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Journal of Natural Sciences Research ISSN 2224-3186 (Pa er! ISSN 222"-#$21 (%nline! &ol.3' No.

13' 2#13

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Isolation and Characterization of an Anticonvulsant Principle from Leaf Extract of Pyrenacantha Staudtii
%la(o)e S.%1*.' +a,unle -.%2.' .(eo)e %..3.' +ashola /.%1.' 1. 0e art1ent of Pure an( . lie( -he1istr)' 2a(o,e .,intola 3ni4ersit) of 5echnolog)' %g6o1oso' Nigeria. 2. 0e art1ent of -he1istr)' Jose h .)o 7a6alola 3ni4ersit)' I,e8i 9 .ra,e8i' Nigeria. 3 0e art1ent of -he1istr)' %6afe1i .wolowo 3ni4ersit)' Ile 9 Ife' Nigeria. *: 9 1ail of corres on(ing author; oluola(o)e<)ahoo.co.u, Abstract; Pyrenacantha staudtii was in4estigate( as a otential source of no4el anticon4ulsant (rug nucleus. S)ste1atic isolation of co1 oun(s in the non 9 olar e=tracts (i.e n-he=ane an( (ichloro1ethane! of the lea4es of Pyrenacantha staudtii was carrie( out using colu1n an( thin la)er chro1atogra h). 5he isolate( co1 oun(s were characteri>e( using infra re( (IR! an( nuclear 1agnetic resonance (N?R! s ectrosco ic techni@ues to 6e 6is(8-h)(ro=)l-2-1eth)lnon)l! hthalate an( 6is(8-1eth)lnon)l! hthalate. 5he two co1 oun(s were screene( for anticon4ulsant acti4it) using 1ice. 7is(8-h)(ro=)l-2-1eth)lnon)l! hthalate showe( a recia6le anticon4ulsant acti4it) 6ut 6is(8-1eth)lnon)l! hthalate (i( not. Key words; Pyrenacantha staudtii' Icacinacae' anticon4ulsant Introduction; Pyrenacantha staudtii is an ar6orescent liana in the fa1il) of Icacinacae. It grows u to 6 1 high an( with a (ia1eter of a6out "-1# c1. It is foun( in secon(ar) 8ungles in Southern art of Nigeria an( Aest -a1eroun (7ur,ill' 1$$4!. 5he lant is wi(el) use( in tra(itional 1e(icine for the treat1ent of h) ertension' ulcer' infla11ation' intestinal ain' 6lenorrhoea' hernia' an( inso1nia (0al>iel' 1$$"B .guwa an( %,un8i' 1$86!. Ph)toche1ical stu(ies on the lant re4eale( the resence of .l,aloi(s' gl)cosi(es' sa onins' tannins' fla4onoi(s an( aci(ic co1 oun(s (+alo(un et al' 2##"B .nosi,e et al' 2##8!. In a stu() to 4ali(ate the tra(itional use of the lant in re4ention of 1iscarriages' the leaf e=tract was foun( to e=hi6it significant inhi6itor) effect on o=)tocin in(uce( contractions on rat uterus at a (ose (e en(ent le4el (+alo(un et al' 2##"!. In4estigation of the 1ethanol e=tract of the lant resulte( in isolation of 3-car6o1etho=) )ri(ine (+alo(un an( 3sifo' 2##C! an( 21etho=)l )ri(ine which was shown to ha4e a rela=ant effect on the s1ooth 1uscles of the uterus (+alo(un et al' 2##6!. 5he cru(e a@ueous e=tract of the lant has also 6een shown to ossess analgesic' h) notic an( anticon4ulsant acti4ities (.we et al' 2##"!. In this stu()' we re ort the isolation an( characteri>ation of two co1 oun(s fro1 the leaf e=tracts of the lant one of which ro4e( to 6e a otential anticon4ulsant (rug. Experimental; 5he infra re( s ectra (ata were o6taine( using D7r (isc or nu8ol on Nicolet .4ator +5 9 IR 33# 6) 5her1o :lectronic -or oration. 5he 1E an( 13- Nuclear ?agnetic Resonance (N?R! s ectra were o6taine( fro1 a &arian ?ercur) s ectro1eter o erating at 2## ?E> for 1E an( "# ?E> for 13-. 5he che1ical shift 4alues are re orte( in 1 relati4e to 5?S as internal stan(ar(. .ll sol4ent use( for e=traction an( colu1n chro1atogra h) were /eneral Pur ose Reagent (/PR!' re(istille( 6efore use. /el filtration was erfor1e( using Se ha(e= 2E 9 2# re4iousl) swollen in s ecifie( sol4ent(s! rior to loa(ing of e=tract onto the colu1n (3." = 8." c1!. 5hin la)er chro1atogra h) (tlc! were (one with alu1iniu1 sheet re coate( with nor1al hase silica gel 6# +2"4 (?erc,' #.2# 11 thic,ness!. 5he tlc were run using suita6le sol4ent s)ste1s. S ots were locate( on the (e4elo e( tlc lates 6) 4isuali>ation un(er ultra4iolet light at 2"4 an( 366 n1. Sample collection; 5he lea4es of Pyrenacantha staudtii were collecte( fro1 the re1ises of the 3ni4ersit) of I6a(an in the SouthAestern region of Nigeria' Aest .frica an( i(entifie( at the her6ariu1 of the +e(eral Institute of +orest Research' I6a(an' 4oucher s eci1en was (e osite( with nu16er1#6886. Extraction and Isolation; . 66# g air-(rie( ow(ere( lea4es of Pyrenacantha staudtii was e=tracte( at roo1 te1 erature with "#F a@ueous ethanol for C2 hrs. 5he resulting 1i=ture was filtere( an( concentrate( to (r)ness in vacuo on a rotator) e4a orator to gi4e the cru(e e=tract (C".# gB 11.4F!. 5he cru(e e=tract was re(issol4e( in water an( successi4el) artitione( with n-he=ane' (ichloro1ethane an( eth)lacetate. 5he e=tracts thus o6taine( were concentrate( to (r)ness on a rotator) e4a orator )iel(ing fractions as follows; n-Ee=ane (1#.# gB 1."2F!B 0ichloro1ethane (4." gB #.68F! an( :th)lacetate (2.3 gB #.3"F!. 5he n-Ee=ane e=tract (1#.# g! was su68ecte( to silica gel o en colu1n chro1atogra h) eluting with (ifferent sol4ent co16inations' starting with n-Ee=ane an( a se@uential increase in olarit) 6) 4olu1e 6etween n-Ee=ane' (ichloro1ethane an( eth)lacetate. . total of ninet) eight ($8! fractions of 1" 1l each were collecte( an(

6$

Journal of Natural Sciences Research ISSN 2224-3186 (Pa er! ISSN 222"-#$21 (%nline! &ol.3' No.13' 2#13

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anal)>e( 6) tlc. +ractions with si1ilar tlc rofile were oole( together an( concentrate( to (r)ness in vacuo with a rotator) e4a orator. +i4e fractions co(e( PE.' PE7' PE-' PE0 G PE: were o6taine(. +raction PE: was further urifie( on se ha(e= 2E -2# ("#F toluene-ethanol with gra(ient increase in olarit) ! to affor( co1 oun( 1(P1E:!. 5he (ichloro1ethane e=tract (4." g! was also su68ecte( to silica gel o en colu1n chro1atogra h) with sol4ent co16inations' starting with (ichloro1ethane an( then se@uential increase in olarit) 6) 4olu1e 6etween (ichloro1ethane' eth)lacetate an( 1ethanol. . total of si=t) se4en (6C! fractions of 1" 1l each were collecte( an( anal)>e( 6) tlc. +ractions showing si1ilar tlc characteristics were oole( together an( concentrate( to (r)ness in vacuo with a rotator) e4a orator. :ight fractions co(e( P0.' P07' P0-' P00' P0:'P0+'P0/'P0E were o6taine( an( anal)>e( 6) tlc. +raction P0- was further urifie( through re arati4e thin la)er chro1atogra h) with (ichloro1ethane as the (e4elo ing sol4ent. 5he 6an( with Rf #."4 was scra e(' elute( with (ichloro1ethane' filtere( an( concentrate( to (r)ness to affor( co1 oun( 2 (P01!. Animals; ?ice (weighing 24-2C g! were use( for the e= eri1ent. 5he ani1als were 6re( an( house( un(er stan(ar( en4iron1ental con(itions in the (e art1ent of Phar1acognos) an( 5hera eutics' -ollege of ?e(icine' 3ni4ersit) of I6a(an' Nigeria. 5he) were fe( with stan(ar( (iet an( water a( li6itu1. 5he ani1als were (i4i(e( into four grou s of fi4e each. !S" # induced convulsion$ 5he 1etho( e1 lo)e( is as (escri6e( 6) :lisha et al (1$88!. 5he first grou of ani1als which ser4es as a control was a(1inistere( with 2F 0?S% intra eritoneall) an( the ani1als o6ser4e( for latenc) 1inute of con4ulsion (i.e. ti1e (ela) 6efore the ani1al starts to show s)1 to1s of con4ulsion which inclu(e; 1asticator) 1o4e1ent' hea( no((ing an( 1)oclonic 8er,ing of li16s followe( 6) wil( running an( su6se@uent tonic e=tension of the whole 6o() ! an( 1ortalit). In the other grou s' 2F0?S% (1# 1gH,g! was intra eritoneall) a(1inistere( into the 1ice thirt) 1inutes after a(1inistration of (ifferent concentrations of the isolate( co1 oun(s. 5he ani1als were then o6ser4e( for tonic con4ulsions o4er a 24 hr erio( to note lethalit). %esults and iscussion; -o1 oun( 1 isolate( fro1 the n-Ee=ane e=tract was a )ellow oil with Rf #." in 0ichloro1ethane ; n-Ee=ane (3;1!. 5he infrare( s ectru1 of 1 showe( signals at (I c1-1! 33CC' 2$2C' 1C28' 1"8# an( C44 corres on(ing to % 9 E stretching 4i6ration of h)(rogen 6on(e( alcoholic grou ' - 9 E stretching 4i6ration of al,ane' car6on)l stretching of an ester' - J - stretching (ue to 6en>ene ring an( - 9 E out of lane 6en(ing of 6en>ene -- E res ecti4el). 5he signal at C44 c1-1 is also an in(ication of the 1'2-(isu6stitution of the 6en>ene ring.
O C O CH3 CH3 OH

C O

O CH3 CH3 OH

1 5he 1E G 13- N?R s ectra (ata are resente( in ta6le 1. 5he 13--N?R s ectru1 of the co1 oun( showe( it has a total of twent) eight car6on ato1s co1 rising 4 1eth)l' 12 1eth)lene' 8 1ethine an( 4 @uartenar) car6ons. Eowe4er onl) half of each of the signals are o6ser4a6le in the s ectru1 6ecause of the s)11etrical nature of the 1olecule (ha4ing a lane of s)11etr) across the 6en>ene ring an( se arating the two si(e chains!. 5he 1E N?R s ectru1 also showe( the (ifferent roton en4iron1ent as resente( in ta6le 1 an( hence su orts the structure.

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Journal of Natural Sciences Research ISSN 2224-3186 (Pa er! ISSN 222"-#$21 (%nline! &ol.3' No.13' 2#13

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5a6le1; 1E N?R an( 13- N?R (ata for co1 oun( 1. Position K- ( 1! - t) e CH3 21 11.182 CH3 $1 14.28" CH2 31 23.21" CH2 41 23.$"$ CH2 "1 2$.14# CH 2 61 2$.$4$ CH 2 C1 3#."C4 21 38.$43
CH

KE ( #.$ 1.1 1.2 1.2 1.2 1.2 1.2 2.3 3." 4.2 C."

1!

1 81 4 3 1'2

1

68.38# $$.38# 12$.#22

CH 2

CH

CH

131.116
CH

C.C 1.2
CH

132.664 168.##4
C O

5a6le 2; 1E G 13- N?R (ata for co1 oun( 2 Position K- ( 1! - t) e CH3 81 11.2#4 CH3 $1 14.314 CH2 21 23.236 CH2 31 23.$64 CH2 41 2$.1"3 CH2 "1 2$.$4$ CH2 61 3#."C$ CH2 C1 38.$3$ CH2 11 68.3$6 4 12$.#4# CH 3 131.164 CH 2 132.626 1 168.#"C
C O

KE ( #.$ #.$ 1.3 1.3 1.3 1.3 1.3 1.4 4.2 C.6 C.6 -

1!

. total of 26 car6on ato1s were esta6lishe( in the 13- - N?R' si= of which are 1ethine car6ons at K( 1! 38.$4' 12$.#4 an( 131.16 (-C1' --4' --3!B two @uartenar) car6ons at K( 1! 132.63 an( 168.#6 (--2' --1! an( ea, 1eth)lene car6ons --21' --31' --41 --"1' --61' --11 at K( 1! 23.24' 23.$6' 2$.1"' 2$.$"' 3#."8 an( 68.4# res ecti4el).5he 1olecule is also s)11etrical' hence onl) half of the signals are o6ser4e( in the s ectru1. 5his (ata is also consistent with the co1 oun( 6is(8 - 1eth)lnon)l! hthalate.
O CH3 C O CH3

C O

O

CH3

CH3

2 5a6les 3 to " gi4e the result of the anticon4ulsant e= eri1ent. 5a6le 3 shows that the a4erage latenc) of 1inute C1

Journal of Natural Sciences Research ISSN 2224-3186 (Pa er! ISSN 222"-#$21 (%nline! &ol.3' No.13' 2#13

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con4ulsion was three 1inutes on the a4erage while the latenc) of 1inute (eath was four 1inutes. .ll the ani1als in this control grou (ie( gi4en 1##F 1ortalit). In ta6le 4' we ha4e the result of the effect of co1 oun( 1 on the ani1als. .t a (ose of 1 1gH,g' the latenc) 1inute of tonic con4ulsion increase( to fi4e 1inutes when co1 are( with the control an( the ercentage 1ortalit) (ecrease( to 8#F. /enerall)' as the concentration of co1 oun( 1 increases' the latenc) of 1inute tonic con4ulsion an( latenc) of (eath 1inute increase( while the ercentage 1ortalit) (ecrease( co1 are( to control. .t a (ose of 4 1gH,g' the latent 1inute of con4ulsion increase( to twent) 1inutes on the a4erage an( therefore the (rug coul( 6e sai( to 6e anticon4ulsant acti4e at this concentration. 5a6le "' shows that all the rats treate( with co1 oun( 2 (ie( 8ust as in the control e4en though there were 1arginal increases in latent 1inute of tonic con4ulsion an( latent (eath 1inute co1 are( to the control. 5his shows that co1 oun( 2 1a) not 6e effecti4e as an anticon4ulsant (rug. 5a6le 3; .nticon4ulsant acti4it) on control grou 2F 0?S% .ni1al 0ose 1gH,g 2atenc) of 2atenc) of F 1ortalit) F rotecte( 1inute tonic (eath 1inute con4ulsion 1 1# 3 4 1## # 2 1# 2 4 1## # 3 1# 2 4 1## # 4 1# 3 4 1## # " 1# 4 6 1## # .ll the ani1als (ie( 5a6le 4; :ffect of -o1 oun( 1 on 0?S% 9 in(uce( con4ulsion .ni1al 0ose 1gH,g ?inute tonic ?inute (eath F 1ortalit) F rotecte( con4ulsion 1 1 " C 8# 2# 2 1 6 8 8# 2# 3 1 " 6 8# 2# 4 1 " C 8# 2# " 1 " C 8# 2# 4 rats (ie( .ni1al 1 2 3 4 " 0ose 1gH,g 2 2 2 2 2 ?inute tonic con4ulsion 11 12 1# 11 "12 ?inute (eath 16 16 1" 18 16 F 1ortalit) 6# 6# 6# 6# 6# F rotecte( 4# 4# 4# 4# 4# 3 rats (ie( F rotecte( 8# 8# 8# 8# 8# 1 rat (ie(

.ni1al 1 2 3 4 "

0ose 1gH,g 4 4 4 4 4

?inute tonic con4ulsion 16 2# 18 21 2"

?inute (eath 2# 2" 22 26 2$

F 1ortalit) 2# 2# 2# 2# 2#

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Journal of Natural Sciences Research ISSN 2224-3186 (Pa er! ISSN 222"-#$21 (%nline! &ol.3' No.13' 2#13

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5a6le "; :ffect of co1 oun( 2 on 0?S% - in(uce( con4ulsion .ni1al 0ose 1gH,g ?inute tonic ?inute (eath con4ulsion 1 1 " 8 2 1 6 $ 3 1 C 1# 4 1 " C " 1 " 6

F 1ortalit) 1## 1## 1## 1## 1##

F rotecte( # # # # # all rats (ie( F rotecte( # # # # # all rats (ie( F rotecte( # # # # # .ll rats (ie(

.ni1al 1 2 3 4 "

0ose 1gH,g 2 2 2 2 2

?inute tonic con4ulsion " 6 C C 8

?inute (eath $ 12 11 13 1"

F 1ortalit) 1## 1## 1## 1## 1##

.ni1al 1 2 3 4 "

0ose 1gH,g 4 4 4 4 4

?inute tonic con4ulsion 6 C $ 1# 12

?inute (eath 13 14 16 18 1$

F 1ortalit) 1## 1## 1## 1## 1##

%efrences$ .guwa' -.N. an( -.%. %,un8i (1$86!. Preli1inar) Phar1acological stu(ies of Nigerian ?e(icinal lant. J.Ethnol. Pharmacol' 8; "3-63 .nosi,e -...' 3gwu 3.7 an( Nwa,an1a %. (2##8!. :ffect of ethanol e=tract of Pyrenacantha staudtii lea4es on car6ontetrachlori(e in(uce( he ato=icit) in rats. Biokemistri 2#(1!' 1C - 22 .we' :.%B ?a,in(e J.?B %la8i(e ..%B Aa,eel %.D an( .(elo)e ..%. (2##"!; :4aluation of analgesic' anticon4ulsant an( h) notic acti4ities of P. staudtii. Afri. J. Traditional, Complementary Alternative Med. 2; 122 9 128 7ur,ill E.?. (1$$4!. 5he 3seful lants of .frica' Ro)al 7otan) /ar(ens' Dew' 2n( :(ition' &olu1e 2' 2$2 0al>iel J.?. (1$""!. 5he 3seful lants of Aest 5ro ical .frica. Aart1oughs 2t(. /reat 7ritain. P 188 9 1$2 :lisha :B .l-?ali,i S. an( I6rahi1 0. (1$88!. :ffects of Jasmium officinale flower on the -entral Ner4ous S)ste1 of the ?ouse. Int. J. crude dru s !es 26; 221-22C +alo(un .B 3sifoh - an( Nworgu L. (2##"!. Ph)toche1ical .nal)sis an( Inhi6itor) effect of Pyrenacantha staudtii leaf e=tract on Isolate( rat uterus. J. Pharm. " Biores. 2(2!' 1## - 1#3 +alo(un .B 3sifo -. an( Nwogu L (2##6!. S1ooth 1uscle rela=ant acti4it) of 3 9 car6o1etho=) )ri(ine fro1 P. staudtii leaf on isolate( rat uterus. African Journal of Biotechnolo y' "(1#!' 12C1 9 12C3. +alo(un .. an( 3sifo -. (2##C!. Isolation an( characteri>ation of 3-car6o1etho=) )ri(ine fro1 the lea4es of Pyrenacantha staudtii Eutch an( 0al> Icacinaceae. Acta Poloniae Pharmaceutica # $ru !esearch' 63 (3!' 23" 9 23C.

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