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Study on Cytotoxic Activity of Clerodendrum Inerme and Caesalpinia Crista by Brine Shrimp Lethality Bioassay

The science and practice of medicine and its primary source plays an important role in identifying the new molecule of drug through both synthetically and from that of natural origin. Most of the developing countries have adopted traditional medical practice as an integral part of their culture. Historically, all medicinal preparations were derived from plants, whether in the simple form of raw plant materials or in the refined form of crude extracts, mixtures, etc. The aim of this study was to evaluate the Cytotoxic Activity of Clerodendrum inerme (Family: Verbenaceae) and Caesalpinia crista (Family: Leguminosaceae), two widely distributed shrubs of the Sundarbans mangrove forest. A general cytotoxicity of all the extracts of C. inerme and C. crista were determined by Brine Shrimp Lethality Bioassay where the chloroformic extract of bark of C. inerme has the lowest LC50 value of 5μg/ml. The ethanolic and chloroformic extracts of leaf of C. inerme and methanolic extracts of bark of C. crista also exhibited a significant cytotoxic activity with LC50 values of 10μg/ml, 9.10μg/ml and 10μg/ml, respectively. In this investigation 0.5% chloramphenicol was used as the reference which LC50 value was 10μg/ml.
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  International Journal of Innovation and Applied Studies ISSN 2028-9324 Vol. 8 No. 4 Oct. 204! pp.  #4- 80 $ 204 Innovative Space of Scientific %esearc& Journals &ttp'(())).i*ias.issr-*ournals.or+( Corresponding Author: ,d. Jalal ddin  1574 Study on Cytotoxic Activity of Clerodendrum inerme  and Caesalpinia crista  by Brine Shrimp Lethaity Bioassay Md. Jalal Uddin, Md. Shamim Akhter, Kazi Mohammed Didarul Islam, and Md. Morsaline Billah iotec&nolo+/ and enetic 1n+ineerin+ iscipline! &ulna niversit/! &ulna-9208! an+lades& op/ri+&t $ 204 ISS% Journals. 5&is is an open access article distri6uted under t&e Creative Commons Attriution !icense ! )&ic& per7its unrestricted use! distri6ution! and reproduction in an/ 7ediu7! provided t&e ori+inal )or is properl/ cited. A BS! AC! :  5&e science and practice of 7edicine and its pri7ar/ source pla/s an i7portant role in identif/in+ t&e ne) 7olecule of dru+ t&rou+& 6ot& s/nt&eticall/ and fro7 t&at of natural ori+in. ,ost of t&e developin+ countries &ave adopted traditional 7edical practice as an inte+ral part of t&eir culture. istoricall/! all 7edicinal preparations )ere derived fro7 plants! )&et&er in t&e si7ple for7 of ra) plant 7aterials or in t&e refined for7 of crude e:tracts! 7i:tures! etc. 5&e ai7 of t&is stud/ )as to evaluate t&e /toto:ic Activit/ of lerodendru7 iner7e ;<a7il/' Ver6enaceae= and aesalpinia crista ;<a7il/' >e+u7inosaceae=! t)o )idel/ distri6uted s&ru6s of t&e Sundar6ans 7an+rove forest. A +eneral c/toto:icit/ of all t&e e:tracts of . iner7e and . crista )ere deter7ined 6/ rine S&ri7p >et&alit/ ioassa/ )&ere t&e c&lorofor7ic e:tract of 6ar of . iner7e &as t&e lo)est > 0 value of ?+(7l. 5&e et&anolic and c&lorofor7ic e:tracts of leaf of . iner7e and 7et&anolic e:tracts of 6ar of . crista also e:&i6ited a si+nificant c/toto:ic activit/ )it& > 0  values of 0?+(7l! 9.0?+(7l and 0?+(7l! respectivel/. In t&is investi+ation 0. @ c&lora7p&enicol )as used as t&e reference )&ic& > 0  value )as 0?+(7l. # $%&' (S :  /toto:ic Activit/! rine S&ri7p! 5raditional! Sundar6ans! S&ru6s and >et&alit/. 1   ) *! '(+C!)'*   rine S&ri7p >et&alit/ ioassa/ is a recent develop7ent in t&e assa/ procedure for t&e 6ioactive co7pounds and natural product e:tracts! )&ic& indicates c/toto:icit/ as )ell as a )ide ran+e of p&ar7acolo+ical activities e.+. anticancer! antiviral! pesticidal etc. ;Anderson et al. ! 988=. ioactive co7pounds are al7ost al)a/s to:ic in &i+& doses. &ar7acolo+/ is si7pl/ to:icolo+/ at a lo)er dose or to:icolo+/ is si7pl/ p&ar7acolo+/ at a &i+&er dose. 5&us! in vivo  let&alit/ of a si7ple Boolo+ical or+anis7 ;rine s&ri7p napulii= can 6e used as a convenient 7onitor for screenin+ and fractionation in t&e discover/ of ne) 6ioactive natural products ;ui et al  .!990=. Natural product e:tracts! fractions or pure co7pounds can 6e tested for t&eir 6ioactivit/ 6/ t&is 7et&od. 5&is 6ioassa/ is indicative of c/toto:icit/ and a )ide ran+e of p&ar7acolo+ical activit/ of natural products. 5&e 6rine s&ri7p let&alit/ 6ioassa/ )as proposed 6/ ,ic&ael et al. in 9 C and 7odified 6/ ot&ers. Since its introduction! t&is in vivo let&alit/ test &as 6een successivel/ e7plo/ed for providin+ a frontline screen t&at can 6e 6aced up 6/ 7ore specific and 7ore sop&isticated 6ioassa/s once t&e active co7pounds &ave 6een isolated. 5&e 6rine s&ri7p let&alit/ 6ioassa/ is rapid ;24 &=! si7ple ;e.+.! no aseptic tec&niDues are reDuired=! easil/ 7astered! ine:pensive! and reDuires s7all a7ounts of test 7aterial ;&isal6erti!  et al.,  993=. 5&us! t&is researc& )or )as desi+ned to evaluate t&e c/toto:ic activit/ of C. inerme  and C. crista  6/ rine S&ri7p >et&alit/ ioassa/ for furt&er e:ploration of t&eir 6ioactive constituents. ,   - A!$ )ALS A*( - $!.'(   ! $S! - A!$ )ALS   5&e Selected plant  Clerodendrum inerme  and Caesalpinia crista  )ere collected fro7 &a+ra7aree! &adpai ran+e! 1astern <orest epart7ent of t&e Sundar6ans on 2 t&  ,arc&! 20 and collected plants sa7ples )ere identified 6/ t&e  -d/ 0aa +ddin -d/ Shamim A2hter #a3i -ohammed (idaru )sam and -d/ -orsaine Biah )SS* : ,,68,4 9o/  *o/ 4 'ct/ ,14 1575 e:perts of an+lades& National er6ariu7! ,irpur! &aa. 5&is researc& )or )as carried out fro7 2C t&  ,arc& to  t&  Septe76er! 20 in ,olecular iolo+/ and Ani7al ell ulture >a6orator/ of &ulna niversit/! &ulna! an+lades&. $ ;$ )-$*!AL - A!$ )ALS    Artemia salina  >eac& ;6rine s&ri7p e++s for7 store=! 5a6le salt! ure Nal! S7all tan ;+lass *ar= to +ro) s&ri7p! cover and la7p to attract s&ri7p! ipettes ; 7l! 7l=! ,icro-pipette ;0?l! 200?l ad*usta6le=! 5est tu6e ; 7l=! Volu7etric flas ;07l=! 5)een-80! Spoon! 1lectric )ater 6lo)er to produce current! 1lectric 6ul6 to produce &eat! Stand to &old t&e 6ul6! etri dis&! 5est tu6e stand! eaer ; liter=! p 7eter! &lora7p&enicol! Alu7inu7 foil! eter+ent! i+ital 6alance! Vorte: 7ac&ine. ; $;A A!)'* '< - A!$ )ALS   ollected plant sa7ples )ere )as&ed 6/ distilled )ater ;E= to re7ove undesira6le 7aterials and e:cess of )ater )as drained off. 5&e leaves and ste7s )ere separated fro7 eac& ot&er and t&e/ )ere sliced into s7all pieces. 5&e sliced 7aterials )ere )ei+&ed 6/ 6alance. 5&e sliced leaves and ste7s )ere dried for fe) da/s under sunli+&t )it& s&ado). After t&at t&e dried sa7ples )ere po)dered separatel/ 6/ +rindin+ 7ac&ine and t&en .4 +7 po)dered leaf of   C. inerme  )as taen into clean flat-6otto7ed +lass container and soaed in 200 7l of 9 @ et&anol! .4 +7 po)dered leaf of   C. inerme  and 2.8 +7 po)dered 6ar of C. crista  )as taen into clean flat-6otto7ed +lass container and soaed in 200 7l of a6solute 7et&anol and .4 +7 po)dered leaf and 0.#2 +7 po)dered 6ar of   C. inerme  and 2.8 +7 po)dered 6ar of C. crista  )as taen into clean flat-6otto7ed +lass container and soaed in 200 7l of a6solute c&lorofor7. 5&e containers )it& its contents )ere sealed and ept for a period of # da/s acco7pan/in+ occasional s&ain+ and stirrin+. 5&e )&ole 7i:ture t&en under)ent a coarse filtration 6/ a piece of clean! )&ite cotton 7aterial. 5&en it )as filtered t&rou+& E&at7an filter paper. 5&e filtrates o6tained )ere evaporated under ceilin+ fan and in a )ater- 6at& until dried. It rendered concentrates of +reenis&! 6ro)nis&! reddis& and so7eti7es 6lacis& in color. 5&e concentrates )ere desi+nated as crude e:tract of 9 @ et&anol! 7et&anol and c&lorofor7 of C. inerme  and  C. crista . . A!C.)*= '< B )*$ S . )-;   C0+7   Sea salt ;pure Nal 20+7 and ta6le salt 40+7= )as )ei+&ed accuratel/! dissolved in distilled )ater to 7ae one liter and t&en filtered off to +et a clear solution. Sea )ater )as taen in t&e s7all tan and s&ri7p e++s )ere added to t&e one side of t&e tan and t&e side )as covered. 5&e e++s )ere allo)ed for t)o da/s ;24-3C &rs= to &atc& and 7ature as nauplii ;larvae=. 5&e &atc&ed s&ri7ps )ere attracted to t&e la7p t&rou+& t&e perforations in t&e da7 and t&e/ )ere taen for 6ioassa/. ; $;A A!)'* '< S !'C# S 'L+!)'*   #7+ of dried et&anolic! 7et&anolic and c&lorofor7ic e:tracts of leaves and c&lorofor7ic e:tract of 6ars of C. inerme, 7et&anolic and c&lorofor7ic e:tracts of 6ars of C. crista  )ere taen into 07l individual volu7etric flas. 5&e e:tracts )ere dissolved in .4 7l sea )ater and one drop 5)een-80 and ad*usted to final concentration of F+(Fl. As standard 0. @ sterile &lora7p&enicol e/e drop )as used )&ic& concentration )as ?+(?l. ; 'C$(+ $   One &undred clean test tu6es )ere taen! 4 of )&ic& )ere for t&e sa7ples in seven different concentrations ;t)o test tu6es for eac& concentration=! 4 for positive control ;t)o test tu6es for eac& concentration= and 2 for ne+ative control. 5&en 47l of sea)ater )as +iven to eac& of t&e test tu6es. Eit& t&e &elp of t&e 7icropipette specific volu7es ; ! 0! 20! 40! 80!C0 and 320?l= of sa7ples )ere transferred fro7 t&e stoc solutions to t&e test tu6es. ifferent concentration of c&olora7p&enicol ; ! 0! 20! 40! 80! C0 and 320?l= )ere taen in t&e rest of t&e 4 test tu6es )&ic& are used as positive control and finall/ t&e volu7e is 7aintained at 07l in eac& test tu6e ;sa7ple! positive control and ne+ative control= usin+ sea )ater. So! t&e concentration of sa7ples and c&lora7p&enicol 6eca7e 2. ! ! 0! 20! 40! 80 and C0?+(7l respectivel/. <inall/ )it& t&e &elp of a asteur pipette! 0 live s&ri7ps nauplii )ere taen into eac& of t&e test tu6es ;,e/er et al.,  982=. After 24 &rs! t&e test tu6es )ere o6served and t&e nu76ers of survived nauplii in eac& test tu6e )ere counted and t&e results )ere noted. <ro7 t&is! t&e percenta+e of let&alit/ of 6rine s&ri7p nauplii )as calculated at eac& concentration for eac& sa7ple.  Study on Cytotoxic Activity of Clerodendrum inerme  and Caesalpinia crista  by Brine Shrimp Lethaity Bioassay )SS* : ,,68,4 9o/  *o/ 4 'ct/ ,14 157> S !A!)S!)CAL A *AL%S)S   / usin+ t&ese follo)in+ eDuations Arit&7etic ,ean! Standard eviation and Standard 1rror )ere calculated' 1D. ;=   Arit&7etic ,ean ;  X  = G n X  ∑  E&ere! H G Su77ation of O6served Value n = No. of O6servation   1D. ;2=   Standard eviation ;S= G n X  X   2 )(  − ∑  E&ere!  GIndividual Value  X  G ,ean Value n = No. of O6servation 1D. ;3=   Standard 1rror ;S1= G )1(  − nSD  E&ere! S G Std. eviation n  G No. of O6servation 8   $S+L!S A*( ( )SC+SS)'*S   In t&e c/toto:icit/ assa/ 6ased on t&e let&alit/ of 6rine s&ri7p! t&e crude e:tract of C.inerme  and C.crista  s&o)ed let&alit/ indicatin+ t&e 6iolo+ical activit/ of t&e co7pound present in t&e e:tract. %esults found in t&is 6ioassa/ are ta6ulated to t&e ne:t pa+es. ale #. $esults %or c&toto'ic e%%ect o% chloramphenicol    S   a   7   p    l   e   4   o   n   c .    ;   ?   +    (   7    l    =   5   r   e   a   t   7   e   n   t  -      5   r   e   a   t   7   e   n   t  -   2   A   v   + .   n   o   o    f   a    l   i   v   e   s    &   r   i   7   p   s   a   7   p    l   e   N   e   +   a   t   i   v   e   c   o   n   t   r   o    l    ;   a    l   i   v   e    =   S   t    d .    d   e   v   i   a   t   i   o   n   S   t    d .   e   r   r   o   r   ,   o   r   t   a    l   i   t   /    ;   @    =   >   4        0     ;   ?   +    (   7    l    = 2. 9 9 9 0 0 0 0 0 # 8 #. 0.#0#0# 0. 2 0 0 0 0 20 4 4 4 0 0 C0 40 3 3 3 0 0 #0 80 0 0 0 0 0 00 C0 0 0 0 0 0 00  -d/ 0aa +ddin -d/ Shamim A2hter #a3i -ohammed (idaru )sam and -d/ -orsaine Biah )SS* : ,,68,4 9o/  *o/ 4 'ct/ ,14 1577 ale (. $esults %or c&toto'ic e%%ect o% the methanolic e'tract o% lea% o% C. inerme    S   a   7   p    l   e   4   o   n   c .    ;   ?   +    (   7    l    =   5   r   e   a   t   7   e   n   t  -      5   r   e   a   t   7   e   n   t  -   2   A   v   + .   n   o   o    f   a    l   i   v   e   s    &   r   i   7   p   s   a   7   p    l   e   N   e   +   a   t   i   v   e   c   o   n   t   r   o    l    ;   a    l   i   v   e    =   S   t    d .    d   e   v   i   a   t   i   o   n   S   t    d .   e   r   r   o   r   ,   o   r   t   a    l   i   t   /    ;   @    =   >   4        0     ;   ?   +    (   7    l    = 2. 8 # #. 0 0.#0#0# 0. 2 3C. # # # 0 0 30 0 # C C. 0.#0#0# 0. 3 20 C . 0.#0#0# 0. 4 40 4 4. 0.#0#0# 0. 80 4 2 3 .4424  #0 C0 2  . 0.#0#0# 0. 8 ale ). $esults %or c&toto'ic e%%ect o% the ethanolic e'tract o% lea% o% C. inerme    S   a   7   p    l   e   4   o   n   c .    ;   ?   +    (   7    l    =   5   r   e   a   t   7   e   n   t  -      5   r   e   a   t   7   e   n   t  -   2   A   v   + .   n   o   o    f   a    l   i   v   e   s    &   r   i   7   p   s   a   7   p    l   e   N   e   +   a   t   i   v   e   c   o   n   t   r   o    l    ;   a    l   i   v   e    =   S   t    d .    d   e   v   i   a   t   i   o   n   S   t    d .   e   r   r   o   r   ,   o   r   t   a    l   i   t   /    ;   @    =   >   4        0     ;   ?   +    (   7    l    = 2. C # C. 0 0.#0#0# 0. 3 0 C C C 0 0 40 0 0 0 0 20 4 C .4424  0 40 4 3 3. 0.#0#0# 0. C 80 3 2 2. 0.#0#0# 0. # C0  0 0. 0.#0#0# 0. 9 ale *. $esults %or c&toto'ic e%%ect o% the chloro%ormic e'tract o% lea% o% C. inerme    S   a   7   p    l   e   4   o   n   c .    ;   ?   +    (   7    l    =   5   r   e   a   t   7   e   n   t  -      5   r   e   a   t   7   e   n   t  -   2   A   v   + .   n   o   o    f   a    l   i   v   e   s    &   r   i   7   p   s   a   7   p    l   e   N   e   +   a   t   i   v   e   c   o   n   t   r   o    l    ;   a    l   i   v   e    =   S   t    d .    d   e   v   i   a   t   i   o   n   S   t    d .   e   r   r   o   r   ,   o   r   t   a    l   i   t   /    ;   @    =   >   4        0     ;   ?   +    (   7    l    = 2. C 8 # 0 .4424  30 9.0 C C C 0 0 40 0 4 4. 0.#0#0# 0. 20 4 4 4 0 0 C0 40 3 3 3 0 0 #0 80    0 0 90 C0 0  0. 0.#0#0# 0. 9
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