Sakia Ferdousy 1, Md Atiar Rahman 1*, Md Mamun Al-Amin 2, Jannatul Aklima 1 and J. M. Kamirul Hasan Chowdhury 1

Transcription

1 Ferousy et l. Clinil Phytosiene (216) 2:17 DOI /s ORIGINAL CONTRIBUTION Antioxitive n neuroprotetive effets of Lee mrophyll methnol root extrts on izepm-inue memory impirment in mnesi Wistr lino rt Open Aess Ski Ferousy 1, M Atir Rhmn 1*, M Mmun Al-Amin 2, Jnntul Aklim 1 n J. M. Kmirul Hsn Chowhury 1 Astrt Bkgroun: A neurologil isorer is eoming one of the mjor helth prolems worlwie. Syntheti rugs for neurologil isorers often proue unwnte effets while tritionl plnt- erive rugs offer unique strtegy in omting the neurologil iseses. This stuy investigte how the ntioxitive properties of Lee mrophyll root extrts integrtively help s neuroprotetive n ntimnesi in niml moel. Methos: Lee mrophyll root methnol extrt (LM) ws ssesse y open fiel test, hole ross test, elevte plus mze test n thiopentl soium inue sleeping time test for sreening neurophrmologil effet on Wistr lino rts using the LM t 1 & 2 mg/kg oy weight. Antimnesi effet of LM ws stuie on izepm inue nterogre mnesi in Wistr lino rts. Morris wter mze test ws performe to evlute whether the lerning n memory of rts re impire y mnesi proue y izepm. The ntioxitive enzymes superoxie ismutse, tlse, glutthione peroxise n oxitive iomrkers mlonilehye, nitri oxie, vne oxition protein prout from the hippompus of epitte nimls were mesure to ssess the ntioxitive potentil of the extrt. Results: LM ws foun to show signifint (p <.5) reution in loomotor tivity n inrese in the urtion of sleeping of nimls. Morris wter mze test showe tht nimls trete with LM signifintly (p <.5) erese the men espe lteny ompre to nimls in izepm group. LM ws lso foun to reue the ontent of mlonilehye, nitri oxie n vne oxition protein prout n to inrese the tivities of superoxie ismutse, tlse n glutthione peroxise in hippompus. Conlusion: Our stuies suggest tht ntioxitive pity of LM in hippompus my ugment the ntimnesi effets in nterogre mnesi. Keywors: Lee mrophyll, Dizepm, Amnesi, Antioxint, Htikn Arevitions: LM, Lee mrophyll root methnol extrt; BW, Boy weight; CNS, Centrl Nervous system; NC, Norml ontrol; PC, Positive ontrol; EPM, Elevte plus mize; GABA, γ mino utyri i; ROS, Retive oxygen speies; SOD, Superoxie ismutse * Corresponene: tir@u.. 1 Deprtment of Biohemistry & Moleulr Biology, University of Chittgong, Chittgong 4331, Bnglesh Full list of uthor informtion is ville t the en of the rtile 216 The Author(s). Open Aess This rtile is istriute uner the terms of the Cretive Commons Attriution 4. Interntionl Liense ( whih permits unrestrite use, istriution, n reproution in ny meium, provie you give pproprite reit to the originl uthor(s) n the soure, provie link to the Cretive Commons liense, n inite if hnges were me.

2 Ferousy et l. Clinil Phytosiene (216) 2:17 Pge 2 of 11 Bkgroun Humn neurologil isorers hve pose n ever inresing thret to puli helth re. Amnesi is severe isruption of memory without efiits in intelligene, ttention, pereption or jugment. Anterogre mnesi les to prtil or omplete inility to rell the reent pst eventully result from mge to the hypothlmus n thlmus n the surrouning ortil strutures. Reent stuies suggeste tht oxitive stress is linke to severl neurologil isorers s well s mnesi [1]. Therefore, investigtion of therpies is n urgent whih meliortes the neurologil isorers n mnesi y relieving oxitive stress. Current therpeuti options, suh s interventionl proeures, surgery, n syntheti rug, re limite in their ility to improve neurl funtion euse they fil to repir mge neurons or improve neurl regenertion [2]. They sometimes use setion n myorelxtion [3]. Due to suh filure of therpies in neuronl mge repir n severe verse effets of ntimnesi rugs, lterntive tretments s herl or plnt-erive rugs re inresingly eing use to llevite the ffetive isorers [4]. Similr therpeuti effets with lesser sie effets re onsiere s e vntge of these rugs. Espeilly the nturl ntioxitive flvonois possessing nxiolyti effet is the prime hoie s neuroprotetive n ntimnesi [5]. Lee mrophyll (Htikn) is hereous shru primitively ientifie s Inin hitt lso known s Hstikrnpls, Hthikn or Htkn (Elephnts er) elonging to Leeee fmily. It is roun 9 m in height with swithy rnhes n perennil tuerous roots. It is istriute to the reltively hotter prts of Ini, from the Estwrs of Gnges, Bihr, Bengl, Assm, the Teri n its ontiguous plins n lso in the western Ini from Konkn south-wrs [6]. In Bnglesh, the plnt is foun in Chittgong, Rjshhi, Ntore n other northern prts of the ountry [7]. The plnt hs een oserve useful in ner [8], rthritis, goiter n tetnus [9], urolithisis [1], hepti mge [11] n miroil infetion [12]. This reserh investigte whether ntioxitive effets of LM ontriute s neuroprotetive s well s ntimnesi in izepminue niml moel. Methos Colletion n ientifition of plnt mterils The plnt mteril, Lee mrophyll roots were ollete from ultivte re of Bnglesh Counil of Sientifi n Inustril Reserh (BCSIR), Rjshhi in Mrh, 214. The plnt smple ws ientifie n txonomilly uthentite s Lee mrophyll y Dr. Sheikh Bokhter Uin, Professor, Deprtment of Botny, University of Chittgong. A smple speimen hs een preserve s the ession numer ACCU-211/7 for future referene. Preprtion of plnt extrt The roots of Lee mrophyll were wshe n hoppe into smll piees, oven-rie t 45 C. Drie mterils were groun into power y mehnilly prepre Willy mill. Resulting power (6 g) ws then eftte with n-hexne extrtion. Deftte mterils were issolve in 99 % istille methnol n left for h t room temperture (23 ±.5) C with osionl stirring. The superntnt for repetitive extrtion ws filtrte through heeseloth n Whtmn filter pper no 1 n the filtrtes were ollete to get rie uner reue pressure (temperture elow 5 C) using rottory vuum evportor (RE2, Biy sterling Lt, UK). The onentrte extrts were ollete in Petri ish n llowe to ir ry for omplete evportion. Blk-green semisoli rue (39 g, yiel 6.5 %, W/W) ws preserve t 4 C for further use. Experimentl nimls n mintenne Six to seven weeks ol ult Wistr lino rts verge weight ± 5.87 g of either sex were selete for neuroprotetive tivities. The rts were proure from the niml house of Bnglesh Counil of Sientifi n Inustril Reserh (BCSIR), Chittgong-422, Bnglesh. They were helthy n limtize for week to stnr lortory onitions efore the strting the experiment. Throughout the experimentl perio, the nimls were house iniviully in polyronte ges ( m 3 ) t room temperture (23 ± 2) C, reltive humiity (6 7 %) n were expose to 12:12 h light: rk yle. They were supplie with stnr pellet iet n wter liitum. All niml experimenttions were mintine n rrie out with the guielines of Animl Ethis Review Bor of Fulty of Biologil Sienes, University of Chittgong (pprovl no. AERB/ FBS/UC/5, 215). Drugs n hemils Tween-8 ws purhse from Shrl S. L., Spin. Dizepm ws kinly onte y Opsonin Phrmeutils Lt., Bnglesh. Thiopentl soium (Genisi, Squre phrmeutils Lt.) ws purhse from lol mrket. All other hemils use in this reserh were of nlytil regent gre until unless speifie with itionl referene. Aute toxiity test n osge justment Alimtize Wistr lino rts mintine in the mentione lortory onition were use for ute toxiity stuy. Five nimls reeive single orl ose of.5, 1., 1.5, n 2. g/kg Boy Weight (BW) of LM. Animls

3 Ferousy et l. Clinil Phytosiene (216) 2:17 Pge 3 of 11 were kept over-night fsting prior to ministrtion the LM. After ministrtion, foo ws withhel for further 3 to 4 h. Iniviul niml ws kept in lose oservtion uring the first 3 min fter osing, perioilly first 24 h (speil ttention for the first 4 h), therefter for perio of 3 ys to reor the elye toxiity. One ily ge sie oservtion inluing hnges in skin n fur, eyes n muous memrne, respirtory n irultory rte, utonomi n entrl nervous system (CNS) hnges were oserve. The effetive therpeuti ose ws tken s one tenth of the mein lethl ose (LD 5 >2. g/kg) [13]. This is n estlishe n wiely use moel supporte y numer of reserhers [14, 15]. Experimentl proeure for neurophrmologil tests To investigte neurophrmologil tivity open fiel test, hole ross test, elevte plus mze (EPM) test n thiopentl soium-inue sleeping time tests were performe. For eh of these tests, the nimls were ivie into four groups nmely norml ontrol (NC), positive ontrol (PC) n tretment ontrol Lee mrophyll root methnol extrt 1 mg/kg BW (LM1) n tretment ontrol Lee mrophyll root methnol extrt (LM2) group. Eh group ontins six to eight rts. NC group reeive orl ministrtion of vehile (1 % Tween 8 in wter). PC group reeive Dizepm (referene stnr) t the ose of 1 mg/kg BW, intrperitonelly. The ose of izepm for the ssessment of neurologil ehvior is selete from litertures [16, 17]. LM1 n LM2 group reeive LM orlly t 1 n 2 mg/kg BW respetively. Neurophrmologil n ehviorl tests re grphilly presente in Fig. 1. Open fiel test The experiment ws performe oring to the metho esrie y Gupt et l. [18]. The open fiel pprtus onsiste of wooen floor of hlf squre meter with series of squres eh lterntively olore lk n white. The pprtus hve wll of 4 m height. Animls were trete with NC, PC, LM1 & LM2 n were ple in the mile of the open fiel. The numers of squres visite y the nimls were ounte. It ws ounte for 3 min t, 3, 6, 9 n 12 min following the tretments. The neurologil pprtuses were thoroughly lene with 7 % isopropnol fter eh tril. Hole-ross test The metho ws rrie out s esrie y Tkgi et l. [19]. For this experiment, ge ws use hving size of m with fixe prtition in the mile. A hole of 3 m imeter ws me t height of 7.5 m in the enter of the ge. After tretment with NC, PC, LM1 n LM2 the nimls were llowe to ross the hole from one hmer to nother n the numers of pssge of rt through the hole from Fig. 1 Grphil presenttion of the neurophrmologil n ehviorl tests performe in this reserh

4 Ferousy et l. Clinil Phytosiene (216) 2:17 Pge 4 of 11 one hmer to other ws ounte. It ws ounte for perio of 3 min t, 3, 6, 9 n 12 min following the tretments. EPM test The EPM test ws rrie out y the protool introue y Wlf n Frye with slight moifition [2]. The EPM pprtus onsists of two open rms (5 1 m) n two lose rms (5 l5 m) riting from pltform (5 5 m) to form plus sign figure. The pprtus ws situte 4 m ove the floor. The open rms eges were.5 m in height to keep the rts from flling n the lose-rms eges were 15 m in height. Sixty min fter ministrtion of the test extrt n rugs, eh niml ws ple t the enter of the mze fing one of the enlose rms. During 5 min test perio, the numer of open n enlose rms entries n the time spent in open & enlose rms ws reore. Entry into n rm ws efine s the point when the niml ples ll four pws onto the rm. The proeure ws onute in soun ttenute room; oservtions me from n jent orner. Thiopentl soium inue sleeping time test The metho ws rrie out s esrie y Fujimori n Co [21]. Briefly, 3 min fter NC, PC, LM1 n LM2 ministrtion, thiopentl soium (t 4 mg/kg oy weight ose) ws ministere to eh rt to inue sleep. The nimls were oserve for the ltent perio (time etween thiopentl ministrtion n loss of righting reflex) n urtion of sleep (i.e. time etween the loss n reovery of righting reflex) inue y thiopentl soium. Investigtion for nti-mnesi effet The experiment ws rrie out s esrie y Wng et l. [22]. The nimls were ivie s erlier. NC group reeive istille wter only. PC group ws trete with izepm t 5 mg/kg oy weight orlly. LM1groupreeiveizepmt5mg/kgBWn LM t the oses of 1 mg/kg BW. LM2 group reeive izepm t 5 mg/kg BW n LM t the oses of 2 mg/kg BW. In this stuy, izepm ws ministere to inue nterogre mnesi in nimls n the ose of izepm ws selete from literture [23]. The stuy ws esigne to exmine nti-mnesi effet of test smple on izepm inue mnesi. The nimls were trete ily in the evening. After 12 h, the lerning n memory of rts ws evlute. Aquisition n retrievl trils using Morris wter mze The Morris wter mze is one of the most wiely use tsks in ehviorl neurosiene for stuying the psyhologil proesses n neurl mehnisms of sptil lerning n memory, whih re mjor funtions of the hippompus [24, 25]. The wter mze ws evelope y Rihr Morris n ple nvigtion in the wter mze is now often use s generl ssy of ognitive funtion, for exmple for testing the impt of vrious isturnes of the nervous system e.g. niml moels of stroke, ging, neuroegenertive isese, or the potentil impt of novel therpeuti rugs [26]. It hs, therefore, een extensively use for the evlution of sptil memory efiit inue y Benzoizepines [27]. This moel suitly omines the evlution of oth sptil memory n loomotor tivity. Therefore, it n e use for the evlution of rugs tht not only ffet sptil memory ut lso impir loomotor tivity. The wter mze onsiste of irulr tnk, 11 m in imeter n with wll 4 m in height. A white pltform (1 m in imeter) ws ple insie, n the tnk ws fille with wter (22 ± 1 C), until the top of the pltform ws sumerge 1 m elow the wter s surfe. Before eginning quisition trining, eh rt reeive pre-trining session tht onsiste of pling the rt on the pltform where it h to sty t lest 15 s, followe y 3 s swimming perio, n ening y severl trils of liming onto the pltform until eh sujet ws le to lim without help. This nonsptil proeure ws require to voi onfusion etween proeurl spets of the tsk n susequent sptil performne [28]. The following y, the rts were given five quisition sessions with 24 h istne one from nother, eh quisition onsiste of four trils per y with n intertril intervl of 1 min. Throughout the ourse of this quisition perio, the hien pltform remine in the sme fixe position for ll rts. In the present stuy, qurnt Q3 ws esignte s the trget qurnt. In the eginning of eh tril, the rts were relese with its he fing the wll t one of four points lote long the perimeter of the mze, ritrrily esignte s N, S, E, n W. The orer of the strting points ws etermine rnomly, n eh strting point ws use only one per session. One rt lote the pltform, it ws llowe to remin there for 3 s efore eing remove from the tnk. If rt file to lote the pltform within 6 s, it ws mnully guie to it. Men espe lteny (the time etween eing ple in the wter n fining the hien pltform) ws lulte whih is onsiere s n inex of quisition. After five sessions of quisition trining, susequent tril ws onute in whih the pltform ws remove from the pool n the rt h to swim gin for 6 s with no opportunity to espe. This is the retrievl tril stuy whih evlute how well the nimls h lerne n onsolite the pltform lotion uring the 5 ys of trining. The perentge of time spent

5 Ferousy et l. Clinil Phytosiene (216) 2:17 Pge 5 of 11 in trget qurnt y the niml ws lulte. The time spent y the niml in the trget qurnt (Q3) while serhing for the hien pltform ws reore s n inex of retrievl. Assys of Superoxie ismutse (SOD), Ctlse (CAT), Glutthione peroxise (GSH-Px), Mlonilehye (MDA) n vne oxition of protein prout (AOPP) estimtion Twelve hours fter evlution of lerning n memory, the rts were srifie through hloroform nesthesi n trnsril perfusion with ie-ol norml sline (1 ml/1 g BW). The entire intt rin ws refully remove n ple on n ie-hille ish for lening. The hippompus ws rpily remove n weighe. Then the hippompus were homogenize in 4 volumes of.1 mol/l ie phosphte uffer (ph 7.4) n entrifuge t 2, g for 3 min t 4 C. SOD tivity in the hippompus ws etermine y the metho of Misr n Friovih [29] se on uto oxition of epinephrine. Briefly, n liquot of.25 ml ie ol hloroform ws e to.1 ml of superntnt followe y ing.15 ml ie ol ethnol. The mixture ws entrifuge t 3 rpm for 1 min t 4 C. Then.2 ml of superntnt ws tken n 1.5 ml ronte uffer,.5cc EDTA, n.8 ml istille wter ws e. Retion ws strte y ing.4 ml epinephrine. Chnge in sorne ΔOD/min t 48 nm ws re for 3 min. The results were expresse in terms of nmol/min/mg protein. CAT tivity in the hippompus ws mesure t 37 C y following the rte of ispperne of H 2 O 2 t 24 nm [3]. To initite the CAT tivity,.2 ml of tissue homogente/.2 ml of serum ws e to the retion mixture ontining 1.9 ml of 1 mm Phosphte uffer (ph 7.),.1 ml of enzyme extrt, n 1. ml of.75 % H 2 O 2 solution. CAT tivity ws ssye in 1. min t 24 nm. GSH-Px tivity in the rin ws estimte oring to the metho esrie y Ellmn [31]. The retion mixture ontine.1 M phosphte uffer (ph 7.), 1 mm EDTA, 1 mm glutthione (GSH), 1 mm NN3, 1 unit of glutthione reutse, 1.5 mm NADPH n.1 ml of homogente/serum. After inution for 1 min t 37o C, H2O2 ws e to eh smple t finl onentrtion of 1 mm. GPX tivity ws mesure s the rte of NADPH oxition t 34 nm. The MDA ontent, quntittive mesurement of lipi peroxition, ws ssye in the form of thiorituri i retive sustnes y the metho of Wills [32]. Collete superntnt ws inute in solution ontining 8.1 % SDS, 2 % eti i (ph 3.5),.8 % TBA (Thiorituri i) for 1 h t 95 C. After ooling t RT, the smples were well mixe with utnol/pyriine mixture (15:1, v/v), n the queous phse ws refully ollete y spirtion. Asorne of the queous phse ws foun out t 532 nm. MDA ontent ws expresse s nmol/mg protein. Avne protein oxition prout (APOP) ws estimte y the metho followe y Sle et l. [33]. Briefly, two ml of plsm ws ilute 1 : 5 in PBS:.1 ml of 1.16 M potssium ioie ws then e to eh tue, followe y.2 ml eti i fter 2 min. The sorne of the retion mixture ws immeitely re t 34 nm ginst lnk ontining 2 ml of PBS,.1 ml of KI, n.2 ml of eti i. The hlormine-t sorne t 34 nm eing liner within the rnge of 1 mmol/l, AOPP onentrtions were expresse s μmol L 1 hlormine- T equivlents The umultion of nitrite, n initor of nitri oxie (NO) proution ws etermine y olorimetri ssy s esrie y Green et l. [34]. Aliquots of 5 μl of superntnts were inute with 5 μl of Griess regent, onsisting of 1 % sulfnilmie,.1 % N-(1-nphthylethyleneimine) ihyrohlorie n 2.5 % ortho-phosphori i. After 1 min t room temperture, the optil ensity ws mesure t 54 nm. Nitrite onentrtions were lulte y omprison to lirtion urve otine with soium nitrite stnr solutions. Sttistil nlysis Dt is expresse s mens ± SEM. All t re presente s the men ± SD of five nimls. Dt ws nlyze with One Wy Anlysis of Vrine (ANOVA) using sttistil softwre pkge (SPSS for Winows, version 21, IBM Corportion, NY, USA) followe y Tukey s-hsd multiple rnge post-ho test. Vlues were onsiere signifintly ifferent t p <.5. Results Neurophrmologil effet of LM Open fiel test In the experiment, the extrt showe notiele erese in loomotion in the test nimls from the seon oservtion perio to lst stuy perio t oth ose levels. The epressnt tion ws inrese with time n notiele result ws foun t 12 min of test smple ministrtion. Test nimls showe signifint erese in numer of movements t the osges of 1 n 2 mg/kg (1 ± 2.83, 8 ± 2.12, respetively) s ompre to 13 ± 1.41 in the NC group t 12 min of ministrtion of LM). Figure 2 shows the ose epenent CNS epressnt tivities whih were sttistilly signifint (p <.5) ompre to NC n PC groups. Hole ross test In hole ross test, the niml trete with LM showe ose epenent reution in the loomotor tivity. At LM 2, numer of holes rosse (1.5 ±.71) ws omprle with tht of stnr rug izepm (1.5 ±.71). The extrt

6 Ferousy et l. Clinil Phytosiene (216) 2:17 Pge 6 of 11 movements of Numer Control Dizepm LM1 LM2 min 3 min 6 min 9 min 12 min Fig. 2 Effet of LM on loomotor tivity in open fiel test for Wistr lino rts. Dt re shown s Men ± SD for triplite. Dt were nlyze y one-wy ANOVA followe y Tukey s post ho test (SPSS, Version 21.,NY) for multiple omprisons. Vlues with ifferent supersript letters over the lines for given prmeter re signifintly ifferent from eh other group of nimls. Vlues were onsiere s signifint with p<.5 proue reution in spontneous motor tivity, n this effet my e ttriute to CNS epression of stnr rug izepm (1.5 ±.71). LM1 lso reue the loomotor effet ut the vlues were not signifint ompre to PC group. The CNS ws foun to e epresse through the oservtion perio to 12 min (Fig. 3). EPM test LM t oth the oses inrese the perentge of entries into the open rms (Fig. 4). The perentge of time spent into open rms lso inrese t the LM2 mg/ kg oses (16 ±.8) s ompre to the NC group (1.51 ± 1.72). There ws lso signifint inrese in urtion of time (42 ± 2.83) for LM2, into open rm, whih ws sttistilly signifint s ompre to tht (35.5 ± 3.54) of referene group. Thiopentl soium inue sleeping time test Thiopentl soium inue sleeping time test revele tht oth the oses inue sleep t rpi stge s ompre to NC. In ition, oth oses of LM prolonge the urtion of sleeping time (115 ± 1.6, 142 ± 1.6 respetively) in omprison to NC (1 ± 14.14). As test smple erese ltent perio (onset of sleep) n inrese the urtion of sleep, the LM showe ose epenent setive effets whih were sttistilly signifint (p <.5). Figures 5 n 6 represent the tivity in thiopentl soium inue sleeping time test. Effet of LM on izepm-inue mnesi The men espe lteny for the trine rts erese uring the ourse of the 5-y trils. There ws no notiele ifferene foun in the men espe lteny etween ny two groups on the first, seon n thir y. However, on the fourth n fifth y, the men espe lteny of the PC group ws longer thn tht of the ontrol group (P <.1). Compre with the PC group, the espe lteny of rts trete with LM erese to prominent egree (P <.1, Fig. 7). At the lst y of tril, men espe lteny of LM2 ws 2.41 ±.32, whih ws less thn the NC n PC group (3.25 ±.44 1 Control Dizepm LM1 LM2 Numer of hole rosse min 3 min 6 min 9 min 12 min Fig. 3 Effet of LM on loomotor tivity in hole-ross test for Wistr lino rts. Dt re shown s Men ± SD for triplite. Dt were nlyze y one-wy ANOVA followe y Tukey s post ho test (SPSS, Version 21., NY) for multiple omprisons. Vlues with ifferent supersript letters over the lines for given prmeter re signifintly ifferent from eh other group of nimls. Vlues were onsiere s signifint with p<.5

7 Ferousy et l. Clinil Phytosiene (216) 2:17 Pge 7 of Durtion of Open rm entry (se) Perentge of time spent into Open rm entry (%) Control Stnr LM1 LM2 Fig. 4 Effet of LM on urtion of open rm in elevte plus mze (EPM) test for Wistr lino rts. Dt re shown s Men ± SD for triplite. Dt were nlyze y one-wy ANOVA followe y Tukey s post ho test (SPSS, Version 21.,NY) for multiple omprisons. Vlues with ifferent supersript letters over the rs for given prmeters re signifintly ifferent from eh other group of nimls. Vlues were onsiere s signifint with p<.5 (se) perio Ltent Control Dizepm LM1 LM2 Fig. 6 Effet of Lee mrophyll on ltent perio in thiopentl soium inue sleeping time test for Wistr lino rts. Dt re shown s Men ± SD for triplite. Dt were nlyze y one-wy ANOVA followe y Tukey s post ho test (SPSS, Version 21., NY) for multiple omprisons. Vlues with ifferent supersript letters over the rs for given prmeter re signifintly ifferent from eh other group of nimls. Vlues were onsiere s signifint with p <.5 n 2.96 ±.76, respetively). In the proe tril stuy, the time spent in the trget qurnt ws signifintly shorter in PC group s ompre to tht in the NC group. The rts in the LM groups spent more time in the trget qurnt thn the PC group (Fig. 8). Effet of LM on SOD, CAT, Glutthione peroxise, MDA, AOPP, NO tivity in hippompus Compre with the NC group, SOD, tlse n GSH- Px tivities were signifintly erese following izepm ministrtion. Tretment with LM llevite the hnges of SOD, tlse n GSH-Px tivities Fig. 9 (-). In omprison with the NC group, the MDA ontent in izepm-trete mie inrese. However, LM signifintly ttenute the inrese of MDA ontent inue y izepm (Fig. 8). In se of AOPP, LM hs sleep (min) Durtion of Control Dizepm LM1 LM2 Fig. 5 Effet of Lee mrophyll on urtion of sleep in thiopentl soium inue sleeping time test for Wistr lino rts. Dt re shown s Men ± SD for triplite. Dt were nlyze y one-wy ANOVA followe y Tukey s post ho test (SPSS, Version 21.,NY) for multiple omprisons. Vlues with ifferent supersript letters over the rs for given prmeter re signifintly ifferent from eh other group of nimls. Vlues were onsiere s signifint with p <.5 ose-epenently erese AOPP in test groups. However, LM2 erese the ontent of AOPP t vlue of ± 7.42 whih ws sttistilly signifint (p <.5) to PC group. Level of nitri oxie in NC group is 4.22 ± LM2 s well signifintly erese nitri oxie level (Figs. 1 n 11). Disussion In our present stuy neurophrmologil effets of LM hve een evlute through open fiel test, holeross test, EPM n thiopentl soium inue sleeping time test on Wistr lino rts. The most importnt step in evluting rug tion on the CNS is to oserve the ehvior of the test nimls [35]. Open fiel test n hole-ross tests were rrie out to evlute loomotor tivity of nimls. Inrese in loomotor effet is onsiere s n inrese in lertness n erese in loomotor tivity inite setive effet [36]. Present stuy revele tht the LM showe ose epenent CNS epressnt tivities. In Open fiel test, oth LM1 n LM2 showe notiele erese in loomotion in the test nimls from the seon oservtion perio to the en of stuy perio. In hole-ross test, LM showe ose epenent reution in the loomotor tivity. But mximum loomotor reution ws shown y LM2. Different nxiolyti, setive-hypnoti rugs re eluite their tion through Gmm-mino-utyri i (GABA). GABA sutype A (GABAA) is the mjor inhiitory neurotrnsmitter in the entrl nervous system [37]. Therefore it is possile tht, this extrt my t y potentiting GABAergi inhiition in the CNS vi memrne hyperpolriztion whih les to erese in the firing rte of ritil neurons in the rin. It my lso e ue to iret tivtion of GABA reeptor y the extrt

8 Ferousy et l. Clinil Phytosiene (216) 2:17 Pge 8 of 11 3 Control Dizepm LM1 LM2 lteny (se) en espe M st y 2n y 3r y 4th y 5th y Fig. 7 Effet of Lee mrophyll on men espe lteny in quisition tril for Wistr lino rts. Dt re shown s Men ± SD for triplite. Dt were nlyze y one-wy ANOVA followe y Tukey s post ho test (SPSS, Version 21.,NY) for multiple omprisons. Vlues with ifferent supersript letters over the rs for given prmeter re signifintly ifferent from eh other group of nimls. Vlues were onsiere s signifint with p <.5 [38]. As LM erese loomotor tivity in nimls, it inites CNS epressnt tivity. Anxiolyti effet of rug moleules is evlute y EPM test whih is well-estlishe moel for ssessing neurophrmologil responses of roents [39]. The effet is oserve when the experimentl rug inreses open rms entries without ltering the totl numer of rm entries. In our present stuy, oth of the oses of LM inrese the perentge of entries of rts into the open rms. But LM2 showe signifint inrese in the perentge of time spent in the open rms of the mze. This ws slightly lrger thn the effets oserve following tretment with the referene nxiolyti rug izepm. These results inite n nxiolyti-like tivity of LM. Thiopentl t pproprite ose inues hypnosis y potentiting GABA meite postsynpti inhiition through llosteri moifition of GABA A type (GABAA) reeptors. In the urrent reserh, oth LM1 n LM2 potentite sleep inue y thiopentl suggesting tht the roots of the plnt possess sleep inuing property. Aitionlly, sustnes whih possess CNS epressnt tivity either erese the time for onset of sleep or prolong the urtion of sleep or oth [4]. This is onsistent with our stuy where LM1 n LM2 signifintly inrese the urtion of sleep. Similr finings were reore y Fujimori n Co [21] who propose tht the enhnement of hypnosis is goo inex of CNS epressnt tivity meite y GABAA reeptor (s esrie erlier). The reeptor omplex is involve in vrious neuropsyhologil isorers suh s epilepsy, epression, Prkinson s n Alzheimer s isese [41]. Mny flvonois suh s isoflvones S-( )-equol, 4 -hyroxy-7-methoxyisoflvone n 3,7-ihyroxyisoflvone, were foun to e ligns for GABAA reeptors in the CNS; whih le to the hypothesis tht they t s Time spent in trget qurnt (se) Fig. 8 Effet of Lee mrophyll on time spent in trget qurnt in retrievl tril for Wistr lino rts. Dt re shown s Men ± SD for triplite. Dt were nlyze y one-wy ANOVA followe y Tukey s post ho test (SPSS, Version 21., NY) for multiple omprisons. Vlues with ifferent supersript letters over the rs for given prmeter re signifintly ifferent from eh other group of nimls. Vlues were onsiere s signifint with p<.5

9 Ferousy et l. Clinil Phytosiene (216) 2:17 Pge 9 of 11 SOD tivity /3se GP (µmol/mg protein) () () Control Dizepm LM1 LM2 Control Dizepm LM1 LM Control Dizepm LM11 LM2 Control Dizepm LM1 LM2 Fig. 9 Effet of Lee mrophyll on superoxie ismutse (SOD) tivity (); tlse tivity (); Glutthione peroxise (GP) (); n Mlonlehye (MDA) () in hippompus for Wistr lino rts. Dt re shown s Men ± SD for triplite. Dt were nlyze y one-wy ANOVA followe y Tukey s post ho test (SPSS, Version 21., NY) for multiple omprisons. Vlues with ifferent supersript letters over the rs for given prmeters re signifintly ifferent from eh other group of nimls. Vlues were onsiere s signifint with p<.5 Ctlse tivity (U/min) MDA (nmol/mg protein) () () enzoizepine-like moleules [42, 43]. This is supporte y their ehviorl effets in niml moels of nxiety, setion n onvulsion [3]. Sine Lee mrophyll ws foun to possess flvonois, lklois, tnnins n terpenois, ehviorl effets in niml moels of nxiety n setion oul e supporte y ifferent oses LM. Benzoizepines (BZs) inue nterogre mnesi in oth humn n nimls [44]. In this reserh, the effet of LM on izepm inue mnesi ws investigte using Morris wter mze sle for ehviorl experiments. Morris wter mze is use to test sptil memory of the rts [45]. It is most wiely use tsks in ehviorl neurosiene for stuying psyhologil proesses. In our stuy, the nimls in the izepm group took signifintly longer time to lote the hien pltform on ys 4 n 5, whih inite oxition protein (µm/ml) Avne protein Control Dizepm LM1 LM2 Fig. 1 Effet of Lee mrophyll on vne oxition protein prout (AOPP) in hippompus for Wistr lino rts. Dt re shown s Men ± SD for triplite. Dt were nlyze y one-wy ANOVA followe y Tukey s post ho test (SPSS, Version 21., NY) for multiple omprisons. Vlues with ifferent supersript letters over the rs for given prmeter re signifintly ifferent from eh other group of nimls. Vlues were onsiere s signifint with p <.5

10 Ferousy et l. Clinil Phytosiene (216) 2:17 Pge 1 of 11 mg/protein oxie Nitri Control Dizepm LM1 LM2 Fig. 11 Effet of Lee mrophyll on nitri oxie (NO) in hippompus for Wistr lino rts. Dt re shown s Men ± SD for triplite. Dt were nlyze y one-wy ANOVA followe y Tukey s posthotest (SPSS, Version 21., NY) for multiple omprisons. Vlues with ifferent supersript letters over the rs for given prmeter re signifintly ifferent from eh other group of nimls. Vlues were onsiere s signifint with p<.5 tht izepm ministrtion impire sptil lerning. By y 6, we ssesse memory retention in proe tril whih involve removing the hien pltform from the pool n mesuring the length of time the nimls spent in the trget qurnt. The proe tril revele tht izepm-trete rts spent signifintly less time swimming in the trget qurnt. Therefore, the stuy showe tht izepm resulte in long lsting sptil memory efiits s well [46]. On the ontrst, LM ttenute the sptil lerning n memory impirments s ompre to nimls trete y izepm lone. These finings suggeste tht o-ministrtion of LM with izepm signifintly meliorte izepm-inue nterogre mnesi. It is resonle to speulte tht this effet is eing meite y the GABAergi system. The moleulr events unerlying the ntimnesi effet of LM nee to e iserne to not only sreen the tive onstituents of Lee mrophyll ut n filitte evelopment of novel trgets to enhne memory [47]. Free rils n relte moleules re often lssifie together s retive oxygen speies (ROS). The formtion of ROS in nerve ells re numerous, therefore, ells hve to mintin n effetive ntioxint system in orer to protet themselves ginst ROS overlo n susequent mge [48]. During oxitive stress, ROS proution inreses n surpsses the pity of enogenous free ril svengers suh s SOD, tlse n GSH-Px. In ition, the rin is espeilly sensitive to oxitive stress euse it utilizes high levels of oxygen n ontins lrge mounts of esily peroxiizle ftty is. It ws lso reporte tht formtion of ROS ws involve in neurotoxiity of mny xenoiotis [49]. In this reserh, izepm use signifint hnge in MDA, NO, AOPP ontent n SOD, tlse n GSH-Px tivities in hippompus. However, o-ministrtion of LM with izepm not only reue MDA ontent ut lso inrese SOD, tlse n GSH-Px tivities in hippompus of rts. The oserve effet my e lying in the ril svenging properties of LM ontining polyphenolis whih re estlishe for their ntioxitive nture. The present finings, therefore, emonstrte the orreltion etween ntimnesi effets of LM ginst izepminue mnesi n its ntioxitive pity [5], ut the usl reltionship remins to e etermine in the future work. Conlusion The results of this stuy provie sientifi support for the use of LM in the neurologil isorers. It lso revele the CNS epressnt, nxiolyti n setive effets of LM whih meliorte izepm-inue mnesi in niml moels. It hs signifintly inrese ntioxitive enzymes superoxie ismutse, tlse n glutthione peroxise in the hippompus of rin. It lso ereses oxitive iomrkers suh s lipi peroxition, nitri oxie n vne oxition protein prout. Therefore, the use of this plnt in the reution of oxitive stress ssoite - neuroegenertion is eome evient y this stuy lthough further investigtion of the hemil onstituents of this plnt is neessry to justify their involvement in the oserve tions. Soure of support Authors wish to thnk University of Chittgong to llote fun for onuting this reserh uner UGC-Den reserh grnt (Ref.5448/ Res/Pln/Pu/CU/213). Authors ontriution MAR n JA hve plnne n esigne the reserh. SF hs ollete smple, performe the experimentl works in lortory, nlyze the t n written the mnusript. MAR hs lso enevore t nlysis, interprettion of the results, revision of the whole mnusript with grmmtil heking n tehnil esign. MMAA n JMKHC hve ssiste the lortory nlysis of the work. All uthors re n pprove the finl mnusript. Competing interest The uthors elre tht they hve no ompeting interest. Author etils 1 Deprtment of Biohemistry & Moleulr Biology, University of Chittgong, Chittgong 4331, Bnglesh. 2 Deprtment of Phrmy, North South University, Bshunhr, Dhk 1229, Bnglesh. Reeive: 7 June 216 Aepte: 12 August 216 Referenes 1. Simpson T, Pse M, Stough C. Bop monnieri s n ntioxint therpy to reue oxitive sress in the ging rin. Evi Bse Complement Alternt Me. 215;215: Wng Z, Wn H, Li J, Zhng H, Tin M. Moleulr imging in tritionl Chinese meiine therpy for neurologil iseses. BioMe Res Int. 213; 213: Kles A, Bixler E, Vel-Bueno A, Soltos CR, Mnfrei RL. Alprzolm: effets on sleep n withrwl phenomen. J Clin Phrmol. 1987;27: Ernst E. Her rug intertions: potentilly importnt ut woefully uner-reserhe. Eur J Clin Phrmol. 2;56:523 4.

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Drug Met Rev. 1995;27: Musvi S, Kkkr P. Pro n ntioxint responses to repete ministrtion of izepm in rt rin. Mol Cell Biohem. 2;26: Sumit your mnusript to journl n enefit from: 7 Convenient online sumission 7 Rigorous peer review 7 Immeite pulition on eptne 7 Open ess: rtiles freely ville online 7 High visiility within the fiel 7 Retining the opyright to your rtile Sumit your next mnusript t 7 springeropen.om