E.O. Nwaichi, C.C. Monago and P.I. Amaraegbulem

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1 Glol Journl of Environmentl Reserh 7 (1): 08-12, 2013 ISSN X IDOSI Pulitions, 2013 DOI: /idosi.gjer Effets of Athrospir pltensis, Monodor myristi nd Helinthus nnus on the Rte of Polymeriztion, Sikle Cell Reversion nd Oxygen Affinity of Sikle Cell Hemogloin E.O. Nwihi, C.C. Mongo nd P.I. Amregulem Deprtment of Biohemistry, Fulty of Siene, University of Port-Hrourt, Port-Hrourt, Nigeri Astrt: The invitro effets of the ynoteri (Athrospir pltensis), spies (Monodor misti) nd the hene (Hellinthus nnuus) on the rte of polymeriztion, sikle ell reversion nd oxygen ffinity of sikle ell hemogloin (HS) ws studied spetophotometrilly using hemolystes of HS ontining erythroytes treted with sodium metisulphite in the presene of the different plnt extrts.all frtions of the three plnt extrts showed sttistilly signifint dereses (p < 0.05) in the rte of polymeriztion therefore inhiited the rte of sikling with inresing time. The Benzene Solule Extrt (BESE) frtion of H. nnuus demonstrted the highest ntisikling tivity (89.15% inhiition of HSS), while the lest ws y the BESE frtion of A. pltensis (45.16 % inhiition of HSS). The rude queous extrt (CAE) frtion of A. pltensis showed the highest nti-oxidnt effet, while CAE frtion of M. myristi gve the lest. The wter solule extrt (WSE) frtion of H. nnuus expressed mximum rte of sikling reversion. Alohol extrts of H. nnuus, X. ethiopi nd M. myristi were found to exhiit more signifint ntisikling tivity reltive to the queous extrts whih is ttriutle to the presene of some lipophilli mino ids present in the loholi frtion of the extrt nd it ould lso e responsile for the oserved high reversion pity ut low ntioxidnt effet of the lipophilli frtion of the plnt extrts. Key words: Sikle Cell Disese Extrt A. Pltensis H. Annuus X. Aethiopi M. Myristi INTRODUCTION nemi is the nme of speifi form of Sikle-ell disese in whih there is homozygosity for the muttion Sikle-ell disese (SCD), or sikle-ell nemi (SCA) tht uses HS [1]. Sikle-ell nemi is lso referred or deprnoytosis, is n utosoml reessive geneti to s "HSS", "SS disese", "hemogloin S" or lood disorder with over dominne, hrterized y red permuttions thereof. In heterozygous people, who hve lood ells tht ssume n norml, rigid, sikle shpe only one sikle gene nd one norml dult hemogloin [1]. Sikling dereses the ells' flexiility nd results in gene, it is referred to s "HAS" or "sikle ell trit". risk of vrious omplitions. The sikling ours Other, rrer forms of Sikle-ell disese inlude euse of muttion in the hemogloin gene nd life sikle-hemogloin C disese (HSC), sikle et-plusexpetny eomes shortened [1]. Sikle-ell disese, thlssemi (HS/ +) nd sikle et-zero-thlssemi usully presenting in hildhood, ours more ommonly (HS/ 0). These other forms of sikle-ell disese re in people (or their desendnts) from prts of tropil nd ompound heterozygous sttes in whih the person hs su-tropil regions where mlri is or ws ommon. only one opy of the muttion tht uses HS nd one One-third of ll indigenous inhitnts of Su-Shrn opy of nother norml hemogloin llele. Afri rry the gene, euse in res where mlri is The term disese is pplied, euse the inherited ommon, there is fitness enefit in rrying only single normlity uses pthologil ondition tht n sikle-ell gene (sikle ell trit). Those with only one of led to deth nd severe omplitions. Not ll inherited the two lleles of the sikle-ell disese, while not totlly vrints of hemogloin re detrimentl, onept resistnt, re more tolernt to the infetion nd thus show known s geneti polymorphism. An pplition of less severe symptoms when infeted [2-4]. Sikle-ell phytomediine n serve s suor to sufferers [1, 5]. Corresponding Author: E.O. Nwihi, Deprtment of Biohemistry, Fulty of Siene, University of Port-Hrourt, Port-Hrourt, Nigeri. 8

2 Glol J. Environ. Res., 7 (1): 08-12, 2013 MATERIALS AND METHODS grms of the smple were soked in 200ml distilled wter t 100 degrees entigrde for 12 hours. The solutions were Chemils: The hemils / regents used inlude: filtered seprtely using whtmn pper no. 1 nd the Butnol (BDH hemils, My nd Bker LTD, Dgenhm filtrte entrifuged t 600 rpm for 10 mins. The superntnt Englnd), Sodium metisulphite (BDH hemils LTD, were olleted in vils nd onentrted t 100 degrees Poole Englnd) nd Hydrohlori id (Sigm Chemil entigrde to get the rude queous extrt (CAEs). Compny, Poole Dorset, UK), Chloroform nd Methnol The other portions of the smples were seprtely (Lilikem Hospil Servies LTD. No. WWLS 211 Ariri soked in 200 ml of hloroform for twenty four hours Intl. MKT. A, Ai Stte), Antiogulnt (EDTA tue (24 h) to detet them nd generte the ft solule (FAS) nyodenz), distilled wter (sint john s lortory Owerri, frtion y filtrtion. The residues fter evportion to Imo stte), 0.9 % phosphte uffer (hemisiene, Owerri, dryness were suspended into 200 ml of methnol for Imo stte), Sodium dihydrogenorthophosphte 24 hours. The filtrtes were entrifuged nd dented to (NH2PO 4.2H2PO 4.2H20) (l. Teh. hemils), Benzene otin the methnol wter extrt [8, 9]. (Hopkin nd Willims, Chdwell heth Essex Englnd) Butnol wter prtitioning ws done with the Sodium hloride (Nl) (BDH), disodium hydrogen methnol extrt of eh of the smples. Extly 20 ml of orthophosphte nhydride (N2HPO 4) (Kentin Limited, 13 distilled wter nd 20 ml of utnol ws dded to the A Okigwe Rod, Owerri, Imo Stte). methnol extrts fter onentrting them. This ws left to stnd for 24 hours nd the two-phse liquid seprted Blood Smples: Blood smples were olleted from into the utnol-solule (BUS) nd wter solule (WAS) onfirmed HSS ptients y the personnel of the frtions respetively. The BUS nd WAS frtions were Hemtology unit of the Federl Medil Center Owerri onentrted y evportion t 80 degrees nd 100 fter due permission for its usge ws grnted y the degrees entigrde respetively. The volumes were Medil Counil of Nigeri. eqully reorded. Spetrophotometri redings were tken from UV-spetrophotometer. Plnt Smples: Athrospir pltensis ws purhsed from For enzene extrts of the smple, one hundred emeforku Nigeri Limited t suit 18 C/o 55 sngn street, (100) grms of the individul smples were seprtely mile one diou Port Hrourt Nigeri. dissolved in 200ml of hloroform for twenty four hours Hellinthus nnuus ws hrvested from frm in (24 h). The residues fter evportion to dryness were ho Port Hrourt, Rivers Stte of Nigeri nd were suspended into 200 ml of methnol for 24 hours. The onfirmed txonomist of the deprtment of plnt Siene filtrtes were entrifuged nd dented to otin the University of Porthrourt, Cho, Rivers Stte, Nigeri. methnol wter extrt. Benzene solule extrt ws done with the Spie Smples: Monodor myristi ws purhsed from methnol extrt of eh of the smples y dding Eke Ukwu Owerri Min Mrket in Imo Stte of Nigeri nd 20ml of enzene to the extrts nd left to stnd for ws onfirmed y otnist of Imo Stte University (24 h) hours. Owerri. Preprtion of the Blood Smples: Portions (0.20 ml) of Smple Preprtion the whole lood smples were used for the Fe /Fe rtio Preprtion of Plnt Extrt: They smples were nd the sikling reversion experiment while the remining seprtely lended, weighed nd two (200g) grms portions were olleted into EDTA tues. smples divided into two equl prts (100g). One portion Erythroytes were isolted from the lood smples y of eh of the divided smples ws used for rude entrifugtion t 10,000 rpm for fifteen seonds (15ses.) queous extrt ion (CAEI) proess nd the others were using the enh entrifuge (Nikel-Eletro entrifuge). sujeted to th-extrtion proedures with utnol Following reful siphoning of the plsm with Psteur [6, 7]. pipette, the erythroytes were y repeted inversion For the rude queous extrtion of Helinthus suspended in volume of isotoni sline (0.9% NCl) nnuus, one hundred (100) grms of the smple were equivlent to the siphoned plsm. The erythroyte soked in 500ml distilled wter t 100 degrees entigrde suspension ws then frozen t 0 C nd susequently for 12 hours, while for rude extrts of Arthrospir thwed to produe hemolyste for the hemogloin pltensis nd Monodor myristi, one hundred (100) polymeriztion experiment. 9

3 Glol J. Environ. Res., 7 (1): 08-12, 2013 Sikle Cell Hemogloin(Hss) Polymeriztion Inhiition estimted y mesuring the sikling rte for n hour t Experiment: The method of Noguhi nd Shether [10] 10 mins intervls, strting from zero time, whih is ws used for the HSS polymeriztion experiment. immeditely fter stirring. Perent (%) sikling ws HSS polymeriztion ws ssessed y the turidity of the lulted s: solution (polymerizing mixture) t 700 nm, using 2% solution of sodium metisulphite s deoxygenting No of sikled ell x 100 gent [11]. Then 4.4 ml of 2% sodium metisulphite Totl no of ells ounted 1 (N2S203), 0.5ml norml sline (0.9% NCl) nd 0.1ml The rte of sikling reversion ws lulted s the hemogloin were pipetted into uvette, shken nd slope of the Curve t zero time. sorne reding tken in spetrophotometer (Unim-spetroni 20) t 700 nm, every two minutes for Perentge fll (%) 30 min. This represents the ontrol. Distilled wter ws Time tken (mins) used s lnk for ll ssys. For the test ssys, 4.4 ml of NB: The time required to reverse 50% sikled ells ws 2% N2S20 3, 0.5 ml of eh extrt nd 0.1 ml hemogloin lulted from the urve y extrpoltion from the 50% (HSS) solution were pipetted in the uvette nd redings reltive sikled ell xis to the time xis. tken s ove. The rtes of hemogloin polymeriztion for the extrts or frtions were estimted y lulting RESULTS AND DISCUSION the hnge in optil density (OD) versus time in minutes. The rtes were expressed s perentges with respet to From the results represented in Tles 1, 2, 3 nd 4, the ontrol. Benzene solule extrts (BESE) of Athrospir pltensis showed the highest rte of polymeriztion, while the ft Determintion Of The Fe /Fe Rtio: The Fe /Fe rtio solule extrt (FSE) frtion showed the lest. Butnol ws determined y the methods of Dvidson nd Henry solule extrt (BUSE) frtion of Monodor myristi [12]. while the oxygen ffinity of hemogloin nd showed highest rte of polymeriztion, while the WSE methemogloin were mesured t 540 nm nd 630 nm frtion of the extrt hs the lest. BUSE frtion of respetively. The pproh employs lysing 0.02 ml whole H. nnus showed the lest rte of polymeriztion. lood in 5.0 ml of distilled wter nd 0.02 ml norml sline. While the BESE extrt of A. pltensis hs the The sorne of hemogloin (H) nd methemogloin highest rte of polymeriztion nd reltive perent (mh) were mesured t 540 nm nd 630 nm to determine polymeriztion ut lest reltive perent inhiition, while the %H nd % mh respetively. This represents the the BESE frtion of H.nnuus hs the lest rte of ontrol. In determining the effet of the extrt on polymeriztion nd reltive perent polymeriztion, ut Fe /Fe rtio, 0.02 ml of eh extrt ws dded to 5.0 ml highest reltive perent inhiition. Among the vrious of distilled wter nd 0.02 ml of lood dded nd potentil ntisikling gents tested, hydroxyure (HU) inuted for 60 min in test tue. hs een the most effetive ompound used for the tretment of ptients with sikle ell disese (SCD). Sikle Cell Reversion Experiment: Sikle ell reversl NIPRISAN (Nix-0699), n ethnol/wter extrt from experiment ws done s desried y Ekeke et l. [13]. indigenous plnts, hs strong ntisikling effet [14]. Freshly olleted HSS lood were diluted in 1:2 rtio with The kinetis of polymeriztion on ddition of % norml sline/ phosphte uffer (0.2 M, ph. 7.2) mirogrm/ml Nix-0699 used six-fold prolongtion of solution nd then inuted with 2% freshly prepred the dely time prior to deoxy-h S polymeriztion when sodium metisulphite in rtio 1:2 for one hour. At the ompred with tht of untreted H S smples [14-16]. end of the time, 1.6 ml of the pre-sikled lood in Expressed nti-sikling tivity on the inhiition is polystyrene tue ws mixed with 0.4 ml of the extrt. omprle to the findings of Pltt [17]. The herl Cells were ounted, tking smple t n intervl with the formul (HF) queous extrt showed the highest help of dropping pipette on slide nd then overed nti-sikling tivity on weight y weight sis of ll the with over slip. Finger pressure ws used to form thin extrts nd frtions tested, giving 71.4% inhiition of lyer nd the slides over oserved using x 40 ojetive sikling t the end of 180 min inution when lens y ounting the numer of sikled nd unsikled ompred with the norml sline ontrol. CAE frtion of ells in different fields on the slide. This proedure ws M. myristi hs mximum Fe /Fe rtio, while CAE repeted for ll the smples. The ntisikling rte ws frtion of A. pltensis hs the lest (Tle 2) whih is in 10

4 Glol J. Environ. Res., 7 (1): 08-12, 2013 Tle 1: The Rtes of polymeriztion, reltive perent polymeriztion nd reltive perent inhiition of HSS y the frtions of the smple. Smples Frtion Rtes of Polymeriztion Reltive% polymeriztion Reltive % inhiition ± Athrospir pltensis FSE ± Athrospir pltensis CAE ± Athrospir pltensis WSE ± Athrospir pltensis BUSE ± Athrospir pltensis BESE ± Monodor myristi FSE ± Monodor myristi CAE ± Monodor myristi WSE ± Monodor myristi BUSE ± Monodor myristi BESE ± Helinthus nnuus FSE ± Helinthus nnuus CAE ± Helinthus nnuus WSE ± Helinthus nnuus BUSE ± Helinthus nnuus BESE ± Tle 2: Fe /Fe rtio Fe /Fe rtio CAE BESE WSE A. pltensis 1.55 ± ± ±0.02 M. myristi 1.78 ± ± ±0.03 H. nnuus 1.77 ± ± ±0.04 Vlues re mens ± SE of triplite determintions. Mens in sme row with sme lphets re not signifintly different (P 0.05) Tle 3: Rte of sikle ell reversion Smple BESE CAE WSE A. pltensis ± ± ±0.08 M. myristi ± ± ±0.06 H. nnuus ± ± ±0.04 Vlues re mens ± SE of triplite determintions. Mens in sme row with sme lphets re not signifintly different (P 0.05) Tle 4: % Derese in Fe / Fe in studied smples BESE CAE WSE A. pltensis ± ± ±4.16 M. myristi ± ± H. nnuus ± ±7.03 Vlues re mens ± SE of triplite determintions. Mens in sme row with sme lphets re not signifintly different (P 0.05) greement with the findings of Uwkwe nd Nwoguikpe [15] tht reported highest ntioxidnt tivity in CAE frtion of M. myristi. WSE frtion of A.pltensis hs highest rte of reversion while CAE frtion of H.nnuus hs the lest nd WSE frtion of Athrospir pltensis showed highest rte of reversion, while CAE extrts of H. nnus hs the lest. As stted y Mpin et l. [18] nd Adms et l. [19], the seeds queous extrts exhiited higher perentge reversl of sikling of ll the tested prts. Chrhe et l., lso reported inresed pinful rises [20]. The CAE frtion of A.pltensis hs the highest % derese, while CAE frtion of M. myristi hs the lest (Tle 4). CONCLUSION Alohol extrts of H. nnuus, X. ethiopi nd M. myristi were found to exhiit more sttistilly signifint ntisikling tivity reltive to the queous extrts whih is ttriutle to the presene of some lipophilli mino ids present in the loholi frtion of the extrt nd it ould lso e responsile for the oserved high reversion pity ut low ntioxidnt effet of the lipophilli frtion of the plnt extrts. REFFERENCES 1. Roerts, I. nd M. De Montlemert, Sikle ell disese s prdigm of immigrtion hemtology: new hllenges for hemtologists in Europe. Hemtologi, 92(7): Wellems, T.E., K. Hyton nd R.M. Firhurst, The impt of mlri prsitism: from orpusles to ommunities. Journl of Clinil Investment, 119(9): Khn, M., Protetive effet of Spirulin ginst doxoruiin-indued rdiotoxiity". Phytotherpy Reserh, 19(12): Brdkdjin-Mihu. J., M. Bhuund D. Hurtrel, Neontl sreening for sikle ell disese in Frne. Journl of Clinil Pthology, 62(1):

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