Bioactive Constituents from Triguero Asparagus Improve the Plasma Lipid Profile and Liver Antioxidant Status in Hypercholesterolemic Rats

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1 Int. J. Mol. Si. 213, 14, ; doi:1.339/ijms Artile OPEN ACCESS Interntionl Journl of Moleulr Sienes ISSN Biotive Constituents from Triguero Asprgus Improve the Plsm Lipid Profile nd Liver Antioxidnt Sttus in Hyperholesterolemi Rts Sr Vázquez-Cstill 1, *, Roío De l Puert 2, Mrí Dolores Grí Giménez 2, Mrí Angeles Fernández-Arhe 2 nd Rfel Guillén-Bejrno Biotehnology Deprtment, Instituto de l Grs (C.S.I.C.), Seville 4112, Spin; E-Mil: rguillen@i.es Phrmology Deprtment, Shool of Phrmy, University of Seville, Seville 4112, Spin; E-Mils: puert@us.es (R.D.); mdgr@us.es (M.D.G.G.); rhe@us.es (M.A.F.-A.) * Author to whom orrespondene should e ddressed; E-Mil: svzquez@ig.si.es; Tel.: ; Fx: Reeived: 22 Septemer 213; in revised form: 15 Otoer 213 / Aepted: 21 Otoer 213 / Pulished: 24 Otoer 213 Astrt: We hve previously shown tht the Andlusin-ultivted Asprgus offiinlis L. triguero vriety produes hypoholesterolemi nd heptoprotetive effets on rts. This sprgus is rih soure of phytohemils lthough we hypothesized there would e some of them more involved in these funtionl properties. Thus, we imed to study the effets of sprgus (5 mg/kg ody weight (w)/dy) nd their prtilly purified frtions in flvonoids (5 mg/kg w/dy), sponins (5 mg/kg w/dy) nd dietry fier (5 mg/kg w/dy) on oxidtive sttus nd on lipid profile in rts fed holesterol-rih diet. After 5 weeks tretment, plsm lipid vlues, hepti enzyme tivities nd liver mlondildehyde (MDA) onentrtions were mesured. With the exeption of the sponin frtion (), the dministrtion of lyophilized sprgus (), fier frtion (), nd flvonoid frtion () to hyperholesterolemi rts produed signifint hypolipidemi effet ompre to high-holesterol diet (). In ddition, the nd groups exhiited signifint inrese in enzyme tivity from multiple hepti ntioxidnt systems inluding: superoxide dismutse, tlse, nd gluthtione redutse/peroxidse s well s derese in MDA onentrtions ompred to group. These results demonstrte tht triguero sprgus possesses iotive onstituents, espeilly dietry fier nd flvonoids, tht improve the plsm lipid profile

2 Int. J. Mol. Si. 213, nd prevent hepti oxidtive dmge under onditions of hyperholesterolemi. Keywords: triguero sprgus; flvonoids; fiers; sponins; rts; diet-indued hyperholesterolemi; plsm lipids; hepti ntioxidnt enzymes 1. Introdution It is widely epted tht hyperholesterolemi, elevted low-density lipoprotein (LDL-) holesterol onentrtion nd hypertriglyeridemi re mjor risk ftors for the development of theroslerosis nd rdiovsulr disese [1]. Persistent hyperholesterolemi results from n inrese in the prodution nd seretion of LDL tht prolongs its period of irultion, whih inreses oxidtive stress nd leds to the oxidtive modifition of LDL- to ox-ldl- [2,3]. It is desirle to lower totl plsm holesterol through non-phrmeutil strtegies, suh s onsuming foods ontining iotive ompounds with hypoholesterolemi effets. Mny vegetles nd funtionl food supplements hve een used for the primry prevention of rdiovsulr diseses. Indeed, mny studies hve een onduted to identify the tive ingredients responsile for the lipid-lowering properties of plnt foods. Thus, for exmple, dietry fier nd polyphenols from vegetle produts, suh s oo nd penut skins, hve een found to positively ffet serum lipid nd lipoprotein profiles in hyperholesterolemi rts [4,5]. Cultivted triguero sprgus omes from the wild utohthonous sprgus (Asprgus offiinlis L.) from Huétor-Tjr (HT) in Andlusi, Spin. It is importnt to point out tht there re studies tht hve suggested tht triguero HT sprgus ould e hyrid etween ultivted diploid vrieties of Asprgus offiinlis nd the wild speies A. mritimus [6], lthough some other uthors did not find evidene to onfirm this ft [7]. This speies ontins iotive onstituents suh s dietry fier, polyphenols, sponins, sterols, oligoshrides, rotenoids nd mino ids, ll of whih my ontriute to the funtionl properties of this vegetle. Among the ompounds with ntioxidnt tivity, sprgus ontins lrge mount of polyphenols, minly flvonoids [8,9]. The flvonoid rutin onstitutes 6% 8% of the totl phenoli ontent of purple nd green sprgus extrts, nd rutin ould e diretly relted to the ntioxidnt properties of sprgus [1]. Moreover, triguero sprgus is rih in dietry fier, prtiulrly frutns (inulin nd frutooligoshrides) nd ell wll polyshrides ontining feruli id residues, whih lso provide potentil helth enefits [11]. The mjor sponin in green sprgus is protodiosin, ut studies hve shown tht the sponin profile in this sprgus vriety is distint; they re derived from furostn type steroidl genin hving single ond etween rons 5 nd 6 of the B ring, while protodiosin hs doule ond etween C5 nd C6 [12]. In ddition, the totl sponin ontent of this speies is higher thn tht of other ommeril hyrids [12]. Conerning the hypolipidemi effet of this vegetle, other uthors hve previously investigted the holesterol-lowering properties nd heptoprotetive effets of Asprgus remosus nd Asprgus offiinlis yproduts lso in niml models [13,14]. Moreover, in preliminry investigtion, we demonstrted tht orl supplementtion with 5 mg/kg ody weight of triguero sprgus ws le to prevent inreses in plsm lipid onentrtions nd protet ginst the liver oxidtive dmge produed y high-holesterol diet in rts [15]. This sprgus is rih soure of

3 Int. J. Mol. Si. 213, phytohemils lthough we hypothesized there would e some of them more involved in these funtionl properties. Thus, we imed to study the effets of prtilly purified frtions in flvonoids, sponins nd dietry fier from the Asprgus offiinlis L. triguero vriety on plsm lipid ontents nd hepti ntioxidnt enzyme tivities, in n experimentl model of diet-indued hyperholesterolemi in rts. This study is therefore ment to lrify the sis for interest in the onsumption of the speifi sprgus triguero, whih hs unique omposition ompred to other ommeril green sprgus. 2. Results 2.1. Mjor Biotive Constituents in the Asprgus Frtions The mjor iotive onstituents in the sprgus frtions re presented in Tle 1. The three min tive onstituents were dietry fier, flvonoids nd steroidl sponins. Dietry fier ws present t high levels in oth nd frtions, lthough its ontent in ws it higher thn in. The ontent in flvonoids in (1 mg/g dry mtter) ws lmost ten times higher thn in (1.3 mg/g dry mtter). The rihness in flvonoids of the purified frtion () ws not high; lthough it ws ten times greter thn in. The sponin ontent ws pproximted in oth frtions (5 nd 4.4 mg/g dry mtter for nd, respetively) nd the purified frtion () presented elevted onentrtion in steroidl sponins (8 mg/g dry mtter). Tle 1. Mjor iotive onstituents (mg/g dry mtter) in the sprgus frtions. Biotive onstituents Dietry fier 565 ± ± Flvonoids 1 ± ±.1 11 ± Sponins 5 ± ±.4-8 ± 19.8 (Triplites vlues: men ± SD); (lyophilized sprgus), (fier frtion), (flvonoid frtion) nd (sponin frtion) Animl Food Intke nd Weight Gin The rts in the group hd higher verge food intke thn the rest of the groups (p <.5), vrile whih depends on the ompred experimentl group (Tle 2). The dministrtion of sprgus frtions produed redution in ody weight gin ut this ws espeilly signifint for nd t the end of the experimentl period, (28% (p <.5) nd 51% (p <.1), respetively).

4 Int. J. Mol. Si. 213, Tle 2. Weight gin nd verge food intke in experimentl nimls. Groups Initil ody weight Finl ody weight Weight gin (g) Averge food intke (g/dy) ± ± ± ± 1.9 d ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.3 (ontrol stndrd diet), (high-holesterol diet), nd groups fed plus (5 mg/kg), (5 mg/kg), (5 mg/kg) nd (5 mg/kg) for five weeks. Eh vlue represents men ± stndrd devition (n = 6). One-wy ANOVA followed y Bonferroni post test. Vlues with different letters re signifintly different from eh other (p <.5) Plsm Lipid Profile nd Atherogeni Index (AI) Vlues In this experimentl model, the nimls fed the exhiited inresed plsm TG, TC, LDL- nd deresed HDL- (p <.5) ompred with the rts whih were fed the ontrol stndrd diet (), (Figure 1). The rts of the, nd groups, ut not of the group, showed signifintly improved profiles (redued TC, TG nd LDL- onentrtions) in reltion to the group (p <.5), highlighting the nd groups, whih were le to restore the lipid onentrtions to the norml vlues of the group. An inresed HDL- onentrtion ws only oserved with the group, lthough this inrese ws not signifint. However, the lulted therogeni index (AI) vlue for this group ws signifintly different (p <.5) from tht of the group. Figure 1. Plsm lipid onentrtions from rts fed (ontrol stndrd diet), (high-holesterol diet), nd groups fed plus (5 mg lyophilized sprgus/kg), (5 mg fier frtion/kg), (5 mg flvonoid frtion/kg) nd (5 mg sponin frtion/kg) for five weeks. Effets on (1) totl holesterol (TC); (2) high-density lipoprotein holesterol (HDL-); (3) low-density lipoprotein holesterol (LDL-); (4) triglyerides (TG) nd (5) therogeni index (AI) lulted s (TC-HDL)/HDL. Eh vlue represents men ± SD from six rts. One-wy ANOVA followed y Bonferroni post test. Vlues with different letters re signifintly different from eh other (p <.5) TC (mg/dl) HDL- (mg/dl) FS

5 Int. J. Mol. Si. 213, Figure 1. Cont. LDL- (mg/dl) TG (mg/dl) d d 4 2 AI Liver Antioxidnt Evlution CAT, SOD, GPx, GR enzyme tivities in livers from rts fed plus lyophilized sprgus nd sprgus frtions for 5 weeks ompred to the group re represented (Figure 2A). Orl dministrtion of nd ws le to reverse the redued enzyme tivities indued y the high-holesterol diet. CAT tivity ws signifintly elevted in the group, nd SOD, GPx, nd GR tivities were signifintly inresed in the nd groups ompred to the group. The liver MDA onentrtions mesured in the group were signifintly higher thn those in the group (Figure 2B). The, nd groups, ut not the group, showed signifint derese (p <.5) in liver MDA onentrtion ompred to rts fed the, nd this differene ws greter in the nd groups. Figure 2. (A) Hepti ntioxidnt enzyme tivities in livers from rts fed (ontrol stndrd diet), (high-holesterol diet), nd groups fed plus (5 mg lyophilized sprgus/kg), (5 mg fier frtion/kg), (5 mg flvonoid frtion/kg) nd (5 mg sponin frtion/kg) for 5 weeks. Effets on (1) tlse (CAT); (2) superoxide dismutse (SOD); (3) glutthione peroxidse (GPx); (4) glutthione redutse (GR). Eh vlue represents men ± SD from six rts. (B) Mlondildehyde ontents (MDA) in livers from rts fed (ontrol stndrd diet), (high-holesterol diet), nd groups fed plus (5 mg lyophilized sprgus /kg), (5 mg fier frtion/kg), (5 mg flvonoid frtion/kg) nd (5 mg sponin frtion/kg) for five weeks. Eh vlue represents men ± SD from six rts. One-wy ANOVA followed y Bonferroni post test. Vlues with different letters re signifintly different from eh other (p <.5). A CAT mu/mg prot SOD U/mg prot d *** d 2.

6 Int. J. Mol. Si. 213, Figure 2. Cont. A GPx mu/mg prot GR mu/mg prot B nmoles MDA/1mg Disussion Rts fed diet rih in holesterol re often used s n experimentl model for dietry hyperlipidemi; these rts exhiit inresed plsm TG, TC, LDL-, deresed irultion of HDL- nd n overweight stte [16]. In our experiments, redued food intke ws oserved in ll the groups of nimls tht reeived sprgus smples ompred to the. This effet ws orrelted with signifint derese in niml ody weight gin in the nd groups; however, in this sense, no signifint effet ws found in the or groups. The protetive effet produed y the sprgus dministrtion ginst -indued overweight is in ordne with epidemiologil studies inditing tht the onsumption of fier-rih vegetles prevents the development of oesity [17]. After the period of tretment, triguero sprgus nd its mjor iotive frtions, nmely flvonoids nd fiers, showed strong hypotriglyeridemi nd hypoholesterolemi effets, reduing plsm TG, TC nd LDL- ontents. In ddition, inresed HDL- onentrtion ws only oserved with the group, nd lthough this inrese ws not signifint, the group lso showed low vlue of AI (p <.5) suggesting tht might hve n nti-therogeni effet tht would e enefiil for rdiovsulr helth. Severl studies hve demonstrted tht high levels of HDL- re ssoited with lower inidene of rdiovsulr diseses due to the inhiition of LDL oxidtion nd protetion of endothelil ells from the ytotoxi effets of oxidized LDL [18,19]. Previous studies hve demonstrted the hypolipidemi tion of sprgus [13,14,2], though they did not study the iologil tivities of the onstituents seprtely. From our results it n e dedued tht the dietry fier present in nd t 55% nd 61% respetively, might e one of the min onstituents ountle for the lipid-lowering effet of the sprgus. It is well known tht fier is le to derese plsm LDL- onentrtions y interrupting holesterol nd ile id sorption nd inresing LDL reeptor tivity. In ft, dietry fier hs een reported to interfere with holesterol sorption nd enterohepti ile irultion nd to use the depletion of hepti holesterol

7 Int. J. Mol. Si. 213, pools [4,21]. In ddition, our results show tht, s fiers, the flvonoids from sprgus (1% dry mtter) might ontriute to the holesterol-lowering effet. Flvonoids re known to down-regulte serum holesterol ontent, inhiiting holesterol synthesis nd inresing the expression of LDL reeptors [22]. Zhu et l. [14], who ssyed n ethnoli nd n queous extrt from Asprgus offiinlis y-produts, disussed tht the hypolipidemi effet ould e due to the mixture of fier, flvonoids nd sponins. However, we ould not detet modifitions in ny of the prmeters evluted fter the dministrtion of the sponin purified frtion (). It is possile tht the seleted dose of sponin ws too low to e effetive, lthough dose whih ws not too high ws deliertely hosen to void the known toxi effets of these phytohemils [23]. Visvdiy nd Nrsimhhry s investigtions reported tht steroid-type sponins seemed to e minly responsile for the plsm holesterol-lowering effet in rts fed n due to the redution in the sorption of holesterol [13]. However, these uthors ssyed the roots of Asprgus remosus with high steroidl sponin ontent (8.8% dry mtter) nd this is not omprle with our muh smller quntity of sponins in the (.5% dry mtter). The onsumption of n indues dmge to the rt liver due to oxidtive stress used y the exess of holesterol in the diet. This oxidtive stress dmges the ells due to n inrese in free rdils nd onsequent lipid peroxidtion [24]. More reently, it hs een reported tht n N-utnol A. offiinlis extrt, ontining 52% sponin nd 2.3% flvonoids, ws le to derese the liver MDA ontent in mie whih were fed high-ft diet [2]. From our results, we ould dedue tht flvonoids seem to e the min ompounds involved in this effet, sine the (11% in flvonoids) nd the re the most effetive frtions in preventing liver peroxidtion. This finding is resonle euse it is known tht flvonoids re ple of reduing lipid peroxidtion due to their svenging free rdil properties [25]. The preventtive peroxidtion effet oserved with the, lthough not highly signifint, my e due to its ontent in ertin flvonoids, nd to the ft tht the fier of this sprgus vriety ontins other phenolis, suh s feruli id residues in the polyshrides of the ell wll [11]. Hepti enzymes suh s tlse, superoxide dismutse, glutthione redutse nd glutthione peroxidse ontriute to the ntioxidnt defensive mehnisms of the ody. Dereses in the tivities of these ntioxidnt enzymes were oserved in hyperlipidemi rts from the group, ompred to those of the rts in the group. However, the tivities of these enzymes inresed in ll of the groups tht reeived sprgus smples, espeilly in the nimls tht reeived the nd the frtions. Similr findings demonstrted tht flvonoids were ple of stimulting the enzymti tivities of CAT nd SOD [26,27]. On the other hnd, our results gree findings tht demonstrted tht sprgus ws le to inrese the tivities of CAT nd SOD liver enzymes, restoring the ntioxidnt defense system [13,14]. In summry, the dministrtion of Asprgus offiinlis L., triguero vriety, ws shown to improve the plsm lipid profile nd prevent hepti oxidtive dmge under hyperholesterolemi onditions. The mjor iotive onstituents present in this sprgus speies, suh s dietry fier nd ntioxidnt polyphenols, espeilly flvonoids, seem to e highly responsile for this protetive effet. These results suggest tht the onsumption of triguero sprgus, euse of its omposition in iotive omponents, might e enefiil for rdiovsulr helth nd thus would e onsidered s n effetive funtionl food.

8 Int. J. Mol. Si. 213, Experimentl Setion 4.1. Plnt Extrtion nd Anlysis All sprgus smples were supplied y the Food Biotehnology Group of the CSIC (Seville, Spin). Asprgus spers were hrvested from n experimentl field in Huétor-Tájr (Andlusi, Spin). A portion of this mteril ws lyophilized nd ground to fine powder to yield the lyophilized sprgus (). Aording to ptented method [28], nother liquot of the mteril ws extrted with hot wter nd filtered; the residue ws lyophilized nd ground to fine powder to yield 55% of the originl dry mteril of fier purified frtion () with 61% ontent in dietry fier determined y grvimetri nlysis [29]. The wter extrt ws loded into n XAD16 olumn previously equilirted with wter nd the olumn ws wshed with 4% EtOH (4 olumn volumes) to yield 1.4% of the originl dry mteril of prtilly purified flvonoid frtion () with 11% ontent in flvonoids. Flvonoids were determined s previously desried [8]. Finlly, the olumn ws wshed with 9% EtOH (4 olumn volumes) to yield.5% of the originl dry mteril of sponin purified frtion () with 8% ontent in sponins. Steroidl sponins were quntified using spetrophotometri method [3] Animls nd Diets Thirty-six mle Wistr rts, weighing g eh, were purhsed from the Centrl Animl House of Esprtins (Seville, Spin) nd housed in n ir-onditioned room t 25 ± 3 C nd 65% 7% reltive humidity with 12 h light-drk yle. The protool used in this study ws pproved y the Ethis Committee for Animl Experimenttion of the University of Seville (Spin), sed on the reommendtions of the Europen Commission (21/63/EU). The nimls were rndomly ssigned to six groups (n = 6). Group ws fed ontrol stndrd diet (Glol Diet 214 from Hrln Lortories, Hrln Interfun Iéri, S.L., Brelon, Spin), whih ontins nutritionl dditives, inluding Vitmin E (12 IU/kg), Vitmin A (6 IU/kg) nd Vitmin D3 (6 IU/kg). The rest of the nimls were fed high holesterol diet (): the sme ontrol stndrd diet mentioned ove supplemented with 1% holesterol nd.2% holi id, during 2 weeks to evoke hyperholesterolemi. After this time, the experimentl groups reeived the plus the different sprgus frtions dissolved in their drinking wter during 5 weeks: 5 mg/kg ody weight (w) of the whole lyophilized sprgus (), 5 mg/kg w of the fier frtion (), 5 mg/kg w of the flvonoid frtion () nd 5 mg/kg w of the sponin frtion (). The doses were hosen sed on the knowledge of the dietry fier, flvonoid nd sponin ontents present in nd with n im to hieve similr or higher intke of iotive onstituents thn tht provided y the dministrtion of dose of 5 mg/kg w of. In order to monitor their intke of food nd drink, the rts were housed individully in ges nd were given d liitum nd offered different dose frtions (,, nd ) dissolved in 3 ml of wter per dy; the nimls onsumed pproximtely 95% of the prepred queous smples eh dy. The food intke ws reorded dily, nd the ody weight ws mesured efore the strt of the experiment nd every week until the end of the experiment.

9 Int. J. Mol. Si. 213, At the end of the experimentl period, we kept the nimls fsted for 12 h, with ess to pure wter. The rts were nesthetized with hlorl hydrte (12% in sline), injeted intrperitonelly nd their lood ws drwn y rdi punture nd heprinized. To determine the plsm lipid profile, lood smples were entrifuged t 4g (4 min, 4 C) to otin the plsm. The rts were euthnized y injeting hlorl hydrte t 24% in sline. The livers were olleted, weighed nd rinsed with physiologil sline. All smples were stored t 8 C until nlyzed Plsm Lipid Profiles The onentrtions of TC, TG nd HDL- in plsm were determined y enzymti olorimetri methods using ommeril kits (Spinret, Spinret, S.A., Giron, Spin). LDL- ws mesured ording to the proedures desried y Friedewld et l. [31]. The therogeni index (AI) ws defined s (TC-HDL-)/ HDL-) nd ws lulted for eh group Tissue Preprtion nd Liver Antioxidnt Evlution Liver homogentes (1% w/v) were prepred in.25 M surose, 1 mm EDTA, 1 mm DL-dithiothreitol nd 15 mm Tris-HCl (ph 7.4) nd were homogenized with Polytron homogenizer (Qigen) t 2 rpm using glss equipment on ie. Eh homogente ws entrifuged t 8g for 2 min t 4 C. The tissue protein onentrtion ws determined ording to the Brdford method [32]. The superntnt ws used to determine hepti enzyme tivity. Ctlse tivity (CAT) ws mesured using the method desried y Aei [33]. An enzymti retion ws initited y dding n liquot of 2 µl of the homogenized liver nd the sustrte (H 2 O 2 ) to onentrtion of.5 M in medium ontining 1 mm phosphte uffer, ph 7.4. Chnges in sorne were reorded t 24 nm. CAT tivity ws lulted in terms of nmol H 2 O 2 onsumed/min/mg of protein nd expressed in mu/mg protein. Superoxide dismutse (SOD) tivity ws estimted using the xnthine-oxidse-ytohrome method pulished y MCord nd Fridovih [34]. This method is sed on ytohrome oxidtion y superoxide nion nd its dismuttion y SOD. Briefly, 1 µl of the liver homogente were mixed with potssium phosphte uffer (5 mm) (ph 7) with EDTA (.1 mm), ytohrome (1 µm), xnthine (5 µm), tlse (1 U) nd finlly xnthine-oxidse (.4 U) whih triggered the retion. The tivity ws mesured t 55 nm. One unit ws determined s the needed enzyme to inhiit the redution of ytohrome y 5%. The SOD tivity ws expressed s U/mg of protein. Glutthione peroxidse (GPx) tivity ws ssyed with oupled enzyme system in whih GSSG redution ws oupled to NADPH oxidtion y glutthione redutse [35]. The ssy mixture ontined 5 mm potssium phosphte (ph 7), 1 mm EDTA, 1 mm NN3, 1 mm GSH,.2 mm NADPH, 1 U glutthione redutse nd 2 µl of liver homogente. After 5 min pre-inution (2 25 C), the retion ws initited y the ddition of.25 mm H 2 O 2. The derese in the sorne t 34 nm ws followed spetrophotometrilly. GPx tivity ws lulted in terms of nmol H 2 O 2 onsumed/min/mg of protein nd expressed in mu/mg of protein. Glutthione redutse (GR) tivity ws determined spetrophotometrilly y mesuring NADPH oxidtion t 34 nm [36]. The retion mixture ontined potssium phosphte (1 mm) (ph 7.6),.5 mm EDTA, 2 mm KCI,.1 mm NADPH nd 1 µl of liver homogente. After 5 min of

10 Int. J. Mol. Si. 213, pre-inution t 3 C the retion ws initited y the ddition of 1mM GSSG. GR tivity ws lulted in terms of nmol H 2 O 2 onsumed/min/mg of protein nd expressed s mu/mg of protein. Lipid peroxidtion ws estimted ording to the method reported y Esteruer nd Cheesemn [37]. Briefly, 5 µl of liver homogente ws mixed with 1% (w/v) utylhydroxytoluene in eti id, 8% (w/v) sodiumlurylsuphte in distilled H 2 O, 2% (v/v) eti id in distilled H 2 O (ph 3.4), nd.8% (w/v) thiorituri id in distilled H 2 O nd then inuted for 3 min t 9 C then ooled in ie for 5 min. After tht, N-utnol ws dded, vortexed nd entrifugted t 4 rpm for 1 min. The upper orgni phse ws olleted nd the sorne ws red t 532 nm. The mlondildehyde (MDA) onentrtions in eh of the liver smples were determined from stndrd urve generted from 1,1,3,3-tetrethoxypropne nd expressed s nmol of MDA formed per 1 mg of tissue Sttisti Setion The results were expressed s the men ± stndrd devitions (n = 6). The dt were evluted y nlysis of vrine (ANOVA), nd differenes in men vlues mong groups were ssessed using the Bonferroni post-ho tests in GrphPd Prism softwre version 5.1. p <.5 ws onsidered to indite sttistilly signifint differene. 5. Conlusions These results show tht ultivted Asprgus offiinlis L. possesses iotive onstituents, espeilly fier nd flvonoids, tht help to improve the plsm lipid profile nd prevent hepti oxidtive dmge under hyperholesterolemi onditions. Aknowledgments This study hs een supported y funds from the Comisión Interministeril de Cieni y Tenologí (Spnish CICYT) (Projet AGL /ALI). The uthors re grteful to the Food Biotehnology Group of the CSIC, Seville, Spin, for supplying the sprgus smples. Conflits of Interest The uthors delre tht there re no onflits of interest. Referenes 1. Ross, R. Atheroslerosis: An inflmmtory disese. N. Engl. J. Med. 1999, 34, Steinerg, D. Low density lipoprotein oxidtion nd its pthologil signifine. J. Biol. Chem. 1997, 272, Tiwri, A.K. Nturl produt ntioxidnts nd their therpeuti potentil in mitigting peroxidtive modifition of lipoproteins nd theroslerosis: Reent development. J. Med. Arom. Plnt Si. 1999, 21,

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