HPLC quantification of phenolic content and assessment of methanolic extract of Antiaris africana for toxicological study

Transcription

1 Vol. 15(9), pp , 2 Mrh, 2016 DOI: /AJB Artile Number: 36363D ISSN Copyright 2016 Author(s) retin the opyright of this rtile Afrin Journl of Biotehnology Full Length Reserh Pper HPLC quntifition of phenoli ontent n ssessment of methnoli extrt of Antiris frin for toxiologil stuy Omotyo B. Ilesnmi 1,2 *, Tolulope M. Olleye 1, Afolbi C. Akinmolun 1 n Tiwo T. Alwoe 3 1 Deprtment of Biohemistry, Shool of Sienes, Feerl University of Tehnology Akure, Ono stte, Nigeri. 2 Deprtment of Biologil Sienes, Fulty of Siene, Feerl University Otuoke, Byels ste. 3 Deprtment of Chemil Siene, Fulty of Siene, Feerl University Otuoke, Byels Stte. Reeive 29 June, 2015; Aepte 17 Deember, 2015 The stuy ws ime t evluting the toxiologil n ntioxint tivities of Antiris frin Engl. (fmily Moree), tht is use in Nigeri n other West Afri ountries s pne for the tretment of severl ilments. The methnoli extrt of A. frin (MEA) obtine ws nlyse for ntioxint tivities in vitro n sreene for vrious phytohemils present. Phenoli n flvonoi ontents were etermine followe with high performne liqui hromtogrphy -ioe-rry etetion (HPLC-DAD) fingerprinting of phenoli ontent. Furthermore, the sub-ute toxiity of MEA ws etermine vi orl ministrtion of vrying oses for 14 onseutive ys (0,,, 200 n 400 mg/kg) in rts. After orl ministrtion for 14 onseutive ys in mle rts, the toxiity effet ws ssye by etermining sprtte minotrnsferse (AST) n lnine minotrnsferse (ALT) for hepti funtion; ure n retinine for renl funtion; retinine kinse (CK) for ri funtion; n lipi profile. HPLC results showe tht the mjor phenolis present re queretin, rutin, ffei i, grli i n queretin. MEA ws ble to svenge iphenyl piryl hyrzyl, hyroxyl n nitri oxie rils n prevent lipi peroxition inue by ferrous sulphte t ll onentrtion teste. The toxiology investigtion showe tht t low oses, A. frin is non-toxi, while t high oses; it is moertely toxi to the nimls. In onlusion, A. Afrin is generlly non-toxi; however, re must be tken in ministrtion t higher oses. Key wors: Toxiology, HPLC, phytohemils, Antiris frin. INTRODUCTION Herbl meiine is gining groun s the tretment of hoie in the western worl (Dey n De, 2015). Most ountries in Afri n other eveloping ountries rely on its usge for their primry helthre (Eisenberg et l., 1998; Worl Helth Orgniztion, 2008). Nigeri hs been plnning on integrting herbl meiine s egree progrm into the tertiry institution urriulum (Vngur, 2014). This evelopment is not surprising, looking t the *Corresponing uthor. E-mil: ilesnmi.omotyo@gmil.om. Tel: Author(s) gree tht this rtile remins permnently open ess uner the terms of the Cretive Commons Attribution Liense 4.0 Interntionl Liense

2 Ilesnmi et l. 321 ft tht tritionl meiine hs plye n importnt role in isese tretment sine the emergene of mn s evolution (Ahm et l., 2013; Pnkj et l., 2009). Most rugs hve their origin from nturl soures, either they re iretly isolte from plnts or the isolte ompoun is moifie to improve its effiy (Fu et l., 2013). Most nturl ompouns re mjorly lssifie s lklois, sterois, tnnins, phenoli ompouns, flvonois n sponin (Bishnu et l., 2009). Antiris frin is plnt foun in vrious prts of Nigeri n West Afri. It is ommonly lle Ooro, Oriro or ko Iroko in the South West prt of Nigeri; Frin Loko in the North; n Ojinwu in the South Est. The plnt is lrge tree usully bout 15 to 20 m high, but it n grow sometimes up to 40 m, n hs white ltex n lternte issymmetri leves (Berg et l., 1985; Berhut, 1979), with hevy flt rown n blothy grey n white brk. The flowers re smll with greenish white olor tht proues re velvety fruits (Gill, 1992). It hs wie usge both in inustry (timber mking) n tritionl meiine. Vrious prt of the plnt suh s leves, stems n brks re ethnobotnilly use in the tretment of vrious iseses suh s rheumti n respirtory infetion (Gill, 1992; Mnn et l., 2003), epilepsy, lumbrgo, skin irritnt, purgtive, hest pin (Okogun et l., 1976), syphilis (Berhut, 1979), throt infetion, leprosy, ner (Kuete et l., 2009), n nervous isorers in the northern prt of Nigeri (Moronkol n Fruq, 2013). However, the report on the inustril usge hs overshowe its ethno phrmologil usge s more works re publishe on it s ompre to the ltter. This hs le to erth of report on the sientifi rtionle behin its ethnophrmrologil usge. Therefore, this experiment ws onute to give sientifi insight to its tritionl usge. MATERIALS AND METHODS Chemils Queretin, rutin, sorbi i, tnni i, Folin-Ciolteu regent, soium rbonte, luminum hlorie, potssium ette, eoxyribose, 1,1, iphenyl 2,2- piryl hyrzyl (DPPH), were obtine from Sigm (Chemil Co, St. Louis, MO, USA). ALT, AST, retinine (CREA), ure, CK, totl holesterol, triglyerie n high ensity lipoprotein kits were from Rnox. All other regents were of nlytil gre. All UV Vis mesurements were reore on Shimzu UV Plnt olletion n preprtion of extrt Leves of A. frin were ollete t Forest Reserh Institute of Nigeri (FRIN) gren n uthentite t the herbrium of Botny Deprtment, University of Ibn, Nigeri by Mr. Esimkhir (vouher number M). The leves were ir rie n pulverize. The powere smple (1.23 kg) ws merte in 5 L of 80% methnol for 72 h n then filtere. The filtrte ws onentrte n then lyophilize to obtin the 81.1 g methnol extrt of A. frin (MEA) use for the stuy. This ws store in n mber bottle n refrigerte. Phytohemil sreening Extrts were phytohemilly sreene for the presene of lklois, sponins, tnnins, phlobtnnins, nthrquinones, flvonois, sterois, n terpenois using stnr lbortory proeures (Sofowor, 1993; Trese n Evns, 1985). Estimtion of totl phenoli ontent The totl phenoli ontent of the extrt ws estimte oring to moifie proeure of Singleton et l. (1999). Briefly, eionise wter (0.5 ml) n 125 μl of Folin Collteu regent were e to 125 μl of MEA issolve in istille wter. The mixture ws llowe to stn for 6 min before ing 1.25 ml of 7% (w/v) N 2CO 3 solution. The retion mixture ws then llowe to stn for itionl 90 min before tking the bsorbne t 760 nm ginst the blnk. The tnni i stnr urve ws prepre by ing 125 μl of tnni i issolve in istille wter (2, 4, 8 n 10 µg/ml finl onentrtions) in lieu of extrt. The mount of totl phenolis ws expresse s tnni i equivlents (TAE, mg tnni i/g smple) through the librtion urve of tnni i. Estimtion totl flvonoi ontent Flvonoi ontent ws estimte using the luminum hlorie olorimetri metho (Chng et l., 2002). The plnt extrt in methnol (1 g/ml) ws mixe with 0.1 ml of 10% luminum hlorie (w/v), 0.1 ml of 1 M potssium ette n 2.8 ml istille wter. The mixture ws llowe to stn t room temperture for 30 min. The bsorbne of the retion mixture ws re t 415 nm. Results were expresse s mg/g queretin equivlent (QE). Quntifition of ompouns in extrt by HPLC-DAD HPLC fingerprinting n reverse phse hromtogrphi nlyses were rrie out uner grient onitions using gilent elipse plus C 18 olumn (4.6 x 1 mm) pke with 5 μm imeter prtiles. Quntifition of phenols involve mobile phse me up of wter ontining 2% eti i (A) n methnol (B). The omposition of B ws vrie strting from the more polr (5% of B) until 2 min n inrese grully till % of B s esribe by the metho of Sbir et l. (2012), with slight moifition. All hromtogrphy opertions were rrie out t mbient temperture n in triplite. High performne liqui hromtogrphy (HPLC- DAD) ws performe with Shimzu Prominene Auto Smpler (SIL-20A) HPLC system (Shimzu, Kyoto, Jpn), equippe with Shimzu LC-20AT reiproting pumps onnete to DGU 20A5 egsser with CBM 20A integrtor, SPD-M20A ioe rry etetor n LC solution 1.22 SP1 softwre. Determintion of reuing power Vrying mounts of the extrt (10 to 800 µg) in 1 ml of istille wter were mixe with 2.5 ml of phosphte buffer (0.2 M, ph 6.6) n 2.5 ml potssium ferriynie (1%). The mixture ws inubte t C for 20 min. A portion (2.5 ml) of 10% TCA ws e to the mixture whih ws then entrifuge t 3000 rpm for 10 min. The upper lyer of the solution (2.5 ml) ws mixe with 2.5 ml of istille wter n 0.5 ml of 0.1% FeCl 3 n the bsorbne ws mesure t 700 nm. Inrese bsorbne of the retion mixture inite inrese reuing power.

3 322 Afr. J. Biotehnol. Nitri oxie ril svenging ssy µg of MEA ws e in the test tubes to 1 ml of soium nitroprussie solution (25 mm) n the tubes inubte t 37 C for 2 h. An liquot (0.5 ml) of the inubtion solution ws remove n ilute with 0.3 ml of Griess regent (1% sulphnilmie in 5% H 3PO 4 n 0.1% nphthylethyleneimine ihyrohlorie). The bsorbne of the hromophore forme ws immeitely re t 570 nm ginst istille wter s blnk with tehin ( µg) use s stnr. Results were expresse s perentge ril svenging tivity (RSA). Potentil to inhibit eoxyribose egrtion (eoxyribose ssy) µg of smple in µl of istille wter ws e to solution ontining 200 µl KH 2PO 4 KOH ( mm), 200 ul eoxyribose (15 mm), 200 µl FeCl 3 (0 µm) n µl EDTA (1 mm) in test tube n llowe to mix. The retion ws initite by ition of µl H 2O 2 (10 mm) n µl sorbi i (1 mm). The retion mixture ws inubte t 37 C for 1 h. At the en of the inubtion perio, 1 ml of 1% w/v TBA ws e to eh mixture followe by the ition of 1 ml of 2.8% w/v TCA. The solution ws hete in wter bth t 80 C for 20 min to evelop the pink olore MDA-(TBA) 2 ut. After ooling, the solution ws entrifuge n the bsorbne of the superntnt mesure t 532 nm ginst istille wter s blnk. Results were expresse s the perentge inhibition of eoxyribose egrtion. Inhibition of Fe 2+ /sorbte inue lipi peroxition Liver homogente from rt ws prepre by removing liver immeitely fter srifie. The liver ws rinse in ie-ol 1.15% KCl to remove bloo stin, blotte n weighe. The weighe tissue ws then homogenize in four volumes of ie-ol 0.1 M phosphte buffer; ph 7.4. The retion mixture ontining 0.1 ml of liver homogente in 30 mm tris buffer, 0.16 mm ferrous mmonium sulphte, 0.06 mm sorbi i n ifferent mount of the extrt (10-0 µg), ws inubte for 1 h t 37 C. The resulting thiobrbituri retive speies (TBARS) ws mesure by the metho of Vrshney n Kle (1990). An liquot (0.4 ml) of the retion mixture ws mixe with 1.6 ml of 0.15 M tris-kcl buffer to whih 0.5 ml of 30% TCA ws e. Then 0.5 ml of 0.75% TBA ws e n ple in wter bth for 30 min t 85 C, fter whih it ws oole in ie n entrifuge t room temperture for 3 min t 3000 g. The bsorbne of the ler superntnt ws mesure ginst referene blnk of istille wter t 532 nm. Animl hnling n tretment Animls Albino rts (Wistr strin) ge four weeks were obtine from the niml house of the Lgos University Tehing Hospitl n fe with ommerilly vilble stnr pellete fee n wter libitum throughout the perio of experiment. All niml experimentl protools onforme to the interntionl guie for the re n use of lbortory nimls (Ntionl Reserh Counil, 2011). Rts were rnomly ivie into five groups with six nimls per group. Group I (ontrol) reeive norml sline (0.9% NCl) orlly. Groups II, III, IV n V reeive MEA (,, 200 n 400 mg/kg/y, respetively) orlly for 14 onseutive ys. Twenty four hours fter the lst ministrtion, bloo smple ws ollete vi ervil islotion for the evlution of mrkers of oxitive stress n hepti, renl n ri funtions. There were no observble physil hnges in nimls ministere MEA s ompre with the ontrol throughout the urtion of the experiment. ASSAY Serum olletions At the en of 14-y perio, bloo ws obtine vi ri punture uner light hlorohyte nesthesi. Bloo ws ollete in serum bottles. The bloo ws entrifuge t 4000 rpm t 4 C for 10 min to obtin the serum, whih ws store t -20 C until nlysis for biohemil prmeters. Biohemil estimtions The tivities of lbumin, AST, ALT, ALP, CK, CREA, ure, triglyerie (TG), totl holesterol (TC), high ensity lipoprotein (HDL) n low ensity lipoprotein (LDL) were estimte using ssy kits from Rnox Lbortories Lt., UK oring to the instrutions of the mnufturer. Sttistil nlysis Results re expresse s men ± stnr evition. Differenes between groups were etermine by one-wy nlysis of vrine (ANOVA) using SPSS softwre pkge for winows. Post ho testing ws performe for inter-group omprisons using the lest signifint ifferene (LSD) test n p-vlue < 0.05 ws onsiere signifint. RESULTS HPLC nlysis HPLC fingerprinting of A. Afrin extrt revele the presene of the grli i (t R = min; pek 1), tehin (t R = min; pek 2), hlorogeni i (t R = min; pek 3), ffei i (t R = min; pek 4), ellgi i (t R = min; pek 5), epigllotehin (t R = min; pek 6), rutin (t R = min; pek 7), isoqueritrin (t R = min; pek 8), queritrin (t R = min; pek 9), queretin (t R = min; pek 10) n kempferol (t R = min; pek 11) (Figure 1 n Tble 3). Phytohemil sreening revele the presene of sponin, tnnin, phlobtnnin, flvonoi n terpenoi in the methnol extrt of A. frin (MEA) leves. Phytohemil sreening lso showe tht lkloi is bsent in the MEA (Tble 1) while quntifition of totl phenoli ontent TPC ws ±13.18 mg TAE/g extrt n totl flvonois ws ±9.28 mg QE/g extrt (Tble 2). HPLC DAD revels the presene of glli i, tehin, hlorogeni i, ffei i, ellgi i, epigllotehin, rutin, isoqueritrin, queretin n kempferol. Cffei i is the most bunnt phenoli ompoun (35.97 ± 0.02 mg/g extrt) n rutin is the most bunnt flvonoi (30.37 ± 0.04 mg/g extrt) (Tble 3 n Figure 1).

4 Ilesnmi et l. 323 Figure 1. Representtive high performne liqui hromtogrphy profile of Antiris frin extrt: glli i (pek 1), tehin (pek 2), hlorogeni i (pek 3), ffei i (pek 4), ellgi i (pek 5), epigllotehin (pek 6), rutin (pek 7), isoqueritrin (pek 8), queritrin (pek 9), queretin (pek 10) n kempferol (pek 11). Chromtogrphi onitions re esribe in the methos setion. Tble 1. Phytohemil onstituents of Antiris frin. Phytohemil Alkloi Sponin Tnnin Phlobtnnins Anthrquinone Flvonoi Terpenois Observtion Absent Present Present Present Absent Present Present The ntioxint tivity of plnts is generlly ttribute to the presene of phytohemils present. Free ril svenging n inhibition of TBARS re one of the importnt ssys for the etermintion of ntioxint tivity of plnt extrts. Hene, in orer to explore n unerstn these possible mehnisms, severl ntioxint ssys inluing NO, DPPH n OH ril svenging ssys were performe n evlution of the ntioxint tivities of the results onfirme tht this plnt hs bro rnge of ntioxint properties, inluing substntil inhibition of lipi peroxition. Anlysis of the free ril svenging of NO (Figure 2), DPPH (Figure 3) n OH (Figure 4) of the extrts revele onentrtion-epenent ntiril tivity resulting in the onversion of the rils to non- ril form. MEA signifintly svenge hyroxyl ril genertion s observe in the perentge inrese in prevention of eoxyribose egrtion t ll onentrtion. Also, MEA showe onentrtion-epenent nti- DPPH ril svenging bility. It thus ppers tht the extrts possess hyrogen onting bility n t s ntioxint. However, the svenging bility of queretin, known ntioxint use s positive ontrol ws greter thn tht of the extrts. The ntioxint tivity ws further nlyze by the TBARS metho, whih is use to quntify lipi peroxition tht orrespons to ell membrne mge use by oxitive stress. The Fe (II) inue stimultion in brin TBARS levels. The MEA t ll teste onentrtion ws ble to prevent lipi peroxition s observe in the reution in the mount of TBARS forme (Figure 5), though it ws not s potent s the referene rug (queretin). Reuing power bility One of the key tivities of ntioxint is their bility to onte eletrons n thus reuing rils to less tive speies. This is mesure by the reution of Fe 3+ to Fe 2+ by onting n eletron. Amount of Fe 2+ formtion n be monitore spetrophotometrilly. At onentrtion 400 μg/ml, the methnoli extrt ( ± 0.194) showe greter bsorbne thn sorbi i ( ± 0.338). Reuing power of the MEA is shown in Figure 6. Renl funtion inies Figure 7 shows the effet of MEA on renl funtion. All oses of MEA h no signifint effet on ure level in the serum s ompre to the ontrol (P<0.05), with the ure level flling between ± 0.4 n ± 0.72 mg/l, exept 200 mg/kg ure level of 6.20 ± 1.51 mg/l, while the retinine level ws signifintly erese in the serum s ompre to the ontrol (P<0.05) with the onentrtion flling between 0.46 ± 0.04 to 0.58 ± 0.06 mg/l. Hepti n ri funtion Figures 8 n 9 revel the AST, ALT n CK tivity respetively in nimls ministere MEA. There ws no signifint ifferene in the tivity of AST t ll oses s ompre to the ontrol, with the tivity flling between ± 2.8 to ± 3.5U/I. The serum ALT tivityvries with eh oses of extrt ministere when ompre to the ontrol. MEA t n mg/kg with 20.4 ± 0.19 n 14.8 ± 0.34 U/I, respetively ws not signifintly ifferent from the ontrol (P<0.05), while 200 n 400 mg/kg of MEA showe signifint elevtion in the tivity of ALT with 98.0 ± 0.41 n ± 0.33, respetively s ompre to the ontrol (P<0.05).

5 % NO svenge 324 Afr. J. Biotehnol. Tble 2. Effet of MEA on serum lipi profile. Group CHOL (mg/l) HDL (mg/l) TG (mg/l) LDL (mg/l) Control.28± ± ± ±3.71 mg/kg 41.94±6.43* 4.01±1.97* 17.45± ±6.12* mg/kg 37.02±3.04* 5.34±1.93* 21.94±7.48* 27.67± mg/kg 30.22±1.76* 11.95±4.72* 26.12±2.70* 13.05±4.17* 400 mg/kg 23.78±4.80* 9.30±1.40* 29.00±1.92* 8.67±3.03* Vlues re expresse s men ± stnr error of the men. *Signifintly ifferent from ontrol. Tble 3. Composition of Antiris frin extrt. A. Afrin ompouns Composition mg/g % LOD (µg/ml) LOQ (µg/ml) Glli i ± Ctehin ± 0.02b Chlorogeni i 8.36 ± Cffei i ± Ellgi i ± 0.03e Epigllotehin 4.51 ± 0.01f Rutin ± Isoqueritrin * 2.14 ± 0.03g Queritrin * ± 0.01b Queretin ± 0.03e Kempferol ± 0.02h Results re expresse s men ± stnr evitions (SD) of three etermintions. Averges followe by ifferent letters iffer by Turkey test t p < 0.05.*Quntifie ws queretin. LOD, Limit of etetion; LOQ, Limit of quntifition b b e rut 25 rut onentrtion (µg/ml) Figure 2. Nitri oxie (NO.- ) ril svenging tivity (RSA) of methnoli extrt of Antiris Afrin (MEA). Vlues represent the men ± S.E.M. of the vlues of inhibition in vitro; n = 3 experiments in uplite. P < 0.05 versus ontrol. Vlues with similr lphbets re not signifintly ifferent.

6 % inhibition of eoxyribose egrtion Ilesnmi et l. 325 Figure 3. DPPH svenging tivity of MEA. Vlues represent the men ± S.E.M. of the vlues of inhibition in vitro, n = 3 experiments in uplite. P < 0.05 versus ontrol. Vlues with similr lphbets re not signifintly ifferent. 1 e f 0 b onentrtion (µg/ml) Q 25 Q Figure 4. Inhibition of eoxyribose oxition (hyroxyl ril svenging tivity) by methnoli extrt of Antiris frin (MEA). Vlues represent the men ± S.E.M. of the vlues of inhibition in vitro; n = 3 experiments in uplite. P < 0.05 versus ontrol. Vlues with similr lphbets re not signifintly ifferent. Aministrtion of MEA t ll oses generlly h no signifint effet on CK level, with the tivity flling between ± 2.88 n U/I (P<0.05). Lipi profile Tble 2 summrizes the effet of vrying oses of MEA on lipi profiles in nimls. It shows tht MEA ws ble to signifintly erese totl holesterol, triglyerie n LDL- level in ose epenent mnner when ompre to the ontrol; while, ose epenent elevtion of HDL level when ompre to the ontrol ws lso observe. DISCUSSION The present stuy is esigne to investigte the

7 Absorbne (700nm) MDA (nmole/g tissue) 326 Afr. J. Biotehnol f b e onentrtion (µg/ml) Q 25 Q Figure 5. Effet of methnoli extrt of Antiris Afrin (MEA) on FeSO 4 inue lipi peroxition. Vlues represent the men ± S.E.M. of the vlues of inhibition in vitro; n = 3 experiments in uplite. P < 0.05 versus ontrol. Vlues with similr lphbets re not signifintly ifferent b AA onentrtion (µg/ml) AA Figure 6. Reuing power of methnoli extrt of Antiris frin (MEA). Vlues represent the men ± S.E.M. of the vlues of inhibition in vitro, n = 3 experiments in uplite. P < 0.05 versus ontrol. Vlues with similr lphbets re not signifintly ifferent. phytohemil onstituents present n the biohemil effet of orl ministrtion of MEA on the biomrkers of orgn toxiity. A. frin (AA) is plnt tht is ommonly use in Nigeri n other prt of West Afrin ountries s pne in the tretment of severl ilments (Kuete et l., 2009). The lol usge of AA in

8 ACTIVITY (U/I) Conentrtion (mg/l) Conentrtion (mg/l) Ilesnmi et l A * 1.5 B * * * * * 0 Control mg/kg mg/kg GROUP 200mg/kg 400mg/kg 0.0 Control mg/kg mg/kg GROUP 200mg/kg 400mg/kg Figure 7. The effet of MEA on mrker of renl toxiity A (Ure onentrtion); B (CREA onentrtion). Vlues re expresse s men ± stnr error of the men. *Signifintly ifferent from ontrol (P<0.05). 1 ** ** AST ALT 0 Control mg/kg mg/kg group 200mg/kg 400mg/kg Figure 8. Effets of MEA on mrkers of heptotoxi enzyme. Vlues re expresse s men ± stnr error of the men. *Signifintly ifferent from ontrol (P<0.05). the tretment of severl ilments hs been going on for ges pst in ifferent prt of Nigeri, however there hs been no sientifi report on the phytohemils present in it n proper sientifi report on its sfety oses upon orl ministrtion of the plnt. Phytohemil omponents suh s lklois, polyphenols, flvonoi, ri glyosie n sponin hve been reporte to be responsible for both phrmologil n toxi tivities in plnts (Akinmolun et l., 2010; Aggrwl et l., 2006). This neessitte preliminry investigtion to ientify the vrious phytohemils present, ssess its ntioxint potentils in vitro n possible toxiities of AA. Biomrkers of toxiity inlue ALT n AST for ytotoxiity n isturbne in hepti funtion; ure n retinine for isturbne in renl funtion; retinine kinse is for ri funtion n lipi profile s link to riotoxiity, ibetes n obesity. Phytohemil sreening revele the presene of sponins, tnnins, phlobtnnins, flvonois n terpenois. Phenoli ompouns re not foun in nimls, mjorly synthesize by plnts, they re seonry metbolites erive from the shikimte-phenylpropnoisflvonois pthwys. Flvonois, one of the lrgest groups of polyphenols hve been reporte to be of helth

9 ACTIVITY (U/I) 328 Afr. J. Biotehnol. 1 0 Control mg/kg mg/kg GROUP 200mg/kg 400mg/kg Figure 9. Effet of MEA on retinine kinse tivity. Vlues re expresse s men ± stnr error of the men. *Signifintly ifferent from ontrol (P<0.05). benefit to humn. Severl reports hs shown tht flvonoi possess ntioxitive, nti-inflmmtory, ntimirobil, rioprotetive n neuroprotetive effets (Petti n Sully, 2009; Asensi et l., 2011; Tmlli et l 2013) nti-llergeni, nti-therogeni, ntiinflmmtory, nti-mirobil, nti-thromboti, rioprotetive n vsoiltory effets (Petti n Sully, 2009; Asensi et l., 2011). These metbolites re si to be useful to plnt itself but n be toxi to nimls, inluing mn. Sponin is known ntinutritionl phytohemils tht possess the potentil to reue the uptke of ertin nutrients inluing holesterol n gluose t the gut through intrlumenl physiohemil intertion or other yet unientifie tivity (Prie et l., 1987), suggesting possible uses in the tretment of ibetes n riovsulr relte iseses; while, tnnin, phlobtnnin flvonoi n terpenois re polyphenoli ompouns tht hve been reporte to be responsible for most biologil tivity in plnts (Al-Sereiti et l., 1999). Attk of lipi n its erivtives by rils is one of the mehnisms by whih oxitive stress tke ple, leing to both ell membrne mge n ytotoxiity. Thus the bility of plnt extrt to inhibit lipi peroxition is regre s key initor of ntioxint tivities. Our t shows tht MEA inhibit lipi peroxition in onentrtion epenent mnner using brin homogente. Hyroxyl ril svenging pity of n extrt is iretly relte to its ntioxint tivity (Bbu et l., 2001). The bility of extrts to quenh hyroxyl rils seems to be iretly relte to the prevention of propgtion of the proess of lipi peroxition n they seem to be goo svengers of tive oxygen speies, thus terminting or reuing the rte of retion. MEA svenge the generte hyroxyl rils s observe in the prevention of 2-eoxy-2-ribose egrtion in onentrtion epenent mnner. The ntioxint potentil of plnt extrts n lso be etermine by the bility of plnt extrt to reue ferri ion to ferrous ion (Fe 3+ to Fe 2+ ) by eletron ontion terme reuing power (RP). The mount of Fe 2+ forme is now monitore spetrophotometrilly (Ebrhimzeh et l., 2008). Severl reserhers hve reporte iret orreltion between ntioxint tivities n reuing power of ertin plnt extrt (Akinmolun et l., 2010; Koni et l., 2010; Olleye et l., 2010). MEA showe reuing power t ll onentrtion. In the sfe ose stuy in rts given the MEA orlly t oses rnging from -400 mg/kg, ll the nimls in eh group showe no behviourl ifferene, though there ws n initil response to the MEA on first ministrtion whih ws normlize few hours lter. One of the key initors of verse retion to rugs n hemils is ltertion in boy weight (Sellers et l., 2007; Rz et l., 2002; Teo et l., 2002; Tofovi n Jkson, 1999). Aministrtion of MEA showe no signifint hnge in boy weight (not reporte). Biologil mrkers like enogenous enzymes hve been shown n estblishe to be orgn-speifi n n lek from mge or n injure orgn (Kubvt n Asq, 2009). Hepti funtion hs been monitore by the evlution of the levels of ALT n AST in onjuntion with biohemil nlytes suh s holesterol, retine kinse n retinine in the serum. The tivity of plnt extrt to lower the serum level of AST n ALT is one of the mehnism of meiting heptoprotetive or

10 Ilesnmi et l. 329 heptotoxiity effet of plnt extrt (Aewusi n Afolyn, 2010). Any mge to the hepti orgn shows n inrese in the serum level of AST n ALT (Olleye et l., 2010; Ahsn et l., 2009; Fkurzi et l., 2008; Liu et l., 2006). From the experiment, there ws vrying observtion in the serum level of AST n ALT s ompre to the ontrol; MEA showe no toxi effet on AST tivity t ll ministere oses, while the effet on ALT tivity showe tht n mg/kg osges were not toxi, however, ministrtion of 200 n 400 mg/kg ws toxi s observe in the signifint elevtion in serum ALT tivity. AST is generl mrker of toxiity, thus its lekge is not only epenent on hepti isturbnes, it n be implite in other tisuues s well. However ALT is mjor biomrker of hepti ytotoxiity (Jmes et l., 1993), thus its inrese tivity t 200 n 400 mg/kg is sign of toxiity of MEA t high osges. One of the risk ftors implite in the ourrene of oronry isese is high level of holesterol (hyperholesterolemi). Reently, it hs been reporte tht the onset of riovsulr events n be well ontrolle fter reuing the serum LDL-holesterol level using severl therpeuti gents (Cheng et l., 2004). MEA use signifint erese in the serum levels of triglyeries, totl holesterol, LDL holesterol, but inrese HDL-holesterol. One of the mehnism of rugs use in the tretment of riovsulr isese is in their bility to lower lipi level in the bloo (Nofer et l., 2001). Thus, it n be eue tht MEA possess hypolipiemi potentil n n be potentil rug for tretment of riovsulr relte iseses. This might be linke to probble role of MEA in lipi metbolism n lerne from the boy. The effet of ifferent plnt speies on lipi lowering hs been reporte s key ftor in their meiinl use (Kono et l., 1992; Niu n Thippeswmy, 2002; Devi n Shrm, 2004). Polyphenol is one of the most bunnt phytohemil present in plnts; it is bunnt in vrious prt of the plnts, suh s the leves, roots, stems n sees. One of the key fetures of the therpeuti uses of polyphenol is bse on its ntioxint properties, nti-inflmmtory, ntitumor n ntimirobil tivities (Xu et l., 2012; Wng et l., 2009). Phenolis re ompouns possessing one or more romti rings with one or more hyroxyl groups. They re broly istribute in the plnt kingom n re the most bunnt seonry metbolites of plnts, with more thn 8,000 phenoli strutures urrently known, rnging from simple moleules suh s phenoli is to highly polymerize substnes suh s tnnins (Di n Mumper, 2010; Hrborne n Willims, 2000). The min lsses of polyphenols, bse on their struture, re phenoli is, flvonois, stilbenes, n lignns. Flvonois ount for pproximtely two-thirs of the phenolis in our iet (Sthishkumr et l., 2008) n one of the most bunnt polyphenols known, it n be lssifie into six mjor groups; flvonols, flvones, flvnones, flvn-3-ols, nthoynins n isoflvones. In support of this,our results shows tht flvonoi is the most bunnt polyphenols present in A. frin. Polyphenols rise from the ommon intermeite phenyllnine, or preursor, shikimi i. Flvonois re the group of polyphenols most stuie n more thn 4,000 hve been ientifie n tegorize into six sublsses: flvonols, flvones, flvnones, flvn-3-ols, nthoynins n isoflvones. The interest in flvonois rises from stuies tht hve shown tht onsumption of foo rih in it protets ginst mny hroni iseses suh s liver mge, hroni kiney injury, ner, riovsulr iseses n neuroegenertive iseses. This protetive property is ttribute to their ntioxint, nti-inflmmtory n metl helting bilities (Di n Mumper, 2010). A vriety of phenoli ompouns, in ition to flvonois, re foun in fruit, vegetbles n mny herbs. Phenoli ompouns (suh s ffei, ellgi n feruli is, sesmol n vnillin) hve been reporte to hve n inhibitory on theroslerosis isese (Deker, 1995). HPLC fingerprinting revels the presene of queretin, rutin, isoqueretirin, ffei i n glli i. The presene of these hemil onstituents in this plnt, is n inition tht the plnt, if properly sreene, oul yiel rugs of phrmeutil signifine. Conlusion The orl ministrtion of MEA for 14 onseutive ys t oses rnging from -400 mg/kg showe no eth mong the nimls. In ition, no signifint effet ws observe in the niml behvior n tivities throughout the urtion of ministrtion, lso no verse effet ws observe in the orgn n boy weights of the nimls. From the biohemil prmeters, it n be eue tht MEA is sfe t low oses n signifintly reue holesterol level s evient from signifint reution in LDL prmeters. Conflit of Interests The uthors hve not elre ny onflit of interests. REFERENCES Aewusi EA, Afolyn AJ (2010). A review of nturl prouts with heptoprotetive tivity. J. Me. Plnts Res. 4(13): Aggrwl BB, Ihikw H, Groi P, Weersinghe P, Sethi G, Bhtt ID, Pney MK, Shishoi S, Nir MG (2006). From tritionl yurvei meiine to moern meiine: ientifition of therpeuti trgets for suppression of inflmmtion n ner. Expert Opin. Ther. Trgets 10: Ahm M, Chung PL, Gbriel AA, Nur NI, Hshim MA, Hor SY, Lee FA, Mun FY (2013). Sfety ssessment of stnrise methnol extrt of Cinnmomum burmnnii. Phytomeiine 20: Ahsn MR, Islm KM, Bulbul IJ (2009). Heptoprotetive tivity of Methnol Extrt of some meiinl plnts ginst rbon tetrhlorie-inue heptotoxiity in rts. Eur. J. Si. Res.

11 330 Afr. J. Biotehnol. 37(2): Akinmolun AC, Obuotor EM, Frombi EO (2010). Evlution of Antioxint n Free Ril Svenging Cpities of Some Nigerin Inigenous Meiinl Plnts. J. Me. Foo 13(2): Al-Sereiti MR, Abu-Amer KM, Sen P (1999). Phrmology of rosemry (Rosmrinus offiinlis Linn.) n its therpeuti potentils. Inin J. Exp. Biol. 37: Asensi M, Orteg A, Men S, Fei F, Estrel JM (2011). Nturl polyphenols in ner therpy. Crit. Rev. Clin. Lb. Si 48: Bbu BH, Shylesh BS, Pikkl J (2001). Antioxint n heptoprotetive effet of Alnthus iiifous. Fitoterpi 2: Berg CC, Hijmn MEE, Weerenburg JCA (1985). Flore u Cmeroun: Morées. Pris: Muséum Ntionl ʼHistoire Nturelle, Lbortoire e Phnérogmie. Berhut J (1979). Flore Illustree u Senegl: Diotyleones, Tome VI: Linées à Nymphéées. Dkr: Ministère u Développement Rurl et e lʼhyrulique. Bishnu J, Sunil L, Anuj S (2009). Antibteril property of ifferent meiinlplnts: Oimum sntum, Cinnmomum zeylnium, Xnthoxylum rmtum norignum mjorn. Kthmnu Univ. J. Si. Eng. Tehnol. 5(1): Di J, Mumper RJ (2010). Plnt Phenolis: Extrtion, Anlysis n Their Antioxint n Antiner Properties. Moleules 15: Deker EA (1995). The role of phenolis, onjugte linolei i, rnosine, n pyrroloquinoline quinone s nonessentil ietry ntioxints. Nutr. Rev. 53: Devi R, Shrm DK (2004). Hypolipiemi effet of ifferent extrts of Cleroenron olebrookinum Wlp in norml n high-ft iet fe rts. J. Ethnophrmol. 90: Dey A, De JN (2015). Neuroprotetive therpeutis from botnils n phytohemils ginst Huntington s isese n relte neuroegenertive isorers. J. Herb. Me. 5:1-19. Ebrhimzeh MA, Hosseinimehr SJ, Hmiini A, Jfr M (2008). Antioxint n free ril svenging tivity of Feijo sllowin fruits peel n leves. Phrmologyonline 1:7-14. Eisenberg DM, Dvis RB, Ettner SL, Appel S, Wilkey S, Vn Rompy M, Kessler RC (1998). Trens in lterntive meiine use in the Unite Sttes, : results of follow-up ntionl survey. JAMA 280(18): Fkurzi S, Hiruszh I, Nnthini U (2008). Moring oleifer Lm. prevents etminophen inue liver injury through restortion of glutthione level. Foo Chem. Toxiol. 46(8): Fu J, Di L, Lin Z, Lu H (2013). Houttuyni ort Thunb: A Review of Phytohemistry n Phrmology n Qulity Control. Chin. Me. 4: Gill LS (1992). Ethnomeil uses of plnts in Nigeri. University of Benin Press, Benin City, Nigeri, 12pp. Hrborne JB, Willims CA (1992). Avnes in flvonoi reserh sine Phytohemistry 55: Jmes RC, Hrbison RD, Roberts SM (1993). Phenylpropnolmine Potentition of Aetminophen-Inue Heptotoxiity: Eviene for Glutthione-Depenent Mehnism. Toxiol Appl Phrmol. 118(2): Koni MZ, Kremer D, Gruz J, Strn M, Bisev G, Kosle I (2010). Antioxint n ntimirobil properties of Moltki petre (trtt.)griseb. flower, lef n stem infusions. Foo Chem. Toxiol. 48: Kono S, Shinhi K, Ike N, Yni F, Imnishi K (1992). Green te onsumption n serum lipi profiles: ross-setionl stuy in northern Kyushu, Jpn. Prev. Me. 21: Kubvt JB, Asq SM (2009). Role of Si orifoli L. leves on biohemil n ntioxint profile uring myoril injury. J. Ethnophrmol. 124: Kuete V, Vouffo B, Mbveng AT, Vouffo EY, Sigt RM, Dongo E (2009). Evlution of Antiris frin methnol extrt n ompouns for ntioxint n ntitumor tivities. Phrm. Biol. 47(11): Liu CT, Chung PT, Wu CY, Weng YM, Wenlung C, Tseng CY (2006). Antioxitive n in vitro heptoprotetive tivity of Bupleurum koi lef infusion. Phytother. Res. 20(11):3-8. Mnn A, Gbte M, N-Umr A (2003). Meiinl n Eonomi Plnts of Nupeln. Jube-Evns Books n Publitions, ISBN , Bi, Niger Stte, Nigeri, 276 p. Moronkol DO, Fruq UZ (2013). Chemil Constituents of the Lef, Stem-brk n Root Essentil oils of Antiris Afrin. J. Appl. Phrm. Si. 3(6): Niu KA, Thippeswmy NB (2002). Inhibition of humn low ensity lipoprotein oxition by tive priniples from spies. Mol. Cell. Biohem. 229: Nofer JR, Kehrel B, Fobker M, Levku B, Assmnn G, von Ekrstein A (2002). HDL n rterioslerosis: beyon reverse holesterol trnsport. Atheroslerosis 161:1-16. Okogun IJ, Spiff AI, Ekong DEU (1976). Triterpenois n betines from the ltex n brk of Antiris frin. Phytohemistry 15: Olleye MT, Akinmolun AC, Ogunboye AA, Akinhunsi AA (2010). Antioxint tivity n heptoprotetive property of lef extrts of Boerhvi iffus Linn ginst etminophen-inue liver mge in rts. Foo Chem. Toxiol. 48: Pnkj G, Abhishek C, Kushik P (2009). Lbortory evlution of rue extrtsof Cinnmomum tml for potentil ntibteril tivity. Eletroni J. Biol. 5(4): Prie KR, Johnson TI, Fenwik GR (1987). The hemil n biologil signifine of sponins in foos n feeing stuff. Crit. Rev. Foo Si. Nutr. 26: Rz M, Al-Shbnh OA, El-Hiyh TM, Al-Mje AA (2002). Effet of prolonge vigbtrin tretment on hemtologil n biohemil prmeters in plsm, liver n kiney of Swiss lbino mie. Si. Phrm. 70: Sbir SM, Ahm SD, Hmi A, Khn MQ, Athye ML, Sntos DB, Boligon AA, Roh JBT (2012). Antioxint n heptoprotetive tivity of ethnoli extrt of leves of Soligo mirogloss ontining polyphenoli ompouns. Foo Chem. 131(3): Sthishkumr T, Bskr R, Shnmugm S, Rjsekrn P, Ssivm S, Mniknn V (2008). Optimiztion of flvonois extrtion from the leves of Tbernemontn heynen Wll. using L16 Orthogonl esign. Nt. Si. 6(3): Sellers RS, Morton D, Mihel B, Roome N, Johnson JK, Yno BL, Perry R, Shfer K (2007). Soiety of Toxiologi Pthology position pper: orgn weight reommentions for toxiology stuies. Toxiol. Pthol. 35: Sofowor A (1993). Meiinl plnts n Tritionl meiine in Afri. Spetrum Books Lt, Ibn. Teo S, Stirling D, Thoms S, Hobermn A, Kiorpes A, Khetni V (2002). A 90-y orl gvge toxiity stuy of Δ-methylphenite n Δ,lmethylphenite in Sprgue Dwley rts. Toxiology 179: Tofovi SP, Jkson EK (1999). Effets of long-term ffeine onsumption on renl funtion in spontneously hypertensive hert filure prone rts. J. Criovs. Phrmol. 33: Vngur (2014). Avilble t Vrshney R, Kle RK (1990). Effet of lmoulin ntgonist on rition inue lipi peroxition in mirosomes. Int. J. R. Biol. 58: Worl Helth Orgniztion (2008). Tritionl Meiine (esse ). Xu T, Li D, Jing D (2012). Trgeting Cell Signling n Apoptoti Pthwys by Luteolin: Crioprotetive Role in Rt Criomyoytes Following Ishemi/Reperfusion. Nutrients 4(12):