Preprints (www.preprints.org) NOT PEER-REVIEWED Poste: 2 Septemer 2016 Artile Cytotoxi Ativity of Vernoni mespilifoli Less Use in the Folk Meiine in the Estern Cpe Provine, South Afri Jeremih Oshiomme Unuofin, Glori Aeronke Otunol * n Anthony Jie Afolyn Meiinl Plnts n Eonomi Development (MPED) Reserh Centre, Deprtment of Botny, University of Fort Hre, Alie 5700, South Afri; unuofinjeremih@gmil.om (J.O.U.); folyn@ufh..z (A.J.A.) * Corresponingene: gotunol@ufh..z; Tel.: +27-40-602-2320 Astrt: Vernoni mespilifoli is wiely use in folk meiine in the Estern Cpe Provine, South Afri. The im of this stuy ws to evlute the iologil tivity of the etone, queous n ethnol extrts of Vernoni mespilifoli using rine shrimp hthility n lethlity ssy. The result showe hthing suess in this orer: queous extrt (48.6%) > etone extrt (38.2%) > ethnol extrt (26.8%). The LC50 of the lethlity ssy were in this orer: etone extrt (67.8 µg/ml) > queous extrt (132 µg/ml) > ethnol extrt (383 µg/ml). Aoring to Meyer s toxiity inex (using rine shrimps), LC50 < 1000 µg/ ml is toxi. Therefore, the results of the three solvent extrts oul e si to e toxi s o hve LC50 < 1000 µg/ ml. However, the toxiity of the rue extrts oul suggest or onfer some ntitumor properties, hene further in vitro, in vivo n ntitumour ssys re reommene to further sustntite these lims. Keywor: Vernoni mespilifoli; Artemi slin; toxiity; iologil tivity; hthility; lethlity 1. Introution The pivotl role meiinl plnts n tritionl helth systems ply in solving the helth re prolems of the worl is gining inresing ttention. As result of this reirth of interest, reserh on meiinl plnts is rising impressively t the interntionl level, prtiulrly eveloping ountries where tritionl meil prtie is imie s n essentil prt of their ulture [1]. Biotive ompouns present in meiinl plnts re responsile for their effiy [2]. These ompouns re minly seonry metolites n they inlue lklois, essentil oils, tnnins n resins to mention few, whih funtion either in their originl form or in semi-syntheti form [3]. In spite of these iotive ompouns exhiiting therpeuti potentil, there is insuffiient knowlege out their toxiogeni effets when onsume in lrge mount [4]. Mny reserh stuies t present fous on oth phrmology n toxiity of meiinl plnts use y humns to promote sfety with the use of plnt prouts for the tretment of vrious ilments [5]. To this en, it is of gret importne to verify the phrmologil qulities of herlerive remeies n lso their level of toxiity ontrry to the puttive view of the inouity/inouousness of nturl prouts [6]. Vrious ssys re eing employe for the reserh of potentil toxiity of herl extrts se on ifferent iologil moels, suh s in vivo ssys on lortory nimls. Brine Shrimp Lethlity Assy (BSLA) hs gine reognition s n lterntive iossy tehnique to sreen the toxiity of lge [7], of entl mterils [8], toxiity of hevy metls [9] n metl ions [10], toxiity of nnoprtiles [11], s well s sreening of mrine nturl prouts
Preprints (www.preprints.org) NOT PEER-REVIEWED Poste: 2 Septemer 2016 2 of 12 [12], the toxiity plnt extrts [13, 14, 15, 16]. It lso inites ytotoxiity of myri of phrmologil tivities suh s ntimiroil, ntivirl, pestiil n nti-tumor of ompouns [13, 14]. Vernoni mespilifoli Less. populrly known s Uhlunguhlungu (Xhos) mong the inigenous people of the Nkonkoe Muniiplity of the Estern Cpe of South Afri is one of the five Southern Afrin speies of the Vernoni genus tht is enemi or ner-enemi to this suontinent [17]. It is liming shru tht is 0.6 9.0 m tll, with pinntely-veine leves, eplete reeptle with otuse involurl rts, n white to violet florets [17]. Vernoni mespilifoli is ommonly foun in the Estern Cpe, Kwzulu-Ntl, Limpopo, Mpumlng n Western Cpe provines of South Afri [18]. It is use in the Estern Cpe of South Afri for ethno-meiinl mngement of weight loss n hypertension [19] n lso for the tretment of hert wter isese in gots [20]. Although V. mespilifoli is use for ethno-meiinl purposes there is limite knowlege out it toxiity level. This stuy im to investigte the ytotoxi tivity of the rue extrts of Vernoni mespilifoli using rine shrimp moel. 2. Mterils n methos 2.1 Mterils The whole plnt prts use for this for this stuy ws ollete in June 2015 from its nturl hitt in the wil t Zihlhleni Villge Mipse, Nkonkoe Rurl Estern Cpe, South Afri whih lies t Ltitue 32 51 41.846 S n Longitue 27 10 59.318 E. The plnt ws uthentite y Mr. Tony Dol of Selmr Shonln Herrium, Rhoes University, South Afri, n vouher speimen (Unuofin Me, 2015/1) ws prepre n eposite t the Giffen Herrium, University of Fort Hre. 2.2 Preprtion of extrts The whole plnt ws rinse with eionise wter n gently lotte with pper towel to remove the wter n susequently oven-rie (LABOTEC, South Afri) t 55 C for 72 hours until onstnt weight ws hieve. The rie smple ws then groun into power (Polymix PX-MFC 90D Switzerln) n store t 4 C till neee for nlyses. The groun smple (200g) ws weighe into 3 seprte onil flsks ontining (2 L) etone, ethnol, n wter respetively n shken in n oritl shker (Oritl Inutor Shker, Gllenkmp) for 48 hours. The rue extrts were filtere uner pressure using Buhner funnel n Whtmn No. 1 filter pper. The etone n ethnol extrts were further onentrte to ryness to remove the solvents uner reue pressure using rotry evportor (Strike 202 Steroglss, Itly), while the queous filtrte otine ws onentrte using freeze ryer (Vir Tis enhtop K, Vir Tis Co., Griner, NY). 2.3 A. slin hthing ssy The metho esrie y [21] ws employe with little moifitions. Five petri ishes ontining 30 ml of the extrts were prepre in filtere se wter y issolving them in minute mount of the prent solvents to yiel two-fol ilution of onentrtions (1, 0.5, 0.25, 0.125 n 0.0625 mg/ml). A positive ontrol ws lso prepre y issolving potssium ihromte in sewter in the sme onentrtions s the plnt extrts. Se wter lone ws use s the negtive ontrol. The setup ws llowe to stn for 30 minutes to llow the solvents to evporte. Ten (10) A. slin ysts were stoke in eh of the petri ishes ontining 30 ml of the prepre two-fol onentrtions (1 to 0.0625 mg/ml) of the plnt frtions n positive
Preprints (www.preprints.org) NOT PEER-REVIEWED Poste: 2 Septemer 2016 3 of 12 ontrol. The petri ishes were prtly overe, inute t 30 C n uner onstnt illumintion for 72 hours. The numer of free nuplii in eh petri ish ws ounte fter every 24 hours till the en of 72 hours. The perentge of hthility ws lulte y ompring the numer of hthe nuplii with the totl numer of ysts stoke. 2.4 A. slin lethlity ssy A. slin ysts were hthe in se wter n 10 nuplii were pipette into eh petri ish ontining the two-fol onentrtions of the extrts n ontrol s in the hthility ssy esrie ove. The petri ishes were then exmine n the numer of living nuplii (tht exhiite movement uring severl seons of oservtion) ws ounte fter every 24 hours n the set up ws llowe to stn for 72 hours uner onstnt illumintion. The perentge of mortlity (M %) ws lulte s: Mortlity (%) = (Totl nuplii Live nuplii) 100 / Totl nuplii. 2.5 Dt nlysis The perentge hthility suess n mortlity t otine from the 5 ifferent onentrtions of eh frtion n ontrol experiments were use to onstrut the oseresponse urves. These were use to etermine their orresponing LC50 vlues. The LC50 ws tken s the onentrtion require for prouing 50% mortlity of the nuplii. LC50 vlues were etermine from the est-fit line otine y regression nlysis of the perentge hthility n lethlity versus the onentrtion. The sttistil nlysis ws one on MINITAB version 17 for Winows. One-wy nlysis of vrine (ANOVA) followe y Fisher s Lest Signifint Differene (for mens seprtion) ws use to test the effet of onentrtion n time of exposure of the plnt extrts on the hthility suess of the ysts n mortlity of lrve respetively. 3.0 Results n Disussion 3.1 Brine shrimp hthility Brine shrimp hthility test ws use to etermine the iologil tivity of Vernoni mespilifoli. The hthing suess of A. slin inute with ifferent plnt extrts n ontrol is s shown in Figure. The se wter exhiite signifintly higher (P < 0.05) hthing suess (71%) thn the solvent extrts n the positive ontrol (potssium ihromte) (5.4%). The hthing suess of the ysts in the etone, queous n ethnol extrts were 38.2%, 48.6% n 26.8% respetively n were signifintly ifferent (P < 0.05) from eh other. The hthing suess of A. Slin ysts inute with queous extrts h the highest hthing suess n presumly lest toxi of the solvent extrts use (Figure 1). This oul explin why most tritionl herl meiines re prepre using wter s solvent euse it is not or less toxi. This oul lso suggest why the yst showe more resistnt to hthing in the etoni n ethnol extrts thn in the queous extrts. This resistnt oul e ttriute to the permeility rrier provie y the ysts [22, 23].
Preprints (www.preprints.org) NOT PEER-REVIEWED Poste: 2 Septemer 2016 4 of 12 80 70 e Hthing suess (%) 60 50 40 30 20 10 0 Aqueous extrt Aetone extrt Ethnol extrt Potssium ihromte Se wter Figure 1: Perentge hthing suess of A. slin ysts inute in ifferent solvent extrts n ontrols. *The vlues re mens of five onentrtions for eh plnt extrt/ontrol ± SD of three replites. Brs with ifferent letters re signifintly ifferent (P < 0.05). The A. slin emryos re highly efenseless to toxins t erly evelopmentl stges [24, 25, n 26]. In the rine shrimp hthility ssy, the hthing suess signifintly erese with inresing onentrtions of the rue extrts in ose epenent mnner. The hthing suess of the yst ws lso evlute t ifferent onentrtions n the result is epite in Figure 2. The queous extrt exhiite its mximum hthing suess t 0.5 mg/ml (76%). The etone n ethnol extrts h similr hthing pttern, with the highest hthing suess oserve t 0.125 mg/ml (Figure 2). The overll est perentge hthing suess potentil of ysts ws oserve with yst inute t 0.5 mg/ml in the queous extrt whih ws signifintly (P > 0.05) greter when ompre with other solvent use. Potssium ihromte showe the lowest hthing suess. At 0.0625 mg/ml, there ws no signifint ifferene in the hthing suess of queous n potssium ihromte. There ws lso no signifint ifferene in the hthing suess of ysts inute t 0.125 mg/ml in oth the queous n etone extrts. The etone n ethnol extrts h no signifint ifferene in their hthing suess t 0.25 mg/ml. At onentrtions of 0.25 mg/ml -1 mg/ml for potssium ihromte, no hthing ws oserve. Also, ysts inute in 1 mg/ml of etone n ethnol extrts i not hth. The inhiitory effet of Vernoni mespilifoli rue extrts oul hve ttriute to the toxi ompouns present in the extrts tht hve oviil property. Also reports hve shown tht seonry metolites in plnts oul hve effet on the emryoni evelopment [27].
Preprints (www.preprints.org) NOT PEER-REVIEWED Poste: 2 Septemer 2016 5 of 12 90 Hthility suess (%) 80 70 60 50 40 30 20 10 0 0.0625 0.125 0.25 0.5 1 Conentrtion (mg/ml) Aqueous extrt Aetone extrt Ethnol extrt Potssium ihromte Figure 2: Effet of vrying onentrtions on hthing suess (%) of A. slin ysts. *Vlues re mens ± SD of three replites of the onentrtions for eh plnt extrt/ontrol. Set of rs with ifferent letters re signifintly ifferent (P > 0.05). The results of the effet of exposure time on hthility showe tht the sensitivity of A. slin yst to the plnt extrts ws strongly epenent upon exposure perio (Figure 3). A similr tren ws oserve with the queous n etone extrts s there ws signifint ifferenes in hthing suess from 24 h to 48 h (P > 0.05). The lowest hthing suess ws oserve fter 24 h tretment in ll the extrts. This result is in line with reports from [26, 27] whih stte tht A. slin is extremely suseptile to toxin uring its erly stge of evelopment. The etone n ethnol extrts experiene mximum hthing t 48 h with 45% n 32% hthing suess respetively, fter whih eth set in for the lrey hthe nuphlii. The queous extrt h the highest hthing suess t 60 hours. Also in Figure 3 there ws 1.3 n 1.1 fol erese respetively in hthe yst from 48 to 72 h in oth etone n ethnol extrts while queous extrt erese signifintly y 1.1 fol fter 60 h (P<0.05). Se wter exhiite optimum hthing t 36 h n remine the sme throughout the experiment. Cysts inute in potssium ihromte erese signifintly y 3.5-fol fter 36 hours (P<0.05), followe y no hthing of yst fter 48 hours.
Preprints (www.preprints.org) NOT PEER-REVIEWED Poste: 2 Septemer 2016 6 of 12 90 80 % Hthility 70 60 50 40 30 20 10 0, 24 36 48 60 72 Time (h) Aqueous extrt Aetone extrt Ethnol extrt Se Wter Potssium ihromte Figure 3: Effet of time (h) on the hthing suess of A. slin ysts. *Vlues re mens ± SD of three replites for eh plnt extrt/ontrol. Set of rs with ifferent letters re signifintly ifferent (P< 0.05). 3.2 Brine shrimp lethlity Brine shrimp ytotoxiity test is onsiere s preliminry ssessment of toxiity. This ssy etermines lethl onentrtion of tive ompouns suh s hevy metls, pestiies, n meiines in rine meium [28, 29, 30] n hs een employe to etermine toxiity of vrious tive ompouns euse of it is relile, rpi n very onvenient to rry out [13, 31, 21]. The perentge mortlity of A. slin lrve (nuplii) inute in ifferent solvent extrts of Vernoni mespilifoli n ontrols re shown in Figure 4. There ws high mortlity of nuplii inute in oth the queous n etoni extrts lthough there ws no signifint ifferene in their perentge mortlity, wheres the nuplii inute with potssium ihromte h signifintly greter mortlity when ompre to ll the extrts n se wter (P < 0.05). The ethnoli extrt n se wter showe mortlity of 49.60% n 0% respetively.
Preprints (www.preprints.org) NOT PEER-REVIEWED Poste: 2 Septemer 2016 7 of 12 120 100 Mortlity (%) 80 60 40 20 0 Aqueous extrt Aetone extrt Ethnol extrt Se wter Potssium ihromte Figure 4: Perentge mortlity of A. slin nuplii inute in ifferent plnt extrts n ontrols. *The vlues re mens of five onentrtions for eh plnt extrt/ontrol ± SD of three replites. Brs with ifferent letters re signifintly ifferent (P < 0.05). The effet of ifferent onentrtions of the plnt extrts on the mortlity of lrve is shown in Figures 5. The egree of mortlity of nuplii ws in onentrtion-epenent fshion. The highest mortlity ws oserve in ll the extrts t 1 mg/ml ompre to potssium ihromte whih showe mximum mortlity (100%) t 0.125 mg/ml. There ws no signifint ifferene (P<0.05) in perentge mortlity of the nuplii etween the queous extrt n etone extrt t onentrtions of 0.125, 0.25 n 1 mg/ml. At 0.125 n 0.5 mg/ml, queous n ethnol extrts lso exhiite no signifine ifferene in perentge mortlity (P<0.05). At 1 mg/ml, there ws no signifine ifferene in perentge mortlity etween queous n etone extrts n lso etween the ethnoli extrt n potssium ihromte (P<0.05) in Figure 5. The results revele tht the effet of vrying onentrtions of ll the plnt extrts on the mortlity of lrve ws in onentrtion epenent fshion, therefore it n e postulte tht though these re toxiologil t, this plnt possesses phrmologil tivity se on the osge ministere [21, 32].
Preprints (www.preprints.org) NOT PEER-REVIEWED Poste: 2 Septemer 2016 8 of 12 120 Mortlity (%) 100 80 60 40 20 0 0.0625 0.125 0.25 0.5 1 Conentrtion (mg/ml) Aqueous extrt Aetone extrt Ethnol extrt Potssium Dihromte Figure 5: Perentge mortlity of A. slin ysts inute t ifferent onentrtions of the plnt extrts n ontrol. * The vlues re mens of three replites ± SD (t ifferent hours). Set of rs with ifferent letters re signifintly ifferent (P < 0.05). All extrts were sreene t five ifferent onentrtions viz. 62.5, 125, 250, 500 n 1000 μg/ml n oserve for their toxi effet on A. slin from 24-72 h. Potssium ihromte ws use s stnr [33]. The perentge mortlity ue to exposure time is s shown in Figure 6. The result revele tht the perentge mortlity ws time epenent; the longer the exposure of nuplii to the plnt extrts, the greter the mortlity. It ws oserve tht etween 24-72 h of exposure of the nuphlii to queous, etone n ethnol extrts, there ws 2.94, 3.41 n 2.5-fol inrese in the mortlity of nuphlii respetively. The nuphlii inute in se wter i not ie throughout the urtion of the experiment. Generlly, the mortlity of nuplii ws signifintly similr when inute with the ethnol, etone, n the queous extrts t 24 h n ws signifintly higher thn se wter (P<0.05) (Figure 6). The mortlity of nuplii inute in these plnt extrts inrese exponentilly with time with the highest mortlity oserve t 72 hours with ll the extrts. The nuplii ttin the seon n thir instrs of their life yle within 48 hours hene revel their gretest sensitivity to toxins t this time [34, 35]. However, the finings of this stuy inite tht the mximum sensitivity ws rehe fter 72 hours of exposure.
Preprints (www.preprints.org) NOT PEER-REVIEWED Poste: 2 Septemer 2016 9 of 12 120 Mortlity (%) 100 80 60 40,, e 20 0 24 36 48 60 72 Time (h) Aqueous extrt Aetone extrt Ethnol extrt Se Wter Potssium ihromte Figure 6: Perentge mortlity of A. slin ysts inute t ifferent time urtions in the plnt extrts n ontrols. * The vlues re mens ± SD of 3 replites (of ll the onentrtions) for eh plnt extrt/ontrol ± SD. Set of rs with ifferent letters re signifintly ifferent (P < 0.05). Aoring to Meyer et l [13] n Bstos et l [36] the rine shrimp lethlity were interprete in orne to the riterion y Meyer toxiity inex tht LC50 vlues greter thn 1000 μg/ml (1 mg/ml) re onsiere non-toxi, LC50 vlues equl/greter thn 500 μg/ml (0.5 mg/ml) ut not greter thn 1000 μg/ml re onsiere to hve wek toxiity while those hving LC50 vlues less thn 500 μg/ml re onsiere toxi. The LC50 vlues were lulte s 132 µg/ml for queous extrt, 67.8 µg/ml for etone extrt n 383 µg/ml for the ethnol extrts, respetively (Tle 1). All of the rue extrts of V. mespilifoli were foun to e lethl with LC50 <1 mg/ml (Tle 1) n hene it oul e employe s promising lterntive in the tretment n mngement of tumor, s rine shrimp lethlity test now serves s n initor for the preliminry sreening of iotivity inluing for ntiner [37]. Tle 1: Lethl ose onentrtion (LC 50 ) of etone, ethnol n queous extrts of Vernoni mespilifoli ginst Brine Shrimp Smple Regression eqution LC 50 (µg/ml) Toxiity sttus R 2 (%) Aqueous Y = 19.476ln(x) + 89.4 132 Toxi 97.4 extrt Aetone extrt Y = 33.161x + 47.75 67.8 Toxi 85.8 Ethnol extrt Y = 88.667x + 16.042 383 Toxi 96.3 Potssium ihromte Y= 1.4427247ln(x) + 101 <0.100 Toxi 50 *LC50 is the onentrtion (µg/ml) of the plnt extrts n positive ontrol (Potssium ihromte) suffiient to otin 50% of inhiition of nuplii mortlity of A. slin, respetively. R 2 is the oeffiient of etermintion of the regression eqution.
Preprints (www.preprints.org) NOT PEER-REVIEWED Poste: 2 Septemer 2016 10 of 12 4.0 Conlusion This stuy showe tht the ifferent rue extrts of Vernoni mespilifoli were lethl (LC50 < 1 mg/ml) in the rine shrimp lethlity ssy. Further in vivo n in vitro stuies with ifferent humn ell lines is require to sertin the ntitumor tivity of this plnt n lso the isoltion of the le ompoun responsile for this tivity, might e utilize for the evelopment of novel ntiner rug. Aknowlegments: The uthors wish to knowlege the finnil support of Ntionl Reserh Fountion (NRF) (Grnt No. 85294) n Govn Meki Reserh Development Centre, University of Fort Hre, South Afri. Author Contriutions: Jeremih Oshiomme Unuofin, Glori Aeronke Otunol n Anthony Jie Afolyn oneive n esigne the experiments; Jeremih Oshiomme Unuofin performe the experiments; Jeremih Oshiomme Unuofin n Glori Aeronke Otunol nlyze the t; Anthony Jie Afolyn ontriute regents/mterils/nlysis tools; Jeremih Oshiomme Unuofin wrote the pper. Authorship must e limite to those who hve ontriute sustntilly to the work reporte. Conflits of Interest: The uthors elre no onflit of interest. Referenes 1. Roinson, H.; Funk, V.A. Gymnnthemum koekemoere (Composite, Vernoniee), new speies from South Afri. PhytoKeys 2014, 36, 59 65. 2. Rimono, D., von Sten, L., Foen, W., Vitor, J.E., Helme, N.A., Turner, R.C., Kmuni, D.A. n Mnym, P.A. Re List of South Afrin Plnts. Strelitzi 25. South Afrin Ntionl Bioiversity Institute, Pretori. 2009. 3. Afolyn, A.J.; Meie, B.O. Ethnootnil stuy of meiinl plnts use s ntioesity remeies in Nkonkoe Muniiplity of South Afri. Phog. Mg. 2010, 2, 11, 368-373. 4. Dol, A.P.; Coks, M.L. Tritionl veterinry meiine in the Alie istrit of the Estern Cpe Provine, South Afri. S. Afr. J. Si. 2001, 97, 375 379. 5. Frnsworth, N. R.; Soejrto, D. D. Glol importne of meiinl plnts. In: Akerele, O., Heywoo, V., n Synge, H. (Es.), The Conservtion of Meiinl Plnts. Cmrige University Press, Cmrige: 1991, 25-51. 6. Slim, A. A.; Chin, Y. W.; Kinghorn, A. D. Drug isovery from plnts. Springer, Berlin, Heielerg New York. 2005, p. 379. 7. Rsool Hssn, B. A. Meiinl plnts (importne n uses). Phrmeut. Anl. At 2012, 3, 139. http://x.oi.org/10.4172/2153-2435.1000e139 8. Melnie, J. C. Herl remeies: verse effets n rug intertions. Am. Fm. Physiin 1999, 59, 5, 1239-1244. 9. Prr, L.A.; Yher, R.S.; Sriñs, I.G.; Buel, L.I. Comprtive stuy of the ssy of Artemi slin L. n the estimte of the meium lethl ose (LD50 vlue) in mie, to etermine orl ute toxiity of plnt extrts. Phytome. 2001, 8, 395-400. 10. Firenzuoli, F.; Gori, L. Herl meiine toy: linil n reserh issues. Evi. Bse Complement. Alternt. Me. 2007, 4, 1, 37 40. 11. Myorg, P.; Pérez, K.R.; Cruz, S.M.; Cáeres, A. Comprison of iossys using the nostrn rustens Artemi slin n Thmnoephlus pltyurus for plnt extrt toxiity sreening. Brs. J. Phrm. 2010, 20, 897-903. 12. Pelk, M.; Dnzl, C.; Distler, W.; Petshelt, A. A new sreening test for toxiity testing of entl mterils. J. Dent. 2000, 28, 341-345.
Preprints (www.preprints.org) NOT PEER-REVIEWED Poste: 2 Septemer 2016 11 of 12 13. Mrtínez, M.; Del Rmo, J.; Torreln, A.; Díz-Myns, J. Effet of mium exposure on zin levels in the rine shrimp Artemi prthenogeneti. Aquulture 1999, 172, 315-325. 14. Kokkli, V.; Ktrmos, I.; Newmn, J. D. Monitoring the effet of metl ions on the moility of Artemi slin nuplii. Biosensors 2011, 1, 36-45. 15. Murer-Jones, M.A., Love, S.A., Meierhofer, S., Mrquis, B.J., Liu, Z., Hynes, C.L. Toxiity of nnoprtiles to rine shrimp: An introution to nnotoxiity n interisiplinry siene. J. Chem. Eu. 2013, 90, 475-478. 16. Crllo, J.L.; Hernánez-In, Z.L.; Pérez, P.; Grí-Grávlos, M.D. A omprison etween two rine shrimp ssys to etet in vitro ytotoxiity in mrine nturl prouts. BMC Bioteh. 2002, 2, 17-23. 17. Meyer, B.N.; Ferrigni, N.R.; Putnm, J.E.; Josen, L.B.; Nihols, D.E.; MLughlin, J.L. Brine Shrimp: A onvenient generl iossy for tive plnt onstituents. Plnt Mei 1982, 45, 31-34. 18. MLughlin, J.L.; Rogers, L.L.; Anerson, J.E. The use of iologil ssys to evlute otnils. Drug Inf. J. 1998, 32, 513-524. 19. Moshi, M.J.; Innoent, E.; Mgul, J.J.; Otieno, D.F.; Weisheit, A.; Mzi, P.K.; Nono, R.S.O. Brine shrimp toxiity of some plnts use s tritionl meiines in Kger Region, north western Tnzni. Tnz. J. H. Res. 2010, 12, 63-67. 20. Shrm, N.; Gupt, P.C.; Singh, A.; Ro, C.V. Brine shrimp Biossy of Pentpetes phoenie Linn. n Ipomoe rne jq. leves. Der. Phrm. Lett. 2013, 5, 162-167. 21. Otng, WM., Grierson, DS., Nip, RN. Assessment of potentil toxiity of three South Afrin meiinl plnts using the rine shrimp (Artemi slin) ssy. Afr. J. Phrmol. 2013, 7, 1272-79. 22. Awn, K.; Au-Hssn, N. Gentmiin resistne in linil strins of enteroteree ssoite with reue gentmiin uptke. Fol. Miroiol. 1998, 43, 438-440. 23. Au-Shn, B.; Awn, G.; Au-Sfiy, D.; Jrrr, N.; Awn, K. Anti-teril tivities of some plnt extrts utilize in populr meiine in Plestine. Turk. J. Bio. 2004, 28, 99-102. 24. Sleet, R.B.; Brenel, K. Homogeneous popultions of Artemi nuplii n their potentil use for in vitro testing in evelopmentl toxiology. Tertog. Crinog. Mutgen 1985, 5, 41-54. 25. Lewn, L.; Anersson, M.; Morles, P.G. The use of Artemi slin in toxiity. Testing Alterntives L. Anim. 1992, 20, 297-301. 26.Vsonelos, V.; Azeveo, J.; Silv, M.; Rmos, V. Effets of mrine toxins on the reproution n erly stges evelopment of quti orgnisms. Mr. Drugs 2010, 8, 59-79. 27. Suhr, S.; Knhrlplli, V.R.; Rivinrn, V.K.; Prre, S.K.; Chintl, S.; Thtiplly, R. Comprtive toxiity ssessment of three Tephrosi speies on Artemisi slin n niml ell lines. J. Nt. Phrm. 2011, 3, 143-148. 28. Solis, P.N.C., Wright, W., Anerson, M.M., Gupt, M.P., Phillison, J.D. A mirowell ytotoxiity ssy using Artemi slin. Plnt Mei 1993, 59, 50 252. 29. Jki, B.; Burji, H.R.; Stih-er, O. Biologil Sreening of ynoteri for ntimiroil n mollusil tivity, rine shrimp lethlity, n ytotoxiity. Phrm. Biol. 1999, 37, 138 143. 30. Piri, R.; Frosini, A.; Treii, M. R.; Mrgheri, M. C. Biotivity in freeliving n symioti ynoteri of the genus Nosto. J. Appl. Phyol. 2000, 12, 543-547.
Preprints (www.preprints.org) NOT PEER-REVIEWED Poste: 2 Septemer 2016 12 of 12 31. Lhti, K.; Ahtiinen, J.; Rpl, J.; Sivonen, K.; Niemelä, S.I. Assessment of rpi iossys for eteting ynoteril toxiity. Lett. ppl. Miroiol. 1995, 21, 109 114. 32. Kiiti, C.M.; Afolyn, A.J. Antifungl tivity n rine shrimp toxiity ssessment of Buline yssini use in the folk meiine in the Estern Cpe Provine, South Afri. Bnglesh J. Phrmol. 2016, 11, 469-477. 33.Pmj, R.; Arun, P.C.; Prshnth, D.; Deepk, M.; Amit, A., Anjn, M. Brine shrimp lethlity iossy of selete Inin meiinl plnts. Fitoterpi 2002, 73, 508-510. 34. Lewis, G.E.; Testing the toxiity of extrts of Southern Afrin plnts using rine shrimp (Artemi slin). S. Afr. J. Si. 1995, 91, 382-390. 35. Sreejmole, K.L; Rhkrishnn, C.K. Antioxint n ytotoxi tivities of ethyl ette extrt of the Inin green mussel Pern viriis. Asin J. Phrm. Clin. Res. 2013, 6, 197-201. 36. Bstos, M.L.A.; Lim, M.R.F.; Conserv, L.M., Anre, V.S., Roh, E.M.M., Lemos, R.P.L. Stuies on the ntimiroil tivity n rine shrimp toxiity of Zeyheri tuerulos (Vell.) Bur. (Bignoniee) extrts n their min onstituents. Annls Clinil Miroiology n Antimiroils 2009, 8, 16. DOI: 10.1186/1476. 37. Artnti, N.; Firmnsyh, T.; Drmwn, A. Biotivities evlution of Inonesin mistletoe (Denropthoe pentnr (L.) Miq.) leves extrts, J. Applie Phrm. Si., 2012, 1, 24-27. 2016 y the uthors; liensee Preprints, Bsel, Switzerln. This rtile is n open ess rtile istriute uner the terms n onitions of the Cretive Commons y Attriution (CC-BY) liense (http://retiveommons.org/lienses/y/4.0/).