International Journal of Pharmaceutical Sciences and Nanotechnology Volume 3 Issue 1 April June 2010

Research Paper CNS Activity of Ethanol Extract of Wedelia chinensis in Experimental Animals
V. Suresh, .!. "umar, A. Suresh, N.S. "umar, #. Arunachalam an$ ". %masan&ar'
College of Pharmacy, J.K.K. Munirajah Medical Research Foundation, Namakkal, ndia.

A(S) AC)*

The plant Wedelia chinensis was found to be used by different traditional systems and folklore for the treatment of various disorders. The aim of the present study is to investigate the effect on central nervous system (CNS) of the ethanol extract of Wedelia chinensis whole plant in Swiss albino mice and istar rats.The CNS effects were evaluated by general behaviour! exploratory behaviour! muscle relaxant activity and phenobarbitone sodium"induced sleeping time using standard procedures in experimental animal models.The results revealed that the ethanol extract at #$$ and %$$ mg&kg caused a significant reduction in the spontaneous activity (general behavioural profile)! exploratory behavioural pattern ('"ma(e and head dip test)! muscle relaxant activity (rotarod and traction tests)! and significantly potentiated phenobarbitone sodium"induced sleeping time.The results conclude that the extract exhibit CNS depressant activity in tested animal models.

"E+,- .S* Wedelia chinensis) muscle relaxant) phenobarbitone*induced sleeping time) CNS depressant activity /ntro$uction
Wedelia chinensis !"steraceae# is a $erennial her% of a%out &.' to &.( cm height. )ea*es are fleshy, usually +,( cm long and -,. cm /ide, irregularly toothed or serrate, usually /ith a $air of lateral lo%es and o%*iate in sha$e. Flo/ers are yello/, tu%ular in terminal or a0illary head and +,. cm in diameter. 1raditionally the fruits, lea*es and stem are used in child%irth and in the treatment of %ites and stings, fe*er and infection. 1he lea*es are used in the treatment of kidney dysfunction, cold, /ounds and amenorrhea !Mathe/, 2(3'#. 1he lea*es are also used for dyeing hair and for $romoting their gro/th. 1he tonic of the lea*es is used in cough andce$halalgia. 4ecoction of the $lant is used in menorrhagia and skin diseases !Kirtikar and 5asu, 2(6.7 8a0enaet al., 2(39#. 1he $lant has also found its use in inflammations, helmintic diseases and li*er disorders !"nonymous, 2(3'#.1he decoction of the $lant /as e0tensi*ely used %y the tri%es in Kolli :ills of Namakkal 4istrict, 1amilnadu, ndia, to reduce mental tension and also to induce slee$ and the $lant affects CN8 !"nonymous, 2(+3#.1he $lant has %een used as astringent, %itter, acrid, anti,inflammatory andcardiotonic, and treatment of /ounds, seminal /eakness and *iral,he$atitis !Cho$ra, 2(.97 ;aidyaratnam, 2((6#. "n ethanolic !. <# e0tract inhi%its the gro/th of =hrlich>s ascites carcinoma. 1he e0tracts of this $lant ha*e %een tested in e0$erimental animal models for their
* +or correspondence, -. .masanker! /*mail, youmasankar0yahoo.co.in

he$ato$rotecti*e effect !"$erset al., -&&-#, analgesic and anti,inflammatory acti*ity!8ureshkumaret al., -&&9# and androgen su$$ressing acti*ity !)in et al., -&&6#1he alcoholic e0tract of the lea*es /as found to $ossess /ound healing $ro$erties !;ermaet al., -&&37 Mishra et al., -&&(#, antio0idant !;erma and Khosa, -&&3# and li$id $ero0idation inhi%itory acti*ity !;erma and Khosa, -&&(# in rats. 1he $lant is traditionally used to reduce mental tension and to induce slee$and scientifically re$orted to $ossess antio0idant $ro$erty /hich indicates its usefulness in reducing an0iety and stress in emotional conditions. 1herefore, in the light of the traditional and re$orted uses, the $resent study /as undertaken to in*estigate the CN8 acti*ity of the ethanol e0tract of Wedelia chinensis/hole $lant in *ariouse0$erimental animal models.

!aterials an$ !etho$s

0lant material an$ extraction
1he $lant /as collected from Kolli hills in 8alem district, 1amil Nadu, ndia in 4ecem%er -&&9 and identified %y a 1a0onomistfrom 5otanical 8ur*ey of ndia, Coim%atore, 1amilnadu, ndia and the s$ecimens /ere de$osited in the her%arium of 4e$artment of Pharmacognosy, JKK Munirajah Medical Research Foundation College of Pharmacy, Komara$alayam, 1amilnadu, ndia. ?ne kg of coarsely $o/dered $lant material /as successi*ely e0tracted /ith three *olumes of (.< ethanol for 6- h at room tem$erature. 1he /hole e0tract /as collected in a . litre conical flask, filtered, and the sol*ent /as e*a$orated to dryness under reduced $ressure in rotary e*a$orator
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!=yela, Ja$an# at +&,+.@C. 1he /A/ yield of the $re$ared e0tract /as 3.(< /ith res$ect to the dry $o/der. 1he $reliminary $hytochemical grou$ tests of the $lant e0tract /ere done %y standard methods !1rease and =*ans, 2(3'7 Plummer, 2(3.7 Ballis, 2(3.# for the $resence of alkaloids, ter$enoids, steroids, amino acids, fla*onoids, gums, reducing sugars, tannins and sa$onins.

standard drug or $ro$ylene glycol !.mlAkg# as *ehicle control. 1he animals /ere under o%ser*ation for %eha*ioural changes if any, at '& minutes inter*al in the first hour and at one hour inter*als for ne0t + h for the follo/ing $arameters !Mukherjee et al., 2((97 Murugesanet al., 2(((#.

A3areness, alertness an$ spontaneous activity

1he a/areness and alertness /ere recorded %y *isual measure of the animal>s res$onse /hen $laced in different $ositions and its a%ility to orient itself /ithout %um$s or falls !1urner, 2(9.#. 1he normal %eha*iour at resting $osition /as scored as &. 8imilarly little acti*ity !F#, moderate fle0i%ity !FF#, strong res$onse !FFF# and a%normal restlessness !FFFF# /ere recorded. 1he s$ontaneous acti*ity of mice /as recorded %y $lacing the animal in a %ell jar. t usually sho/s a moderate degree of inGuisiti*e %eha*iour. )ess or moderate acti*ity /as scored as FF and strong acti*ity as FFF. f three is slight or little motion, the score /as F /hile the animal slee$s, the score /as ,. =0cessi*e or *ery strong inGuisiti*e acti*ity like constant /alking or running /as scored as FFFF." similar test /as $erformed /ith the same scoring, /hen the animal are remo*ed from the jar and $laced on a ta%le !1urner, 2(9.7 Mukherjee et al., 2((9#.

Animals
8/iss al%ino mice !-&,-.g# and Bistar al%ino rats !2.&, 23&g# of either se0 /ere used. 1hey /ere o%tained from the animal house, J.K.K. Munirajah Medical Research Foundation College of Pharmacy, Komara$alayam, ndia. 1he animals /ere housed in grou$s of 2& $er cage !standard metal cage# $rior to $harmacological studies.1he animals /ere $ro*ided /ith free access to standard diet and /ater ad libitum for at least - /eeks on a 2-A2- h lightAdark cycle. "ll animals /ere fasted o*ernight %efore test /hile $ro*iding ta$ /ater ad libitum. 1he am%ient tem$erature /as --C2@C, e0ce$t $heno%ar%itone sodium, induced slee$ing time e0$eriments, /hich /ere carried out at '&C2@C. Plant e0tracts and standard drug /ere sus$ended in $ro$ylene glycol immediately $rior to the use and gi*en orally 2h %efore the e0$eriments in a dose of . mlAkg %ody /eight in mice and rats. Control animals recei*ed the same dose of *ehicle under the same conditions. njections /ere normally made intra$eritonially unless other/ise mentioned. "ll $rocedures descri%ed /ere re*ie/ed and a$$ro*ed %y the nstitutional "nimal =thicsCommittee ! "=C#.

)ouch, pain an$ soun$ responses

1he touch res$onse /as recorded %y touching the mice /ith a $encil or force$s at a *arious $arts of the %ody !i.e. on the side of the neck, a%domen and groin#.1he $ain res$onse /as graded /hen a small artery clam$ /as attached to the %ase of the tail, and res$onse /as noted."l%ino mice normally utter no sound, so that *ocaliDation may indicate no0ious stimulus.

Chemicals
Chlor$romaDine hydrochloride ! ndus Pharmaceuticals )imited, ndia#, diaDe$am !)u$in )a%oratories )imited, ndia#, $heno%ar%itone sodium !Rhone,Poulenc ndia )imited, ndia#, $ethidine !Ran%a0y )a%oratories )imited, ndia#, as$irin !E8;, Mum%ai, ndia# and $ro$ylene glycol !8R) )a%oratories, ndia# /ere $rocured and used in the study. "ll other chemicals of highest a*aila%le $urity /ere o%tained from Merck, Mum%ai, ndia.

Anal4esic activity
"nalgesic acti*ity /as studied %y using tail immersion and tail,flick methods.

)ail immersion test

8/iss al%ino mice of either se0 /ere di*ided into four grou$s of 2& animals each. Pro$ylene glycol !.mlAkg#, e0tract at the doses of 2&&and -&& mgAkg and $ethidine !.mgAkg# /ere administered intra$eritonially. 1he tail !u$ to .cm# /as then di$$ed into a $ool of /ater maintained at ..C&..@C. 1he time in seconds to /ithdra/ the tail out of /ater /as taken as the reaction time. 1he reading /as taken after '& minutes of the administration of the test drug !Hosh, 2(3+#.

Acute toxicity in animals

For acute to0icity studies, the test e0tract in the doses 2&&, -&&, +&&, 3&& and 29&& mgAkg /ere administered in fi*e grou$s of 2& mice each. 1he mortality rates /ere o%ser*ed after 6- hours. 1he )4.&/as determined using the gra$hical methods of )itchfield and Bilco0on !2(+(#.

#eneral 2ehavioural metho$s

=*aluation of general %eha*ioural $rofile /as $erformed %y the method of 4i0it and ;arma !2(69#. Fifty adult al%ino mice /ere di*ided into fi*e grou$s. 1he first three grou$s of animals /ere administered /ith the e0tract at the doses of 2&&, -&& and '&&mgAkg intra$eritonially. 1he last t/o grou$s recei*e either chlor$romaDine !.mgAkg# as

)ail flic& test

Bistar rats of either se0 /eighing 2.& , 23&g /ere di*ided into + grou$s of 2& animals each. 1he tail of the rat /as $laced on the nichrome /ire of an analgesiometer !1echno, )uckno/, ndia# and the time taken %y the animal to

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/ithdra/ !flick# its tail from the hot /ire /as taken as the reaction time. =0tract at the doses of 2&& and -&& mgAkg and $ethidine !.mgAkg# /ere administered intra$eritonially. Pro$ylene glycol !.mlAkg# /as ser*ed as a control. "nalgesic acti*ity /as measured after '& min of administration of test and standard drugs !Hosh, 2(3+#.

e0tract !2&&, -&& and '&&mgAkg#, *ehicle !. mlAkg, $ro$ylene glycol# and diaDe$am !2&mgAkg# res$ecti*ely. 1he ina%ility to $ut at least one hind foot /as considered as failure in the traction test !RudDiket al., 2(6'#.

otaro$ test
Mice /ere $laced on a horiDontal steel rod !'-mm diameter# rotating at the s$eed of -. r$m. 1he mice ca$a%le of remaining on the to$ for ' min or more, in three successi*e trails /ere selected for the study. 1he selected animals /ere di*ided into fi*e grou$s !nL2&#. Hrou$s /ere injected intra$eritonially /ith the e0tract at 2&&, -&& and '&&mgAkg, $ro$ylene glycol !.mlAkg# and diaDe$am !2&mgAkg# res$ecti*ely. =ach grou$ of animals /as then $laced on the rod at an inter*al of '&, 9&, (&, 2-& and 2.& min. 1he animals failed more than once to remain on the rotating rod for ' min /ere considered as $ositi*e for muscle rela0ation !4unham and Miya, 2(.6#.

Effect on pheno2ar2itone so$ium in$uce$ sleepin4 time

Mice /ere di*ided into + grou$s of 2& animals each. "nimals recei*ed +&mgAkg i.$. $heno%ar%itone sodium '& min after the injection of $ro$ylene glycol !. mlAkg# and the e0tract at the doses of 2&&, -&& and '&& mgAkg. 1he slee$ing time /as recorded, and measured as the time inter*al %et/een the loss and remaining of the light reflu0 !4handiya and Collum%ine, 2(.(7 Mandalet al., -&&2#.

Exploratory 2ehaviour
=0$loratory %eha*iour of the animals /as e*aluated using I,maDe and head di$ tests.

Statistical analysis
1he results /ere e0$ressed as mean C 8.=.M. 8tatistical analysis of difference %et/een grou$s /as e*aluated %y "N?;" follo/ed %y 4unnett>s$osthoc test. 1he Chi, sGuare test /as used for calculating the $ercentage muscle rela0ant acti*ity. " $,*alue less than &.&. /as considered significant.

+5ma6e test
1he test /as $erformed in + grou$s of 2& al%ino rats at '&, 9&, (& and 2-& min after injection of either $ro$ylene glycol !.mlAkg#, e0tract !2&& and -&& mgAkg# and diaDe$am!2& mgAkg# res$ecti*ely. 1he rats /ere $laced in*idually in a symmetrical I,sha$ed run/ay !'' J '3 J 2'cm# for ' min and the num%er of times a rat entered in the arm of the maDe /ith all +ft !an Kentry># /ere counted !Rushton et al., 2(927 Mandalet al., -&&2#.

esults an$ .iscussion

1he $reliminary $hytochemical screening of Wedeliachinensisethanol e0tract /as $erformed %y standard methods and the results indicated the $resence of tannins, ter$enoids, fla*onoids, steroids and reducing sugars.

7ea$ $ip test

Fi*e grou$s of female al%ino mice !nL2&# /ere $laced on the to$ of a /ooden %o0 /ith 29e*enly s$aced holes, '&min after injection of the e0tract !2&&, -&& and '&&mgAkg, *ehicle !.mlAkg, $ro$ylene glycol# and diaDe$am !2&mgAkg# res$ecti*ely. 1he num%er of times that each animal di$$ed the head into the hole /as counted for a $eriod of ' min !4orr et al., 2(62#.

)oxicity stu$y
1he $lant e0tract /as found to %e non,to0ic u$ to doses of 2.9 gAkg and did not cause any death of the animals tested. 1his indicates that the )4.& *alue of the e0tract /as more than 2.9 gAkg.

!uscle relaxant activity

1he effect of e0tract on muscle rela0ant acti*ity /as studied %y usingtraction androtarod tests.

Effect on 2ehavioural profiles

1he results o%tained from the e0$eriments are $resented in 1a%le 2. 1he e0tract affected s$ontaneous acti*ity, sound and touch res$onses at a dose of a%o*e -&&mgAkg and $roduced moderate or slight de$ression relating to a/areness and alertness. :o/e*er, the standard drug chlor$romaDine hydrochloride caused significant de$ression of all these res$onses com$ared /ith ethanol e0tract. 1he results indicate that the e0tract influences general %eha*ioural $rofiles, as e*idence in the s$ontaneous acti*ity, touch, sound and $ain res$onses.

)raction test
1he screening of the animals /as done %y $lacing the fore$a/s of the male mice in a small t/isted /ire rigidly su$$orted a%o*e a %ench to$. Normally the mice gras$ the /ire /ith the fore$a/s, and $lace at least one hind foot on the /ire /ithin the .sec /hen allo/ed to hang free. 1he test /as conducted on fi*e grou$ of animals !nL2&# /hich /ere $re*iously screened, '& min after the injection of the

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Anal4esic activity
1he results of the analgesic acti*ity %y tail immersion and tail flick methods /ere $resented in 1a%le -. n %oth the tests the reaction time /as significantly increased in treated animals /hen com$ared to control. 1his indicates that the e0tract ha*e sho/n significant analgesic acti*ity com$ared /ith the control in a dose de$endent manner. 1he acti*ity may %e due to its action on central ner*ous system,similar to that of $ethidine.

%eha*iour com$ared /ith controls !1a%le '#. n head di$ test, there /as a significant reduction in the head ti$ res$onses occurred in mice treated /ith the e0tract, com$ared /ith the control !1a%le +#.

Effect on pheno2ar2itone so$ium5in$uce$ sleepin4 time

1he e0tract significantly $otentiated the $heno%ar%itone sodium,induced slee$ing time at the doses studied, /ith res$ect to the control !1a%le .#. 1he $otentiation of$heno%ar%itone sodium,induced slee$ing time is $ossi%ly through a CN8 de$ressant action !4unham NB al., 2(.6# or a tranGuiliDing action !Mandal et al., -&&2#.

Exploratory 2ehaviour potentials

n the I,maDe test, the animals treated /ith the e0tract in tested doses ha*e sho/n a marked decrease in e0$loratory Table 1 /ffect of ethanol extract of
Behaviour

edeliachinensis on general behavioural profiles in mice and rats (n12$).

Wedeliachinensis extract (mg/ g! 2$$ #$$ 33 333 333 33 333 3 %$$ 333 333 33 3333 333 3333 3333 333 333 3333 3333 3333 * * * * * * 3 3 3 3 33 3 "hlor#roma$ine (% mg/ g! Pro#ylene glycol (% ml/ g!

Spontaneous activity 4lertness 4wareness Sound response Touch response 5ain response

6epression levels, *! no effect) 3! slight) 33! moderate) 333! strong) 3333! very strong

Table & 4nalgesic effect of Wedeliachinensis on tail flick and tail immersion tests in mice and rats.
Treatment 5ropylene glycol 4spirin W. chinensis extract 'ose 7 ml&kg 2$$ mg&kg 2$$ mg&kg #$$ mg&kg Tail flic test ((eaction time in sec! #.#78$.29 9.#$8$.2; #.7$8$.2% %.$#8$.$: Tail immersion test ((eaction time in sec! #.%#8$.2: 9.9;8$.2# #.9;8$.$# %.$78$.$#

<alues are mean 8 S./.! n12$. 4ll the data are significant at 5=$.$$2 vs. control! Students t*test.

Table ) /ffect of Wedeliachinensis on exploratory behaviour ('*ma(e test) in rats.

*x#eriment 5ropylene glycol 6ia(epam W. chinensis extract 'ose 7 ml&kg 2$ mg&kg 2$$ mg&kg #$$ mg&kg Number of entries after treatment (min! )+ >.# 8 $.# %.$ 8 $.2 :.9 8 $.$7 7.$ 8 $.# ,+ >.% 8 $.# %.2 8 $.2 :.9 8 $.$7 7.$ 8 $.# -+ >.% 8 $.# %.# 8 $.2 :.: 8 $.$7 7.2 8 $.# 1&+ >.9 8 $.# %.% 8 $.2 :.? 8$.$7 7.2 8 $.#

<alues are the number of entries in % min (mean 8 S./.! n12$). 4ll the data are significant at 5=$.$$2 compared with control.

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Table . /ffect of Wedeliachinensis on exploratory behaviour (head dip test) in mice.

*x#eriment 5ropylene glycol 6ia(epam W. chinensis extract 'ose 7 ml&kg 2$ mg&kg 2$$ mg&kg #$$ mg&kg %$$ mg&kg /ead di# test >> 8 2.$ %# 8 #.$@@@ ?2 8 %.$@ :$ 8 #.$@@ %% 8 2.$@@@

<alues are number of head dips in % min (mean 8 S./.! n12$) @5 = $.$7! @@5=$.$2 and @@@5=$.$$2 compared with control

Table % /ffect of

edeliachinensis on phenobarbitone sodium*induced sleeping time.

*x#eriment 'ose 7 ml&kg 2$$ mg&kg #$$ mg&kg %$$ mg&kg Slee#ing time (min! :9 8 $ ?$ 8 #.$@ ;# 8 %.$@ 22> 8 %.$@

5ropylene glycol W. chinensis extract

<alues are mean 8 S./.! n12$. 5=$.$$2 compared with control

Effect on muscle relaxant activity

n the traction test, the mice treated /ith the e0tract sho/ed a significant failure in traction at all the doses tested. 1he result from the rotarod test sho/ed that the e0tract significantly reduced the motor co,ordination of the tested animals. "ll these results su$$ort the use of this $lant>s decoction %y the tri%es in Kolli :ills of Namakkal 4istrict, 1amilnadu, ndia, to reduce mental tension and also to induce slee$ !"nonymous, 2(+3#.

Cho$ra RN.#lossar! of Indian Medicinal Plants, Council of 8cientific and ndustrial Research, Ne/ 4elhi, 2(.9, $$. -.3. 4andiyaPC and Collum%ine.8tudies on Acoruscalamus ! #M some $harmacological action of the *olatile oil. $ Pharmacol %&p Ther" 129* '.','.( !2(.(#. 4i0it ;K and ;arma KC. =ffect of essential oil of the lea*es of 'lumealacera 4C on central ner*ous system. Indian $"Pharmacol"11* 6,22 !2(69#. 4orrM, 8tien%erg:, 1omkie/ieD M, Joyee 4, Prosolt R4 and 8ummerfield ". Persistence of dose related factor in mice. (ature"231* 2-2,2-' !2(62#. 4unham NB and Miya 18. " note on sim$le a$$aratus for detecting neurological deficit in rats and mice. $ Am Pharm Assoc Am Pharm Assoc )'altim*" 8:* -&3,-&( !2(.6#. Hhosh MN. +undamental of %&perimental pharmacolo,!, -nd ed. 8cientific %ook "gency, Calcutta, 2(3+. $$. 2.'. Kritikar KR and 5asu 54. 2(6.. ndian Medicinal Plants, ;ol . nd - ed, 5ishen 8ingh Mahendra Pal 8ingh, 4ehradun, $$. 2'9+,9. )in FM, Chen )R, )in =:,Ke FC, Chen :I, 1sai MJ and :siao PB. Com$ounds from Wedeliachinensis synergistically su$$ress androgen acti*ity and gro/th in $rostate cancer cells. Carcino,enesis"21M -.-2N-.-( !-&&6#. )itchfield J1 and Bilco0on F. 2(+(." sim$lified method of e*aluating dose effect e0$eriments. $ Pharmacol %&p Ther" (9, ((,22'. Mandal 8C, 4hara "K andMaiti 5C. 8tudies of $sycho$harmacological acti*ity of "ndrogra$his $articulate e0tract. Ph!tother Res"19M -.',-.9 !-&&2#.

Conclusion
1he $ossi%le CN8 acti*ity of ethanolic e0tract of Wedeliachinensis/as in*estigated %y common $sycho$harmacological tests. 1he reduction in e0$loratory %eha*iour in animals is similar /ith the action of other CN8 de$ressant agents. " significant lack in motor co, ordination and muscle rela0ant acti*ity /as also noted in animals treated /ith crude e0tract. 1he results altogether indicates that the e0tract sho/s CN8 de$ressant acti*ity.

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Mathe/KM.+lora of Tamilnadu-carnatic. 1he Ra$inat :er%arium, 8t. Jose$hs College, 1richira$alli, 2(3', Part, , $$. '(-. Mishra H, 8inha R, ;erma N, Khosa R), Harg ;K and 8ingh P. :e$ato$rotecti*e acti*ity of alcoholic and aGueous e0tracts of Wedeliachinensis, Pharmacolo,!online"1M '+.,'.9 !-&&(#. Mukherjee1, 8aha K, 5alasu%ramanium R, Pal M and 8aha 5P. 8tudies of $sycho$harmaco,logical acti*ity of (elumbanucifera,aertn. RhiDome =0tract. $" %thnopharmacol" 98M 9',96 !2((9#. Murugesan 1, Hhosh ), 4as J, Pal M and 8aha 5P. CN8 acti*ity of $ussiaeasuffruticusa)inn.e0tracts in rat and mice. Pharm Pharmacol Comm" 9* 99',999 !2(((#. Plummer 4 .An Introduction to Practical 'iochemistr!, -nded, 1ata McHra/,:ill Pu%lishing Co. )td, Ne/ 4elhi, 2(3., $$. 2'9,2+'. RudDik "4, :ester J5, 1ang ":, 8ta/ RN and Friis B. The 'en-odia-epines. Ra*en Press, Ne/ Iork, 2(6', $$.-3., -(6. Rushton R, 8tein%erg : and 1inson C.Modification of the effects of an "m$hetamine %ar%iturate mi0ture %y the $ast e0$erience of rats !I,sha$ed Run/ay#. (ature"1;2M .'',.'. !2(92#.

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