Olarewaju M. Oluba. Oluba Lipids in Health and Disease (2019) 18:12

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1 Olu Lipids in Helth nd Disese (219) 18:12 RESEARCH Gnoderm terpenoid extrt exhiited nti-plsmodil tivity y mehnism involving redution in erythroyte nd hepti lipids in Plsmodium erghei infeted mie Olrewju M. Olu Open Aess Astrt Biotive omponents of Gnoderm luidum hs reently gined intense reserh ttention due to their limed nutritionl nd mediinl properties. Thus, the terpenoid extrt from the fruit odies of G. luidum (GT) ws evluted for tivity ginst Plsmodium erghei in mie in two seprte experiments. In ddition, the effets of the extrt on erythroyte nd hepti lipids s well s liver HMG-CoA redutse tivity efore nd fter the tretments were lso ssessed. Mie with estlished infetion were dministered nd 25 mg/kg/dy GT lone nd in omintion with hloroquine (), in either se two seprte ontrols designted: ( mg/kg hloroquine) nd (1 ml DMSO) were lso inluded. Tretment ws dministered orlly for 12 dys nd prsitemi determined every three dys. Perentge survivl ws signifintly inresed to 87% from 66% due to omintion of GT with ompred to GT lone nd to 75% from 62% when GT25 ws dministered with ompred to GT25 lone. Erythroyte triglyerides, totl holesterol (TC), LDL nd phospholipids ontents were signifintly lower in GT + -treted mie ompred to lone nd. Similrly, hepti TC nd phospholipid levels were signifintly lower in the GT + -treted mie ompred to lone nd nd HMG-CoA redutse tivity in the liver ws signifintly inhiited due to dministrtion of GT +. Dt from this study suggest tht the nti-plsmodil tion of GT ould involve mehnisms ssoited with its hypolipidemi tivity. It ws lso demonstrted tht hloroquine, when dministered in omintion with GT, potentites its urtive effet in P. erghei-infeted mie. Keywords: Gnoderm luidum, Terpenoid extrt, Antiplsmodil tivity, Hypolipidemi tivity, Heptoprotetion Introdution Mlri remins mjor puli helth hllenge ffeting. illion people in 16 ountries nd territories. Aout 216 million mlri ses re reported to our nnully 81% of whih our in the Afrin su-region [1]. Out of out 48, deths resulting from mlri in 2, n estimted 9% were tred to su-shrn Afri [1]. Nigeri with out 51 million ses nd Correspondene: olu.olrewju@lmu.edu.ng; olumike2@yhoo.o.uk Deprtment of Biologil Sienes, Food Sfety nd Toxiology Reserh Unit, Environment nd Tehnology Reserh Cluster, College of Siene nd Engineering, Lndmrk University, P.M.B, Omu-Arn, Kwr Stte 1, Nigeri 27, deths ontriutes pproximtely % of Afrin nnul mlri urden. Aording to WHO report out 97% of the totl Nigerin popultion (pproximtely 175 million) is t risk of mlri infetion [1]. The rising prolem of resistne to ville hemotherpies nd prolem of rerudesene of mlri fter tretment with rtemisinin stress the need for new ntimlril gents [2]. The lssil drugs quinine nd rtemisinin re oth plnt derivtives otined from Cinhon speie [] nd Artemisi nnu [4] respetively, suggesting tht other effetive drugs might e plnt-derived. Plnts nd fungi produts hve formed mjor omponent of foods nd therpeuti gents of The Author(s). 219 Open Aess This rtile is distriuted under the terms of the Cretive Commons Attriution 4. Interntionl Liense ( whih permits unrestrited use, distriution, nd reprodution in ny medium, provided you give pproprite redit to the originl uthor(s) nd the soure, provide link to the Cretive Commons liense, nd indite if hnges were mde. The Cretive Commons Puli Domin Dedition wiver ( pplies to the dt mde ville in this rtile, unless otherwise stted.

2 Olu Lipids in Helth nd Disese (219) 18:12 Pge 2 of 9 indigenous Afrin ommunities for enturies. In reent times, inresed reserh ttention hs foused on phytohemils extrted from plnts with trditionlly limed mediinl properties in order to eluidte the sientifi sis for their oserved tivities nd to seek new led ompound from them whih ould e developed into therpeuti drugs with enhned effiy nd miniml side effets [5]. The rtionle for the serh of ntimiroil ompounds from fungi is tht oth fungi nd humns shre ommon miroil pthogens suh s Esherihi oli nd Stphyloous ureus, therefore we n enefit from their defense strtegies ginst these miroorgnisms [6]. There hs een puity of informtion on Afrin Gnoderm nd mjority of mediinl investigtions on Gnoderm speies hve een rried out on speies tht hve een isolted from other prts of the world. Sine speies within the Gnodermtee fmily ll hve ommon evolutionry history, it is not unlikely tht Afrin Gnoderm my ontin similr ompounds to those isolted elsewhere. Gnoderm luidum is n edile mushroom with long history of use s mediinl her in Orientl ountries [7]. The extrt hs een investigted for its helth enefits in preventing rdiovsulr disese, ner, nd miroil infetion, nd for its lipid nd gluose lowering, nti-inflmmtion, nti-oxidnt, nti-prsite, nd multiple orgn protetion effets [8 ]. Its long history of usge with no ttendnt toxiity represents the desired end result in the development of effetive therpeuti interventions. In West Afri, mny helth lims hve een mde on the effet of Gnoderm speies on immune system. Trditionl medil prtitioners usully onsider Gnoderm s nturl immune regultor [9]. Biotive omponents of the mediinl mushroom, Gnoderm luidum hs reently gined intense reserh ttention due to their limed nutritionl nd mediinl properties. Experimentl evidenes reveled tht the terpenoids re the most importnt iologilly tive sustnes in Gnoderm luidum due to their vrying phrmologil pplitions [8, 9]. Terpenoids extrted from Gnoderm luidum hve een demonstrted to exhiit nti-hypertensive [8], hypoholesterolemi [1, 11], heptoprotetive [12], nd ytotoxiity ginst numerous ner ell lines [1]. A reent study y Olu et l. [] demonstrted the promising potentil of rude hloroform extrt of Gnoderm luidum fruit ody s n nti-plsmodil gent. Chnges in erythroyte lipid omposition nd the host oxidnt-ntioxidnt sttus re two ritil ssoited events involved in the pthogenesis of mlri [16]. During the intrerythroyti stge of the prsite development, out 5 to 1 merozoites re produed every 24 h. A orresponding inrese in metoli tivity with onomitnt memrne turn-over rte is required to sustin the growing prsite t this stge. Lied et l. [17] hve shown tht Plsmodium fliprum infetion indues six-fold inrese erythroyte phospholipid ontent. In order to meet up with its high requirement for phospholipids, the infeted erythroytes ontin phospholipid synthesizing enzymes [18]. Thus, potent inhiitors of plsmodil phospholipid synthesis ws previously hrterized s potentil trget for nti-mlril hemotherpy due to its ruil role to the prsite survivl [19]. The mehnism(s) ontriuting to the nti-plsmodil tivity of Gnoderm luidum extrt remins unler. Inresing evidene indites tht hnges in tissue lipid ontent my e implited in the proess [12]. The present study ws imed t extrting the terpenoid frtion of Gnoderm luidum fruit ody nd evlution of its potentil nti-plsmodil tivity when dministered lone or in omintion with hloroquine ginst Plsmodium eghei in mie. Mterils nd methods Fungl mteril Gnoderm luidum fruit odies were olleted from forest reserve t Ipele, Ondo Stte, Nigeri nd otnilly identified y Dr. Soji Fkoy, myologist in the Deprtment of Biologil Sienes, Joseph Ayo Blol University, Ikeji-Arkeji, Nigeri. Speimen smple ssigned vouher numer 11 ws deposited in institution herrium for referene purpose. Preprtion of gnoderm terpenoid extrt (GT) G. luidum fruit ody thoroughly wshed with len sterile wter ws ir-dried t room temperture for two weeks. The dried smples were ut into smller piees nd milled into powder using mehnil grinder. The terpenoid extrtion ws rried out ording to the method desried y Weng et l. [2] with little modifitions. Briefly, the powdered fungl mteril (2 g) ws first extrted y reflux using 5% ethnol for 24 h t room temperture. The resulting mixture ws filtered using Whtmn numer 1 filter pper nd the omined filtrte onentrted in vuo t 5 C. The onentrted extrt ws prtitioned etween hloroform nd wter. The omined hloroform lyer ws extrted with 5% NHCO solution. The omined NHCO frtion ws idified to ph with 2 N HCl under ie-ooling nd then extrted with hloroform. The omined hloroform lyer ws evported under redued pressure to give mixture of idi ompounds herein referred to s Gnoderm terpenoid extrt (GT). This ws stored in drk irtight ontiner t 4 C until used for the experiment.

3 Olu Lipids in Helth nd Disese (219) 18:12 Pge of 9 Quntifition of totl terpenoid ontent Totl terpene ontent of the GT extrt ws quntified spetrophotometrilly sed on the method desried y Go et l. [21] using gnoderi id A s stndrd. The totl triterpenoid ontent ws expressed s milligrms of gnoderi id A equivlents per grm of GT. Animls nd tretment Eighty-four (84) Swiss mle mie (etween 7 nd 9 weeks old) proured from the niml re filities of Institute for Advned Medil Reserh nd Trining (IAM- RAT), University Tehing Hospitl, Idn, Nigeri were used for the study. The nimls were kept in stndrd mie ges under ontrolled environment nd with unrestrited ess to food nd wter. Approvl for the study ws grnted y Joseph Ayo Blol University Reserh nd Ethis Committee with pprovl numer JABU/REC/AS. All niml experimentl proedures were rried out in strit ompline with the rules guiding the Cre nd Use of Lortory Animls s ontined in the Ntionl Institutes of Helth mnul [22]. Prsite inoultion The mlril prsite Plsmodium erghei (NK-65 strin) ws otined from the Institute for Advned Medil Reserh nd Trining (IAMRAT), UCH, Idn, Nigeri. Gnoderm terpenoid extrt tretment Two sets of niml experiments were rried out. In the first experiment intended to sertin the effet of GT terpenoid extrt lone, 2 mle mie were inoulted intrperitonelly (i.p.) with.2 ml 1 5 P. erghei-infeted red lood ells suspended in PBS. Three dys fter prsite inoultion, mie in eh group were fsted overnight, weighed nd their prsitemi determined. Therefter, they were rndomly ssigned into four experimentl groups (n =8) designted:, dministered mg/kg gvge hloroquine hydroxyhloroquine sulfte (Aspen Phrm, Johnnesurg, South Afri); GT, reeived mg/kg Gnoderm terpenoid extrt; GT 25, reeived 25 mg/kg Gnoderm terpenoid extrt;, reeived 1 ml DMSO. The extrt ws dissolved in DMSO in order to keep the onentrtion nd dose similr. Tretment ws dministered one dily for 12 onseutive dys during whih they were oserved dily for survivl nd their prsitemi level determined every three dys. In nother seprte experiment, intended to sertin the effet of GT extrt dministered in omintion with,52 mle mie were inoulted intrperitonelly (i.p.) with.2 ml 1 5 P. erghei-infeted red lood ells suspended in PBS. Three dys fter prsite inoultion, mie in eh group were fsted overnight, weighed nd their prsitemi determined. Therefter, they were rndomly ssigned into four experimentl groups (n = 1) designted:, dministered mg/kg gvge hloroquine hydroxyhloroquine sulfte; GT +, reeived omintion of mg/kg Gnoderm terpenoid extrt nd mg/kg gvge hloroquine hydroxyhloroquine sulfte; GT 25 +, reeived omintion of 25 mg/kg Gnoderm terpenoid extrt nd mg/ kg gvge hloroquine hydroxyhloroquine sulfte;, reeived 1 ml DMSO. Before ommenement of the extrt tretment, five mie from eh group were srified nd their lood smples olleted nd nlyzed to otin the seline level of eh prmeter monitored. After 12-dy tretment, the nimls were fsted overnight, weighed nd their prsitemi estimted from their til lood smples efore een euthnized nd then srified y ervil dislotion. Upon srifie, lood smple ws olleted from eh niml into plin smple ottles, liver, kidney, spleen nd rin were quikly exise, lotted on tissue pper, lered of fts nd weighed. Serum preprtion Blood smples olleted in plin tues were left stnding for h to lot nd then entrifuged t 5 rpm for 1 min t 4 C. Serum from the entrifuged lood smple ws olleted y sution using psture pipette into sterile plin smple ottles nd stored t 4 C for further nlysis. Tissue preprtion Liver homogente ws prepred y weighing 2 g of lened tissue nd homogenized using.1 M phosphte uffer fter whih it ws entrifuged t 5 rpm (4 C) for 1 min to otin the superntnt whih ws susequently used s tissue homogente. Erythroytes nd liver lipids were extrted with hloroform-methnol (2:1 v / v ) ording to the method desried y Folh et l. [2]. Biohemil nlysis Erythroyte nd hepti Phospholipids ws estimted using the proedure desried y Stewrt [24]. Triglyeride onentrtion ws determined following the protool desried y Crr et l. [25]. Totl holesterol onentrtion ws estimted ording to the method of Allin et l. [26], nd HDL-holesterol ording to Wrmik et l. [27]. LDL-holesterol ws evluted using Friedewld s eqution [28]. Asprtte minotrnsferse (AST) nd lnine minotrnsferse (ALT) tivities were determined using Rndox dignosti kits (Rndox Lortory Limited, Antrim, UK).

4 Olu Lipids in Helth nd Disese (219) 18:12 Pge 4 of 9 Estimtion of -hydroxy--methyl-glutryl CoA (HMG- CoA) redutse tivities The rtio of the onentrtion of -hydroxymethyl-glutryl CoA (HMG CoA) to mevlonte in the liver ws used s mesure of the tivity of HMG-CoA redutse [29]. Sttistil nlysis Results re shown s men ± SEM. Men of the vrious groups were ompred y using One-Wy Anlysis of Vrine (ANOVA) followed y Turkey s multipleomprisons. Results with p <.5 were onsidered signifint. Results From the 2 g powdered smple G. luidum used in this study, 45. g GT ws extrted. This trnsltes to 2.27 g GT per g G. luidum powder. Body weight hnge ws signifintly higher in mie dministered hloroquine lone nd hloroquine in omintion with mg/kg Gnoderm terpenoid extrt ompred with infeted untreted mie (Tle 1). The oserved differene in ody weight hnge etween mie dministered hloroquine nd those dministered GT t mg/kg ws not signifint. The reltive liver, kidney, spleen nd rin weights were not signifintly different etween mie dministered GT ( nd 25 mg/kg) ompred to hloroquine (Tle 1). The survivl rte of the mie in response to Plsmodium erghei hllenge ws signifintly inresed from 66 to 87% due to omined dministrtion of Gnoderm terpenoid extrt t mg/kg nd ompred with when the sme dose of the extrt ws dministered lone nd from 62 to 75% due to dministrtion of the extrt t 25 mg/kg in omintion with ompred with when the extrt ws dministered lone (Fig. 1 nd, respetively). Signifint redution in perentge prsitemi from 21.7 to 1.5% due to omintion of Gnoderm extrt t mg/kg with hloroquine (Fig. 1 nd d, respetively). Erythroyte triglyeride (Fig. 2), totl holesterol (Fig. 2), LDL-holesterol (Fig. 2d) nd totl phospholipid (Fig. 2e) onentrtions were signifintly lower in GT + -treted mie ompred to infeted ut untreted mie (). No signifint ltertion ws oserved in erythroyte HDL-holesterol level etween the treted mie groups nd the untreted mie (Fig. 2). Liver holesterol (Fig. ) nd Phospholipid (Fig. ) onentrtions were signifintly lower in mie dministered GT + ompred to infeted ut untreted mie. -Hydroxymethylglutryl-CoA redutse (HMG-CoA redutse) tivity expressed s rtio of [HMG-CoA]/[mevlonte] ws signifintly higher mie dministered Gnoderm terpenoid extrt ( nd 25 mg/kg) in omintion with ompred to those dministered lone nd the infeted ut untreted mie (Fig. ). Serum sprtte minotrnsferse (AST) (Fig. 4) nd lnine minotrnsferse (ALT) (Fig. 4) tivities were signifintly lower in GT + -treted nd -treted mie groups ompred to infeted ut untreted mie. Disussion Gnoderm luidum hs een onsumed for severl yers in ountries suh s Jpn, Chin nd North Kore due to its mny reognized helth enefits []. In Nigeri nd her neighoring West Afrin sttes, mushroom re onsumed for their nutritionl enefit rther thn for their mediinl vlues. The omponent prts of this mushroom inluding the myelium, fruit odies nd spores hve een demonstrted to exhiit different iotive properties inluding ntioxidnt [], ntitumor [9], nti-inflmmtory [1], ntidieti [1], ntimiroil nd ntimlril [12 14] tivities, nd they re urrently eing deployed in the mngement of diseses suh s ner hypertension), immunosuppression nd hyperholesterolemi [2]. Bsed on the positive ttriutes of mushroom onsumption on niml nutrition nd helth, inrese wreness on the inlusion of mushroom in our dily mel is wrrnted. As suh, reserh dt demonstrting the potentil ttriutes of these mushrooms in niml nd humn studies will go long in hieving this set gol. The 2.27% yield of GT otined in this study is higher ompred to 1.67% reported y Go et l. [21]. The present study, demonstrted the nti-plsmodil tivity of Gnoderm terpenoid extrt dministered either singly or in omintion with hloroquine in enhning the Tle 1 Body weight nd reltive orgn weights of Plsmodium erghei-infeted mie dministered omintion of Gnoderm terpenoid extrt nd hloroquine for period of 12 dys GT + GT 25 + Body weight hnge (g) 12. ± ±.7, 8.6 ± ±.2 Reltive orgn weight Liver.8 ±.1.8 ±.1.8 ±.1.1 ±.1 Kidney. ±.1. ±.1. ±.1.4 ±.1 Spleen.7 ±.1.7 ±.1.7 ±.1.1 ±.1 Brin. ±.1. ±.1. ±.1. ±.1 Vlues re expressed s mens ± SEM of 5 or 8 determintions. Vlues in the sme rows rrying different supersript re sttistilly signifint (p <.5)

5 Olu Lipids in Helth nd Disese (219) 18:12 Pge 5 of 9 Perent survivl (%) GT GT25 5 Perent survivl 5 Prsitemi (%) 5 1 Time (dy) GT25 GT d Prsitemi (%) 5 1 Time (dy) Time (dy) Time (dy) Fig. 1 Perentge survivl of Plsmodium erghei infeted mie dministered Gnoderm terpenoid extrt (GT) lone () nd omintion of GT plus hloroquine () with their orresponding prsitemi profile () nd (d) respetively. Vlues re mens ± SEM of 5 or 8 determintions survivl rte of mie infeted with P. erghei in ddition to drstilly inhiiting the prsite growth nd survivl in mie. Signifint improvement in ody weight oserved in the infeted mie dministered Gnoderm terpenoid extrt in omintion with hloroquine further ttested to the positive helth sttus of the niml. The effiy of triterpenes extrted from different fungi s potentil nti-plsmodil gents hs een reported y severl uthors. In study y Goulrt et l. [], terpenoid relted moleules suh s frnesol, nerolidol, limonene nd linlool were demonstrted to inhiit the prolifertion of P. fliprum in vitro t the intrerythroyti stges. In similr study, Jordão et l. [4] demonstrted tht nerolidol inhiits the de novo synthesis of the isopreni hin tthed to the enzoquinone ring during the intrerythroyti stges of P. fliprum. Limonene, triterpene ws lso reported to inhiits protein isoprenyltion in the sme stges of the prsite [5]. Hlogented monoterpenes extrted from the mrine red lg, Plomium ornutum (Plomiee) exhiited signifint nti-plsmodil tivity ginst hloroquine-sensitive strin of P. fliprum [6]. Similrly, the eremophilne sesquiterpenoids, erklesmins A nd C, isolted from Berklesminum nigropile, were reported to exhiit in vitro ntiplsmodil tivity with IC 5 vlues of 6. nd 5.4 μg, respetively [7]. Lnostnes isolted from the ethyl ette extrt of Gnoderm luidum ws reported to exhiit in vitro nti-plsmodil tivity with IC 5 vlues of etween 6 to 2 μm [8]. In seprte study, grihomronne D, nother lnostne from Grini ymos (Clusiee) showed seletive tivity ginst P. fliprum with IC 5 of 7.7 μm) [9]. Results from this study showed tht erythroytes lipids inluding triglyerides, holesterol, LDL-holesterol nd phospholipid were signifintly lower in infeted mie dministered Gnoderm terpenoid extrt in omintion with hloroquine ompred with infeted ut untreted mie. The oservtion tht this trend ws not repeted in mie treted with hloroquine lone seems to suggest tht the this hypolipidemi tion ould e ttriuted to Gnoderm terpenoid extrt. Severl studies hve reported the hypolipidemi tivity of different extrts of Gnoderm speies. Olu et l. [12] hd erlier oserved positive orreltion etween serum nd liver lipoprotein holesterol onentrtion nd prsitemi level in Plsmodium erghei infeted mie treted with queous extrt of Gnoderm luidum. The mlri prsites hve high requirement for holesterol nd phospholipids for its survivl in the humn host [4]. Cirulting HDL-holesterol prtiles nd erythroyti memrne re the potentil soures of holesterol nd phospholipids for these prsites [4]. Erythroyte

6 Olu Lipids in Helth nd Disese (219) 18:12 Pge 6 of 9 Conentrtion (mg/dl) Conentrtion (mg/dl) 5 Conentrtion (mg/dl) Dy (post-infetion) d Conentrtion (mg/dl) Dy (post-infetion) e Conentrtion (mg/dl) Dy (post-infetion) Dy (post-infetion) Dy (post-infetion) Fig. 2 Erythroyte triglyeride (), totl holesterol (), HDL-holesterol (), LDL-holesterol (d) nd phospholipid (e) of Plsmodium erghei infeted mie dministered terpenoid extrt of Gnoderm luidum in omintion with hloroquine for period of 12 dys. Vlues re mens ± SEM of 5 or 8 determintions. Brs rrying different lphets re signifint (p <.5) phospholipids ontent hs een demonstrted to inrese 5 folds following mlril infetion [41]. During the lte stge of the prsite development, infeted erythroytes ontin 5 times more phospholipids thn uninfeted ells [17]. Vil et l. [42] lso reported tht the infeted erythroytes ontin phospholipid synthesizing enzymes. Thus, potent inhiitors of plsmodil phospholipid synthesis ws previously hrterized s potentil trget for nti-mlril hemotherpy due to its ruil role to the prsite survivl [19]. It ws lso importnt to evlute the totl holesterol nd phospholipid ontents of the liver euse the exoerythroyti stge of the mlri prsite life yle ours in the liver. It hs een shown tht therpeuti gents trgeted t this stge of the prsite life yle will go long wy in preventing linil episodes of mlri. Results from this study showed tht Gnoderm terpenoid extrt does not only exert its hypolipidemi tion in the erythroytes ut lso in the liver. Liver totl holesterol nd phospholipid ontents were signifintly redued due to dministrtion of the extrt in P. erghei infeted mie. Similr to wht otined in the erythroyte, hloroquine dministrtion does not ffet liver phospholipid nd totl holesterol onentrtions. Thus, suggesting tht hloroquine my e ting through other mehnisms other thn redution in hepti lipids. In ddition, hepti HMG-CoA redutse, the rte limiting enzyme in de novo holesterol synthesis mesured s

7 Olu Lipids in Helth nd Disese (219) 18:12 Pge 7 of 9 Conentrtion (mg/g tissue) Conentrtion (mg/g tissue) Dy (post-infetion) Dy (post-infetion) Ativity ([HMG CoA]/[mevlonte]) Dy(post-infetion) Fig. Liver totl holesterol onentrtion (), phospholipid onentrtion () nd HMG-CoA redutse tivity () of Plsmodium erghei infeted mie dministered terpenoid extrt of Gnoderm luidum in omintion with hloroquine for period of 12 dys. Vlues re mens ± SEM of 5 or 8 determintions. Brs rrying different lphets re signifint (p <.5) Ativity (U/mL) Ativity (U/mL) Dy (post-infetion) Dy (post-infetion) Fig. 4 Serum sprtte minotrnsferse, AST () nd lnine minotrnsferse, ALT () tivities of Plsmodium erghei infeted mie dministered terpenoid extrt of Gnoderm luidum in omintion with hloroquine over period of 12 dys. Vlues re mens ± SEM of 5 or 8 determintions. Brs rrying different lphets re signifint (p <.5). Legend: Note: : Infeted ontrol, infeted mie dministered 1 ml dimethyl sulfoxide (DMSO); GT : Infeted mie dministered Gnoderm terpenoid extrt t mg/kg ody weight/mouse/dy; GT 25 : infeted mie dministered Gnoderm terpenoid extrt t 25 mg/kg ody weight/mouse/dy;, Infeted mie dministered hloroquine t mg/kg ody weight/mouse/ dy; Note: : Infeted ontrol, infeted mie dministered 1 ml dimethyl sulfoxide (DMSO); GT + : Infeted mie dministered omintion of Gnoderm terpenoid extrt t mg/kg plus mg/kg hloroquine/mouse/dy; GT 25 + : Infeted mie dministered omintion of Gnoderm terpenoid extrt t 25 mg/kg plus mg/kg hloroquine/mouse/dy

8 Olu Lipids in Helth nd Disese (219) 18:12 Pge 8 of 9 rtio of HMG-CoA onentrtion to mevlonte onentrtion ws signifintly higher in the extrt treted mie. This is inditive of the ft tht the extrt inhiits the onversion of the sustrte, HMG-CoA to mevlonte y the enzyme HMG-CoA redutse. Tht is the extrt inhiits hepti holesterogenesis. This oservtion grees with the report of Wng et l. [4] who hd erlier demonstrted tht lnostne triterpenes isolted from the fruit odies of Gnoderm leuoontextum from Tiet, inhiited HMG-CoA redutse tivity in vitro in rt pig mirosomes. In nother study, Hjjj et l. [44] reported tht 26-oxygenosterols isolted from G. luidum inhiited the enzyme lnosterol 14α-demethylse whih tlyzes the onversion of 24,25-dihydrolnosterol to holesterol in humn hepti ell lines. In order to sertin the potentil toxiity of Gnoderm terpenoid extrt used in this study, serum AST nd ALT tivities were mesured. Dt otined showed tht mie dministered Gnoderm terpenoid extrt hd signifintly lower AST nd ALT tivities ompred with untreted ontrol. The enzymes AST nd ALT re memrne-ound enzymes nd re routinely used s iomrkers to estimte the extent of dmge to the liver. Inrese levels of this enzymes in the serum or plsm is inditive of potentil dmge to hepti ells thus ringing out their lekge to the plsm. Thus, high serum AST nd ALT tivities re reognized mrkers of ellulr dmge nd funtionl integrity of liver ell memrne [45, 46]. In the present study, Gnoderm terpenoid extrt ws oserved to protet the liver ginst P. erghei-indued dmge. This is demonstrted in the signifint redution in serum AST nd ALT tivities in P. erghei-infeted mie treted with the GT extrt. In ddition, the reltive liver, kidney, spleen nd rin weights of mie treted with the extrt ws not different from tht otined for hloroquine-treted mie. This oservtion is in greement with the reports y severl uthors. Olu et l. [12, 14] reported tht rude queous nd ethnoli extrts of G. luidum showed heptoprotetion ginst P. erghei-infeted mie. Similrly, Wu et l. [47] showed tht Gnoderm triterpenoids demonstrted heptoprotetion ginst oxidtive dmge indued y tert-utyl hydroperoxide in humn hepti HepG2 ells. Conlusion This study demonstrted the ntiplsmodil tivity of Gnoderm terpenoid extrt through its redution in prsitemi nd thus improved survivl rte in mie infeted with P. erghei. It ws lso demonstrted tht hloroquine potentites the urtive effet of Gnoderm terpenoid extrt on P. erghei in mie. Furthermore, Gnoderm terpenoid extrt exhiited signifint hypolipidemi effet through redution in infeted erythroyte lipids (triglyerides, totl holesterol, LDL-holesterol nd phospholipid) nd this is suggested s possile mehnism for its nti-plsmodil tivity. Arevitions ALT: Alnine minotrnsferse; AST: Asprtte minotrnsferse; : Chloroquine; DMSO: Dimethylsulfoxide; GT: Gnoderm terpenoid extrt; HDL: High-density lipoprotein; HMG-CoA: -hydroxy -methylglutryl- CoA; IMRAT: Institute for Advned Medil Reserh nd Trining; INF- CTR: Infeted ontrol; LDL: Low-density lipoprotein; PBS: Phosphte uffered sline; UCH: University College Hospitl Aknowledgements The gift of Plsmodium erghei y the Institute for Advne Medil Reserh nd Trining (IAMRAT), University College Hospitl, Idn, Nigeri is well knowledged. Funding The study reeived no externl funding. Avilility of dt nd mterils All dt nd mterils were ville in this study. Author s ontriutions OMO is responsile for the design, experimentl nlyses, dt nlysis nd interprettion nd drfting of the mnusript. The uthor red nd pproved the finl mnusript. Ethis pprovl nd onsent to prtiipte Approvl for the study ws grnted y Joseph Ayo Blol University Reserh nd Ethis Committee with pprovl numer JABU/REC/AS. Consent for pulition Not pplile. Competing interests The uthor delres tht he\she hs no ompeting interests. Pulisher s Note Springer Nture remins neutrl with regrd to jurisditionl lims in pulished mps nd institutionl ffilitions. Reeived: 5 June 218 Aepted: 18 Deemer 218 Referenes 1. World Helth Orgniztion. World helth sttistis. World Helth Orgniztion; 2. ISBN Ridley RG. Chemotherpeuti hope on the horizon for Plsmodium vivx mlri? Pro Ntl Ad Si. 22;99(21): Philipson JD, Wright CW. Antiprotozol ompounds from plnts soure. Plnt Med. 199;57: Klymn DL, Lin AJ, Aton N, Sovill JP, Hoh JM, Milhous WK, Theohrides AD, Doek AS. Isoltion of rtemisinin (qinghosu) from Artemisi nnu growing in the United Sttes. J Nturl Prod. 1984;7(4): Crgg GM, Newmn DJ. Nturl produt drug disovery in the next millennium. Phrm Biol. 21;9(sup1): Zjwiony KJ. Biologilly tive ompounds from Aphyllophorles (polypore) fungi. J Nt Prod. 24;67: Wsser SP, Cotes P, Blkmn M, Crgg G, Levine M, Moss J, White J. 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