Original Article Antiatherogenic Potential of Nigella sativa Seeds and Oil in Diet-Induced Hypercholesterolemia in Rabbits

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1 Hindwi Pulishing Corportion Evidence-Bsed Complementry nd lterntive edicine Volume 2011, rticle D , 8 pges doi: /ecm/neq071 Originl rticle ntitherogenic Potentil of Nigell stiv Seeds nd Oil in Diet-nduced Hypercholesterolemi in Rits Ghny l-nqeep, 1, 2, 3 del S. l-zuiri, 4, 5 znh smil, 1, 2 Zulkhiri Hj mom, 1 nd Norhizn ohd Es 1 1 Deprtment of Nutrition nd Dietetics, Fculty of edicine nd Helth Sciences, Universiti Putr lysi, 43400, Serdng, Selngor, lysi 2 Lortory of oleculr Biomedicine, nstitute of Bioscience, Universiti Putr lysi, 43400, Serdng, Selngor, lysi 3 Deprtment of Food Science & Technology, Fculty of griculture, University of Sn, Sn, Yemen 4 Lortory of Cncer Reserch KN-UP, nstitute of Bioscience, Universiti Putr lysi, 43400, Serdng, Selngor, lysi 5 Deprtment of Biochemistry nd oleculr Biology, Fculty of edicine nd Helth Sciences, University of Sn, Sn, Yemen Correspondence should e ddressed to Ghny l-nqeep, eco-sondos@yhoo.com Received 2 pril 2010; ccepted 20 y 2010 Copyright 2011 Ghny l-nqeep et l. This is n open ccess rticle distriuted under the Cretive Commons ttriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. Nigell stiv or Blck seed (N. stiv L.) is trditionlly used for severl ilments in mny iddle Estern countries. t is n nnul herceous plnt tht elongs to the Rnuculce fmily with mny eneficil properties s ntitumor, ntidietic, ntihypertensive, ntioxidtive nd nticteril. This work ttempted to study the effect of N. stiv seeds powder nd oil on therosclerosis in diet-induced hypercholesterolemic (HC) rits in comprison with simvsttin (ST). Twenty-five dult New Zelnd mle white rits, weighing kg, were divided into five groups; norml group (, n = 5) nd four hypercholesterolemic groups (n = 20): positive control () nd three HC groups force fed diet supplemented with 1000 mg Kg 1 ody weight of N. stiv powder (NSP), 500 mg Kg 1 ody N. stiv oil (NSO) nd 10 mg Kg 1 ST for 8 weeks. Feeding HC rits with N. stiv either in powder or oil forms ws shown to significntly reduce (P <.05) totl cholesterol (TC) nd low-density lipoprotein cholesterol (LDLC) levels nd enhnce high-density lipoprotein cholesterol (HDL) levels fter tretment for 2, 4, 6 nd 8 weeks compred to the group. Plque formtion ws significntly inhiited while the intim: medi rtio ws significntly reduced in the NSP nd NSO supplemented groups compred to the group. n conclusion, tretment of HC rits with N. stiv seeds powder or oil showed hypocholesterolemic nd ntitherogenic crdioprotective properties. 1. ntroduction Complementry nd lterntive edicine (C), including herl medicine, is populr in the generl popultion worldwide [1]. Nigell stiv L., commonly known s lck seeds hve een used for nutritionl nd medicinl purposes in mny iddle Estern countries nd other prts of the world [2, 3]. Seeds oil of oth N. stiv nd Neem hs een used trditionlly in si nd the iddle Est to tret mny diseses nd severl virl diseses [4, 5]. Recently, reserchers hve tken interest into the seeds in different forms: the seed itself, the seed extrct, its oil nd its voltile sustnces. Studies on N. stiv seednditsoilhveprovidedscientificsupport for the trditionl use of the seed nd its oil for tretment of rheumtism, immune stimultion, dietes, cncer nd relted inflmmtory diseses [6]. Nigell stiv seeds ctive constituents, for exmple voltile oil nd thymoquinone, showed protection ginst nephrotoxicity nd heptotoxicity induced y either diseses or chemicls [7]. The seed oil hs nti-inflmmtory, nlgesic, ntipyretic, ntimicroil nd ntineoplstic ctivity [7]. Evidence concerning the hypocholesterolemic effect of N. stiv seeds in nimls nd humn is inconclusive. Hert diseses remin one of the leding cuses of deth worldwide [8]. Severl pulictions produced from different lortories included recommendtions for reduction in cholesterol consumption s mens of preventing these diseses [8]. These recommendtions hve gined support s the role of hypercholesterolemi in the incidence of coronry hert diseses hs een estlished. n ddition,

2 2 Evidence-Bsed Complementry nd lterntive edicine most of the studies in this re shifted the ttention to the wys of lowering plsm cholesterol. However, few studies hve een crried out in vivo to investigte the hypocholesterolemic properties of N. stiv seeds nd their oil. Nigell stiv seeds oil nd thymoquinone hve een shown to hve hypocholesterolemic ctivity in rts [6, 9]. There is no ville study, which exmines the potentil of N. stiv in powder form nd s prt of the diet on hypercholesterolemi. Furthermore, to our knowledge, no pulished study hs investigted the ntitherogenic enefits of N. stiv seeds nd oil. Therefore the im of this study ws, to investigte the hypocholesterolemic nd ntitherogenic properties of N. stiv seeds nd the extrcted oil on dietinduced hypercholesterolemic in rits. 2. ethods 2.1. Nigell stiv Seeds Collection nd Extrction. Nigell stiv seeds were purchsed from locl herl grocery from Tiz city in the Repulic of Yemen. The seeds were clened nd kept t 4 C in the Lortory of oleculr Biomedicine, nstitute of Bioscience, University Putr lysi, lysi. The seeds were ground using n electric grinder (Ntionl, model X-915 C Jpn). Homogenized nd ground smples (100 g) were soked overnight with n n-hexne (Fisher Scientific Co Ltd, Ottw, Cnd) t rtio of 1 : 5 (w/v) nd filtered using the Whtmn pper (Fisher Scientific Co Ltd). The solvent ws evported in vcuum rotry evportor (Buchi, Flwil, Switzerlnd) t 40 C. The crude oil smples were comined, weighed, nd stored t 30 C until nlysis Experimentl nimls nd Diet. Twenty-five mle New Zelnd white rits weighing kg were used for this study. They were housed individully in stinless steel mesh-ottomed cges nd were fed initilly stndrd rits chow pellets for 1 week for dpttion. ll rits were mintined t lmost constnt environmentl conditions throughout the study t C nd 12 h 1ight : drk cycle. This study ws crried out ccording to the guidelines pproved y the niml Cre nd Use Committee (CUC) of Fculty of edicine nd Helth Sciences, Universiti Putr lysi Diet ngredients nd Preprtion. The diet used ws otined from i nnsur (lysi), contining: soyen mel (15%), corn (30%), plm kernel mel (36%), soyen oil (2%), strch (10%), molsses (2%), minerl mixture (3.5%), vitmins mixture (0.3%), DL-methionine (0.2%), clcium cronte (CCO 3 ), (0.5%) nd clcium phosphte (CHPO 4 ) (0.5%) (Tle 1). Diet ws mixed nd prepred in the lortory of nutrition in niml Sciences Deprtment, University Putr lysi. The ingredients (soyen, corn nd PKC) were ground using n electric grinder (nesty 3001 UK), weighed nd mixed using n electric mixer (SPR, 107). The minerl nd vitmin mixtures, strch (10%) nd oil nd molsses were mixed with cholesterol (1%) nd dried in n oven t C overnight. Tle 1: Diet ingredient prepred for the experimentl rits. ngredient ixture of diet (%) Soyen 15 Corn 30 Plm kernel mel 36 Strch 10 olsses 2 Corn oil 2 Vitmins mixture 0.3 inerl mixture 3.5 DL-methionine 0.2 CCO CHPO Totl 100 The vitmin premix provided (mg kg 1 feed): thimin 60, rioflvin 22.5, nicinmide 152, clcium pntothente 56, choline chloride 2000, inositol 1000, folic cid 8.5, iotin 1, pyridoxine-hydrochloride 22.5, p- minoenzoic cid 500, Vitmin B , DL-tocopheryl cette 50, mendione 4, retinyl cette nd retinyl plmitte 30 ( U), choleclciferol 30 (3000 U), Vitmin C 400. The minerl premix provided (mg kg 1 feed): sodium citrte dihydrte, FeSO 4 7H 2 O 900, no 2 140, Kl(SO 4 ) 2 12H 2 O 200, ZnSO 4 H 2 O 125, KBr 20, NiSO 4 6H 2 O 8.5, CuSO 4 5H 2 O 100, CoSO 4 7H 2 O 5, N 2 oo 4 2H 2 O 5, K 5, s 2 O 3 0.2, NF 8.5, N 2 B 4 O 7 10H 2 O 5, N 2 SeO 3 5H 2 O Chemicls nd Regents. Cholesterol nd Sudn 1V were purchsed from Sigm-ldrich, St Louis, issouri, US nd Simvsttin ws purchsed from Rnxy, Phrmnig Logisttics Sdn Bhd T, while TC, LDL, HDL nd triglycerides (TG) estimtion kits were supplied y Roche Dignostic GmH, d nnheeim, Germny. Formlin ws otined from BHD Chemicls nd Xylene from jx Chemicls, uurn, ustrli, wheres Hemtoxylin nd Eosin (H&E) were purchsed from erck, msterdm, The Netherlnds. solute lcohol ws ought from R& Chemicls, Essex, UK Experimentl Design. The rits were rndomly ssigned to five groups of five nimls ech; negtive control (), received norml diet prepred nd used s reference nd four groups induced with hypercholesterolemi (n = 20) in which rits were fed norml diet supplemented with 1% cholesterol for 3 weeks. Hypercholesterolemic rits were further sudivided into four groups: positive control () nontreted group, cholesterol diet supplemented with 1000 mg kg 1 N. stvi seeds in powder form (NSP), cholesterol diet supplemented with 500 mg kg 1 N. stiv seeds oil (NSO) nd group force-fed cholesterol diet supplemented with 10 mg kg 1 dy 1 simvsttin (ST) (dissolved in 4 ml distilled wter nd ws given orlly) for 8 weeks. The mount of food nd wter ws recorded dily, while the ody weight ws recorded every 2 weeks Lipid Profiles nlysis. Blood smples from the er mrginl vein of the rits were tken efore nd fter 1.0% cholesterol dministrtion for 3 weeks, nd fter 2, 4, 6 nd 8

3 Evidence-Bsed Complementry nd lterntive edicine 3 weeks of tretment. nlysis of lipid profiles ws crried out using Hitchi nlyzer ortic nner Surfce Lesion Formtion. t the end of the experiment, rits were dissected nd orts were removed, opened longitudinlly nd were prepred for ccurte detection nd estimtion of lipid deposits in the intim following the method reported y Prsd [10]. Photogrphs of the inner surfce of the ort were nlyzed for the plque re using imge-nlysis softwre. The ortic strips were immersed in 10% formlin for 24 h nd rinsed riefly in 70% ethnol. The tissues were then immersed in Herxheimer s solution contining Sudn V (5 g), ethyl lcohol 70% (500 ml) nd cetone (500 ml) t room temperture for 15 min nd were wshed under running wter for 1 h. The orts were plced on plstic templtes nd the luminl surfces were photogrphed using digitl cmer. The totl nd therosclerotic res of the intiml surfce of the ort were mesured in squre millimeters using n imgenlysis softwre. The ortic plques were ssessed lindly, in which the scorer does not know the smples to which group it elong, smples previously coded. The extent of therosclerosis ws expressed s percentge of the luminl surfce covered y therosclerotic chnges [11]. Histologicl nlysis of the nerest prt of ort to the hert ws cut, leled nd fixed in 10% formlin for few dys nd prepred for light microscopy y dehydrting the tissue smples in n scending series of lcohol solution for 14 h in n utomtic tissue processor mchine (TP1020). The tissues were locked, nd cut using microtome (Letiz Wetzlr 1512) to 4-μm sections, psted on slides nd dried on hot plte t Cfor30minndthenkeptt37 C. H&E were used to stin the tissue sections. Plque ccumultion ws nlyzed for the verge determintion of the thickness of the intim, medi nd intim : medi rtio for three rits per group using n imge-nlysis system Sttisticl nlysis. Dt were presented s group mens ± SD nd were nlyzed using SPSS progrm version The differences etween nd hypercholesterolemic groups were tested y independent smple t-test. One-wy NOV followed y Dunnet s Post hoc test ws used to compre the mens of the nd the tretment groups (N. stiv in powder form group, N. stiv oil group nd ST group). 3. Results 3.1. Body Weight. Feeding rits with 1% cholesterolsupplemented diet for 3 weeks resulted in significnt increse (P <.05) in ody weight compred to. Body weight of the nd the groups were shown to e incresed until the end of the experiment. On the other hnd, fter 2 weeks of tretment the ody weight of NSO nd NSP groups were shown to e slightly decresed nd this phenomenon ws continued until the end of experiment. Similr effect on ody weight ws oserved when nimls treted with ST, ut this effect strted fter 4 weeks of tretment. HDL (mmol/l) wk 0 NSP fter cholesterol wk 2 wk 4 wk 6 wk 8 Tretment period (week) NSO ST Figure 1: Chnges in plsm HDL levels in 0 week, fter induction 1% cholesterol for 3 weeks nd fter 2,4, 6 nd 8 weeks of tret. Results re expressed s mens ± SD of five nimls per group. NSP, Nigell stvi seeds in powder form; wk, week. Vlues with the sme superscript letters re not significntly different from ech other t P<.05. Comprison of plsm HDL (mmol L 1 ) vlues t vrious times Hypercholesterolemi nduction in Rits. Feeding rits 1% cholesterol supplemented diet resulted in significnt increse in plsm TC nd LDL levels nd slightly decresed HDL levels (Tles 2 nd 3 nd Figure 1). n ddition, these nimls hd plque formtion in the dominl ort, which led to significnt increse (P <.05) in the thickness of the intim nd the intim : medi rtio in cholesterol group s compred with the group Plsm Lipid Profile Levels fter N. stiv Seeds nd Oil nd ST Tretments in Experimentl Rits. significnt reduction (P <.05) in plsm TC, LDL nd TG levels of NSP-, NSO- nd ST-treted groups were oserved t 2, 4, 6 nd 8 weeks of tretment compred to group (Tles 2 4). Tretment of rits with NSP nd NSO ws shown to hve significnt increse (P >.05) in plsm HDL levels s compred to the group t Weeks 4, 6 nd 8 of tretment. However, throughout the tretment period no significnt differences were noticed in plsm HDL nd TG levels otined from ST nd groups. Plque formtion ws significntly inhiited nd the intim : medi rtio ws significntly reduced (P <.05) in N. stiv tretment groups nd ST group s compred to the group. t should e mentioned tht tretment with ST-induced mortlity in rits (two rits) fter 10 dys of tretment nd incresed liver weight nd its percentge to ody weight ssessment of therosclerotic Plques. Feeding rits 1% cholesterol supplemented diet-induced plque formtion in the dominl ort. The plque re exceeded 10.63± 1.44%, 2.53 ± 1.06%, 2.19 ± 0.88% nd 3.97 ± 0.47% in the, NSP, NSO nd ST groups, respectively (Tle 5). Lipid deposits in the intiml surfce of the ort of ll groups were oserved in which it ws stined

4 4 Evidence-Bsed Complementry nd lterntive edicine Tle 2: Chnges in plsm TC levels (mmol L 1 ) t 0 week, fter feeding of 1.0% cholesterol for 3 weeks nd fter 2, 4, 6 nd 8 weeks of tretment. Group Week 0 HC induction for 3 weeks Tretment period (weeks) ± ± ± ± ± ± ± ± ± ± ± ± 1.3 NSO 0.48 ± ± ± ± ± ± 0.3 NSP 0.84 ± ± ± 3.8 c 4.55 ± ± ± 0.3 ST 0.31± ± ± 1.7,c 6.72 ± ± ± 0.6 Resultsreexpressedsmens±SD of five rits. ll groups received 1% cholesterol dded to the diet except for group. Within column, vlues with the sme superscript letters re not significntly different from ech other t P<.05. Tle 3: Chnges in plsm LDL levels (mmol L 1 ) t 0 week, fter feeding of 1.0% cholesterol for 3 weeks nd fter 2, 4, 6 nd 8 weeks of tretment. Group Week 0 HC induction for 3 weeks Tretment period (weeks) ± ± ± ± ± ± ± ± ± ± ± ±1.6 NSO 0.24 ± ± ± ± ± ±0.7 NSP 0.43 ± ± ± ± ± ± 0.1 ST 0.15 ± ± ± 2.3, 4.95 ± 2.1 c 2.18 ± ± 0.2 Resultsreexpressedsmens± SD of five rits. group ws not given cholesterol. Within column, vlues with the sme superscript letters re not significntly different from ech other t P<.05 Comprison of plsm LDL (mmol L 1 )vluestvriousperiods. rick red when immersed into Sudn V (Figure 2). Plque formtion in the whole re of the ort ws significntly inhiited (P <.05) y N. stiv nd ST tretments s compred to the group. There ws no significnt difference (P >.05) in the whole re exmined etween tretment groups (NSP, NSO nd ST) Quntittive nlysis of Histologicl Dt ntim nd edi Thickness. Thickness of the intim in the group ws significntly higher (P <.05) when compred to group (Tle 6 nd Figure 3), ut no significnt differences were oserved in the thickness of intim of the ort in the tretment groups (NSP, NSO nd ST) compred to. The group hs shown to hve the highest vlue of the intim thickness, ut it ws significntly different only when compred with the NSP nd the NSO groups. On the other hnd, significnt reduction (P <.05) in the intim thickness of the NSP nd the NSO groups in comprison with tht result otined from the STtreted group. The new findings showed tht there were no significnt differences oserved mong ll the treted groups compred to the group ntim : edi Rtio. The intim : medi rtio in the nimls fed cholesterol ws significntly higher (P <.05) compred to the group, (Tle 6 nd Figure 4). The present results showed significnt difference (P <.05) etween the group (71%) nd the tretment groups (26, 33 nd 53% in the NSP, NSO nd ST groups, resp.). However, the intim : medi rtio ws oserved to e significntly NSP NSO ST Figure 2: Representtives photogrphs of the intiml surfces of the orts from the five experimentl groups showing Sudn V-stined lipid deposits. Lipid deposits re stined rick-red. reduced in the NSP nd NSO groups (P <.05) compred to the ST group, while there ws no significnt difference (P <.05) in intim : medi rtio oserved etween the NSO nd the NSP group.

5 Evidence-Bsed Complementry nd lterntive edicine 5 Tle 4:ChngesinplsmTGlevels(mmolL 1 ) t 0 week, fter feeding of 1.0% cholesterol for 3 weeks nd fter 2, 4, 6 nd 8 weeks of tretment. Group Week 0 HC induction for 3 weeks Tretment period (weeks) ± ± ± ± ± ± ± ±± ± ± ± ± 0.1 NSO 0.22 ± ± ± ± ± ± 0.07 NSP 0.34 ± ± ± ± ± ± 0.2 ST 0.32 ± ± ± ± ± ± 0.1 Within column, vlues with the sme superscript letters re not significntly different from ech other t P<.05. Comprison of plsm TG (mmol L 1 ) vlues t vrious times. Tle 5: Percentge of lesion re in the experimentl rits. Group Whole re of the ort (cm 2 ) re of the therom (cm 2 ) %Lesion re 7.53 ± ± ± 1.44 NSP 6.62 ± ± ± 1.06 NSO 7.21 ± ± ±0.88 ST 6.71 ± ± ± 0.47 Effects of N. stiv nd simvsttin tretments on plque formtion in the intrluminl surfce of the dominl ort. Vlues re mens ± SD (n = 5).Within column, vlues with the sme superscript letters re not significntly different from ech other (P <.05). ntim/medi NSP NSO ST Rit groups ntim/medi Figure 3: Theintim : medi rtioofthe different groups compred to the group. NSP, Nigell stvi seeds in powder form. Vlues re mens ± SD (n = 5). Vlues with the sme superscript letters re not significntly different from ech other (P <.05). 4. Discussion Complementry nd lterntive medicine (C) hs gined worldwide populrity over the pst 20 yers [12, 13]. Nigell stiv seeds hve een used for nutritionl nd medicinl purposes in mny iddle Estern countries nd other prts of the world [1, 14, 15]. The seeds re considered nturl food dditive nd condiment. However, they re typiclly consumed mixed with honey, nd in king products or pstries. t is evident tht for most of the c hypocholesterolemic drugs to e effective, they must e used for severl weeks. This my expose the ptients to severl side effects, especilly liver injury [16]. Thus, reserch hs focused on the use of nturl products of plnt origin for the prevention of hert diseses. Our results showed tht ody weight of the group ws significntly incresed until the end of the experiment compred to the, wheres the N. stiv tretment groups (NSO nd NSP) showed slight decrese in the ody weight during the 8 weeks of the tretment time. These results re similr to wht ws found y Zoui et l. [9], who oserved significnt decrese (P <.05) in ody weight of norml rts tht received dily dose of 1 mg kg 1 of N. stiv fixed oilyorlgvgefor12weeks.nigell stiv tretment of rts (2 g kg 1 dy 1 of the originl seed for 1 week) reported to cuse reduction in the ody weight ccompnied y significnt nd sustined reduction in food intke [11]. ST group showed decrese in ody weight strting from Week 4 of the tretment until the end of the experiment, which might e resulted from the decresed dily food intke y 50% during the tretment time. These findings re similr to wht ws reported y Zgoy et l. [17], where they oserved decrese in food intke with weight loss of mice treted with sttin. Significnt elevtion in plsm TC nd LDL levels nd slight decrese in plsm HDL levels were used s indictors of hypercholesterolemi resulted from feeding rits cholesterol supplemented diet. These findings were in the sme line s with those results reported y Prsd [10]. n ddition, the significnt reduction of TC nd LDL levels nd enhncement of HDL levels due to N. stiv tretments (NSO nd NSP), re in greement with the previous studies s reported y El-Dkhkhni et l. [2, 14], who found tht feeding rts with N. stiv oil (800 g kg 1 dy 1 )orllyfor 4 weeks cused significnt decreses in the serum LDL nd

6 6 Evidence-Bsed Complementry nd lterntive edicine Tle 6: ximum thickness of the intim, medi nd intim : medi rtio in experimentl rits. Group NSP NSO ST ntim thickness (μm) ±437.68,c, ± d ± , ± ± c,d edi thickness (μm) ± ± ± ± ± ntim/medi 0.34 ± ± ± ± ± 0.04c Vlues re expressed s mens ± SD n = 5 for ll groups. Within column, vlues with the sme superscript letters re not significntly different from ech other (P <.05). ST () () (c) NSP NSO (d) (e) Figure 4: Representtive photogrphs of the microscopic chnges from the five groups stined with E&H stin. TG levels, nd n elevtion of serum HDL levels. Recently, it ws reported tht the petroleum ether extrct of N. stiv significntly reduced plsm TG nd incresed HDL cholesterol [11]. The voltile oil of N. stiv ws oserved to e s efficient s the cholesterol-reducing drug ST [18]. Furthermore, study in hypercholesterolemic rts showed tht feeding rts with N. stiv oil decresed serum TC, TG nd LDL levels [9]. dditionlly, treting rts with n orl dose of 1 ml kg 1 ody weight of N. stiv seeds fixed oil for 12 weeks showed significnt decrese in totl serum TC nd TG [9]. On the other hnd, our previous results [19] showed tht N. stiv seeds oil is rich in vitmin E nd totl ntioxidnt ctivity, which my explin the significnt reduction in plsm TC, LDL levels. s shown y Jorge et l. [20] vitmin E dministered to hypercholesterolemic rits significntly reduced the plsm LDL nd vessel wll oxidtion fter 2 nd 4 dys of tretment, respectively, which ws ssocited with decrese in vessel nd plsm TC levels nd n improvement in endothelil cell functioning fter 6 dys [20]. t ws lso found tht oil extrcted from N. stiv seeds is rich in unsturted ftty cids, which could e responsile for the decrese of TC nd LDL cholesterol levels s reported y other reserchers [10]. The hypocholesterolimic effect of N. stiv seeds nd their oil could e ttriuted to the seeds contents of totl dietry fier (TDF), insolule dietry fier (DF) nd solule dietry fier (SDF) s oserved y lngee et l. [21]. n ddition, it ws found tht severl dietry fiers significntly decrese plsm cholesterol levels in humn sujects nd therey my reduce the risk of coronry hert diseses [22]. The present study demonstrted tht ST tretment (10 g kg 1 dy 1 ) significntly decresed TC nd LDL levels when compred to the group over the tretment period nd these findings re in greement with those results otined y other reserchers [23, 24]. The results otined from this study showed tht feeding rits with 1.0% of cholesterol supplemented to their diet induced significnt increse in the lesions s compred to norml rits. n ddition, our results showed tht the plque formtion in ll tretment groups ws significntly inhiited s compred to the group. The reduction in therogenesis cused y N. stiv seeds could e ttriuted to

7 Evidence-Bsed Complementry nd lterntive edicine 7 nti-therogenic potentil of NSP nd NSO in rits 1% cholesterol supplemented diet for 8 weeks Norml diet NSP nd NSO Simvsttin Lipid profiles %lesionre intim/medi Lipid profiles %lesionre intim/medi Lipid profiles %lesionre intim/medi Norml lipid profiles intim/medi rtio therosclerotic plque formed therosclerotic plque inhiited therosclerotic plque inhiited NO therosclerotic chnges Figure 5: hypotheticl digrm to present the nti-therogenic potentil of Nigell stiv seeds nd oil. their high content of vitmin E, since incresed consumption of vitmin E is inversely correlted with the development of the coronry hert diseses [16]. Furthermore, vitmin E supplementtion significntly reduced therosclerotic lesions in the scending ort of diet-induced hypercholesterolemic rits [25]. n ddition to vitmin E, the ntitherogenic enefits of N. stiv tretment (NSP nd NSO) my lso e ttriuted to the ctive constituent of N. stiv seeds (Thymoquinone) s reported recently y Rghe et l. [26] s well s to the high content of unsturted ftty cids, where it hs een shown tht incresed consumption of polyunsturted ftty cids improves endothelium dependent relxtion nd protects ginst the development of therosclerotic crdiovsculr diseses [10]. Feeding rits with 1% cholesterol supplemented dietinduced therosclerosis; however, the histologicl exmintion of norml rit reveled tht the ort wll hs uniform thickness with no ulging in the lumen, nd the intim ws intct without ny interruption in contrst to the hypercholesterolemic rits. Dt from histopthologicl exmintion of N. stiv fed rits reveled significnt inhiition of ortic therosclerotic chnges when compred with the ort of the group. n generl N. stiv tretment (NSP nd NSO) showed significnt decrese in intim : medi rtio compred to ST group. t ws reported tht, ST-reduced therosclerotic plque size nd significntly incresed the plque content of vsculr smooth muscle cells nd collgen nd reduced inflmmtion contriuting to therosclerotic plque [27]. ST decresed the intim thickness nd significntly decresed the intim : medi rtio y 42 nd 25%, respectively, compred to the group [27]. The relevnce of these finding s shown in Figure 5 depict the eneficil role of N. Stiv powder nd oil in preventing the development of therosclerosis. n conclusion, this study points out to the importnce of N. stiv seeds nd oil in reducing the rteril wll lipid deposition, totl cholesterol nd LDL levels nd consequently the therogenesis indicting its potentil helth vlue. Funding University Putr lysi (grnt numer 62166). cknowledgments The uthors thnk Universiti Putr lysi for the finncil support for this reserch project nd Phrmnig Logisttics Sdn Bhd for their supply of simvsttin. References [1] B. Sd, H. zizeh, nd O. Sid, Trdition nd perspectives of r herl medicine: review, Evidence-Bsed Complementry nd lterntive edicine, vol. 2, no. 4, pp , [2]. El-Dkhkhny,. Brkt,. d El-Hlim, nd S.. ly, Effects of Nigell stiv oil on gstric secretion nd ethnol induced ulcer in rts, Journl of Ethnophrmcology, vol. 72, no. 1-2, pp , [3] O. Ghosheh,. Houd, nd P. Crooks, High performnce liquid chromtogrphic nlysis of the phrmcologiclly ctive quinones nd relted compounds in the oil of the lck seed (N. stiv L.), Journl of Phrmceuticl nd Biomedicl nlysis, vol. 5, pp , 1990.

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