Phytochemical constituents and antioxidant activities of the whole leaf extract of Aloe ferox Mill

Olubunmi Abosebe Wintola and Anthony Jide Afolayan

Abstract
Background:
Aloe ferox Mill. (A sph odela cea e) is u sed in Sou t h A fr ica for t h e t r ea t m en t of con st ipa t ion a m on g v a r iou s a ilm en t s. Despit e t h e ex t en siv e st u dies con du ct ed on t h e a n t iox ida n t a ct iv it ies of t h e lea f g el a n d pu lp ex t r a ct of t h e pla n t , t h er e is n o in for m a t ion on t h e a n t iox ida n t pr oper t ies of t h e w h ole lea f ex t r a ct of t h e species.

Materials and Methods:

T h e a n t iox ida n t a ct iv it ies of et h a n ol, a cet on e, m et h a n ol a n d a qu eou s ex t r a ct s of A. ferox w er e in v est ig a t ed spect r oph ot om et r ica lly a g a in st 1 ,1 - diph en y l-2 -picr y lh y dr a zy l (DPPH), 2 ,2 -a zin o-bis(3 et h y lben zt h ia zolin e-6 -su lfon ic a cid) (A BT S) dia m m on iu m sa lt , h y dr og en per ox ide (H 2O 2), n it r ic ox ide (NO), lipid per ox ida t ion a n d fer r ic r edu cin g pow er . T ot a l ph en ols, fla v on oids, fla v on ols, pr oa n t h ocy a n idin s, t a n n in s, a lk a loids a n d sa pon in s w er e a lso det er m in ed u sin g t h e st a n da r d m et h ods.

Results:
T h e per cen t a g e com posit ion s of ph en ols (7 0 .3 3 ), fla v on ols (3 5 .2 ), pr oa n t h ocy a n idin s (1 7 1 .0 6 ) a n d a lk a loids (6 0 .9 ) w er e sig n ifica n t ly h ig h in t h e a cet on e ex t r a ct , follow ed by t h e et h a n ol ex t r a ct w it h v a lu es of 7 0 .2 4 , 1 2 .5 3 , 7 6 .7 a n d 2 3 .7 6 r espect iv ely , w h ile t h e lea st com posit ion w a s fou n d in t h e a qu eou s ex t r a ct . Mor eov er , bot h fla v on oids a n d sa pon in s con t en t s w er e a ppr ecia bly h ig h in bot h m et h a n ol a n d et h a n ol ex t r a ct s, w h ile ot h er s w er e v er y low . T a n n in s lev els w er e, h ow ev er , n ot sig n ifica n t ly differ en t ( P > 0 .0 5 ) in a ll t h e solv en t ex t r a ct s. A t 0 .5 m g /m l, t h e fr ee r a dica l sca v en g in g a ct iv it y of t h e m et h a n ol, a cet on e a n d et h a n ol ex t r a ct s sh ow ed h ig h er in h ibit ion a g a in st A BT S, h y dr og en per ox ide a n d n it r ic ox ide r a dica ls. W h er ea s, sca v en g in g a ct iv it y of t h e ex t r a ct s a g a in st DPPH* a n d lipid per ox ida t ion w er e obser v ed a t a con cen t r a t ion of 0 .0 1 6 a n d 0 .1 1 8 m g /m l r espect iv ely in com pa r ison t o t h e bu t y la t ed h y dr ox y lt olu en e (BHT ), g a llic a cid a n d r u t in . T h e fer r ic r edu cin g pot en t ia l of t h e ex t r a ct s w a s con cen t r a t ion depen den t a n d sig n ifica n t ly differ en t fr om t h a t of v it a m in C a n d BHT .

Conclusion:
T h e pr esen t st u dy sh ow ed h ig h lev el of r a dica l sca v en g in g a ct iv it y by et h a n ol a n d m et h a n ol w h ole lea f ex t r a ct s of A. ferox w it h h ig h er a n t iox ida n t a ct iv it ies t h a n a cet on e a n d a qu eou s ex t r a ct s. T h e sig n ifica n t differ en ces sh ow t h a t t h e w h ole lea f ex t r a ct cou ld be u sed a s a pot en t a n t iox ida n t in m edicin e a n d food in du st r ies.
Key words: Aloe

ferox , a n t iox ida n t , a sph odela cea e, fr ee r a dica ls, ph y t och em ica ls

INTRODUCTION
Fr ee r a dica ls a r e ch em ica l en t it ies t h a t ca n ex ist sepa r a t ely w it h on e or m or e u n pa ir ed elect r on s pr odu ced fr om v a r iou s bioch em ica l r ea ct ion s.[1 ,2 ] T h ey occu r con t in u ou sly in t h e cells a s a r esu lt of en zy m a t ic a n d n on --en zy m a t ic r ea ct ion s t o differ en t m olecu les in t h e body .[3 ] T h ey a r e g en er a t ed a s a

r esu lt of im ba la n ce bet w een for m a t ion a n d n eu t r a liza t ion of pr o-ox ida n t s in t h e body m et a bolic pr ocess.[4 ,5 ] Ex a m ples of t h ese a r e r ea ct iv e ox y g en species (ROS) or r ea ct iv e n it r og en species (RNS) r a dica ls w h ich in clu de su per ox ide a n ion s, sin g let ox y g en , h y dr og en per ox ide a n d h y dr ox y l r a dica ls. T h ese r a dica ls ca n ca u se v a r iet y of pa t h olog ica l effect s su ch a s pr ot ein da m a g e, DNA m u t a t ion , ca r cin og en esis, a g in g , ca r diov a scu la r a n d n eu r o-deg en er a t iv e disea ses. T h ey a r e im plica t ed a s a r esu lt of ox ida t iv e st r ess, occa sion a l lea k a g es fr om con t in u ou s ex posu r e t o ch em ica ls, con t a m in a n t s a n d ex og en ou s fa ct or s.[2 ,6 ,7 ] T h e pa t h olog ica l disr u pt ion of t h ese r a dica ls in t h e h u m a n cells cou ld be pr ev en t ed by qu en ch in g t h e u psh ot of ca t a ly t ic a ct iv it ies.[5 ,7 ] Sy n t h et ic dr u g s su ch a s bu t y la t ed h y dr ox y t olu en e (BHT ), r u t in , a n d bu t y la t ed h y dr ox y l a n isole (BHA ) a r e com m on ly u sed. How ev er , t h ey h a v e been r epor t ed t o ca u se a dv er se side effect s su ch a s t ox icit y , cell da m a g e, in fla m m a t ion s, a n d a t h er oscler osis in a n im a ls a n d h u m a n s.[2 ,8 ] In v iew of t h e in cr ea sin g r isk fa ct or s, t h er e h a s been a g loba l t r en d t ow a r d t h e u se of n a t u r a l su bst a n ces pr esen t in m edicin a l pla n t s a s t h er a peu t ic a n t iox ida n t a g en t s.[3 ] Sev er a l st u dies h a v e t h er efor e dem on st r a t ed t h a t pla n t s pr odu ce pot en t ph y t och em ica ls w it h st r on g a n t iox ida n t a ct iv it ies a n d t h u s r epr esen t a n im por t a n t sou r ce of n a t u r a l a n t iox ida n t s.[9 ] Mor eov er , t h e u se of n a t u r a l pr odu ct s of pla n t or ig in h a s been pr oposed beca u se t h ey a r e n a t u r a l a n t iox ida n t s w it h r ich sou r ces of m et a bolit es. T h ey con t a in bioa ct iv e ch em ica ls su ch a s ph en ols, a lk a loids a n d lig n in w h ich a r e pot en t r a dica l t er m in a t or s t h a t ca n h elp in r edu cin g t h e r isk of ca n cer , t ox icit y , in fla m m a t ion a n d ca r diov a scu la r disea ses.[1 0 ,1 1 ] Aloe ferox (Ca pe a loe), loca lly ca lled ik h a la in X h osa , is w idely dist r ibu t ed in Fr ee St a t e, Lesot h o, W est er n a n d Ea st er n Ca pe of Sou t h A fr ica . It is a per en n ia l, sin g le-st em m ed sh r u b, r ea ch in g 2 3 m in h eig h t , w it h t h ick su ccu len t lea v es bea r in g br ow n spin es on t h e m a r g in . For cen t u r ies, t h e lea f ex u da t e of A. ferox h a s been u sed t h er a peu t ica lly for t h e t r ea t m en t of con st ipa t ion .[1 2 ] T oda y , t h e pla n t is r epu t ed for it s a n t isept ic, la x a t iv e, m oist u r izin g , clea n sin g a n d a n t i-in fla m m a t or y pr oper t ies. T r a dit ion a lly , t h e fr esh a n d dr ied w h ole lea f ex t r a ct of t h e pla n t is u sed dir ect ly a s a n in fu sion a n d decoct ion for t h e t r ea t m en t of v a r iou s disea ses su ch a s sk in ca n cer , g a st r oin t est in a l disor der , in fla m m a t ion , bu r n s a n d psor ia sis.[1 3 ,1 4 ] T h e lea f g el h a s been ex t en siv ely docu m en t ed t o h a v e sig n ifica n t la x a t iv e,[1 5 ] a n t i-in fla m m a t or y ,[1 6 ] w ou n d h ea lin g ,[1 3 ] sex u a lly t r a n sm it t ed disea ses cu r e,[1 7 ] con t r ol of g a st r oin t est in a l pa r a sit e,[1 8 ] im m u n ost im u la n t , a n t iox ida n t , a n t it u m or a n d a n t idia bet ic a ct iv it ies.[1 9 ] Fu r t h er m or e, t h e lea f g el a n d pu lp ex t r a ct of t h e pla n t h a v e sev er a l ph a r m a colog ica l effect s su ch a s a n t im icr obia l a n d a n t it u m or pr oper t ies.[2 0 ,2 1 ] W e pr ev iou sly r epor t ed t h e la x a t iv e a n d t ox icolog ica l effect of t h e a qu eou s w h ole lea f ex t r a ct of A. ferox in t h e t r ea t m en t of con st ipa t ion .[2 2 ,2 3 ] Despit e t h e ex t en siv e st u dies con du ct ed on t h e a n t iox ida n t a ct iv it ies of t h e lea f g el a n d pu lp ex t r a ct of A. ferox ,[1 9 ,2 4 ] t h er e is n o in for m a t ion in t h e scien t ific lit er a t u r e on t h e a n t iox ida n t pr oper t ies of t h e w h ole lea f ex t r a ct of t h e species. T h er efor e, t h e pr esen t st u dy w a s a im ed a t det er m in in g t h e qu a n t it a t iv e ph y t och em ica l con st it u en t s, fr ee r a dica l sca v en g in g a n d a n t iox ida n t a ct iv it ies of t h e w h ole lea f ex t r a ct of A. ferox u sin g v a r iou s solv en t s.

MATERIALS AND METHODS

Collection of plant materials and preparation of extracts
Fr esh m a t u r e w h ole lea v es of A. ferox w er e collect ed in Ju n e 2 0 0 9 a t Nt sela m a n zi a r ea of t h e Ea st er n Ca pe Pr ov in ce of Sou t h A fr ica . T h e pla n t w a s a u t h en t ica t ed by Pr of. D. S. Gr ier son a t t h e Depa r t m en t of Bot a n y , Un iv er sit y of For t Ha r e, a n d a v ou ch er specim en (W in t ola Med. 2 0 0 9 /0 1 ) w a s deposit ed a t t h e Giffen h er ba r iu m of t h e Un iv er sit y . T h e lea v es w er e ov en dr ied t o con st a n t w eig h t a t 4 0 C a n d m illed t o a h om og en eou s pow der . T h e pow der ed pla n t m a t er ia l (2 0 0 g ) w a s ex t r a ct ed sepa r a t ely in dist illed w a t er , a cet on e, m et h a n ol a n d et h a n ol on a sh a k er (St u a r t Scien t ific Or bit a l Sh a k er , Essex , UK) for 4 8

h . Ea ch ex t r a ct w a s filt er ed u sin g a Bu ch n er fu n n el a n d W h a t m a n No. 1 filt er pa per . T h e filt r a t e obt a in ed w it h w a t er w a s fr ozen a t 4 0 C a n d dr ied for 4 8 h u sin g a fr eeze dr y er (V ir T is ben ch t op K, V ir T is Co., Ga r din er , NY , USA ) t o g iv e a y ield of 3 1 .2 g of dr ied ex t r a ct . T h e ot h er ex t r a ct s w er e in div idu a lly con cen t r a t ed t o dr y n ess u n der r edu ced pr essu r e a t 4 0 C u sin g a r ot a r y ev a por a t or . A cet on e, m et h a n ol a n d et h a n ol ex t r a ct s y ielded 1 9 .5 g , 2 5 .6 g a n d 2 0 .6 g , r espect iv ely . T h e r esu lt in g ex t r a ct s w er e r econ st it u t ed w it h t h eir r espect iv e solv en t s t o g iv e t h e desir ed con cen t r a t ion s u sed in t h e st u dy .

Chemicals used
T h e follow in g ch em ica ls w er e u sed for t h e v a r iou s ex per im en t s: 1 ,1 -diph en y l--2 --picr y lh y dr a zy l (DPPH), 2 ,2 -a zin o-bis(3 -et h y lben zt h ia zolin e-6 -su lfon ic a cid) (A BT S), v a n illin , a lu m in iu m ch lor ide (A lCl 3), pot a ssiu m a cet a t e (CH 3CO 2K), fer r ic ch lor ide (FeCl 2), BHT , a scor bic a cid, r u t in , Folin -Cioca lt eu r ea g en t , sodiu m ca r bon a t e (Na 2CO 3), ph osph a t e bu ffer , pot a ssiu m fer r icy a n ide [K 3Fe(CN) 6 ], t r ich lor oa cet ic a cid (T CA ), 2 -t h ioba r bit u r ic a cid (T BA ), g la cia l a cet ic a cid (CH 3COOH), sodiu m n it r opr u sside (Na 2[Fe(CN) 5NO]2 H 2O). T h ey w er e pu r ch a sed fr om Mer ck , Ga u t en g , Sou t h A fr ica . A ll ot h er ch em ica ls u sed w er e of a n a ly t ica l g r a de.

Determination of total phenols

T h e a m ou n t of ph en ol in t h e w h ole lea f ex t r a ct s of A. ferox w a s det er m in ed spect r oph ot om et r ica lly u sin g t h e m odified m et h od of W olfe et al.,[2 5 ] w it h Folin -Cioca lt eu r ea g en t . A n a liqu ot of t h e ex t r a ct w a s m ix ed w it h 5 m l Folin -Cioca lt eu r ea g en t (pr ev iou sly dilu t ed w it h w a t er a t a con cen t r a t ion of 1 :1 0 v /v ) a n d 4 m l (7 5 g /l) of sodiu m ca r bon a t e. T h e t u bes w er e v or t ex ed for 1 5 s a n d left t o st a n d for 3 0 m in a t 4 0 C for color dev elopm en t . A bsor ba n ce w a s t h en m ea su r ed a t 7 6 5 n m u sin g t h e A JI-C0 3 UV V IS spect r oph ot om et er . Resu lt s w er e ex pr essed a s m g /g of t a n n ic a cid equ iv a len t u sin g t h e ca libr a t ion cu r v e: Y = 0 .1 2 1 x , R2 = 0 .9 3 6 5 1 2 , w h er e x is t h e a bsor ba n ce a n d Y is t h e t a n n ic a cid equ iv a len t .

Estimation of flavonoids
T ot a l fla v on oid con t en t w a s det er m in ed u sin g t h e m et h od of Or don Ez et al.[2 6 ] A v olu m e of 0 .5 m l of 2 % A lCl 3 et h a n ol solu t ion w a s a dded t o 0 .5 m l of t h e sa m ple solu t ion . A ft er 1 h a t r oom t em per a t u r e, t h e a bsor ba n ce w a s m ea su r ed a t 4 2 0 n m . A y ellow color in dica t ed t h e pr esen ce of fla v on oids. Pla n t ex t r a ct s w er e ev a lu a t ed a t a fin a l con cen t r a t ion of 0 .1 m g /m l. T ot a l fla v on oid con t en t w a s ca lcu la t ed a s m g /g of qu er cet in u sin g t h e follow in g equ a t ion ba sed on t h e ca libr a t ion cu r v e: Y = 0 .0 2 5 5 x , R2 = 0 .9 8 1 2 , w h er e x is t h e a bsor ba n ce a n d Y is t h e qu er cet in equ iv a len t .

Total flavonols
T ot a l fla v on ol con t en t w a s det er m in ed by a dopt in g t h e pr ocedu r e descr ibed by Ku m a r a n a n d Ka r u n a k a r a n .[2 7 ] T h e r ea ct ion m ix t u r e con sist ed of 2 .0 m l of t h e sa m ple, 2 .0 m l of A lCl 3 pr epa r ed in et h a n ol a n d 3 .0 m l of (5 0 g /l) sodiu m a cet a t e solu t ion . T h e a bsor ba n ce a t 4 4 0 n m w a s m ea su r ed a ft er 2 .5 h a t 2 0 C. T ot a l fla v on ol con t en t w a s ca lcu la t ed a s m g /g of qu er cet in equ iv a len t fr om t h e ca libr a t ion cu r v e u sin g t h e equ a t ion : Y = 0 .0 2 5 5 x , R2 = 0 .9 8 1 2 , w h er e x is t h e a bsor ba n ce a n d Y is t h e qu er cet in equ iv a len t .

Determination of total proanthocyanidin

T ot a l pr oa n t h ocy a n idin w a s det er m in ed ba sed on t h e pr ocedu r e of Oy edem i et al.[5 ] T o 0 .5 m l of 1 m g /m l of t h e ex t r a ct solu t ion w a s a dded 3 m l of v a n illin -m et h a n ol (4 % v /v ) a n d 1 .5 m l of h y dr och lor ic a cid a n d v or t ex ed. T h e m ix t u r e w a s a llow ed t o st a n d for 1 5 m in a t r oom t em per a t u r e a n d t h e

a bsor ba n ce w a s m ea su r ed a t 5 0 0 n m . T ot a l pr oa n t h ocy a n idin con t en t w a s ev a lu a t ed a t a con cen t r a t ion of 0 .1 m g /m l a n d ex pr essed a s ca t ech in equ iv a len t (m g /g ) u sin g t h e ca libr a t ion cu r v e equ a t ion : Y = 0 .5 8 2 5 x , R2 = 0 .9 2 7 7 , w h er e x is t h e a bsor ba n ce a n d Y is t h e ca t ech in equ iv a len t .

Determination of tannins
T a n n in det er m in a t ion w a s don e a ccor din g t o t h e m et h od of A OA C[2 8 ] w it h som e m odifica t ion s. T o 0 .2 0 g of t h e sa m ple w a s a dded 2 0 m l of 5 0 % m et h a n ol. T h is w a s sh a k en t h or ou g h ly a n d pla ced in a w a t er ba t h a t 8 0 C for 1 h t o en su r e u n ifor m m ix in g . T h e ex t r a ct w a s filt er ed in t o a 1 0 0 -m l v olu m et r ic fla sk , follow ed by t h e a ddit ion of 2 0 m l of dist illed w a t er , 2 .5 m l of Folin -Den is r ea g en t a n d 1 0 m l of 1 7 % a q. Na 2CO 3 a n d w a s t h or ou g h ly m ix ed. T h e m ix t u r e w a s m a de u p t o 1 0 0 m l w it h dist illed w a t er , m ix ed a n d a llow ed t o st a n d for 2 0 m in . T h e blu ish -g r een color dev eloped a t t h e en d of t h e r ea ct ion m ix t u r e of differ en t con cen t r a t ion s r a n g es fr om 0 t o 1 0 ppm . T h e a bsor ba n ce of t h e t a n n ic a cid st a n da r d solu t ion s a s w ell a s sa m ple w a s m ea su r ed a ft er color dev elopm en t a t 7 6 0 n m u sin g t h e A JIC0 3 UV -V IS spect r oph ot om et er . Resu lt s w er e ex pr essed a s m g /g of t a n n ic a cid equ iv a len t u sin g t h e ca libr a t ion cu r v e: Y = 0 .0 5 9 3 x 0 .0 4 8 5 , R2 = 0 .9 8 2 6 , w h er e x is t h e a bsor ba n ce a n d Y is t h e t a n n ic a cid equ iv a len t .

Determination of alkaloids
A lk a loids w er e qu a n t it a t iv ely det er m in ed a ccor din g t o t h e m et h od of Ha r bor n e.[2 9 ] T w o h u n dr ed m illilit er s of 1 0 % a cet ic a cid in et h a n ol w a s a dded t o 5 g pow der ed pla n t sa m ple, cov er ed a n d a llow ed t o st a n d for 4 h . T h e filt r a t e w a s t h en con cen t r a t ed on a w a t er ba t h t o on e-fou r t h of it s or ig in a l v olu m e. Con cen t r a t ed a m m on iu m h y dr ox ide w a s a dded dr opw ise t o t h e ex t r a ct u n t il t h e pr ecipit a t ion w a s com plet ed a n d t h e w h ole solu t ion w a s a llow ed t o set t le. T h e collect ed pr ecipit a t es w er e w a sh ed w it h dilu t e a m m on iu m h y dr ox ide a n d t h en filt er ed. T h e r esidu e w a s dr ied a n d w eig h ed. T h e a lk a loid con t en t w a s det er m in ed u sin g t h e for m u la : % a lk a loid = fin a l w eig h t of t h e sa m ple / in it ia l w eig h t of t h e ex t r a ct 1 0 0 .

Determination of saponins
Qu a n t it a t iv e det er m in a t ion of sa pon in s w a s don e u sin g t h e m et h od of Oba don i a n d Och u k o.[3 0 ] T h e pow der ed sa m ple (2 0 g ) w a s a dded t o 1 0 0 m l of 2 0 % a qu eou s et h a n ol a n d k ept in a sh a k er for 3 0 m in . T h e sa m ples w er e h ea t ed ov er a w a t er ba t h for 4 h a t 5 5 C. T h e m ix t u r e w a s t h en filt er ed a n d t h e r esidu e r e-ex t r a ct ed w it h a n ot h er 2 0 0 m l of 2 0 % a qu eou s et h a n ol. T h e com bin ed ex t r a ct s w er e r edu ced t o a ppr ox im a t ely 4 0 m l ov er t h e w a t er ba t h a t 9 0 C. T h e con cen t r a t e w a s t r a n sfer r ed in t o a 2 5 0 m l sepa r a t or y fu n n el a n d ex t r a ct ed t w ice w it h 2 0 m l diet h y l et h er . T h e et h er la y er w a s disca r ded w h ile t h e a qu eou s la y er w a s r et a in ed a n d t o w h ich 6 0 m l n -bu t a n ol w a s a dded. T h e n -bu t a n ol ex t r a ct s w er e w a sh ed t w ice w it h 1 0 m l of 5 % a qu eou s sodiu m ch lor ide. T h e r em a in in g solu t ion w a s h ea t ed on a w a t er ba t h . A ft er ev a por a t ion , t h e sa m ples w er e dr ied in t h e ov en a t 4 0 C t o a con st a n t w eig h t . T h e sa pon in con t en t w a s ca lcu la t ed u sin g t h e for m u la : % sa pon in = fin a l w eig h t of sa m ple/in it ia l w eig h t of ex t r a ct s 1 00.

Antioxidant assay
T h e a n t iox ida n t a ct iv it ies of t h e w h ole lea f ex t r a ct s of A. ferox w er e det er m in ed u sin g DPPH, A BT S, r edu cin g pow er , lipid per ox ida t ion , n it r ic ox ide a n d h y dr og en per ox ide. Determination of ferric reducing power of the extracts T h e r edu cin g pow er of t h e w h ole lea v e ex t r a ct of A. ferox w a s ev a lu a t ed a ccor din g t o t h e m et h od descr ibed by A iy eg or o a n d Ok oh .[3 1 ] T h e m ix t u r e con t a in in g 2 .5 m l of 0 .2 M ph osph a t e bu ffer (pH 6 .6 ) a n d 2 .5 m l of K 3Fe(CN) 6 (1 % w /v ) w a s a dded t o 1 .0 m l of t h e

ex t r a ct s a n d st a n da r ds (0 .0 2 5 0 .5 m g /m l) pr epa r ed in dist illed w a t er . T h e r esu lt in g m ix t u r e w a s in cu ba t ed for 2 0 m in a t 5 0 C, follow ed by t h e a ddit ion of 2 .5 m l of T CA (1 0 % w /v ), w h ich w a s t h en cen t r ifu g ed a t 3 0 0 0 r pm for 1 0 m in . 2 .5 m l of t h e su per n a t a n t w a s m ix ed w it h 2 .5 m l of dist illed w a t er a n d 0 .5 m l of FeCl 3 (0 .1 % w /v ). T h e a bsor ba n ce w a s t h en m ea su r ed a t 7 0 0 n m a g a in st bla n k sa m ple. In cr ea sed a bsor ba n ce of t h e r ea ct ion m ix t u r e in dica t ed h ig h er r edu cin g pow er of t h e pla n t ex t r a ct . DPPH radical scavenging assay T h e m et h od of Liy a n a -Pa t h ia n a a n d Sh a h idi[3 2 ] w a s u sed for t h e det er m in a t ion of sca v en g in g a ct iv it y of DPPH fr ee r a dica l. DPPH (1 m l, 0 .1 3 5 m M) pr epa r ed in m et h a n ol w a s m ix ed w it h 1 .0 m l of a qu eou s ex t r a ct r a n g in g fr om 0 .0 2 5 t o 0 .5 m g /m l. T h e r ea ct ion m ix t u r e w a s v or t ex ed t h or ou g h ly a n d left in t h e da r k a t r oom t em per a t u r e for 3 0 m in . T h e a bsor ba n ce w a s m ea su r ed spect r oph ot om et r ica lly a t 5 1 7 n m . T h e sca v en g in g a bilit y of t h e pla n t ex t r a ct w a s ca lcu la t ed u sin g t h e equ a t ion : DPPH sca v en g in g a ct iv it y (%) = [(A bs con t r ol A bs sa m ple)]/(A bs con t r ol)] 1 0 0 , w h er e A bs con t r ol is t h e a bsor ba n ce of DPPH + m et h a n ol a n d A bs sa m ple is t h e a bsor ba n ce of DPPH r a dica l + sa m ple (sa m ple or st a n da r d). ABTS radical scavenging activity T h e m et h od descr ibed by A deda po et al.[1 ] w a s a dopt ed for t h e det er m in a t ion of A BT S a ct iv it y of t h e pla n t ex t r a ct . T h e w or k in g solu t ion w a s pr epa r ed by m ix in g t w o st ock solu t ion s of 7 m M A BT S a n d 2 .4 m M pot a ssiu m per su lfa t e in equ a l a m ou n t s a n d a llow ed t o r ea ct for 1 2 h a t r oom t em per a t u r e in t h e da r k . T h e r esu lt in g solu t ion w a s fu r t h er dilu t ed by m ix in g 1 m l A BT S + solu t ion w it h 6 0 m l m et h a n ol t o obt a in a n a bsor ba n ce of 0 .7 0 6 0 .0 0 1 u n it s a t 7 3 4 n m a ft er 7 m in u sin g a spect r oph ot om et er . T h e per cen t a g e in h ibit ion of A BT S
+

by t h e ex t r a ct w a s ca lcu la t ed fr om t h e

follow in g equ a t ion : % in h ibit ion = [(A bs con t r ol A bs sa m ple)]/(A bs con t r ol)] 1 0 0 . Nitric oxide scavenging activity T h e m odified m et h od descr ibed by Oy edem i et al.[5 ] w a s u sed t o det er m in e t h e n it r ic ox ide r a dica l sca v en g in g a ct iv it y of a qu eou s a n d ot h er solv en t ex t r a ct s of A. ferox . A v olu m e of 2 m l of 1 0 m M of sodiu m n it r opr u sside pr epa r ed in 0 .5 m M ph osph a t e bu ffer ed sa lin e (pH 7 .4 ) w a s m ix ed w it h 0 .5 m l of pla n t ex t r a ct s, g a llic a cid a n d BHT in div idu a lly a t 0 .0 2 5 0 .5 m g /m l. T h e m ix t u r e w a s in cu ba t ed a t 2 5 C for 1 5 0 m in . 0 .5 m l of t h e in cu ba t ed solu t ion w a s m ix ed w it h 0 .5 m l of Gr iess r ea g en t [1 .0 m l su lfa n ilic a cid r ea g en t (0 .3 3 % pr epa r ed in 2 0 % g la cia l a cet ic) a cid a t r oom t em per a t u r e for 5 m in w it h 1 m l of n a ph t h y len edia m in e dich lor ide (0 .1 % w /v )]. T h e m ix t u r e w a s in cu ba t ed a t r oom t em per a t u r e for 3 0 m in , follow ed by t h e m ea su r em en t of t h e a bsor ba n ce a t 5 4 0 n m . T h e a m ou n t of n it r ic ox ide r a dica ls in h ibit ed by t h e ex t r a ct w a s ca lcu la t ed u sin g t h e follow in g equ a t ion : NO r a dica l sca v en g in g a ct iv it y (%) = [(A bs con t r ol A bs sa m ple)]/(A bs con t r ol)] 1 0 0 , w h er e A bs con t r ol is t h e a bsor ba n ce of NO r a dica ls + m et h a n ol a n d A bs sa m ple is t h e a bsor ba n ce of NO r a dica l + ex t r a ct or st a n da r d. Hydrogen peroxide scavenging activity Hy dr og en per ox ide sca v en g in g a ct iv it y of t h e pla n t ex t r a ct w a s est im a t ed u sin g t h e m odified m et h od descr ibed by Oy edem i et al.[5 ] Pla n t ex t r a ct (4 m g /m l) pr epa r ed in dist illed w a t er a t v a r iou s con cen t r a t ion s w a s m ix ed w it h 0 .6 m l of 4 m M H 2O 2 solu t ion pr epa r ed in ph osph a t e bu ffer (0 .1 M pH 7 .4 ) a n d in cu ba t ed for 1 0 m in . T h e a bsor ba n ce of t h e solu t ion w a s m ea su r ed a t 2 3 0 n m u sin g t h e Biom a t e t h er m ospect r on oic, a g a in st a bla n k solu t ion con t a in in g t h e pla n t ex t r a ct w it h ou t H 2O 2. T h e a m ou n t of h y dr og en per ox ide r a dica l in h ibit ed by t h e ex t r a ct w a s ca lcu la t ed u sin g t h e follow in g equ a t ion : H 2O 2 r a dica l sca v en g in g a ct iv it y = [(A bs con t r ol A bs sa m ple)]/(A bs con t r ol) 1 0 0 , w h er e A bs con t r ol is t h e a bsor ba n ce of H 2O 2 r a dica ls + m et h a n ol a n d A bs sa m ple is t h e a bsor ba n ce of H 2O 2 r a dica l + sa m ple or ex t r a ct or st a n da r d. Estimation of lipid peroxidation A m odified t h ioba r bit u r ic a cid-r ea ct iv e species (T BA RS) a ssa y descr ibed by Da sg u pt s a n d De[3 3 ] w a s u sed t o m ea su r e t h e lipid per ox ide for m ed, u sin g eg g -y olk h om og en a t es a s lipid-r ich m edia . Eg g h om og en a t e (0 .5 m l, 1 0 % in dist illed w a t er , v /v ) a n d 0 .1 m l of t h e w h ole ex t r a ct

of A. ferox w er e m ix ed in a t est t u be a n d t h e v olu m e w a s m a de u p t o 1 m l w it h dist illed w a t er . Fin a lly , 0 .0 5 m l FeSO 4 (0 .0 7 M) w a s a dded t o t h e m ix t u r e a n d in cu ba t ed for 3 0 m in t o in du ce lipid per ox ida t ion . T h er ea ft er , 1 .5 m l of 2 0 % a cet ic a cid (pH a dju st ed t o 3 .5 w it h Na OH) a n d 1 .5 m l of 0 .8 % T BA (w /v ) (pr epa r ed in 1 .1 % sodiu m dodecy l su lfa t e) a n d 0 .0 5 m l 2 0 % T CA w er e a dded, v or t ex ed a n d h ea t ed in a boilin g w a t er ba t h for 6 0 m in . A ft er coolin g , 5 .0 m l of n -bu t a n ol w a s a dded t o ea ch t u be a n d cen t r ifu g ed a t 3 0 0 0 r pm for 1 0 m in . T h e a bsor ba n ce of t h e or g a n ic u pper la y er w a s m ea su r ed a t 5 3 2 n m . For t h e bla n k , 0 .1 m l of dist illed w a t er w a s u sed in pla ce of t h e ex t r a ct .

Statistical analysis
W h er e a pplica ble, t h e r esu lt s w er e ex pr essed a s m ea n st a n da r d dev ia t ion (SD) of t h r ee r eplica t es a n d w er e su bject ed t o a n a ly sis of v a r ia n ce u sin g t h e st u den t Min it a b r elea se v er sion 1 2 , W in dow s 9 5 . Sig n ifica n t lev els w er e t est ed a t P < 0 .0 5 .

RESULTS
Phytochemical constituents
Ph y t och em ica l a n a ly sis con du ct ed on t h e w h ole lea f ex t r a ct s of A. ferox r ev ea led t h e pr esen ce of ph en ols, fla v on oids, fla v on ols, pr oa n t h ocy a n idin s, t a n n in s, a lk a loids a n d sa pon in s [T a ble 1 ]. Most pla n t s a r e u sed for t h e t r ea t m en t of disea ses beca u se of t h e pr esen ce of t h ese ph y t och em ica ls w h ich h a v e been r epor t ed t o h a v e h ig h m edicin a l v a lu es. T h e con cen t r a t ion s of ph en ols in t h e differ en t solv en t ex t r a ct s w er e sig n ifica n t ly differ en t fr om ea ch ot h er . T h is g iv es a n in dica t ion t h a t solv en t s possess differ en t ex t r a ct in g ca pa cit y for ph en ol. T h e h ig h con t en t of ph en ol in t h e w h ole lea f ex t r a ct s of A. ferox , obser v ed in t h e a cet on e a n d et h a n ol ex t r a ct , m a y be a con t r ibu t in g fa ct or t ow a r d it s a n t iox ida n t a ct iv it y .
Tab l e 1 Phy toc hemi c al anal y si s of di f f er ent sol v ent ex tr ac ts of the w hol e l eaf of A. ferox

T h e fla v on oid con t en t in t h e w h ole lea f ex t r a ct s in t h e v a r iou s solv en t s w a s in t h e or der : m et h a n ol > et h a n ol > a cet on e > a qu eou s ex t r a ct s. T h e fla v on oid con t en t in t h e m et h a n ol ex t r a ct w a s sig n ifica n t ly h ig h er t h a n t h e et h a n ol ex t r a ct . Mea n w h ile, t h e a qu eou s a n d a cet on e ex t r a ct s w er e n ot sig n ifica n t ly differ en t fr om ea ch ot h er in t h is a spect . T h e a cet on e ex t r a ct possessed h ig h fla v on ol con t en t w h ich w a s sig n ifica n t ly differ en t fr om t h e ot h er solv en t ex t r a ct s ev a lu a t ed in t h is st u dy , a n d t h is m a y be lin k ed t o t h e pola r it y of t h e solv en t u sed. T h is w a s follow ed by t h e et h a n ol, m et h a n ol a n d a qu eou s ex t r a ct s in t h is or der , bu t t h ese w er e n ot sig n ifica n t ly differ en t fr om ea ch ot h er . A m oder a t e a m ou n t of fla v on ols w a s obser v ed in t h e m et h a n ol a n d a qu eou s ex t r a ct s, bu t t h er e w a s a n a ppr ecia ble a m ou n t in t h e a cet on e a n d et h a n olic ex t r a ct s. T h e a cet on e ex t r a ct con t a in ed h ig h pr oa n t h ocy a n idin con t en t , follow ed by t h e et h a n ol, m et h a n ol a n d a qu eou s ex t r a ct s [T a ble 1 ]. T a n n in con t en t w a s g en er a lly low in a ll t h e solv en t ex t r a ct s of A. ferox com pa r ed t o a ll ot h er ph y t och em ica ls. A lk a loid con t en t of t h e w h ole lea f ex t r a ct of A. ferox follow ed t h e t r en d: a cet on e > et h a n ol > m et h a n ol > a qu eou s ex t r a ct s, a s sh ow n in T a ble 1 . T h e a cet on e ex t r a ct ex h ibit ed h ig h con cen t r a t ion of a lk a loids w h ich w a s sig n ifica n t ly differ en t fr om ot h er solv en t ex t r a ct s. T h e per cen t a g e con cen t r a t ion of sa pon in s in t h e et h a n ol ex t r a ct w a s sig n ifica n t ly h ig h er t h a n ot h er solv en t s, bu t low er in t h e a qu eou s ex t r a ct .

Antioxidant activities of the extracts

T h e ex t r a ct s sh ow ed h ig h per cen t a g e in h ibit ion a ct iv it y in a ll t h e fr ee r a dica l sca v en g in g m odels ev a lu a t ed in t h e pr esen t st u dy . Ferric reducing power T h e pot en t ia l of t h e pla n t ex t r a ct s t o r edu ce Fe 3+ t o Fe 2+ by elect r on t r a n sfer is a n in dica t ion of t h eir a n t iox ida n t a bilit y . T h e r edu cin g pow er of t h e ex t r a ct s in com pa r ison w it h t h e st a n da r ds (v it a m in C a n d BHT ) is pr esen t ed in Fig u r e 1 . T h e obser v ed ch a n g e of y ellow color of t est solu t ion t o v a r iou s sh a des of blu e a n d g r een , depen din g on t h e con cen t r a t ion of t h e ex t r a ct , is a n in dica t ion of it s a n t iox ida n t a ct iv it y . T h e fer r ic r edu cin g a ct iv it ies of t h e ex t r a ct s w er e sig n ifica n t ly low er t h a n t h e st a n da r d dr u g s u sed in t h is or der : v it a m in C > BHT > et h a n ol > a cet on e > m et h a n ol > a qu eou s ex t r a ct s. A t 0 .5 m g /m l, t h e a bsor ba n ce v a lu es of a cet on e, m et h a n ol a n d et h a n ol ex t r a ct s w er e com pa r a ble w it h v it a m in C a n d BHT a s depict ed in Fig u r e 1 .

Fi gur e 1 Reduc i ng pow er of di f f er ent sol v ent ex tr ac ts of A. ferox

DPPH radical scavenging activity T h e DPPH sca v en g in g pot en t ia l of t h e w h ole lea f ex t r a ct s of A. ferox w a s con cen t r a t ion depen den t a s sh ow n in Fig u r e 2 . T h e per cen t a g e in h ibit ion of DPPH by t h e v a r iou s solv en t ex t r a ct s a n d t h e st a n da r d dr u g s w a s r ecor ded in decr ea sin g or der : BHT > r u t in > m et h a n ol > et h a n ol > a cet on e > a qu eou s ex t r a ct . T h e % in h ibit ion of BHT a n d r u t in w a s sig n ifica n t ly differ en t fr om t h e ex t r a ct s. T h ou g h t h e a qu eou s ex t r a ct sh ow ed t h e lea st in h ibit ion , it w a s n ot sig n ifica n t ly differ en t fr om t h e ot h er solv en t s. T h e IC 50 v a lu e of m et h a n ol ex t r a ct w a s 0 .0 8 6 m g /m l, a cet on e a n d et h a n ol ex t r a ct s w it h sim ila r v a lu e of 0 .2 8 8 m g /m l a n d t h a t of a qu eou s ex t r a ct w a s 0 .5 1 7 m g /m l.
Fi gur e 2 DPPH r adi c al sc av engi ng ac ti v i ty of the w hol e l eaf ex tr ac t of A. ferox ob tai ned usi ng di f f er ent sol v ents

ABTS radical scavenging activity T h e per cen t a g e in h ibit ion of A BT S r a dica l by t h e v a r iou s solv en t ex t r a ct s of A. ferox is sh ow n in Fig u r e 3 . T h e % in h ibit ion of A BT S by t h e v a r iou s solv en t ex t r a ct s w a s con cen t r a t ion depen den t a n d com pa r ed fa v or a bly w ell w it h t h e BHT a n d r u t in . T h e sca v en g in g a ct iv it y in t h e v a r iou s solv en t s w a s in t h e or der : r u t in > m et h a n ol > a cet on e > et h a n ol > BHT > a qu eou s ex t r a ct s. T h e a cet on e a n d et h a n ol ex t r a ct s com pa r ed fa v or a bly w ell w it h BHT bu t low er t h a n t h e m et h a n ol ex t r a ct [ Fig u r e 3 ]. T h er e w a s a sig n ifica n t differ en ce in t h e % in h ibit ion of A BT S
+

a t t h e differ en t

con cen t r a t ion s a n d solv en t s u sed. T h e m et h a n ol ex t r a ct a n d BHT h a d IC 50 v a lu es of 0 .0 2 a n d 0 .0 2 4 m g /m l, r espect iv ely , follow ed by a cet on e a n d et h a n ol ex t r a ct s a t 0 .0 3 3 a n d 0 .0 6 2 m g /m l, r espect iv ely . T h e a qu eou s ex t r a ct ex h ibit ed t h e lea st sca v en g in g a ct iv it y (0 .1 7 3 m g /m l) in com pa r ison w it h ot h er solv en t ex t r a ct s.

Fi gur e 3 A BTS r adi c al sc av engi ng ac ti v i ty of the w hol e l eaf ex tr ac t of A. ferox ob tai ned usi ng di f f er ent sol v ents

Nitric oxide inhibition T h e r a dica l sca v en g in g a ct iv it y of t h e w h ole lea f ex t r a ct of A. ferox w a s r eflect ed a s t h e % in h ibit ion of n it r ic ox ide [Fig u r e 4 ]. T h e a ct iv it ies of t h e v a r iou s ex t r a ct s w er e in t h e or der : BHT > r u t in > m et h a n ol > et h a n ol > a qu eou s > a cet on e ex t r a ct s. T h e % in h ibit ion of n it r ic ox ide by t h e v a r iou s solv en t ex t r a ct s differ ed sig n ifica n t ly fr om t h ose of BHT a n d r u t in . T h e IC 50 v a lu es of 0 .0 2 3 , 0 .0 2 4 , 0 .0 1 6 a n d 0 .0 1 7 m g /m l w er e r ecor ded for t h e m et h a n ol ex t r a ct , et h a n ol ex t r a ct , BHT a n d r u t in r espect iv ely , w h ile a cet on e a n d a qu eou s ex t r a ct s h a d t h eir a ct iv it ies a t 0 .0 7 7 a n d 0 .0 7 4 m g /m l r espect iv ely .

Fi gur e 4 N i tr i c ox i de r adi c al sc av engi ng ac ti v i ty of the w hol e l eaf ex tr ac t of A. ferox ob tai ned usi ng di f f er ent sol v ents

Hydrogen peroxide inhibition T h e sca v en g in g a ct iv it y of t h e w h ole lea f ex t r a ct of A. ferox a g a in st h y dr og en per ox ide is sh ow n in Fig u r e 5 . T h e sca v en g in g a ct iv it ies of t h e pla n t in v a r iou s solv en t s w er e in t h e or der : a cet on e < et h a n ol < g a llic a cid< m et h a n ol < BHT < a qu eou s ex t r a ct . T h e per cen t a g e in h ibit ion s of h y dr og en per ox ide by t h e v a r iou s solv en t s w er e con cen t r a t ion depen den t a n d differ ed sig n ifica n t ly fr om ea ch ot h er .

Fi gur e 5 Hy dr ogen per ox i de r adi c al sc av engi ng ac ti v i ty of the w hol e l eaf ex tr ac t of A. ferox ob tai ned usi ng di f f er ent sol v ents

Lipid peroxidation T h e sca v en g in g of lipid per ox ides by t h e w h ole lea f ex t r a ct of A. ferox , BHT a n d g a llic a cid is pr esen t ed in Fig u r e 6 . T h e % in h ibit ion of lipid per ox ides by t h e v a r iou s solv en t ex t r a ct s w a s r ecor ded in in cr ea sin g or der : g a llic a cid > BHT > m et h a n ol > a qu eou s > et h a n ol > a cet on e ex t r a ct . T h e % in h ibit ion of lipid per ox ida t ion by t h e v a r iou s solv en t s did n ot differ sig n ifica n t ly fr om t h e st a n da r d BHT a s com pa r ed t o g a llic a cid. T h e da t a sh ow ed a sig n ifica n t differ en ce fr om t h e solv en t ex t r a ct u sed. Ga llic a cid h a d t h e h ig h est in h ibit or y a ct iv it y a g a in st lipid per ox ida t ion , w it h a n IC 50 v a lu e of 0 .1 1 8 m g /m l, w h ile BHT , a qu eou s, a cet on e, m et h a n ol, et h a n ol ex t r a ct s w er e a ct iv e w it h IC 50 v a lu es of 1 .1 8 6 , 1 .8 3 7 , 1 .4 9 2 , 0 .9 3 0 a n d 1 .2 7 0 m g /m l, r espect iv ely .
Fi gur e 6 Li pi d per ox i dati on sc av engi ng ac ti v i ty of the w hol e l eaf ex tr ac t of A. ferox ob tai ned f r om di f f er ent sol v ents

DISCUSSION
Ph en olic com pou n ds a r e k n ow n t o ex h ibit st r on g a n t iox ida n t a ct iv it ies, w h ich h a v e dir ect a n t iox ida n t pr oper t ies du e t o t h e pr esen ce of h y dr ox y l g r ou ps, w h ich a ct a s h y dr og en don or .[3 4 ] A ddit ion a lly , t h ey a r e fou n d t o be effect iv e in sca v en g in g fr ee r a dica ls a s a r esu lt of t h eir r edox pr oper t ies t h a t a llow t h em t o a ct a s r edu cin g a g en t s.[5 ] T h e ph y t och em ica ls in v est ig a t ed, a s sh ow n in t h is st u dy , h a v e been r epor t ed t o possess st r on g a n t iox ida n t a ct iv it ies du e t o t h eir a bilit y t o a dsor b, qu en ch fr ee r a dica ls a n d decom pose per ox ides g en er a t ed in t h e sy st em .[1 ]

Fla v on oids a r e h y dr ox y la t ed ph en olics a n d a r e pot en t w a t er -solu ble a n t iox ida n t s w h ich h elp in r a dica l sca v en g in g a n d pr ev en t ion of ox ida t iv e cell da m a g e. T h ey h a v e been r epor t ed t o possess st r on g a n t iox ida n t a ct iv it ies.[3 5 ,2 4 ] T h e con cen t r a t ion in t h e w h ole lea f ex t r a ct s of A. ferox w a s low . How ev er , m et h a n ol ex t r a ct s h a d m or e fla v on oids t h a n t h e ot h er solv en t ex t r a ct s in v est ig a t ed in t h is st u dy . Pr oa n t h ocy a n idin s a r e g r ou p of poly ph en olic biofla v on oids w h ich h a v e a pr ot ect iv e effect in elim in a t in g h y dr ox y l r a dica ls.[3 6 ] Pr oa n t h ocy a n idin con t en t w a s r ela t iv ely h ig h in t h e a cet on e ex t r a ct . T h is is in a g r eem en t w it h t h e fin din g s of Loot s et al.[2 4 ] w h o r epor t ed h ig h er con cen t r a t ion of pr oa n t h ocy a n idin s (poly ph en ols) in t h e lea f g el ex t r a ct of A. ferox . A ccor din g t o Loot s et al.[2 4 ], A. ferox t h a t con t a in s ph en olic com pou n ds m a y ser v e a s a pot en t ia l sou r ce of bioa ct iv e a g en t s in t h e t r ea t m en t of ca n cer a n d ot h er deg en er a t iv e disea ses. Sev er a l ph en olic com pou n ds in clu din g t a n n in s a r e in h ibit or s of m a n y h y dr oly t ic en zy m es su ch a s pr ot eoly t ic m a cer a t in g en zy m es u sed by pla n t a g a in st pa t h og en s.[3 7 ] T a n n in s a r e k n ow n t o be u sefu l for t h e pr ev en t ion of ca n cer a s w ell a s t r ea t m en t of in fla m ed or u lcer a t ed t issu es.[3 8 ,3 9 ] How ev er , t a n n in lev el w a s low a n d n ot sig n ifica n t ly differ en t in com pa r ison t o ot h er solv en t ex t r a ct s u sed in t h is st u dy . A lk a loids h a v e been a ssocia t ed w it h m edicin a l pu r pose for cen t u r ies a s a g en t s possessin g a n a lg esic, a n t im a la r ia l, a n t isept ic a n d ba ct er icida l a ct iv it ies, bu t cou ld be t ox ic t o cells.[1 0 ] In t h is st u dy , t h e h ig h con t en t of a lk a loids in t h e a cet on e, et h a n ol a n d m et h a n ol ex t r a ct s of A. ferox cou ld be r espon sible for it s m u ch a ccla im ed m edicin a l v a lu e. Sa pon in s in m edicin a l pla n t s a r e r espon sible for m ost biolog ica l effect s r ela t ed t o cell g r ow t h a n d div ision in h u m a n s a n d h a v e in h ibit or y effect on in fla m m a t ion .[3 8 ,4 0 ] T h e lev el of sa pon in con t en t in t h e solv en t ex t r a ct s of t h e w h ole lea f of A. ferox w a s qu it e m oder a t e. How ev er , et h a n ol ex t r a ct pr odu ced m or e sa pon in s t h a n ot h er solv en t s. T h er e w a s n o sig n ifica n t differ en ce in sa pon in con t en t in a ll t h e v a r iou s solv en t ex t r a ct s in v est ig a t ed in t h is pr esen t st u dy . T h e a bilit y of a su bst a n ce t o a ct a s a n a n t iox ida n t depen ds on it s st r en g t h t o r edu ce ROS by don a t in g h y dr og en a t om .[4 1 ] T h e r edu cin g ca pa cit y of a com pou n d g en er a lly depen ds on t h e pr esen ce of r edu ct a n t s w h ich ex h ibit a n t iox ida t iv e pot en t ia l by br ea k in g t h e fr ee r a dica l ch a in a n d don a t in g h y dr og en a t om .[4 2 ] It h a s been fou n d t h a t t h e t r a n sfor m a t ion of Fe 3+ t o Fe 2+ occu r r ed in t h e pr esen ce of pla n t ex t r a ct .[4 3 ] A. ferox w h ole lea f ex t r a ct s sh ow ed t h e r edu ct iv e ca pa bilit ies w h en com pa r ed t o a scor bic a cid a n d BHT . T h e r edu cin g pow er of t h e ex t r a ct s w a s con cen t r a t ion depen den t a n d t h e a n t iox ida n t a ct iv it ies in a ll t h e solv en t s u sed w er e com pa r a ble w it h v it a m in C a n d BHT . DPPH is a r ela t iv ely st a ble fr ee r a dica l sca v en g er w h ich con v er t s t h e u n pa ir ed elect r on s t o pa ir ed on es by h y dr og en pr ot on don a t ion . Sca v en g in g of DPPH r a dica l in t h is st u dy in dica t es t h e pot en cy of t h e pla n t ex t r a ct s in don a t in g h y dr og en pr ot on t o t h e lon e pa ir elect r on of t h e r a dica ls. Beca u se t h e in h ibit ion w a s m or e a t a h ig h er con cen t r a t ion in a ll t h e solv en t ex t r a ct s, it cou ld be su g g est ed t h a t t h e pla n t ex t r a ct s con t a in com pou n ds ca pa ble of don a t in g pr ot on s t o t h e fr ee r a dica ls. T h e m et h ods h a v e pr ov en t h e effect iv en ess of t h e ex t r a ct s in a con cen t r a t ion -depen den t m a n n er .[4 1 ] T h e w ea k a ct iv it y obser v ed in t h e DPPH r a dica l sca v en g in g a ssa y m a y be a s a r esu lt of t h e low lev el of fla v on oid con t en t s in t h e pla n t ex t r a ct s.[5 ] How ev er , t h e a bilit y of t h e pla n t ex t r a ct s t o sca v en g e DPPH r a dica l cou ld a lso r eflect it s a bilit y t o sca v en g e a n d in h ibit t h e for m a t ion of A BT S +. T h e sca v en g in g a ct iv it y of A BT S
+

by t h e pla n t ex t r a ct s w a s fou n d t o be h ig h er t h a n t h a t of DPPH

r a dica l. T h e solu bilit y of ex t r a ct s in differ en t t est in g sy st em s a n d r a dica l r ea ct iv it y con fir m in g t h e r em ov a l of odd elect r on a r e believ ed t o be r espon sible for t h e h ig h er sca v en g in g a ct iv it y of A BT S. T h e sca v en g in g a ct iv it y of A BT S
+

r a dica l by t h e pla n t ex t r a ct cou ld im ply t h a t t h e ex t r a ct s m a y be u sefu l

for t r ea t in g r a dica l r ela t ed pa t h olog ica l da m a g e, especia lly a t h ig h er con cen t r a t ion s.[1 1 ] Nit r ic ox ide

is a n im por t a n t ch em ica l m edia t or g en er a t ed by en dot h elia l cells, m a cr oph a g es a n d n eu r on s a n d is in v olv ed in t h e r eg u la t ion of v a r iou s ph y siolog ica l pr ocesses. Ex cess con cen t r a t ion of n it r ic ox ide is a ssocia t ed w it h sev er a l disea ses.[4 1 ,4 4 ] Ox y g en r ea ct s w it h t h e ex cess n it r ic ox ide t o g en er a t e n it r it e a n d per ox y n it r it e a n ion s, w h ich a ct a s fr ee r a dica ls.[4 5 ] In t h e pr esen t st u dy , t h e ex t r a ct sh ow ed a m oder a t e a ct iv it y by com pet in g w it h ox y g en t o r ea ct w it h n it r ic ox ide a n d t h u s in h ibit s t h e g en er a t ion of t h e a n ion s. Hy dr og en per ox ide is a n im por t a n t ROS beca u se of it s a bilit y t o pen et r a t e biolog ica l m em br a n es. How ev er , it m a y be t ox ic if con v er t ed t o h y dr ox y l r a dica l in t h e cell.[4 6 ] T h e h y dr ox y l r a dica l is a n ex t r em ely r ea ct iv e fr ee r a dica l for m ed in biolog ica l sy st em s. It h a s been im plica t ed a s a h ig h ly da m a g in g species in fr ee r a dica l pa t h olog y , ca pa ble of da m a g in g a lm ost ev er y m olecu le fou n d in liv in g cells. A m on g t h e ox y g en r a dica ls, t h e h y dr ox y l r a dica l is t h e m ost r ea ct iv e a n d in du ces sev er e da m a g e t o a dja cen t biom olecu les.[4 7 ] Sca v en g in g of H 2O 2 by t h e ex t r a ct s m a y be a t t r ibu t ed t o t h eir ph en olics, w h ich don a t e elect r on t o H 2O 2, a n d t h u s r edu ce it t o w a t er . T h e solv en t ex t r a ct s w er e ca pa ble of sca v en g in g h y dr og en per ox ide in a con cen t r a t ion -depen den t m a n n er . T h is in dica t es t h e pot en cy of t h e pla n t ex t r a ct s in pr ev en t in g t h e join in g of n u cleosides in t h e DNA a n d possible br ea k a g e lea din g t o ca r cin og en esis a n d cy t ot ox icit y .[4 8 ] Fr ee r a dica l in du ced per ox ida t ion in poly u n sa t u r a t ed lipids occu r s eit h er t h r ou g h fer r y lper fer r y l com plex or t h r ou g h OH* r a dica ls.[4 9 ] In t h is st u dy , lipid per ox ida t ion w a s br ou g h t a bou t by eg g -y olk h om og en a t es a s lipid-r ich m edia . T h e t est ed pla n t ex t r a ct s sh ow ed w ea k a n t iox ida n t a ct iv it y w h ich is in dica t ed by t h eir h ig h a bsor ba n ce v a lu es. T h is st u dy sh ow s con cen t r a t ion -depen den t pr ev en t ion of lipid per ox ide g en er a t ion . T h e pr esen t st u dy sh ow s t h e sca v en g in g a ct iv it y of t h e w h ole lea f ex t r a ct s of A. ferox in et h a n ol, m et h a n ol, a cet on e a n d a qu eou s ex t r a ct s. T h e a ct iv it y w a s h ig h in et h a n ol, a cet on e a n d m et h a n ol ex t r a ct s, bu t low in a qu eou s m ediu m , in dica t in g t h eir a n t iox ida n t pot en t ia l. T h e obser v ed r esu lt s su g g est fu r t h er a n a ly ses t o con fir m it s pr oph y la ct ic effect in t h e t r ea t m en t of fr ee r a dica l-m edia t ed disea ses. Most a n t iox ida n t a ct iv it ies depen d on t h e a m ou n t of t h e ph y t och em ica ls pr esen t in t h e pla n t s. A lt h ou g h t h e con t en t s of m ost ph y t och em ica ls ev a lu a t ed a r e n ot v er y h ig h bu t sy n er g ist ica lly boost t h e a n t iox ida n t a ct iv it y of t h e w h ole lea f ex t r a ct s of A. ferox . T h u s t h e pla n t h a d pot en t a n t iox ida n t pr oper t ies t o cu r t a il pr og r ession of r a dica l r ela t ed disea ses a n d t h er eby g iv e cr eden ce t o t h e t r a dit ion a l u sa g e of A. ferox ex t r a ct .

ACKNOWLEDGMENT
T h is r esea r ch w a s su ppor t ed by a g r a n t fr om Gov a n Mbek i Resea r ch a n d Dev elopm en t Cen t r e, Un iv er sit y of For t Ha r e.

Footnotes
Source of Support: Grant from Govan Mbeki Research and Development Centre, University of Fort Hare Conflict of Interest: None declared.

Article information
Pharmacogn Mag. 2011 Oct-Dec; 7(28): 325333. doi: 10.4103/0973-1296.90414 PMCID: PMC3261067 Olubunmi Abosebe Wintola and Anthony Jide Afolayan Department of Botany, University of Fort Hare, Alice 5700, South Africa

Address for correspondence: Prof. A. J. Afolayan, Department of Botany, University of Fort Hare, Alice 5700, South Africa. E-mail: aafolayan/at/ufh.ac.za Received March 26, 2011; Revised May 7, 2011; Accepted November 30, 2011. Copyright : Pharmacognosy Magazine This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. This article has been cited by other articles in PMC. Articles from Pharmacognosy Magazine are provided here courtesy of Medknow Publications

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