Effect of ethanol extracts from red pepper seeds on antioxidative defense system and oxidative stress in rats fed high-fat high-cholesterol diet

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1 Nutrition Reserch nd Prctice (Nutr Res Prct) 1;4(1):11-15 DOI: /nrp Effect of ethnol extrcts from red pepper seeds on ntioxidtive defense system nd oxidtive stress in rts fed high-ft high-cholesterol diet Won-Young Song 1, Kyung-Hyung Ku 2 nd Jeong-Hw Choi 1 1 School of Food science, Interntionl University of Kore, Sngmun-ri, Munsn-eup, Jinju-si, Gyeongnm , Kore 2 Kore Food Reserch Institute, Gyeonggi , Kore Astrct The purpose of the present study ws to investigte the effect of ethnol extrcts from red pepper seeds on the ntioxidtive defense system nd oxidtive stress in rts fed high ft high cholesterol diet. Rts were divided into four experimentl groups which were composed of high ft ㆍ high cholesterol diet group (HF), high ft ㆍ high cholesterol diet with.1% ethnol extrcts from red pepper seeds supplemented group (HEA), high ft ㆍ high cholesterol diet with.2% ethnol extrcts from red pepper seeds supplemented group (HEB) nd high ft ㆍ high cholesterol diet with.5% ethnol extrcts from red pepper seeds supplemented group (HEC). Supplementtion of ethnol extrcts from red pepper seeds groups (HEA, HEB nd HEC) resulted in significntly incresed ctivities of heptic glutthione peroxidse nd ctlse. Heptic superoxide rdicl contents in microsome nd mitochondri were significntly reduced in the groups supplemented with red pepper seeds ethnol extrcts. Heptic hydrogen peroxide content in the mitochondri ws reduced in ethnol extrcts from red pepper seeds supplemented groups. TBARS vlues in the liver were reduced in red pepper seeds ethnol extrcts supplemented groups. Especilly, HEB nd HEC groups were significntly decresed compred to the HF group. Heptic cronyl vlues were significntly reduced in mitochondri in these supplemented groups. These results suggest tht red pepper seeds ethnol extrcts my reduce oxidtive dmge, y ctivtion of ntioxidtive defense system in rts fed high ft ㆍ high cholesterol diets. Key Words: Cholesterol, red pepper seed extrcts, free rdicl, oxidtive stress, ntioxidtive defense system Introduction 2) Currently, the westerniztion of dietry lifestyle is followed y chronic dult diseses including hyperlipidemi, rteriosclerosis, dietes nd hypertension. This is likely ecuse of vrious fctors, such s environmentl pollution nd lck of exercise. Oxidtive stress provoked y imlnce etween free rdicls nd ntioxidtive defense system is mjor cuse of vrious oxidtive diseses, such s cncer, inflmmtion, hert disese, rin disese, decresed immunity nd ging [1]. Rective oxygen species (ROS) re generted s free rdicls in the ody such s superoxide nion, hydroxyl rdicl, nd hydrogen peroxide, nd known to induce mny dult disese, s well s to promote the ging process. The humn ody uses the free rdicl scvenger system s physiologicl defense system to protect tissues from peroxidtive dmge y free rdicls nd lipid peroxides. This defense system is divided into non-enzyme defense system (vitmin E, ß-crotene, etc.) nd n enzyme defense system (superoxide dismutse (SOD), glutthione peroxidse (GSHpx), ctlse, etc.) [2]. It is sid tht in norml physiologicl condition these defense systems protect the ody from oxidtive dmges, however, in pthologicl condition like dietes, the free rdicl scvenger system cn ecome wek wheres the free rdicl production system is promoted resulting in n imlnce etween the systems [3]. Therefore, ttention to the intke of ntioxidnts in order to repress oxidtive stress nd to prevent vrious diseses nd ging hs incresed [4]. Antioxidtive sustnces re widely distriuted in nimls nd plnts. Antioxidnts such s phenol chemicl compounds, flvone derivtives, tocopherol, scoric cid nd selenium in fruits nd vegetles ply key role in the inhiition of ging y delying or preventing the oxidtion of fts, cncer nd crdiovsculr diseses [5]. Being food contining such ioctive mterils, hot pepper, n indispensle spice of Koren diet, cn e referred to representtively. Hot pepper contins phenolic compounds such s scoric cid, cpsnthin, cpsoruin, cryptocpsin, qurcetin, luteolin nd cpsicinoid [6], mny of which hve een reported s eing ntioxidnts [7]. Pepper seeds oil hs out 68~72% linoleic cid, 13~15% plmitic cid, nd 9~11% oleic cid [8]. However, reserches on the ntioxidtive effect of pepper seeds hve een rrely conducted, lthough some studies hve investigted the ntimuttion nd nti-cncer effect of the red pepper seeds [9]. Especilly red pepper seed hs ntimutgenic effects ginst the fltoxin B1 nd MNNG, nd This study ws supported y Technology Development Progrm for Agriculture nd Forestry, Ministry for Agriculture, Forestry nd Fisheries, Repulic of Kore (7). Corresponding Author: Jeong-Hw Choi, Tel , Fx , Emil. jhppychoi@hnmil.net Received: Novemer 23, 9, Revised: Ferury 4, 1, Accepted: Ferury 8, 1 c1 The Koren Nutrition Society nd the Koren Society of Community Nutrition This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution Non-Commercil License ( which permits unrestricted non-commercil use, distriution, nd reproduction in ny medium, provided the originl work is properly cited.

2 12 Antioxidtive effect of red pepper seed ethnol extrct the nticrcinogenic effects in humn cncer cells hve een reported to e high [1]. Therefore, this study ws crried out to investigted the effect of ethnol extrcts from red pepper seed on ntioxidtive system nd oxidtive dmge in the high ft high cholesterol rt. Mterils nd Methods Preprtion of ethnol extrcts from red pepper seed The red pepper seed ws purchsed from the Nmndong Agriculture coopertive. Air-dried red pepper seed ws crushed using grinder (Desung power mixer/grinder DA-28G, Seoul, Kore) nd shttered pepper seeds were used in smples y cold storge. Grinded pepper seeds nd six times 1% ethnol were mixed nd then extrction ws performed t 4 for 24 hours. This process ws repeted three times nd the superntnt ws collected nd filtered with Whtmn No.1 filter pper. The solvent of comined extrcts ws evported under reduced pressure using rotry vcuum evportor (HS-1N, Hnshin Science Co., Kore) nd dried in freeze drier (Bondiro, Ilshin, Kore). Experimentl nimls nd diet Mle Sprgue-Dwley rts weighing 1 ± 1 g were purchsed from KRITC (Dejeon, Kore). Rts were individully housed in stinless steel cges in room with controlled temperture (~23 ) nd lighting (lternting 12 h periods of light nd drk). Rts were divided into four high ft ㆍ high cholesterol groups which were composed of HF group (red pepper seeds Tle 1. Compositions of diets in experiment group (g/kg diet) Groups 1) Ingredients Corn strch Csein Sucrose Corn oil Minerl mixture 2) Vitmin mixture 3) Cellulose DL-methionine Choline Chloride Lrd Cholesterol Red pepper seeds Totl 1, 1, 1, 1, 1) HF: high ftㆍhigh cholesterol diet HEA: high ftㆍhigh cholesterol diet +.1% red pepper seeds ethnol extrct HEB: high ftㆍhigh cholesterol diet +.2% red pepper seeds ethnol extrct HEC: high ftㆍhigh cholesterol diet +.5% red pepper seeds ethnol extrct 2) AIN-76 minerl mixture (g/kg mixture) 3) AIN-76 vitmin mixture (g/kg mixture) ethnol extrct free), HEA group (.1 g red pepper seeds ethnol extrct/1 g diet), HEB group (.2 g red pepper seeds ethnol extrct/1 g diet), nd HEC group (.5 g red pepper seeds ethnol extrct/1 g diet) (Tle 1). The rts fed the experimentl diets for period of 4 weeks. The experimentl design ws pproved y the committee of Kore Interntionl University regrding the cre nd use of lortory nimls. Smple collection nd preprtion Sprgue-Dwley rt liver ws removed nd wshed with ice-cold 9 g/l NCl. A rt liver homogente ws prepred ccording to the descried method of Slter nd Swyer [11]. The liver ws homogenized in Hepes uffer (mmol/l N-2-hydroxyethypiperzine-N -2-ethnesulfonic cid, 1 mmol/l KCl nd 28 mmol/l sucrose, ph 7.4), centrifuged t 3, rpm for 15 min, recentrifuged t 9, rpm for min to remove ny mitochondril contmintion, nd then centrifuged t 41,5 rpm for 6 min to yield the microsoml frction. The superntnt ws further centrifuged t 15, g for 62 min, t 4. Protein in the liver tissues ws determined using method of Lowry et l. [12]. Activities of ntioxidtion enzymes The SOD ctivity ws spectrophotometriclly mesured using modified version of the method developed y Mrklund nd Mrklund [13]. The GSHpx ctivity ws mesured y the method of Lwrence nd Burk [14]. The CAT ctivity ws mesured using Aei s method [15]. Contents of Superoxide rdicl ( 2 - ) nd Hydrogen peroxide (H 2) Superoxide rdicl content ws determined y the method of Azzi et l. [16]. The liver mitochondril hydrogen peroxide level ws mesured y the method of Gy nd Geicki [17]. Mesurement of cronyl vlue nd thiorituric cid rective sustnces (TBARS) The heptic microsome nd mitochondri were determined ccording to the method of Levin et l. [18]. The lipid peroxide in the liver tissues ws determined y mesuring the thiorituric cid rective sustnces (TBARS) ccording to the method of Stoh [19]. Sttisticl nlysis Results were nlyzed y ANOVA nd Tukey's honestly significnt difference test []. Sttisticl significnce is defined s P <.5.

3 Won-Young Song et l. 13 Results Activities of superoxide dismutse (SOD), glutthione peroxidse (GSHpx) nd ctlse (CAT) Tle 2 shows the ctivity of SOD, n ntioxidnt enzyme which reduces superoxide rdicls to H 2, which in turn is excreted s H, sed on the ctivity of GSHpx nd CAT, therey protecting the ody from oxygen toxicity. The result of SOD ctivity in the liver ws greter in the HEA, HEB nd HEC groups thn in the HF group. However, these differences were not significnt mong the experimentl groups. The GSHpx ctivity in the HEA, HEB nd HEC groups were significntly greter thn tht of the HF group. The CAT ctivity in the HEA, HEB nd HEC groups were 14%, 138% nd 152% greter, respectively, thn tht in the HF group. The HEB nd HEC groups were significntly incresed compred to the HF group. Contents of superoxide rdicl ( 2 * - ) nd hydrogen peroxide (H 2) The heptic superoxide rdicl content is shown in Fig. 1. The liver microsoml superoxide rdicl contents in the HEA, HEB nd HEC groups were significntly lower thn tht of the HF group. As well, the heptic mitochondril superoxide rdicl contents in the HEA, HEB nd HEC groups were 35%, 42% Tle 2. Effects of ethnol extrcts from red pepper seeds on heptic superoxide dismutse (SOD), glutthione peroxidse (GSH-px) nd ctlse (CAT) ctivities in rts fed high ft ㆍ high cholesterol diets SOD (unit/mg GSHpx (nmol NADPH/mg CAT (nmol/mg HF ±.147 NS ± ± HEA 1.35 ± ± ±.633 HEB ± ± ± HEC ± ± ± Vlues re the mens ± SE (n=1). Those with different superscript letters re significntly different t P <.5 y Tukey's test. The experimentl groups re the sme s in Tle 1. NS : not significnt Superoxide rdicl (nmoles/mg protein/min microsome mitoc hondri Fig. 1. Effects of ethnol extrcts from red pepper seeds on heptic superoxide rdicl contents in rts fed high ft ㆍ high cholesterol diets. Vlues re the mens ± SE (n=1). Those with different superscript letters re significntly different t P <.5 y Tukey's test. The experimentl groups re the sme s in Tle 1. nd 53% significntly lower, respectively, thn tht in the HF group. The result of heptic mitochondri frction of hydrogen peroxide content is shown in Fig. 2. The content of hydrogen peroxide in the liver mitochondri of the HEC group ws significntly lower thn tht of the HF group. Mesurement of thiorituric cid rective sustnces (TBARS) nd cronyl vlue The result of TBARS contents s n index oxidtive dmge is shown in Fig. 3. These TBARS concentrtions, n index of lipid peroxidtion, were 83%, 74% nd 63% lower in HEA, HEB nd HEC groups, respectively, compred with the HF groups. The HEB nd HEC groups were significntly decresed compred to the HF group. The liver microsoml cronyl vlues (Fig. 4) in the HEA, HEB nd HEC groups were lower thn tht in the HF group, ut these differences were not significnt mong the experimentl groups. The liver mitochondril cronyl vlues in the HEA, HEB nd HEC groups were 34%, 35% nd 37% lower, respectively, thn tht in the HF group. However, there were no significnt differences mong the red pepper seed extrcts supplemented groups. Hydrogen peroxide (nmoles/mg protein/min Fig. 2. Effects of ethnol extrcts from red pepper seeds on mitochondril hydrogen peroxide (H 2O 2) contents in rts fed high ft ㆍ high cholesterol diets. Vlues re the mens ± SE (n=1). Those with different superscript letters re significntly different t P <.5 y Tukey's test. The experimentl groups re the sme s in Tle 1. TBARS (MDA nmole/mg protein) Fig. 3. Effects of ethnol extrcts from red pepper seeds on heptic thiorituric cid rective sustnces (TBARS) vlues in rts fed high ft ㆍ high cholesterol diets. Vlues re the mens±se (n=1). Those with different superscript letters re significntly different t P <.5 y Tukey's test. The experimentl groups re the sme s in Tle 1. c

4 14 Antioxidtive effect of red pepper seed ethnol extrct Cronyl vlue (nmole/mg protein NS microsome mitochondri Fig. 4. Effects of ethnol extrcts from red pepper seeds on heptic cronyl vlue in rts fed high ft ㆍ high cholesterol diets. Vlues re the mens ± SE (n=1). Those with different superscript letters re significntly different t P <.5 y Tukey's test. The experimentl groups re the sme s in Tle 1. NS : not significnt Discussion The purpose of the present study ws to investigte the effect of ethnol extrcts from red pepper seeds on ntioxidtive system nd oxidtive stress of liver in rts fed high ft high cholesterol diet. Oxidtive stress mens the production of highly rective oxygen rdicls tht re toxic to the cells, prticulrly to the cell memrne in which these rdicls interct with the lipid ilyer nd produce lipid peroxides. Endogenous ntioxidnt enzymes (e.g, SOD, GSHpx nd CAT) re responsile for the detoxifiction of deleterious oxygen rdicls. Superoxide dismutse is concerned with the elimintion of 2 -(superoxide) tht develops from nturl sustnces nd is retined in the ody. SOD ctivity is necessry to restrin this development s free rdicl oxidtion is n ostcle nd enemy to the ody [21,22]. The SOD ctivity in the liver ws greter in the HEA, HEB nd HEC groups thn in the HF group. During the reduction of hydrogen peroxide, GSH is converted to n oxidized form, glutthione disulfide (GSSG), nd GSSG is reduced ck to GSH y NADPH-dependnt glutthione reductse [23,24]. Roch et l. [25] showed tht heptic GSH tended to e higher in high ft-fed rts thn in stndrd diet-fed rts. From the current result of oserving the ctivities of the ntioxidtive defense system in the liver, the GSHpx ctivity in the HEA, HEB nd HEC groups ws significntly greter thn tht in the HF group. The CAT ctivity in the HEA, HEB nd HEC groups ws 14%, 138% nd 152% greter, respectively, thn tht in the HF group. These results were considered to e cused y polyphenols within the pepper seeds. The phenolic hydroxyl of the phenolic compounds distriuted widely in plnts ws ound well to mcromolecules such s zymoproteins, nd these compounds hve een recognized s sustnces showing such ioctivity [26]. In study of Ku et l. [27], the totl polyphenol content of the ethnol extrcts of the pepper seeds ws 8. ±.57~33.99 ±.8mg/g. The content ws higher thn those of seeds nd nuts, for instnce, reported y Lee nd Lee [28] in their study exmining the totl polyphenol contents of vegettive foods, showing 27 mg/g of sesme seeds,.83 mg/g of perill seeds,.12 mg/g of pricot stones, 2.6 mg/g of wlnuts,.13 mg/g of pumpkin seeds nd 2.2 mg/g of sunflower seeds. Antioxidtive sustnces otined from nturl mterils re minly phenolic compounds nd flvonoids. Cffeic cid nd chlorogenic cid hve een reported to hve strong ntioxidnt cpcity [29]. ROS ws known to dmge cells y producing lipid peroxides nd y cting on polyunsturted ftty cids of cellulr memrnes [3]. ROS concentrtion ws significntly decresed in the group supplied with ethnol extrcts of pepper seeds compred to the HF group, which is thought to e due to ntioxidtive ctivity y the supply of the pepper seeds cusing inctivted or eliminted free rdicls [31]. This result ws in line with the finding of Sim nd Hn who reported tht greter content of red pepper seeds leds to higher electron donting ility using DPPH [32]. Ki nd Hn reported tht superoxide rdicl is n effective free rdicl scvenger nd such effect is lso contined in the red pepper seed [21]. Lipid peroxidtion cused y free rdicls following oxidtive stress in cell hs een known to produce metolic normlities in n orgnism nd to provoke cncer, muttion, loss of genes nd ging, y dmging DNA [33]. The TBARS concentrtions, n index of lipid peroxidtion, were lower in HEA, HEB nd HEC groups, compred with the HF group. Especilly, HEB nd HEC groups were significntly decresed compred to the HF group. Severl studies hve lso shown elevted lipid peroxidtion products in high ft diet [34,35]. High intke of dietry ft directly enhnced ROS overproduction which incresed lipid peroxidtion [36]. This decrese in the lipid peroxides in the liver tissue resulted from red pepper seed extrct ctivting the function of removing free rdicls, indicting tht the ntioxidtive defense system ws ctivted to reduce the formtion of peroxides. The cronyl vlue concentrtions, n index of protein peroxidtion, were 34%, 35% nd 37% lower in HEA, HEB nd HEC groups, respectively, compred with the HF group. This result ws considered to e the ction of ntioxidtive sustnces such s polyphenols contined y ethnol extrcts of the seed, which suppressed oxidtion of proteins y inhiiting lipid peroxidtion nd finlly repressing the oxidtion of tissues. In conclusion, n ethnol extrct of red pepper seed strengthened the ntioxidtive defense system with incresed ctivity of SOD, GSHpx nd CAT in the liver of high ft high cholesterol diet fed rts. Accordingly, ethnol extrct of red pepper seed ws found to reduce the ccumultion of ROS, such s superoxide rdicl nd H 2, which decrese the genertion of oxidtively dmging sustnces such s TBARS nd the cronyl vlue. Reference 1. Hlliwell B, Gutteridge JMC. Free rdicls, geing, nd disese. In: Free rdicls in iology nd medicine. London. Englnd:

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