EFFECT OF DIETARY TOCOTRIENOLS ON INFECTION AND INFLAMMATION INDUCED LIPOPROTEIN OXIDATION IN HAMSTERS

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1 Interntionl Journl of Phrmcy nd Phrmceuticl Sciences ISSN Vol 3, Issue 3, 211 Reserch Article EFFECT OF DIETARY TOCOTRIENOLS ON INFECTION AND INFLAMMATION INDUCED LIPOPROTEIN OXIDATION IN HAMSTERS M. SALMAN KHAN 1,2 *, AMIR KHAN 1,3, JOHAR IQBAL 1 1*Deprtment of Biochemistry, J N Medicl College, A M University, Aligrh, 222, Indi, 2 Deprtment of Biotechnology, Integrl University, Lucknow 22626, Indi, 3 Deprtment of Biochemicl Sciences, Dolphin (PG) Institute of Biomedicl And Nturl Sciences, Dehrdun 1, Indi. Emil: contctskhn@gmil.com Received: 25 April 211, Revised nd Accepted: 27 My 211 ABSTRACT The tocotrienol isomers (α, β, γ nd δ ) re nturlly occurring nlogues of the Vitmin E fmily found minly in cerel grins nd plm oil, nd re widely used s ntioxidnt nd hypolipidemic gents. We hve initilly investigted the role of mixture of dietry α, β, γ nd δ tocotrienols (Tocomin) in inhiiting the infection/inflmmtion induced lipoprotein oxidtion in hmsters. Syrin hmsters were injected with cteril lipopolyscchride (, 2 μg), zymosn (2 mg), or turpentine (.5 ml) to mimic cute infection, cute systemic inflmmtion, nd cute loclized inflmmtion, respectively. Tocomin (1. mg) ws dministered dily for 1 dys efore nd 12 h fter or 24 h fter turpentine or zymosn injection. Our results demonstrted tht tocotrienols pretretment significntly decresed the levels of plsm lipid peroxidtion products, wheres plsm totl ntioxidnt level ws significntly incresed. In ddition, pretretment with tocotrienols significntly reversed/restored the low density lipoprotein (LDL) nd erythrocytes lipid peroxidtion, including n increse in lg phse of copper induced LDL oxidtion. Moreover, tocotrienols medited significnt meliortion in LDL lysophosphtidylcholine (LPC) content s well s in rylesterse ctivity, indicting potent ntioxidnt property of tocotrienols. In conclusion, our results indicte tht the llevition of inflmmtory conditions nd oxidtive stress s well s inhiition of LDL oxidtion re due to potent free rdicl scvenging properties of dietry tocotrienols nd, thus, cn e used s dietry supplements in the prevention nd tretment of systemic inflmmtory process which might induce therosclerosis. Keywords: LDL Oxidtion, Infection nd Inflmmtion, Tocotrienols INTRODUCTION Recent studies hve suggested link etween therosclerosis nd infection nd inflmmtion 1. Atherosclerosis is multifceted disese process with severl different well defined risks fctors, such s hypercholesterolemi, smoking, hypertension nd dietes. Although some studies hve suggested tht specific infectious gents ply direct role in the vessel wll in the formtion of therosclerotic lesions 2. Both infection nd inflmmtion re ccompnied y systemic host response known s the cute phse response (APR). Acute phse response represents complex rection of the host tht is ccompnied y ltertions in lipid nd lipoprotein metolism tht could e mechnism for enhnced susceptiility to therogenesis. 3. In niml models lipopolyschhride () tretment rpidly increses serum triglyceride (TG), totl cholesterol (TC) nd LDL cholesterol levels, while exhiited decrese in ntitherogenic high density lipoprotein (HDL) cholesterol levels 1. The increse in serum nd lipoprotein lipids in hmsters treted with is pprently due to increse in heptic 3 hydroxy 3 methyl glutryl coenzyme A (HMG CoA) reductse ctivity 4. A specilized feture of infection/inflmmtion is the production of plenty of dmging oxygen free rdicls in the system, which leds to condition of oxidtive stress tht enhnces the peroxidtion of lipids 5. Oxidtive modifiction of lipoproteins plys centrl role in the pthogenesis of therosclerosis 6. Although lipid oxidtion in the vessel wll is thought to occur s result of locl deficiency of endogenous ntioxidnts or n excess of free metl ions, only limited dt support these hypotheses 7 8. Reserch hs shown tht humn therosclerotic plques contin mssive mounts of lipid peroxidtion products, despite the presence of lrge quntities of α tocopherol nd scorte 9. In ddition, vitmin E, despite its effect on reducing oxidtive stress mrkers nd lowering plsm TG levels, ws not effective in inhiiting fom cell formtion in the ortic rch of hyperlipidemicdietic hmster model 1. Results of ex vivo experiment indicte tht supplementtion of lipid solule ntioxidnts to humns 11 nd nimls 6 increses the resistnce of LDL to oxidtion. An increse in serum conjugted diene nd mlondildehyde (MDA) in, zymosn or turpentine treted hmsters ws previously reported 12. In rts, tretment significntly decreses plsm totl ntioxidnts cpcity nd increses MDA level, which were reduced y susequent lipoic cid tretment 13. An increse in erythrocytes MDA concentrtion ws previously reported in treted rts, which were significntly reduced following tretment with vitmin A, C, E or EGB Furthermore, circulting LDL is protected from oxidtive stress y HDL ssocited enzymes, prticulrly proxnse (PON), which destroys iologiclly ctive phospholipids 16. The serum PON ctivity is decresed in rits fter dministrtion of croton oil nd PON depleted HDL is unle to protect LDL from oxidtion in vitro 17. Becuse rective oxygen is generted s prt of host defense nd proxonse protects LDL from oxidtive stress, it my e postulted tht APR my increse LDL oxidtion in vivo 12. Asln et l 18 hve reported significnt decrese in PON nd rylesterse ctivities in H. pylori infected sujects, which seems to e relted to decrese in HDL C, nd in prt, to incresed oxidtive stress nd inflmmtory condition induced y H. pylori infection. Incresed use of nturl products like fruits nd vegetles my protect ginst free rdicl medited LDL oxidtion nd lipid peroxidtion y providing dietry sources of ntioxidnts 19, such s vitmin E. Vitmin E consists of four tocotrienol isomers (α, β, γ, δ ) nd four tocopherol isomers (vitmin E) which re found minly in cerel grins, rice rn nd plm oil nd ll of them re memrne solule ntioxidnts. Pulished reports indicte tht oth in vitro nd in intct memrnes including LDL prticles tocotrienols exert significntly greter protection ginst coronry rtery disese (CAD) Dietry tocotrienols hve een reported to exhiit strong ntioxidnt property in hyperlipidemic nimls nd humns 24. Severl investigtors reported tht tocotrienols hve greter ntioxidnt ctivity thn tocopherols, nd protect more efficiently ginst some free rdicl relted diseses thn does tocopherols However, none of the studies hve reported the inhiitory effect of tocotrienols on infection nd inflmmtion induced LDL oxidtion nd oxidtive stress in hmsters. Therefore, in the present study, we hve exmined the role of Tocomin (tocotrienols) in ttenuting the oxidtive stress in three distinct models of infection nd inflmmtion, which re produced y the dministrtion of, zymosn, or turpentine, tht re well chrcterized inducers of APR.

2 Khn et l. Int J Phrm Phrm Sci, Vol 3, Issue 3, 211, MATERIALS AND METHODS Chemicls Twenty five percent plmvite oil suspension of Tocomin (tocotrienols) contining 6.4 % d α tocotrienol, 1 % d β tocotrienol, 1.2 % d γ tocotrienol, 3.2 % d δ tocotrienol nd 5.7 % d αtocopherol s well s RBD plm olein were supplied s gift from CAROTECH BHD, Chemor, Mlysi. Lipopolyscchrides (E. coli, 55:B5) nd zymosn A were purchsed from Sigm Aldrich (USA). Oil of turpentine ws purchsed from Vsco Drug Lortories (Indi). All other chemicls nd regents used in this study were of nlyticl grde. Animl procedures Mle golden hmsters, weighing out g were purchsed from Centrl Drug Reserch Institute, Lucknow, Indi, were cclimtized for 7 dys. The protocol of the study ws pproved y the niml ethicl committee of the J. N. Medicl College, A M University, Indi. The hmsters were given stndrd pellet rodent chow (Ashirwd Industries, Indi) nd wter d liitum. For the induction of cute systemic infection, cute loclized sterile inflmmtion or cute noninfectious systemic inflmmtion, hmsters were injected with, turpentine oil or zymosn, respectively. Lipopolyscchride, 2 μg in.5 ml sline nd zymosn, 2 mg in.5 ml wrm (5 C) PBS, ph 7., were injected intrperitonelly, wheres, turpentine oil,.5 ml, ws injected sucutneously. Hmsters in group were injected intrperitonelly with.5 ml sline only. Susequently, food ws withdrwn from oth nd treted hmsters 12. Tocomin (1 g) ws prepred in ethnol nd plmvite oil (1:2). In norml group, five hmsters were given 1. ml plmvite oil/hmster/dy, through gstric intution, 1 dys efore nd 24 h fter sline injection. Five hmsters in ech, turpentine or zymosn group were dministered 1. ml plmvite oil/hmster/dy, 1 dys efore nd 12 h fter injection or 24 h fter turpentine or zymosn injection. Five hmsters in ech, turpentine or zymosn Tocomin treted group were given totl 1 mg (5 mg ech in morning nd evening) Tocomin/dy, 1 dys efore nd 12 h fter injection or 24 h fter turpentine or zymosn injection. The dose of Tocomin ws djusted ccording to previous pulished reports 24. At the end of experiment, hmsters in ech group were nesthetized nd lood ws collected y crdic puncture in heprinized tues for plsm seprtion. Mesurement of plsm totl ntioxidnt power (FRAP) nd rylesterse ctivity The method of Benzie nd Strin 33 ws used for mesuring the ferric reducing ility of plsm, the FRAP ssy, which estimte the totl ntioxidnt power. Plsm rylesterse ctivity ws determined y the method of Ayu et l. 34 y using phenyl cette s the sustrte. Isoltion of lipoprotein nd mesurement of lipoprotein oxidtion The precipittion method descried y Wielnd nd Seidel 35 ws used for the isoltion of plsm LDL. The method of Ptsch et l. 36 ws used for the isoltion of HDL. Lipid peroxidtion products in serum were mesured y severl methods. Conjugted diene ws mesured ccording to the method of Corongiu et l. 37. Quntifiction of lipid hydroperoxide ws done ccording to the method Nourooz Zdeh et l. 38. Lipid peroxide contents, mesured s thiorituric cid rective sustnces (TBARS), were ssyed y the method of Ygi 39. Lysophosphtidyl choline (LPC) content in LDL ws determined y Quinn et l. 4. The LPC nd on silic gel pltes ws identified y comigrtion with stndrd, scrped, nd ssyed for phosphorus content s descried y Brtlett 41. Mesurement of ex vivo nd in vitro Cu ++ medited susceptiility of LDL to oxidtion The ex vivo nd in vitro Cu ++ medited susceptiility of isolted LDL to oxidtion ws ssessed y determining the lg phse of conjugted diene formtion using the method of Esteruer et l. 42. Prior to oxidtion studies LDL smple ws dilyzed ginst 5 mm phosphte uffer sline (PBS), ph 7.4, for 12 h. The incution mixture contined 5 μg of LDL in 1. ml of 1 mm PBS, ph 7.. The MDA content in LDL ws ssyed y the method of Niehus nd Smuelsson 43. Mesurement of erythrocytes lipid peroxidtion products nd md relese from intct erythrocytes The determintion of MDA in erythrocytes ws crried out ccording to the method of Stocks nd Dormndy 44. The procedure of Cynmon et l. 45 ws employed for the determintion of MDA relese from intct erythrocytes. The MDA vlues in the smples were clculted y using molr extinction coefficient of M 1 cm 1. Protein estimtion The protein ws determined y the method of Brdford 46 using ovine serum lumin s stndrd. Sttisticl evlution Results were expressed s men ± SD. Sttisticl nlysis of dt ws done y employing two tiled Student t test s descried y Bennet nd Frnklin 47. RESULTS Impct of tocotrienols on plsm totl ntioxidnts nd lipid peroxidtion products We determined the effect of tocotrienol pretretment on plsm totl ntioxidnts nd lipid peroxidtion products. As seen in Fig.1, fter 12 h of or 24 h of turpentine or zymosn injection, plsm totl ntioxidnts ws significntly reduced from vlue of 75 to 3 (61 %), 5 (33 %) or 45 (4 %) μmole/dl, which ws significntly incresed to 61 (16 %), 7 (4 %) or 65 (44 %) μmole/dl in, turpentine or zymosn Tocotrienols pretreted hmsters, respectively. As evident from the dt presented in Fig. 2, Plsm conjugted diene, lipid hydroperoxide nd MDA were significntly incresed y 7 19 %, %, nd %, respectively, in, turpentine or zymosn stressed hmsters, with mximum effect on stressed hmsters, which were significntly reduced y %, % nd %, respectively fter tocotrienols pretretment. These results illustrted tht not only ut other APR inducers, turpentine or zymosn, lso incresed the plsm lipid peroxidtion products. Thus, the dt depicted tht tocotrienols pretretment in stressed hmsters significntly restored the plsm totl ntioxidnts level nd locked the increse in plsm conjugted diene, lipid hydroperoxide nd MDA to level close to norml vlue. Norml, fed.5 ml plmvite oil/hmster/dy, 1 dys efore nd 24 h fter sline injection; N T, fed 1 mg Tocotrienols/hmster/dy, 1 dys efore nd 24 h fter sline injection; L C, T C, Z C,, turpentine or zymosn stressed ; Tocomin treted, fed 1 mg tocotrienols/hmster /dy, 1 dys efore nd 12 h fter injection; or zymosn Tocomin treted, fed 1 mg tocotrienols/hmster/dy, 1 dys efore nd 24 h fter turpentine or zymosn injection. Since, infection nd inflmmtory diseses re chrcterized y excess genertion of oxygen free rdicls, which exhiit erythrocytes memrne lipid peroxidtive dmge in vivo, we hve exmined the cuse of this memrne dmge y quntifying n end product of ftty cid peroxidtion, nmely, MDA. As shown in Fig.3, MDA content of erythrocytes hemolyste ws significntly incresed in, turpentine or zymosn group. Pretretment of, turpentine or zymosn stressed hmsters with tocotrienols significntly prevents the increse in MDA content nd decresed it, in comprison to corresponding, turpentine or zymosn vlue. Furthermore, erythrocytes from, turpentine or zymosn stressed group showed greter susceptiility to hydrogen peroxide induced lipid peroxidtion thn those from norml group. A significnt increse of 19 %, 54 % or 73 % in the relese of MDA in, turpentine or zymosn ws oserved, when compred to norml vlue. Tocotrienols pretretment to these stressed hmsters significntly locked the increse in MDA relese 278

3 [ Khn et l. Int J Phrm Phrm Sci, Vol 3, Issue 3, 211, nd decresed it y 31 %, 2 % or 2 %, respectively. These results demonstrte tht the induction of infection nd inflmmtion in hmsters is ssocited with significnt increse in erythrocytes memrne lipid peroxidtion, which is significntly prevented y the dministrtion of tocotrienols. *µmole/dl Norml treted treted Zymosn Zymosn treted Fig. 1: Antioxidnt impct of Tocomin pretretment on plsm totl ntioxidnts level in three groups of stressed hmsters *Vlues re men ±SD from pooled plsm of 5 hmsters in ech group. Ech vlue re significntly different from norml t p<.1. Ech vlue re significntly different from their respective s t p<.1. Conjugted diene Lipid hydroperoxide MDA *µmole/dl Norml treted treted Zymosn Zymosn treted Fig. 2: Antilipoperoxidtive effect of Tocomin pretretment on plsm conjugted diene, lipid hydroperoxide nd MDA level in three groups of stressed hmsters. *Vlues re men ±SD from pooled plsm of 5 hmsters in ech group. Ech vlue re significntly different from norml t p<.1. Ech vlue re significntly different from their respective s t p< *MDA (nmole/g H) **In vitro MDA relese (Percent) Norml treted treted Zymosn Zymosn treted Fig. 3: Impct of Tocomin pretretment on erythrocyte MDA nd in vitro MDA relese in three groups of stressed hmsters *Vlues re men ± SD from pooled hemolyzte prepred from erythrocytes of 5 hmsters in ech group. ** Vlues re men ± SD from pooled intct erythrocytes of 5 hmsters in ech group. Ech vlue re significntly different from norml t p<.1. Ech vlue re significntly different from their respective s t p<

4 Khn et l. Int J Phrm Phrm Sci, Vol 3, Issue 3, 211, Ex vivo nd Cu ++ medited in vitro oxidtion of LDL in, turpentine or zymosn stressed hmsters pretreted with tocotrienols As seen in Tle 1, the ex vivo se line diene conjugtion (BDC) levels of LDL in, turpentine nd zymosn stressed hmsters were incresed y 1.8, 1.4 nd 1.6 fold, respectively, in comprison to the norml vlue. Pretretment of these stressed hmsters with tocotrienols prtilly locked the in vivo oxidtion of LDL nd reduced their BDC levels y 1.2, 1.2 nd 1.3 fold, respectively, when compred to their respective s. Mximl in vitro Cu ++ medited oxidtion of LDL ws chieved fter 12 h of incution. The mximl conjugted diene vlue of LDL in norml group ws sustntilly incresed y 4.1 fold, when compred to corresponding sl vlues in norml. Compred to corresponding mximl vlues in norml group, LDL ssocited mximl conjugted diene formtion of, turpentine or zymosn hmsters were incresed y 2, 1.4 nd 1.8 fold, respectively. Due to tocotrienols pretretment, the decrese in mximl conjugted diene vlues of LDL in ll the three stressed groups were 1.5, 1.4 nd 1.6 fold, respectively, in comprison to corresponding vlues in, turpentine or zymosn groups. As expected, the lg phse time of LDL oxidtion ws reduced from 9 min in norml group to 3, 6 nd 5 min in, turpentine nd zymosn stressed hmsters. Pretretment of, turpentine or zymosn groups with tocotrienols restored the lg phse time of LDL oxidtion nd incresed them to 6, 72 nd 7 min, respectively. The Tle 1 lso depicted tht the ex vivo se line level of MDA in LDL ws significntly incresed y 2.7, 1.8 nd 2.3 fold, in, turpentine nd zymosn stressed hmsters, respectively, s compred to norml group. Pretretment with tocotrienols locked the in vivo increse in MDA of LDL in ll the three distinct groups of stressed hmsters nd reduced their levels y 1.7, 1.5 nd 1.7 fold, respectively, when compred to corresponding sl vlues in, turpentine nd zymosn stressed hmsters. The mximl MDA content of LDL ws significntly incresed y 11 fold s compred to sl vlue in norml group. In ddition, the mximl MDA vlue in, turpentine nd zymosn group were lso significntly incresed y 2.3, 1.4 nd 2. fold, respectively, when compred to their respective s. Pretretment of, turpentine nd zymosn stressed hmsters with tocotrienols prevented the increse in mximl MDA content of LDL nd reduced them y 1.6, 1.3 nd 1.8 fold, respectively, s compred to their corresponding mximl MDA vlues. Tle 1: Ex vivo nd Cu ++ medited in vitro susceptiility to LDL oxidtion in three groups of stressed hmsters pretreted with Tocomin Group Conjugted diene MDA Bsl Mximl Lg Phse Bsl Mximl Norml * 91.9 (4.1) 9 **4.43± ±.186 (11) 4.79 * (1.8) (2) 3 **11.91±.21 (2.7) 11.8±.22 (2.3) treted * (1.2) # (1.5) 6 **6.91±.23 (1.7) # 67.5±.365 (1.6) 3.63 * (1.4) (1.4) 6 **7.74±.186 (1.8) 69.41±.122 (1.4) treted * (1.2) # (1.4) 72 **5.15±.294 (1.5) # 55.48±.326 (1.3) Zymosn * (1.6) (1.8) 5 **9.95±.29 (2.3) 99.18±.156 (2.) Zymosn treted * (1.3) # 1.2 (1.6) 7 ** 5.91±.292 (1.7) # 54.62±.165 (1.8) The conjugted diene nd MDA vlues re expressed s nmole MDA equivlents/mg protein. *Vlues re otined from LDL isolted from pooled plsm of 5 hmsters in ech group. ** Vlues re men ± SD from LDL isolted from pooled plsm of 5 hmsters in ech group. The lg phse is defined s the intervl etween the intercept of the tngent of the slope of the curve with the time expressed in minutes. Fold increse with respect to sl vlue in norml ; # Fold decrese with respect to sl vlue from their respective s; Fold increse with respect to mximl vlue in norml ; Fold decrese with respect to mximl vlue from their respective s. Significntly different from norml t p<.1. Significntly different from t p<.1. Significntly different from turpentine t p<.1. Significntly different from zymosn t p<.1. The ove results demonstrte mrkedly enhnced ex vivo nd Cu ++ induced in vitro susceptiility of LDL to oxidtive modifiction, which my contriute to the incresed risk ssocited with therogenesis. In ddition, tocotrienols pretretment to stressed hmsters, exhiited potent ntioxidnt effect y preferentilly locking the ex vivo nd in vitro formtion of conjugted diene nd incresed the lg phse time of therogenic LDL. Moreover, these results lso signify tht the host response to infection nd inflmmtion produces LDL tht not only contins more oxidized lipids, ut is lso more susceptile to further oxidtion. Impct on LDL LPC content Lysophosphtidylcholine (LPC) is known to exert severl protherogenic effects nd is mrker for oxidtive modifiction of LDL. Therefore, we hve investigted the possile inhiitory effect of tocotrienols on LDL LPC content in, turpentine or zymosn stressed hmsters. As shown in Fig.4, lysophosphtidylcholine content in LDL ws sustntilly incresed from 5.4 in norml to 41 (712 %), 2 (295 %) nd 31 (51 %) mg/dl in, turpentine nd zymosn stressed group. Pretretment of these stressed hmsters with tocotrienols decresed the LDL LPC content y 38 %, 24 % nd 34 %, when compred to their respective s. These results indicte tht induction of infection nd inflmmtion in hmsters ws ssocited with mrked increse in LDL LPC content, which my e due to increse in plsm PAF AH ctivity. These levels were significntly prevented y the dministrtion of tocotrienols. 28

5 Khn et l. Int J Phrm Phrm Sci, Vol 3, Issue 3, 211, *mg/dl Norml treted treted Zymosn Zymosn treted Fig. 4: Impct of Tocomin pretretment on LDL lysophosphtidylcholine content in three groups of stressed hmsters. *Vlues re men (mg/dl) ± SD from pooled plsm of 5 hmsters in ech group. Significntly different from N C t p<.1. Significntly different from L C t p<.1. Significntly different from T C t p<.1. Significntly different from Z C t p<.1. Regultory effect of tocotrienols pretretment on plm/hdlrylesterse ctivity It is well known tht enzyme rylesterse, lso known s proxonse, is ssocited with plsm HDL. The ntioxidnt ctivity of HDL is elieved to reside in its enzymes, prticulrly rylesterse. Severl reports indicted tht HDL ssocited rylesterse enzyme protects ginst LDL oxidtive modifiction. Therefore, we hve lso investigted the ntioxidtive impct of tocotrienols on enzymtic ctivity of rylesterse in isolted HDL frction of, turpentine or zymosn stressed hmsters. As shown in Fig.5, rylesterse ctivity of norml hmsters ws reduced from vlue of 75 units in plsm to 517 units (27 %) in HDL frction, isolted from plsm of norml hmsters y precipittion method. This decrese in HDL ssocited rylesterse ctivity my e pprently due to decrese in percent recovery (73 %) of HDL prticles, during their frctiontion from plsm. Arylesterse ctivity in plsm nd HDL of stressed hmsters ws significntly reduced from 75 nd 517 units in norml to 468 (34 %) nd 32 units (38 %) in stressed hmsters. Tretment of stressed hmsters with tocotrienols for 1 dys efore nd 12 h fter injection significntly locked the decrese in plsm nd HDL rylesterse ctivity nd incresed them y 3 % nd 31 %, respectively, when compred to corresponding vlues Plsm rylesterse ctivity HDL rylesterse ctivity 2 1 Norml treted treted Zymosn Zymosn treted Fig. 5: Effect of Tocomin pretretment on plsm rylesterse ctivity in three groups of stressed hmsters *Vlues re men ± SD from pooled plsm/hdl of 5 hmsters in ech group. Significntly different from N C t p<.1. Significntly different from L C t p<.1. Significntly different from T C t p<.1. Significntly different from Z C t p<.1. After 24 h of turpentine nd zymosn injection to hmsters, significnt decrese of % nd % in plsm nd HDL rylesterse ctivity ws oserved, when compred to corresponding norml vlues. Pretretment of tocotrienols to turpentine nd zymosn stressed hmsters elevted the rylesterse ctivity in plsm nd HDL y % nd %, respectively. These results indicte tht due to n increse in oxidtive stress, rylesterse ctivity in plsm nd HDL frction ws significntly reduced. Pretretment of stressed hmsters with tocotrienols significntly prevented this decrese in rylesterse ctivity nd restored to level 81 to 97 % of norml vlues. DISCUSSION Plsm lipoprotein oxidtion hs een extensively studied nd it hs een shown tht oxidtive modifiction of plsm lipoproteins plys crucil role in the pthogenesis of therosclerosis 8. Although lipid oxidtion in the vessel wll is thought to occur s result of locl deficiency of endogenous ntioxidnts or n excess of free metl ions, only limited dt support these hypotheses. Becuse plsm contins severl ntioxidnts 11 nd lipoproteins with oxidtive dmge hve een isolted from therosclerotic lesions 8, it is uncler whether oxidized lipoproteins originte in the rteril wll or re produced in the circultion nd then enter the intiml spce. Our dt show tht systemic oxidtion of lipid/lipoprotein prticles 281

6 Khn et l. Int J Phrm Phrm Sci, Vol 3, Issue 3, 211, occurs s prt of the host response to infection nd inflmmtion. Plsm conjugted diene (which mesure the initil phse of lipid peroxidtion), lipid hydroperoxide (intermedite product of lipid peroxidtion) nd MDA (which mesure the degrdtion phse of lipid peroxidtion) ws significntly incresed in hmsters fter, turpentine or zymosn dministrtion. A similr increse in serum conjugted diene nd MDA hs een reported in 3 models of stressed hmsters. However, protective effect of ntioxidnt vitmins ws not investigted 12. The increse in plsm lipid peroxidtion products is ssocited with significnt decline in totl ntioxidnts cpcity of plsm. The former suggests incresed production of oxidnts while the ltter indictes diminished ntioxidnt defense. Both the chnges indicte n existence of profound oxidtive stress. These results re in concordnt with well known prooxidnt properties of, turpentine or zymosn. However, simultneous dministrtion of nd 5 mg Tocomin (tocotrienols) to hmsters, 12 h prior to killing, depicted tht the increse in plsm totl ntioxidnts ws significntly lower (19 %) with no decrese in the levels of conjugted diene nd lipid hydroperoxide except slight decline in MDA, indicting tht pretretment of hmsters with dietry Tocomin, 1 dys prior to dministrtion is essentil (dt not shown). Therefore,, turpentine or zymosn induced oxidtive stress ws not only ttenuted ut significntly reversed/restored in hmsters pretreted with Tocomin. Similr to plsm, MDA content of erythrocytes in, turpentine or zymosn stressed hmsters were significntly incresed. The intct erythrocytes isolted from 3 groups of stressed hmsters exhiited further increse (>4 fold) in susceptiility to hydrogen peroxide induced MDA relese, s compred to the respective sl MDA content. Pretretment of stressed hmsters with Tocomin significntly locked the in vivo s well s in vitro susceptiility of erythrocytes to lipid peroxidtion nd reversed the MDA levels close to norml vlues, indicting potent ntioxidnt property of tocotrienols. Bsed on our results, its seems plusile tht oxygen rdicls formed over nd ove the detoxifying cpcity of erythrocytes cn cuse peroxidtive rekdown of phospholipid ftty cids nd ccumultion of MDA, which my enhnce memrne dmge. Our study suggests tht due to constnt vilility of ntioxidnts, tocotrienols, in the ody of treted hmsters, the integrity of erythrocytes memrne is significntly improved s shown y sustntil protection ginst in vivo nd in vitro H2O2 induced lipid peroxidtion. An increse in erythrocytes MDA concentrtion ws previously reported in treted rts, which were significntly reduced following tretment with vitmin A, C, E or EGB Rective oxygen species nd free rdicls re prt of locl host defense mechnisms, ecuse they ply role in killing invding microorgnisms nd re induced y the sme stimuli tht induce APR. Due to the presence of persistence inflmmtion nd oxidtive stress, lipid peroxidtion ws incresed with concomitnt decline in plsm totl ntioxidnts, which is pprently responsile for cell memrne destruction nd cell dmge. Oxidized LDL cn induce fom cell formtion, nd oxidtive modifiction of LDL is now recognized s n importnt process tht occurs in vivo 8. In contrst to ntive LDL, oxidized LDL prticles initite series of events, including vsculr inflmmtion nd mcrophge fom cell formtion tht re centrl to the therogenic process 26. LDL prticles with greter tendency to ecome oxidized might thus e more likely to prticipte in protherogenic events. There is only one pulished report 12 demonstrting significnt increse in conjugted diene nd lipid hydroperoxide levels in LDL frction from or zymosn treted hmsters. The dt from our study clerly indictes tht during cute infection nd inflmmtion, due to exggerted oxidtive stress nd depletion of plsm ntioxidnts, the ex vivo nd in vitro BDC, lipid hydroperoxide nd MDA level of LDL ws mrkedly incresed in ll the three distinct group of stressed hmsters. Tocomin supplementtion prtilly locked the in vivo nd in vitro LDL oxidizility s seen y decrese in ex vivo nd in vitro level of lipid peroxidtion products nd n increse in their lg phse vlues. Our results lso showed tht LDL LPC content, which is known to exert severl protherogenic effect nd is mrker for oxidtive modifiction of LDL 8, ws sustntilly incresed (4 to 8 fold) in circulting LDL fter, turpentine or zymosn tretment, which indictes tht lipoprotein phospholipids re oxidized in vivo during APR. A similr increse in LDL ssocited LPC content in treted hmsters ws previously reported y Memon et l. 12. Feeding of dietry Tocomin significntly locked the increse in LPC levels of LDL nd reduced them y 1.3 to 1.6 fold, indicting potent hypolipidemic nd ntioxidnt property of Tocomin. Together, these results indicte tht LDL isolted from, turpentine or zymosn treted hmsters ws more susceptile to in vitro Cu ++ ctlyzed oxidtion, which suggests tht cute phse LDL my lone e more susceptile to further oxidtion in the vessel wll. Severl mechnisms could contriute to incresed LDL oxidtion during APR. Proxonse (PON)/rylesterse re HDL ssocited enzyme tht protects LDL nd HDL from oxidtive stress This enzyme destroys oxidized phospholipids tht re iologiclly ctive nd therey prevents the toxic protherogenic effects induced y oxidized LDL nd preserves HDL modifiction y lipid peroxides 28. Vn Lenten et l. 17 hve previously reported tht PON ctivity in HDL decresed in rits following injection of croton oil nd in humns postopertively. Our results demonstrte tht plsm nd HDL rylesterse ctivity significntly decreses (22 % to 38 %) following, turpentine or zymosn dministrtion. This reduction in rylesterse ctivity is consistent with erlier reports in treted hmsters 29 or in H. pylori infected sujects 18. However, in those studies protective effect of ny drug with ntioxidnt property ws not investigted. In our study, pretretment with Tocomin significntly prevented the decrese in rylesterse ctivity nd reversed these levels close to norml vlues. It hs previously een shown tht depletion of PON results in the loss of the ntioxidnt function of HDL, nd ddition of PON to HDL restores the protective function of HDL 3. In ddition, Avirm et l. 31 lso reported tht purified PON is potent inhiitor of in vitro LDL nd HDL oxidtion. These studies strongly suggest n importnt role for PON in preventing the oxidtion of oth LDL nd HDL, nd hence in the protection of therosclerosis. Our results indicting mrkedly enhnced in vivo s well s in vitro oxidizility of therogenic LDL in conjunction with decresed plsm nd HDL rylesterse ctivity during APR is likely to e potentil mechnism for the incresed oxidtion of circulting LDL. A decrese in rylesterse ctivity during cute phse response could therefore e nother fctor linking the APR with incresed therogenesis. Our results indicting strong ntioxidnt impcts of Tocomin in, turpentine or zymosn stressed hmsters re in greement with erlier findings indicting n inhiition in the formtion of conjugted dienes nd MDA y tocotrienol rich frction (TRF) or individul tocotrienols nd tocopherols when fed to rts long with n therogenic diet Support to our results is lso otined from other studies which suggested tht in intct memrnes, including LDL prticles, tocotrienols my hve significntly greter ntioxidnt effect thn tocopherols nd they my provide greter protection ginst CAD Therefore, it ppers tht tocotrienols s TRF or tocotrienols exert their ntioxidnt effect on plsm LDL oxidtion while eing ttched to LDL prticle. Our comined results strongly suggest tht the llevition of oxidtive stress s well s inhiition of LDL oxidtion re due to potent free rdicl scvenging properties of dietry Tocomin (tocotrienols) nd, thus, cn e used s dietry supplements in the prevention nd tretment of systemic inflmmtory process which might induce therosclerosis. ACKNOWLEDGEMENT The uthors like to cknowledge University Grnt Commission (UGC), New Delhi (Indi), for finncil support. This study ws crried out t the Deprtment of Biochemistry, J N Medicl College, A M University, Aligrh, Indi. The uthors like to thnk Dr. Z. H. Beg, for giving guidnce from time to time nd Dr. Asif Ali, Chirmn, for providing fcilities to crry out reserch work. The uthor lso likes to thnk W. H. Leong, vice president CAROTECH, Inc., for kindly providing Tocomin, s gift. 282

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