming growth ftor-1; Timp1, tissue inhiitor of metlloproteinse-1. * Corresponding uthor: A. Suárez, fx þ , emil surez@ugr.es

2 -Cryophyllene redues rt liver firosis 395 British Journl of Nutrition phrmeutil tivity ginst humn ilments suh s inflmmtory diseses (4), CVD (5) nd ner (6). Our group previously reported the heptoprotetive tivity of n ethnol extrt of Rosmrinus tomentosus Huerth-Mort nd Mire (Lmiee) in CCl 4 - nd thioetmide-indued irrhoti rts (7,8). Beuse retive oxygen speies re generted during CCl 4 -indued liver toxiity, we explored the free-rdil-svenging pity of the most frequent terpenes in Rosmrinus spp. essentil oil. The ethnol extrt in Rosmrinus offiinlis ontins vrile mixture of phenoli diterpenes (rnosi id, rnosol nd 12-O-methylrnosi id), ffeoyl derivtives (rosmrini id) nd flvones with other terpenes in lower quntities (9). The present study ontriutes extensive dt on the identifition of -ryophyllene (CAR) s free-rdil-svenging gent nd on its pity to inhiit lipid peroxidtion in vitro. CAR proteted rt livers from CCl 4 toxiity nd redued firosis nd the gene expression of type I ollgen 1 (Col11), trnsforming growth ftor-1 (Tgf1) nd tissue inhiitor of metlloproteinse-1 (Timp1). Ex vivo, CAR proteted CFCS- 2G ells, rt hepti stellte ell (HSC) line, from free rdil dmge nd effetively down-regulted the expression of the Col11 nd Timp1 genes. CAR is voltile sesquiterpene found in lrge mounts in the essentil oils of mny herl drugs, spie nd food plnts, inluding oregno, innmon, lk pepper, rrot, y lurel, grpes, melon, red lush grpefruit, strwerry, guv, ginger, mong others. The present results demonstrted tht CAR is n ntifirogeni ndidte for the prevention nd tretment of hepti firosis. Sine ptients with liver firosis hve n ssoited ondition of mlnutrition, our dt support the need to inrese the onsumption of fruits, vegetles nd whole grins s soure of iotive non-nutrient nturl ompounds to mintin helthy liver sttus. Experimentl methods Chemils All hemils (unless speified) were otined from Sigm- Aldrih Quími S.A., t the highest ville grdes. In vitro ell ulture The CFSC-2G ell line hs similr phenotype to tht of erly-pssge primry HSC (10). CFSC-2G ells were mintined in ulture with minimum essentil medium supplemented with 10 % fetl ovine serum, 0 1 % non-essentil mino ids nd 1 1 % peniillin/streptomyin. For eh experiment, ells were inuted overnight with no dditions nd then treted with pproprite onentrtions of CAR t 24 h efore tretment with n Fe 2þ sorte solution (finl onentrtion mm). Culture medium nd ells were olleted 6 h lter. Animls nd tretments Femle Wistr rts ( g) were housed in ges t onstnt temperture (228C) nd humidity (55 %) with 12 h light 12 h drk yle ( hours). The nimls were hndled in ordne with reommendtions of the Amerin Physiologil Soiety (Counil of Europe, 1982). The nimls were rndomly divided into five groups of six individuls with free ess to wter nd rodent how. To produe ute liver dmge, the experimentl design onsisted in single CCl 4 (0 15 ml/100 g ody weight) intrperitonel injetion. Dose seletion for CAR tretment ws sed on previously pulished studies (11). Similrly to epigllothehin-3- gllte tretment, CAR dministrtion ws strted in nimls sujeted to ute CCl 4 on the dy efore CCl 4 poisoning (12). Body weight ws monitored throughout the experiment. Rts were deprived of food for 12 h, nesthetised nd lood ws olleted from the dominl ort. The liver ws removed, wshed with old sline, weighed nd stored t 2808C. Liver histology nd ollgen ontent A portion of the liver ws fixed in 10 % formldehyde, emedded in prffin nd ut into 5 mm-thik setions. The smples were stined with periodi id-shiff nd fst green neutrl red. A semi-quntittive sore for hepti injury rnging from 0 to 5 ws ssigned to eh rt ording to the presene or sene of heptoyte nerosis, nodulr trnsformtion, firous septe nd iliry hyperplsi. Firosis severity ws sored s norml (0), miniml (1), mild (2), moderte (3), mrked (4) or severe firosis (5). Collgen ontent ws mesured in 15 mm-thik liver setions using olorimetri method sed on the seletive pity of two dyes (Sirius red F3B nd fst green FCF) to ind to ollgen nd non-ollgenous proteins, respetively, in the presene of piri id (13). Mesurement of free rdil-svenging tivity Rdil-svenging tivity ginst the 1,1-diphenyl-2-pirylhydrzyl (DPPH) rdil ws determined y using the method of Mensor et l. (14). Svenging tivity ginst the hydroxyl rdil ws determined y the inhiition of hemiluminesene during linolei id peroxidtion y the Fenton retion, s desried y Xun et l. (15). Svenging tivity ginst the superoxide nion ws ssyed in non-enzymti nd enzymti systems. First, the ompounds were tested to inhiit the photohemil oxidtion of o-dinisidine y rioflvin, s desried y Bridges et l. (16). Seond, the enzymti prodution of the superoxide nion ws ssyed y monitoring the redution in ytohrome (17). The inhiition perentge of superoxide nion genertion ws lulted ording to the formul: perentge inhiition ¼ (A ontrol A smple )/A ontrol 100. The inhiition of the formtion of lipid peroxides y 5-lipoxygense ws performed s desried y Liu & Pn (18). Inhiition of lipid peroxidtion Liver mirosomes were otined s desried y Suárez et l. (19). Lipid peroxidtion ws indued in vitro in liver mirosomes with Fe 2þ sorte solution ( mm). The test ompounds were dded to liver mirosomes

3 396 M. A. Cllej et l. British Journl of Nutrition 30 min efore inititing lipid peroxidtion. Thiorituri idretive sustnes (TBARS) were ssyed in the medium t 30 min fter indution nd expressed s nmol mlondildehyde/mg protein (19). Anlyti determintions Totl iliruin levels were determined with the stndrd 2,5-dihlorophenyldizonium tetrfluoroorte dizo regent, ording to the mnufturer s instrutions (Rohe Applied Siene). Plsm protein ontent ws determined ording to the method of Brdford (20). Ltte dehydrogense (LDH) ws spetrophotometrilly ssyed to test ell viility (Sigm-Aldrih Quími S.A.). Asprtte minotrnsferse (ASAT), lnine minotrnsferse (ALAT), lkline phosphtse nd g-glutmyltrnspeptidse tivities were mesured s desried previously (7,8). Enzyme units were expressed per mg of plsm protein. Gene expression Totl RNA ws extrted from the rt liver tissue or CFSC-2G ells. Briefly, 10 mg of totl RNA were eletrophoresed in grose gels, trnsferred to nylon filters, fixed with UV rosslinker nd hyridised with n - 32 P-deoxyytidine triphosphte-lelled omplementry DNA proe speifi for Col11 or Timp1 mrna nd, sequentilly, with n - 32 P- deoxyytidine triphosphte-lelled oligonuleotide omplementry to glyerldehyde-3-phosphte dehydrogense (Gpdh) mrna. Semi-quntittive PCR ws used to mesure Col11, Tgf1 nd Timp1 gene expression in the rt liver s desried y Pérez et l. (13). Sttistil nlysis All dt re presented s men vlues with their stndrd errors. All vriles were heked for normlity nd homogeneous vrine y using the Kolmogorov Smirnov nd Levene tests, respetively. A two-wy ANOVA ws used to evlute differenes ording to ompound nd onentrtion, nd posteriori Bonferroni tests were rried out for speifi men omprisons. One-wy ANOVA nd post ho men omprison using the Bonferroni test were used to nlyse the effets etween CAR, vitmin E nd ontrol groups, nd to ssess onentrtion effets for speifi ompounds. A vlue of P, 0 05 ws onsidered signifint. Dt nlyses were performed using SPSS for Windows 15.0 (SPSS, In.). Results -Cryophyllene is free-rdil-svenging onstituent of Rosmrinus spp. essentil oil Bsed on pulished dt on essentil oil onstituents in Rosmrinus spp. (9), we first tested the inhiitory pity of -pinene, mphene, -humulene, mphor, orneol, 1,8- ineole nd CAR ginst liver mirosome lipid peroxidtion. Only -humulene nd CAR showed inhiitory pity (dt not shown), whih ws further ssyed in onentrtiondependent test. At ll onentrtions, CAR hd signifintly higher inhiitory tion ginst lipid peroxidtion thn did -humulene, prouol nd vitmin E (Fig. 1). Prouol nd vitmin E were inluded s positive ontrols for syntheti nd nturl ntioxidnt ompounds. CAR showed svenging tivity ginst the hydroxyl rdil (Fenton retion) nd superoxide nion (Rioflvin ssy) in onentrtion-dependent mnner (Tle 1). CAR lso inhiited superoxide niondependent ytohrome redution in the hypoxnthine/ xnthine oxidse system (Tle 1). CAR inhiited lipoxygense-dependent oxidtion of linolei id in similr mnner to -toopherol. CAR showed greter free-rdil-svenging tivity in omprison with prouol nd greter superoxide nion- nd similr hydroxyl rdil-svenging tivities in omprison with -toopherol. Cron tetrhloride-indued liver firosis ws mrkedly suppressed y -ryophyllene A fourth experiment ws designed to determine the protetive effet of CAR on CCl 4 -indued liver injury. Pretretment with CAR signifintly redued plsm ASAT, ALAT nd LDH tivities nd totl iliruin levels in dose-dependent mnner (Tle 2). Representtive imges of liver mirosopi morphology re shown in Fig. 2. Histologil nlysis of the liver of CCl 4 -treted rts reveled mrked firosis with heptoyte nerosis, inflmmtory ell infiltrtion, firosis nd mirovesiulr stetosis. In ontrst, CAR signifintly redued the liver firosis sore. Imge nlysis showed tht CAR redued the liver ollgen ontent (Tle 2) nd signifintly meliorted the histologil struture of the liver ompred with non-treted firoti rts, showing no signs of stetosis, firosis Antioxidnt tivity (%) d 1nM d d 50 nm 1 µm 5 µm Conentrtion Fig. 1. Antioxidnt tivity of prouol ( ), -ryophyllene ( ), -toopherol ( )nd-humulene ( ) on the Fe 2þ /sorte-indued lipid peroxidtion of liver mirosomes, expressed s the perentge inhiition of the prodution of thiorituri id-retive sustnes. Vlues re mens, with their stndrd errors represented y vertil rs (n 6).,,,d Men vlues with unlike letters were signifintly different (P,0 05). d

4 -Cryophyllene redues rt liver firosis 397 Tle 1. Svenging effet of prouol, -toopherol nd -ryophyllene (CAR) t different onentrtions (Men vlues with their stndrd errors, n 6) Prouol -Toopherol CAR Conentrtion (mm) Men SEM Men SEM Men SEM IC 50 DPPH (mg/ml) OH -svenging tivity (%) O 2 2 -svenging tivity (%) , XO inhiition (%) LPO inhiition (%) IC 50, hlf-mximl inhiitory onentrtion; DPPH, 1,1-diphenyl-2-pirylhydrzyl rdil; XO, xnthine oxidse; LPO, 5-lipoxygense.,, Men vlues within row with unlike supersript letters were signifintly different (P,0 05). British Journl of Nutrition or heptoyte nerosis (Fig. 2). Timp1, Tgf1 nd Col11 gene expressions were higher in the liver of CCl 4 -treted rts thn in the liver of norml rts (Fig. 3). Furthermore, the CAR tretment signifintly ttenuted the CCl 4 -indued up-regultion of oth genes. Hene, it ws demonstrted tht CAR protets the rt liver from CCl 4 -indued injury y reduing firosis nd down-regulting Timp1, Tgf1 nd Col11 gene expressions. -Cryophyllene protets CFSC-2G hepti stellte ells ginst free rdil dmge The next step ws to ssess whether CAR proteted hepti CFSC-2G ells ginst oxidtive dmge indued y Fe 2þ H 2 O 2 solution. The degree of ell dmge ws mesured with Trypn lue exlusion ssy, intrellulr TBARS produts nd enzymti mrkers, present in ell medi due to ytoplsmi lekge. Oxidtive dmge redued ell viility to 93 3 % nd inresed intrellulr TBARS produts to 3 72 nmol/mg protein (Tle 3). LDH, lkline phosphtse, g-glutmyltrnspeptidse, ALAT nd ASAT tivities were inresed in ell medi due to free rdil-indued ell dmge. The CAR tretment inresed ell viility nd redued enzyme tivities nd TBARS prodution. The present results indite tht CAR effetively proteted CFSC-2G ells from free rdil-indued ell dmge. -Cryophyllene redues oxidtive stress-indued tivtion of CFSC-2G hepti stellte ells HSC ply key role in response to liver injury. Oxidtive stress tivtes HSC tht eome the min soure of the post-injury extrellulr mtrix deposit tht results in liver firosis. An experiment ws designed to onfirm tht CAR redues HSC tivtion y retive oxygen speies nd down-regultes the mrna of the Timp1 nd Col11 genes. Exposure of CFSC-2G ells to oxidtive dmge up-regulted 2 06-fold the expression of the Col11 gene nd 3 12-fold tht of the Timp1 gene in omprison with untreted ells (Fig. 4). The ddition of CAR signifintly redued the expression of Col11 nd Timp1 in dose-dependent mnner. Disussion CAR is nturl voltile iyli sesquiterpene tht is present in numerous hers, spies nd foods. CAR n e isolted from innmon, love oil nd opi lsm, ll of whih hve een used s nturl remedies. Beuse of its wek Tle 2. Serum iohemistry of helthy rts (ontrol), ron tetrhloride-treted rts (CCl 4 ) nd ron tetrhloride-treted rts reeiving -ryophyllene (CAR) t different onentrtions (Men vlues with their stndrd errors, n 6) Control CCl 4 CAR 2 mg/kg CAR 20 mg/kg CAR 200 mg/kg Men SEM Men SEM Men SEM Men SEM Men SEM ASAT (U/l) ALAT (U/l) LDH (U/l) AP (U/l) BRB (mg/l) Totl protein (mg/l) ASAT, sprtte minotrnsferse; ALAT, lnine minotrnsferse; LDH, ltte dehydrogense; AP, lkline phosphtse; BRB, totl iliruin.,, Men vlues within row with unlike supersript letters were signifintly different (P,0 05).

5 British Journl of Nutrition 398 (A) (B) (C) M. A. Cllej et l. Lipid peroxidtion of ell memrnes is the min dmge used y retive oxygen speies in ells. It is initited y hydroxyl rdils nd superoxide nions, leding to the formtion of peroxyl rdils tht ultimtely propgte the hin retion in lipids. In first step, we explored the ntioxidnt tivities of severl onstituents of Rosmrinus spp. essentil oil. CAR ws the only terpene to show strong inhiitory effet on in vitro lipid peroxidtion (Fig. 1). This inhiition ws signifintly higher thn tht of -humulene, -toopherol or prouol. Given tht different rdil speies initite lipid peroxidtion, we further tested the free rdil-svenging tivity of CAR (Tle 1). Aording to the present results, CAR showed svenging tivity ginst hydroxyl rdil (Fenton retion) nd superoxide nion (rioflvin ssy) in onentrtion-dependent mnner (Tle 1). Its svenging pity ws signifintly higher thn tht of prouol. The svenging properties of -toopherol nd CAR were similr. However, oth sesquiterpenes, CAR nd -humulene filed to svenge DPPH, moleule tht ontins stle free rdil. This result my explin why CAR hs not een previously reported s n ntioxidnt, sine the DPPH test is the most widely used tehnique to hrterise the nti-rdil pity of ompounds. Interestingly, CAR ws le to mrkedly redue the in vitro formtion of lipid peroxides y 5-lipoxygense. Tken together, our dt indited tht CAR is highly effetive hin-reking ntioxidnt in in vitro lipid peroxidtion nd possesses greter svenging tivities ginst retive oxygen speies thn ginst stle orgni rdils. Bsed on these results, we hypothesised tht CAR my protet from oxidtive stress-indued injury. Hepti firosis used y CCl 4 hs een extensively used in experimentl rt models. Hepti responses in rts to ute CCl 4 dministrtion re shown to e similr to humn hepti firosis (26). CCl 4 metolism in the liver stimultes lipid peroxidtion Fig. 2. Protetive effet of -ryophyllene on ron tetrhloride-indued liver dmge. (A) Setions of the liver of helthy rts, (B) ron tetrhloride-treted rts nd (C) ron tetrhloride-treted rts tht reeived 2 mg/kg of -ryophyllene. Originl mgnifition, 200. romti tste, CAR is ommerilly used s food dditive nd in osmetis. The Flvour nd Extrt Mnufturers Assoition grnted CAR the sttus of generlly reognised s sfe, nd it hs een pproved y the US Food nd Drug Administrtion for food use due to its low toxiity (21). CAR hs een shown to hve nti-miroil (22), ntiinflmmtory (4), nninoid (23), nti-rinogeni (24) nd skin penetrtion-enhning properties (25). Our group previously reported the heptoprotetive tivity of n ethnol extrt of R. tomentosus Huerth-Mort nd Mire (Lmiee) in CCl 4 - nd thioetmide-treted irrhoti rts (7,8). The omposition of the ethnol extrt ws unler, ut it ontined most of the voltile terpenes present in its essentil oil, of whih CAR is n importnt omponent (9). mrna reltive onentrtion Firosis Firosis+CAR 2 mg/kg Firosis+CAR 20 mg/kg Firosis+CAR 200 mg/kg Fig. 3. Effet of -ryophyllene (CAR) on the hepti mrna expression of the type I ollgen 1 (Col11, ), trnsforming growth ftor-1 (Tgf1, ) nd tissue inhiitor of metlloproteinse-1 (Timp1, ) genes in ron tetrhloride-indued liver firosis. Rts reeived different onentrtions of CAR. Dt re vlues of mrna expression reltive to glyerldehyde-3-phosphte dehydrogense (Gpdh). Vlues re mens, with their stndrd errors represented y vertil rs (n 3)., Men vlues with unlike letters were signifintly different (P,0 05).

6 -Cryophyllene redues rt liver firosis 399 Tle 3. Protetion of -ryophyllene (CAR) ginst oxidtive stress-indued dmge to CFS-2G hepti stellte ells (Men vlues with their stndrd errors, n 6) Control* Oxidtive dmge -Toopherol (1 mm) -Toopherol (10 mm) CAR (1 mm) CAR (10 mm) Men SEM Men SEM Men SEM Men SEM Men SEM Men SEM TBARS (nmol/mg protein) Cell viility (%) LDH (U/l) ASAT (U/l) ALAT (U/l) d , , 0 5 AP (U/l) , 0 3 g-gt (U/l) d TBARS, thiorituri id-retive sustnes; LDH, ltte dehydrogense; ASAT, sprtte minotrnsferse; ALAT, lnine minotrnsferse; AP, lkline phosphtse; g-gt, g-glutmyl trnspeptidse.,,,d Men vlues within row with unlike supersript letters were signifintly different (P,0 05). * Control ells reeived vehile lone (ethnol). Oxidtive dmge ws indued in ells y ddition of n Fe 2þ /sorte 50 mm/200 mm solution. Cells were inuted with -toopherol or CAR efore oxidtive dmge. British Journl of Nutrition nd the prodution of free rdils (27), using nerosis of heptoytes, induing inflmmtion nd further promoting the progression of hepti firogenesis. In this mehnism, tivtion of Kupffer ells, where 5-lipoxygense is silly expressed in the liver (28), nd the susequent relese of proinflmmtory meditors re n erly step in the pthogenesis of firosis. Sine CAR inhiited in vitro lipid peroxidtion nd 5-lipoxygense tivity, we tested its effet on CCl 4 liver toxiity. Aute poisoning with CCl 4 inresed iliruin onentrtion nd ALAT, ASAT, lkline phosphtse nd LDH tivities in rt plsm, deresed in plsm totl protein vlues, nd produed severe firosis of heptoyte nerosis, inflmmtory ell infiltrtion, firosis nd mirovesiulr stetosis (Tle 2; Fig. 2). CAR demonstrted heptoprotetive tivity, mrkedly restoring the vlues of plsm mrkers of liver dmge nd reduing the degree of stetosis nd the extent of liver firosis. Fold hnge (ritrry units) Oxidtive dmge Oxidtive dmge+car 1 µm Oxidtive dmge+car 10 µm Fig. 4. -Cryophyllene (CAR) redues oxidtive stress-indued tivtion of CFS-2G hepti stellte ells. Dt of mrna expression of the type I ollgen 1 (Col11, ) nd tissue inhiitor of metlloproteinse-1 (Timp1, ) genes reltive to glyerldehyde-3-phosphte dehydrogense (Gpdh) re presented. Oxidtive dmge ws indued with Fe 2þ /sorte 50 mm/200 mm solution. Cells were treted with CAR t the indited onentrtions efore oxidtive dmge. Vlues re mens, with their stndrd errors represented y vertil rs (n 3).,, Men vlues with unlike letters were signifintly different (P,0 05). It is the exuernt deposition of mtrix proteins fter injury tht uses liver firosis nd eventully irrhosis. The mehnism of hepti firogenesis is relted not only to n enhned mtrix iosynthesis ut lso to n inhiition of mtrix rekdown (29). In humn sujets, hroni lohol-indued dmge to the liver signifintly diminishes ollgenolyti tivity in prllel with drmti rise in TIMP-1 nd TGF- levels (30). In the present experiment, CCl 4 poisoning mrkedly inresed the expression of the Col11, Tgf1 nd Timp1 genes. Protetion y CAR ginst CCl 4 -indued liver firosis ws ssoited with redution in liver ollgen ontent, the histology sore nd the expression of the Col11, Tgf1 nd Timp1 genes (Fig. 3). These results indite tht CAR is heptoprotetive ompound tht down-regultes the expression of profiroti genes nd redues hepti histologil lesions. Oxidtive stress promotes the tivtion of HSC tht re the min soure of the post-injury extrellulr mtrix deposit resulting in liver firosis. Oxidtive stress promotes HSC prolifertion nd up-regultes Col11, Tgf1 nd Timp1 gene expressions in HSC (31), nd this proess is modulted y ntioxidnts (32,33). Bsed on our previous results, we hypothesised tht CAR my prevent liver firosis y reduing HSC tivtion. An experiment ws designed to test whether CAR inhiited oxidtive stress-medited HSC tivtion. Retive oxygen speies hve een shown to disrupt ell memrne integrity due to the oxidtion of PUFA, whih uses the spillge of ytoplsmi enzymes. As expeted, exposure of CFCS-2G ells to pro-oxidnt solution redued ell viility nd inresed LDH, ASAT, ALAT, lkline phosphtse nd g-glutmyltrnspeptidse lekge, nd mlonldehyde onentrtion in ulture medi (Tle 3). Oxidtive dmge up-regulted the expression of the Col11 nd Timp1 genes in CFSC-2G ells (Fig. 4). Pretretment of CFSC-2G ells with CAR signifintly redued oxidtive stress-indued dmge in dose-dependent mnner. The min result ws the down-regultion of Col11 nd Timp1 gene trnsription exerted y CAR. In keeping with our hypothesis, the present results onfirmed tht CAR is n effetive inhiitor of ell dmge indued y retive oxygen speies nd n prevent

7 400 M. A. Cllej et l. British Journl of Nutrition oxidtive stress-medited up-regultion of the Col11 nd Timp1 genes in HSC. In summry, we provide the first demonstrtion tht CAR, ompound present in numerous plnts nd foods, is n effetive inhiitor of lipid peroxidtion, proly due to its free rdil-svenging tivity ginst hydroxyl rdils, superoxide nions nd lipid peroxides. Our dt provide novel insights into the mehnisms of CAR in the prevention of liver firosis tht my e relted t lest in prt to its ntioxidnt tivity. Sine CAR is present in numerous plnts, spies nd foods, further experiments re needed to onsolidte the potentil pplition of CAR s iotive nturl ompound tht redues oxidtive stress-medited injury in humn diseses. Aknowledgements None of the uthors hd ommeril interest, finnil interest nd/or other reltionships with phrmeutil, lortory supplies or medil ompnies. The uthors re grteful to the Spnish Ministry of Puli Helth for providing funds during the tenure of reserh. A. 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