FINAL REPORT

PROJECT No 936-5542

INVESTIGATION OF THE CICATRIZANT PROPERTIES OF THE

PLANT EXTRACT SANGRE DE GRADO AND iTS POSSIBLE

UTILIZATION AS A THERAPEUTIC AGENT

PRINCYPPi INVESTIGATOR ABRAHAM J VAISBERG PHD

LABORATORY FOR CELL BIOLOGY AN) VIROLOGY DEPARTMENT OF MICRUBIOLOGY

UNIVERSIDAD PERJANA CAYETANO HEREDIA APARTADO 5045

LIMA--PERU

PERIOD COVERED SEPTEMBER Ist 198 THROUGH

DECEMBER 31st 1985

DE~Ca F S RL CARRIEDJOUT

6 t h ribQLteQn CQ n gC 9 te o

+__) [3

ri z an~dg~cc w~prprIWO~ e o e

report~1~1t t t

d ii n Jre ain pale ~ 1~

charcter z atamp~~onosand -qat a i nL rC)

i ee~ wr f he - I

aoterya W S

ndt e St d nt M r a de Ca me g ck-a_( d- i 1y C in

se on rS ti i n( ie ~ xp rim n 0F h 1lc nic 1 nshy

of~~~ ~ ~ Saguc epineGaoadte~ ~ cIa o t l p r- wo

het~t~nsth ~ a dH_-A-I 5Ck1deIC n Narcos d yla pa ime n h1(-m~ag cThet ichir [ide fe_(pe ts he7pozs 6

~ ~ rmtracarcinogenic~~~ ~ ~ V f angr4rdortI~

1 j1

o rrd

cInte ~a~n1 og~

sp~ge cie ro n

t-Thaeg( -iat 6aoft r a

t o

=7p amp o rea bdf

ei odu

-d Q

ri rtoi hnbpal Ctie

dneai ln nt~e~ e e a dve

fro p-whih cwerwra edf

h_ p tee

ban

t1

o

1

e I

0 1

de na ec a

ren

5

aib ct7irarms~o

a nI drq

63mgI dh Or do aMOegtts

h r I

teie c

-th tr

the plant Taspinewa S b _t 1b h e cIcafriza r ni

th 5a gA de Od t a t 5 ic i- o

RSUL f AND DISCUSSIOQ

ISOLATI6NPU RICflpDN tCHRCTERIZATIOW DU1 10T

1 EXTRACTION PURIFICATION AND QLJANTITAT ION OF TEALKLOI D FROM SANGREDE GRADD-

Yo ~ ~ iptp 4

f teo ol ei~ on ofSag e dG Go organied in~

-ln reer -dcing wit1 h

e e Grdot H se ree1 a6 trw also e tf-aomt esn~re t dif-LrQrt

L mes of the dayF cm othe d i ff ret tec5si n 1 Tjmlooraterepiort f te ncati vri tat eal er itiom

-ndjf75~M~~MSanpgre~f~ t0 ie varesd o

tret trf theres difrnc ewenro e di arnshynothUL1 exratontme F o neac of t is S ingre d r

de~-io te aIk lo o tn n f)n h t te V le oqciatec betwee and 5 Mql a obt

-- etiii a ins wr don m au ig - w i tte

x ted d pCFLlat a a]Laloid ro he San re G ad Sa shy

DE~Ca F S RL CARRIEDJOUT

6 t h ribQLteQn CQ n gC 9 te o

+__) [3

ri z an~dg~cc w~prprIWO~ e o e

report~1~1t t t

d ii n Jre ain pale ~ 1~

charcter z atamp~~onosand -qat a i nL rC)

i ee~ wr f he - I

aoterya W S

ndt e St d nt M r a de Ca me g ck-a_( d- i 1y C in

se on rS ti i n( ie ~ xp rim n 0F h 1lc nic 1 nshy

of~~~ ~ ~ Saguc epineGaoadte~ ~ cIa o t l p r- wo

het~t~nsth ~ a dH_-A-I 5Ck1deIC n Narcos d yla pa ime n h1(-m~ag cThet ichir [ide fe_(pe ts he7pozs 6

~ ~ rmtracarcinogenic~~~ ~ ~ V f angr4rdortI~

1 j1

o rrd

cInte ~a~n1 og~

sp~ge cie ro n

t-Thaeg( -iat 6aoft r a

t o

=7p amp o rea bdf

ei odu

-d Q

ri rtoi hnbpal Ctie

dneai ln nt~e~ e e a dve

fro p-whih cwerwra edf

h_ p tee

ban

t1

o

1

e I

0 1

de na ec a

ren

5

aib ct7irarms~o

a nI drq

63mgI dh Or do aMOegtts

h r I

teie c

-th tr

the plant Taspinewa S b _t 1b h e cIcafriza r ni

th 5a gA de Od t a t 5 ic i- o

RSUL f AND DISCUSSIOQ

ISOLATI6NPU RICflpDN tCHRCTERIZATIOW DU1 10T

1 EXTRACTION PURIFICATION AND QLJANTITAT ION OF TEALKLOI D FROM SANGREDE GRADD-

Yo ~ ~ iptp 4

f teo ol ei~ on ofSag e dG Go organied in~

-ln reer -dcing wit1 h

e e Grdot H se ree1 a6 trw also e tf-aomt esn~re t dif-LrQrt

L mes of the dayF cm othe d i ff ret tec5si n 1 Tjmlooraterepiort f te ncati vri tat eal er itiom

-ndjf75~M~~MSanpgre~f~ t0 ie varesd o

tret trf theres difrnc ewenro e di arnshynothUL1 exratontme F o neac of t is S ingre d r

de~-io te aIk lo o tn n f)n h t te V le oqciatec betwee and 5 Mql a obt

-- etiii a ins wr don m au ig - w i tte

x ted d pCFLlat a a]Laloid ro he San re G ad Sa shy

1 j1

o rrd

cInte ~a~n1 og~

sp~ge cie ro n

t-Thaeg( -iat 6aoft r a

t o

=7p amp o rea bdf

ei odu

-d Q

ri rtoi hnbpal Ctie

dneai ln nt~e~ e e a dve

fro p-whih cwerwra edf

h_ p tee

ban

t1

o

1

e I

0 1

de na ec a

ren

5

aib ct7irarms~o

a nI drq

63mgI dh Or do aMOegtts

h r I

teie c

-th tr

the plant Taspinewa S b _t 1b h e cIcafriza r ni

th 5a gA de Od t a t 5 ic i- o

RSUL f AND DISCUSSIOQ

ISOLATI6NPU RICflpDN tCHRCTERIZATIOW DU1 10T

1 EXTRACTION PURIFICATION AND QLJANTITAT ION OF TEALKLOI D FROM SANGREDE GRADD-

Yo ~ ~ iptp 4

f teo ol ei~ on ofSag e dG Go organied in~

-ln reer -dcing wit1 h

e e Grdot H se ree1 a6 trw also e tf-aomt esn~re t dif-LrQrt

L mes of the dayF cm othe d i ff ret tec5si n 1 Tjmlooraterepiort f te ncati vri tat eal er itiom

-ndjf75~M~~MSanpgre~f~ t0 ie varesd o

tret trf theres difrnc ewenro e di arnshynothUL1 exratontme F o neac of t is S ingre d r

de~-io te aIk lo o tn n f)n h t te V le oqciatec betwee and 5 Mql a obt

-- etiii a ins wr don m au ig - w i tte

x ted d pCFLlat a a]Laloid ro he San re G ad Sa shy

RSUL f AND DISCUSSIOQ

ISOLATI6NPU RICflpDN tCHRCTERIZATIOW DU1 10T

1 EXTRACTION PURIFICATION AND QLJANTITAT ION OF TEALKLOI D FROM SANGREDE GRADD-

Yo ~ ~ iptp 4

f teo ol ei~ on ofSag e dG Go organied in~

-ln reer -dcing wit1 h

e e Grdot H se ree1 a6 trw also e tf-aomt esn~re t dif-LrQrt

L mes of the dayF cm othe d i ff ret tec5si n 1 Tjmlooraterepiort f te ncati vri tat eal er itiom

-ndjf75~M~~MSanpgre~f~ t0 ie varesd o

tret trf theres difrnc ewenro e di arnshynothUL1 exratontme F o neac of t is S ingre d r

de~-io te aIk lo o tn n f)n h t te V le oqciatec betwee and 5 Mql a obt

-- etiii a ins wr don m au ig - w i tte

x ted d pCFLlat a a]Laloid ro he San re G ad Sa shy

lthtou Ar

r1 0Ce e e 0r_ d 0 wpf

ce yreylyla~el d~~sdy 0ri p- d t~ uobf~ n t w f sl l o eparatod l La 7di flft o y w c

1-7~r -g~ 1 a a

un t t~~amp~~OAUVr -

Thn yt om gahy o i ca i Mer

~ ~ L ~~ ~ -- - -I-C)- h a o 3- av4eom t hEAAT UWcTI~ heCoU N r 0SAnD US -DR 1N MEDIUM Fi SSr2)DrE I

~ t a 15Lit~~ EyeumI 0 eCd a Gj dsjr ~ph n f o

~~Dr~~ oa1nM ryc~-ratgapi11 ictew f uo e c e hn xoed t o h

(2511anm)and9i phegaor ract on wCh h-D ItJV fr

by6E Iso 2nd Editi n d ~ -Sta d~w~ aine ag lc6 9 The whF te 4damrpu so aeEpea ot hh 4 o-ec

whe ex P ose shorCl2 ~tji~ 1 ah it4o wavu i- [cor spt oe

an~~~~~ an ~ ~12 ~r weeint neer ethc )1

thre wiho rf 28 V nd C O5 i-esect vely wh e e n

MI

Me~l83 1 -m eb Es rw

PrL)diu qui ni E-b V Isure CMOH ad a

mri rL wseaatoTe ef OUp~r a c~ _W~~~~~~th ~ acein e n ~ hi s lw O a o S a ~ a

Later o thenol $t Nas aebai n re- o eI eoTIk1 grS -h~C sbt~I~e hs ni bEM from e tit onr Io

f ar io - sd h I kal4ad d contentKtendsd toremai r

e I U~

qaL-Thereut ae sowin Fptue-tp ne p otvit qlrIYluh

pk ~rtatif Apo

fae1 0

This reul iniaespo- seiato - n hs ejrs h tec)ni ~U1a 1~I effi ie

b-eimPesue iudCroaorpy

A i meIM P-eSUr c lI4um 4a sd it Ia wIrg Cha1a

turiFLirs

Gi Ia ge IR SN sttoay hI rh dmon~irl ( he taionr~ phsewas25 2

Un aum l 3zsre tm C wippt ees -C-1

e e f pra 0i-n 1dp

aoimdn11 aal a edta5on t~ha I~Ipow alpa0 ma rin t h ep e p au51

Grdofs f Slagrem wa t TFap i a6 66 a~d Vi th E 9 E4d~ a Sm a ft

~etxa rme(F 5 oThe-lrv 1 an5 c9 D~r ) te hl~~r of

of Uni~va str e

hamc af~dip le ri om e a L6i absorptionti~ttael1i seewr r wef n V s

~d~T~1t tero t e ra le d ri ba d maa s eitisht o pius 9 P~beG ot

MRi amete asaC)~e Sh0m

The~gas aspectra performed a yhDr- fo ~ t a~c r a-Drhr e Ob~ranic (hmitrJItiuea]lolc 1- t h1~yr s 1~ ~ bHeUn o req showed armolecula weig thf 6 who~ich ise agrement with1t

m orh~a~wih h fa Tasdpo~e or iten~ 2hL (Persed Pm a c1 6 14 -1 6 1 47

A M 4h yi J4 esetumo yelwakli~ i an d

Grado ahw~ -ngr 7 i ngemnw h hto ap~e

repotedin he ltert~r 01 Shmmaand -J2LM~nio Chm~k

Commu~pp065971)

AL ~~ R IM10 C

~ ~ ~A-ll-E L AV S OfEC WI2I

~ 1-at n l ch 0 H T-ROy

21~~~~~~~~~EXr~9 UiI O0JEA OISG

E VE~0 T_ __ __T ____

YES-r0rm rin o-n d2

-s~ wieter ~ ari1dtoi rac ted o imE

t h enP60letog her-aMaer e C nvcoa oiUTh r 4DLT

~~a aac C ) JaioQ~ra1id j

t ~ wa atan then Pwi q1 Lin

axtrcteewitd thy Iaetatarr5poni0t t alof

~n~preparY s1S1a1g1 wH- 54thn ehastplaerted _g numhd iytdLDciWt chooomut If ka t xra IhPU no Ued hal al~~i uIdbe a

ichsafowhQreLystaet and Lerea ta

aTI RIaATIO -1LAVE Thn aercra lechieri chlo at of extr n

-gel LHF 254 TLOSI n g Ct dl~en Methaofo1 etao 9qy) po i F C -Sn o -se Zi2

ther 9aa~hi TsbdW hc-respots ~sr WimtW pbot thanrfo 63 t aY3 he 5pepttmspohi~~f nrrd Cw1wihL

ISIISS~SBhereeceo th a ft~s shylloQn an NCZ51 of~m~7C a12 poiiora Dreai~ rjduip -f fre e02 gav

~accorin t o~ pr (coi1 t ex2ac Sepraed~rm~Th ato hn The vpias by prepai veo f la h on1s i 1r-a Ip 25ampFthi vie~croatg -si 4-l

crsa inet Thi 2l 6anp)eoie in aorm a71 k i dw o peactv s II g F 4 thn Iayrcroaor5hY

~ ~ eunp~~~~~ ~ a I t--UGing h ytm Dclroehnrehshy

f~ ~ ~ 0 w- 10id h

4anf relthAf ampada ec~ tQ a fl5

gas h~morpe tah J (G-S 4V iar Lampaurf

4CI9O Ur-e i2Lno a t_

1msssecrm l 6LO I~

2 e 3 t 7 fn 0 n h jh-

o iru t

uuit

_

of apoh no i Ts After01co1itin wa o er pramte rs of-the

a ktone~ then Fampor Sioaut

Bign coinet~a ~~

Thas vle simla 1d~~e reore toe

cidedhi OLhrfsn1us I on was fLh athark a oi dD 5wa ~1Si~~tia C ic caM

Obtat2-ne 40 10-0

- i~3 4l 0

4HO

Nm

MI

Me~l83 1 -m eb Es rw

PrL)diu qui ni E-b V Isure CMOH ad a

mri rL wseaatoTe ef OUp~r a c~ _W~~~~~~th ~ acein e n ~ hi s lw O a o S a ~ a

Later o thenol $t Nas aebai n re- o eI eoTIk1 grS -h~C sbt~I~e hs ni bEM from e tit onr Io

f ar io - sd h I kal4ad d contentKtendsd toremai r

e I U~

qaL-Thereut ae sowin Fptue-tp ne p otvit qlrIYluh

pk ~rtatif Apo

fae1 0

This reul iniaespo- seiato - n hs ejrs h tec)ni ~U1a 1~I effi ie

b-eimPesue iudCroaorpy

A i meIM P-eSUr c lI4um 4a sd it Ia wIrg Cha1a

turiFLirs

Gi Ia ge IR SN sttoay hI rh dmon~irl ( he taionr~ phsewas25 2

Un aum l 3zsre tm C wippt ees -C-1

e e f pra 0i-n 1dp

aoimdn11 aal a edta5on t~ha I~Ipow alpa0 ma rin t h ep e p au51

Grdofs f Slagrem wa t TFap i a6 66 a~d Vi th E 9 E4d~ a Sm a ft

~etxa rme(F 5 oThe-lrv 1 an5 c9 D~r ) te hl~~r of

of Uni~va str e

hamc af~dip le ri om e a L6i absorptionti~ttael1i seewr r wef n V s

~d~T~1t tero t e ra le d ri ba d maa s eitisht o pius 9 P~beG ot

MRi amete asaC)~e Sh0m

The~gas aspectra performed a yhDr- fo ~ t a~c r a-Drhr e Ob~ranic (hmitrJItiuea]lolc 1- t h1~yr s 1~ ~ bHeUn o req showed armolecula weig thf 6 who~ich ise agrement with1t

m orh~a~wih h fa Tasdpo~e or iten~ 2hL (Persed Pm a c1 6 14 -1 6 1 47

A M 4h yi J4 esetumo yelwakli~ i an d

Grado ahw~ -ngr 7 i ngemnw h hto ap~e

repotedin he ltert~r 01 Shmmaand -J2LM~nio Chm~k

Commu~pp065971)

AL ~~ R IM10 C

~ ~ ~A-ll-E L AV S OfEC WI2I

~ 1-at n l ch 0 H T-ROy

21~~~~~~~~~EXr~9 UiI O0JEA OISG

E VE~0 T_ __ __T ____

YES-r0rm rin o-n d2

-s~ wieter ~ ari1dtoi rac ted o imE

t h enP60letog her-aMaer e C nvcoa oiUTh r 4DLT

~~a aac C ) JaioQ~ra1id j

t ~ wa atan then Pwi q1 Lin

axtrcteewitd thy Iaetatarr5poni0t t alof

~n~preparY s1S1a1g1 wH- 54thn ehastplaerted _g numhd iytdLDciWt chooomut If ka t xra IhPU no Ued hal al~~i uIdbe a

ichsafowhQreLystaet and Lerea ta

aTI RIaATIO -1LAVE Thn aercra lechieri chlo at of extr n

-gel LHF 254 TLOSI n g Ct dl~en Methaofo1 etao 9qy) po i F C -Sn o -se Zi2

ther 9aa~hi TsbdW hc-respots ~sr WimtW pbot thanrfo 63 t aY3 he 5pepttmspohi~~f nrrd Cw1wihL

ISIISS~SBhereeceo th a ft~s shylloQn an NCZ51 of~m~7C a12 poiiora Dreai~ rjduip -f fre e02 gav

~accorin t o~ pr (coi1 t ex2ac Sepraed~rm~Th ato hn The vpias by prepai veo f la h on1s i 1r-a Ip 25ampFthi vie~croatg -si 4-l

crsa inet Thi 2l 6anp)eoie in aorm a71 k i dw o peactv s II g F 4 thn Iayrcroaor5hY

~ ~ eunp~~~~~ ~ a I t--UGing h ytm Dclroehnrehshy

f~ ~ ~ 0 w- 10id h

4anf relthAf ampada ec~ tQ a fl5

gas h~morpe tah J (G-S 4V iar Lampaurf

4CI9O Ur-e i2Lno a t_

1msssecrm l 6LO I~

2 e 3 t 7 fn 0 n h jh-

o iru t

uuit

_

of apoh no i Ts After01co1itin wa o er pramte rs of-the

a ktone~ then Fampor Sioaut

Bign coinet~a ~~

Thas vle simla 1d~~e reore toe

cidedhi OLhrfsn1us I on was fLh athark a oi dD 5wa ~1Si~~tia C ic caM

Obtat2-ne 40 10-0

- i~3 4l 0

4HO

Nm

Un aum l 3zsre tm C wippt ees -C-1

e e f pra 0i-n 1dp

aoimdn11 aal a edta5on t~ha I~Ipow alpa0 ma rin t h ep e p au51

Grdofs f Slagrem wa t TFap i a6 66 a~d Vi th E 9 E4d~ a Sm a ft

~etxa rme(F 5 oThe-lrv 1 an5 c9 D~r ) te hl~~r of

of Uni~va str e

hamc af~dip le ri om e a L6i absorptionti~ttael1i seewr r wef n V s

~d~T~1t tero t e ra le d ri ba d maa s eitisht o pius 9 P~beG ot

MRi amete asaC)~e Sh0m

The~gas aspectra performed a yhDr- fo ~ t a~c r a-Drhr e Ob~ranic (hmitrJItiuea]lolc 1- t h1~yr s 1~ ~ bHeUn o req showed armolecula weig thf 6 who~ich ise agrement with1t

m orh~a~wih h fa Tasdpo~e or iten~ 2hL (Persed Pm a c1 6 14 -1 6 1 47

A M 4h yi J4 esetumo yelwakli~ i an d

Grado ahw~ -ngr 7 i ngemnw h hto ap~e

repotedin he ltert~r 01 Shmmaand -J2LM~nio Chm~k

Commu~pp065971)

AL ~~ R IM10 C

~ ~ ~A-ll-E L AV S OfEC WI2I

~ 1-at n l ch 0 H T-ROy

21~~~~~~~~~EXr~9 UiI O0JEA OISG

E VE~0 T_ __ __T ____

YES-r0rm rin o-n d2

-s~ wieter ~ ari1dtoi rac ted o imE

t h enP60letog her-aMaer e C nvcoa oiUTh r 4DLT

~~a aac C ) JaioQ~ra1id j

t ~ wa atan then Pwi q1 Lin

axtrcteewitd thy Iaetatarr5poni0t t alof

~n~preparY s1S1a1g1 wH- 54thn ehastplaerted _g numhd iytdLDciWt chooomut If ka t xra IhPU no Ued hal al~~i uIdbe a

ichsafowhQreLystaet and Lerea ta

aTI RIaATIO -1LAVE Thn aercra lechieri chlo at of extr n

-gel LHF 254 TLOSI n g Ct dl~en Methaofo1 etao 9qy) po i F C -Sn o -se Zi2

ther 9aa~hi TsbdW hc-respots ~sr WimtW pbot thanrfo 63 t aY3 he 5pepttmspohi~~f nrrd Cw1wihL

ISIISS~SBhereeceo th a ft~s shylloQn an NCZ51 of~m~7C a12 poiiora Dreai~ rjduip -f fre e02 gav

~accorin t o~ pr (coi1 t ex2ac Sepraed~rm~Th ato hn The vpias by prepai veo f la h on1s i 1r-a Ip 25ampFthi vie~croatg -si 4-l

crsa inet Thi 2l 6anp)eoie in aorm a71 k i dw o peactv s II g F 4 thn Iayrcroaor5hY

~ ~ eunp~~~~~ ~ a I t--UGing h ytm Dclroehnrehshy

f~ ~ ~ 0 w- 10id h

4anf relthAf ampada ec~ tQ a fl5

gas h~morpe tah J (G-S 4V iar Lampaurf

4CI9O Ur-e i2Lno a t_

1msssecrm l 6LO I~

2 e 3 t 7 fn 0 n h jh-

o iru t

uuit

_

of apoh no i Ts After01co1itin wa o er pramte rs of-the

a ktone~ then Fampor Sioaut

Bign coinet~a ~~

Thas vle simla 1d~~e reore toe

cidedhi OLhrfsn1us I on was fLh athark a oi dD 5wa ~1Si~~tia C ic caM

Obtat2-ne 40 10-0

- i~3 4l 0

4HO

Nm

AL ~~ R IM10 C

~ ~ ~A-ll-E L AV S OfEC WI2I

~ 1-at n l ch 0 H T-ROy

21~~~~~~~~~EXr~9 UiI O0JEA OISG

E VE~0 T_ __ __T ____

YES-r0rm rin o-n d2

-s~ wieter ~ ari1dtoi rac ted o imE

t h enP60letog her-aMaer e C nvcoa oiUTh r 4DLT

~~a aac C ) JaioQ~ra1id j

t ~ wa atan then Pwi q1 Lin

axtrcteewitd thy Iaetatarr5poni0t t alof

~n~preparY s1S1a1g1 wH- 54thn ehastplaerted _g numhd iytdLDciWt chooomut If ka t xra IhPU no Ued hal al~~i uIdbe a

ichsafowhQreLystaet and Lerea ta

aTI RIaATIO -1LAVE Thn aercra lechieri chlo at of extr n

-gel LHF 254 TLOSI n g Ct dl~en Methaofo1 etao 9qy) po i F C -Sn o -se Zi2

ther 9aa~hi TsbdW hc-respots ~sr WimtW pbot thanrfo 63 t aY3 he 5pepttmspohi~~f nrrd Cw1wihL

ISIISS~SBhereeceo th a ft~s shylloQn an NCZ51 of~m~7C a12 poiiora Dreai~ rjduip -f fre e02 gav

~accorin t o~ pr (coi1 t ex2ac Sepraed~rm~Th ato hn The vpias by prepai veo f la h on1s i 1r-a Ip 25ampFthi vie~croatg -si 4-l

crsa inet Thi 2l 6anp)eoie in aorm a71 k i dw o peactv s II g F 4 thn Iayrcroaor5hY

~ ~ eunp~~~~~ ~ a I t--UGing h ytm Dclroehnrehshy

f~ ~ ~ 0 w- 10id h

4anf relthAf ampada ec~ tQ a fl5

gas h~morpe tah J (G-S 4V iar Lampaurf

4CI9O Ur-e i2Lno a t_

1msssecrm l 6LO I~

2 e 3 t 7 fn 0 n h jh-

o iru t

uuit

_

of apoh no i Ts After01co1itin wa o er pramte rs of-the

a ktone~ then Fampor Sioaut

Bign coinet~a ~~

Thas vle simla 1d~~e reore toe

cidedhi OLhrfsn1us I on was fLh athark a oi dD 5wa ~1Si~~tia C ic caM

Obtat2-ne 40 10-0

- i~3 4l 0

4HO

Nm

f~ ~ ~ 0 w- 10id h

4anf relthAf ampada ec~ tQ a fl5

gas h~morpe tah J (G-S 4V iar Lampaurf

4CI9O Ur-e i2Lno a t_

1msssecrm l 6LO I~

2 e 3 t 7 fn 0 n h jh-

o iru t

uuit

_

of apoh no i Ts After01co1itin wa o er pramte rs of-the

a ktone~ then Fampor Sioaut

Bign coinet~a ~~

Thas vle simla 1d~~e reore toe

cidedhi OLhrfsn1us I on was fLh athark a oi dD 5wa ~1Si~~tia C ic caM

Obtat2-ne 40 10-0

- i~3 4l 0

4HO

Nm

I STUIES OW0DS-_ _ELUCID0TIC UOFTHEECHONIS 0 F-CTIONshy

ary Qor a 3- ( uI ar A~ P arpa aIes e Po a- fl ayQ i Ie~

eepuic map Pp1 Pt r de9L~i~ ru

thatSar ptf irg~vampac1ci1 e rI ed t mrdeqta Gradoextihtb t ecfc aaQ p0 0

r isoe I on is e hice pip~ 1at I eC3pp f htaH -

Grd 6itosecel prolifera ion cul mirtina2 o

ablowin OLryndio epeiens t e h ac t

~p0 7QtsQince a pine as nso ctlia~ e a aoint ubleiinc vi haopIRovjrn mainly wir~~thbe hlrr ebth for theniynv

i~~ ~~ n itoexermets

an h choryd a easprepaed f4ro it e fo 4bcatrd 4 e 4 oo~ nube lt n ItfoiI1 r~anoaip tm tia0

h typca v~iiih~ cdhr oan6 ici a s an wrdj trea mni rce ts agetrain -a)of23 f w

o ~~ocilatld I5n3 n o thedsh th back ofathe m1 1 d

wegt ewe gramswr~antio-

maig th tretmnt a~we stesaed- TW sur~ig ~ica er ra he f nhtcap a A e-cip t ne iis

thn d p l t d s i I c-A14ht) 8 h ~ s 1ate h

we ght of th mi ce WSMaue n hywreIt ~d groups atrno -oah ve g_ ad h tnad ro o

-the~4 g th e-t~ h u mg Savr of f r swereh n C

v d ~

Iup~ -tL a

wJ~r thiscpr p~s 05hmlnnjof the Solutio~b L tb _L1

TBhous he~a~1vtpeceteg ettty~r~ ee a Mice wer qac f dw h r 4i~4~

force to oFthe ~ ~ he 4 o~th one ore

On eS th ereg t sred r El ofS e an tco oenI 4~~4 aE amp uI4244 r enr r4 rC-s e

4Sffla ch4a4r Te4 1 ta 5 4te444ctd tai aanalye SL u ing the dn- -- TC_ VlC ee sin i-n

444 engt pltQouo5t (Dn to e W Fios~dsia E ioil Lm

4 r4if pr~ntage -or acti tyo~~~inHasC1r-U at~e acodn thyLh

WDS of re cessaryoveag th~efo~an e a td open~ithe I~d oft a

4In~ Tabl we4showi fromn o n t~ hc h

ath zrie exui act

pltLn th results expermenf vn~ie

~testat th c tfsa effecti th cigt1dant4 and th4 e)( rac e r w ic h n m i a w al h e 44lo

4 4 4 gt~ 44 4444f444

he in wa eron Tal 4th SPInge66HC1)a4 And -t4 44Spine4 cho444at had

Ci 4cn z n gt4444E1f e T e irea r 44 sifYer1 lw RESPONSamptr zgtDOSECURVE FOanE CCtiZsN EFFCspr Fl TAIet0 dif cw4 in a p yi gh r4i4ht 4444444on~ 4und4 th com

aspine iaspe active yic rznt prinipl

Sahgr e d~e Gr ado5~ W4e tUdi edthe o ec t oft different4

4 4 44 ~ ~ 44 4 4 4 4~ 4 444A

v d ~

Iup~ -tL a

wJ~r thiscpr p~s 05hmlnnjof the Solutio~b L tb _L1

TBhous he~a~1vtpeceteg ettty~r~ ee a Mice wer qac f dw h r 4i~4~

force to oFthe ~ ~ he 4 o~th one ore

On eS th ereg t sred r El ofS e an tco oenI 4~~4 aE amp uI4244 r enr r4 rC-s e

4Sffla ch4a4r Te4 1 ta 5 4te444ctd tai aanalye SL u ing the dn- -- TC_ VlC ee sin i-n

444 engt pltQouo5t (Dn to e W Fios~dsia E ioil Lm

4 r4if pr~ntage -or acti tyo~~~inHasC1r-U at~e acodn thyLh

WDS of re cessaryoveag th~efo~an e a td open~ithe I~d oft a

4In~ Tabl we4showi fromn o n t~ hc h

ath zrie exui act

pltLn th results expermenf vn~ie

~testat th c tfsa effecti th cigt1dant4 and th4 e)( rac e r w ic h n m i a w al h e 44lo

4 4 4 gt~ 44 4444f444

he in wa eron Tal 4th SPInge66HC1)a4 And -t4 44Spine4 cho444at had

Ci 4cn z n gt4444E1f e T e irea r 44 sifYer1 lw RESPONSamptr zgtDOSECURVE FOanE CCtiZsN EFFCspr Fl TAIet0 dif cw4 in a p yi gh r4i4ht 4444444on~ 4und4 th com

aspine iaspe active yic rznt prinipl

Sahgr e d~e Gr ado5~ W4e tUdi edthe o ec t oft different4

4 4 44 ~ ~ 44 4 4 4 4~ 4 444A

r r~l n hE

-i 5 seve er V1 0 0

4-7 -

Kg2 m1~ETCthF op i cAT nZaTI n S nt-e th5 mICrPel

KJNTCS OFCIARIZA I NTH12 S IN TH RESENCE AND ADSNC ~OTSPNECHLORH-YDRATE

2 ~ txn~n~an~ntration o F_

Fnv s kjo rI~c ar4 n ris nLp i t

Iici~io4 4l phresnce of_ti sip ~ Pe4t

ehded

Fohi A d n(-eteh ic er e divid d-in

Urmp aes mice tent i

1

h Ut5ths he othe i v rc i nedal conroluts t

the jon ea kjc~h o1drace6~cr At1 hus iroe op tob be ofita~ rn

sten 1 t WS) -hL

wi t gr

in e lie-VL ~iprtl~ wasrepatd one Up seaad i m~1e~ a 2 d th yU

1r

wo Ours a aae Ned i Wq Ed~-I~e i t 111- lato hes ) Q tta~ci5 t t i~ a n 1 A ho~iur5 3

boedl9od nedadwahecw~L~amp5 c -th ph It nopatt I 6 b f~e e Ii$5 I

4 t

-re4plce overEKItheOLAS~rf IOf Vr

4 0ug g-tsp the pljpe arvnd o ntamcin an th fI skfinvertd an-o erediu wa adedt pevn te lties f a0

wee l~t e cletadx ons of son nizt The ineHCI

for teath1primntsflrtairj~hpretaamp rfd

Itcwaseimprantc1~ ust-ir- ~in f t t4 0 iL( oe Z

In~ sdond oee w tded o the f feec t ciotaf p nei-111 O

sme ofrOr- exei-tsWl MIae L CO n c~ s ltInthis type of I r Or f iJ nqsarre e eowinrimniTs~a e 6~wa nwhat 047 auldv~iJ th k

ecrv nc~en tra -1 1oo rnm l of~ taspine-HCI~ e t-d sxncnpnriothe4 pmrrcentage o d-atrIiigh~yr f increased Iocnraln thimtseayoh~

1--J-h I shy

eSU~~~~~~ ~~prmns ~ ~ 5 b ad~ ~ aesonin 7 4s a

I114

r a pt i caruase h0

22 EFFECTI OF TASP I NE-HC IbON fTHE MIGRATIOW OF HUMAN FORESKIN FIBROBLASTS

The miral4o of fbolssto th~e W7Owund a _O

andL th~emtod We1olowd wasteoe eirbd by1 LiUr kR

Iine s Wmd 1acingr razor blade ered lr~t kIl pl te nd thenlbli

the right tho ell ~he twas pt amp1ediordpe t T r

tOhe rghtofth liA~ne Pl ats wer thn washd wth PDS in n ordpr~~~~~~~~~~~tc)~~~--t ceII e b m it t e IaII e ninated id d c

a]Og h in -Iero ells werearked~ wit th onto

the nube of c Isln tha crosLed~th I in a h all re- W~r ePCounfted under~~a Phase a t t

OUL o cells ~ wao e l -spine i-iC on the-mill t of the

frbl aststi th cl e L s of the p1t Th elOffil was obser at very Iow 1ocncentrati ons o taspine-1I~ Ta lII hw h eut o f an exper i men ts iIa t n descriIbeoli thprevol4 Table but~ here tJhe i -c i Lill Vwil~

migrtiol the f~lbroblasts at concientrations between A2and C

23 EFF~ECT OF TASPINE7HCI~ ON THE CONTRACTION OF LATTICES MADE OF HUMAN FORESKIN FIBROBLASTS~AND COLLAGENlt~

Won ontraction - isa -aohr~i mot atpl [t

~ci atri zatibprocess that~ we hav StL~ded using i s model sy4stem theEclntractiotn of cel4-colagn lttice described b Y

Deli-and colabaos(rc N ciUA712417

- 4 us

1979

11121 ~

Co 1f ri r Mo Ii ibb ario astt

ll C175r er2iWpr eP l p on eibobt e

b~ate l0 caIgIa5 dise i OlIs o dot t 2iL fct ) hG

r 5) no a u qn~fln 4 Kfcst

coptr I tvi Hank hNM fetald t~oine QImlr~~ shownCC)5m h

jn~P ti[ampI tio Eag 1ti sbaV1t 5 H)

5rLIM IM -~ Iyan f tapnAI1 So h s ia 7n71 k p n i e b h o t

7bt7-IampI a~r were7 added in 0 m4I77f M 7 1 Cinc

44gt7 74 744 n di ed d Am Ed gt a 5h77 J n i- r L tt b dia ef i re

444th d4~ 5 were~ me su i ti 4 e diffn e

The7l s o w i f r n 4ril4i~ f4-rLnn

1)c n atiel sn at L4o n -ach7ag on 4 two

dif4 re4 con4 -n 4to a i -H 444 s i I 4lt4( 14 Fi~ue Iand in~4 Asv pc01 -

744- )p ca~ b w ht44~~c 4

j 4

4l s4e 7 4 of7~7~ Laspill4 I 7 77 i4e _t4 ~ 2

tht 7i lhd4gi~~ca to 744g

the74~ 6- 6 0n -4 7 of f44s L ge I jhiIr -) t ce a on ori d_ - s7i7r

4ke C)44~~ I4447 ed7 th rj at w ic 4

I7t 4e- 4t aL ted 4 s 4 a-e an 774-4-474 atcon 74r o

~~ - a t47e 7 7 44ui n 747444 ~Ath 4 e7f h 4o Latt ce5~ by~

p 7n L h7-l c ~ 4

64 4 4 - j X

T~PN~I1SUDIES TE EFFE9T ICGS ON UTGEIC O S jE D 6RAORAN O

q--PO1TR POETALTE OFhAGE EGAD ADTS

FrTI o po eje u ed t i TEt o Am es Afe tiof~ MLAat0 isb~ on Rsth 77

-c -111 l 74

spheciticaitsin being ruse t fro ahitiin r Griq irOlntba to1 p trophy anwde ait~) r~t L~mwpby1~ T ~d~C tainod ThisoacorL-11a r h~u~a bee aiae rtedt-_to of carcinoges aSMtagL~ by F JAL b

tst ed in) the conentona animafr atns infi mutati fbraness

72- 5135-5139197

iThe m thodrLn seha We U~e~i~s r T 0T Jt)IA T 7 45 oratoyI~~ ofV n N Amefse~ate-a tst

st-a s~~o-interesting tcompoindt Ot t tSe iht ti knovm hi~t 1 Mhnq~L ~ hE varC~o gi opn f or

r) 4 aed n -reiin~p Tid su I sItIt~amp~gues - t- ( Darr Pi d~ r a sa ~tc~apu tio othL

-m tibLthe sm ic h5tris h pro nS1 s hte C b w th

The mleod Le sona e~ ~ -To 2 fmle)S o gra

e~t~I ti f ants r edtovmpai th

m0(w en r I i er on oe

frct ~~

n)te

wr aPtddwidla ia sdi-Va

mi nd w

r er

- dI-iea

Gand s t l

d on~tapn i imshyTheb~4lta

M-~iI fias s5 t n(~W20ch

r~~U~~f~ na-d ia W tr - rqii-d - S

~~nb 00 -eetat pel~ com~clspar ao

Thcqpoid Taspo d5cgml4 an tape -apne7 Cl wa diovd nwtS ndSedb ilrto

~~~~~t h r enrns r wspoe- Am Snred o s yS er-i 1 n e

h 101111-

6 K

14 V MMq ~~ tpE

Vef s ) in ib trv Io i th t hih r-o r nra io 5 Po

obev ta gSr- f de frado a s3IRd

canentraton ofrw 11~ C h~ tetr 14I0 an ~ 2Vj~)For ~an ~f v i hube1 re etat of t5d~ nf -L~ ti

e ospeint toethehee~ Weo~o a CIWbpntnejsu~ bpred no t Lathe concentctionc5 testedidttgenic_

concw~entatiorcn5e of~o ~a de Gradmuand~fTaspif-k oLh hSagre I iTs~~s he wradk e ncr Miadotnoit anr e rre

~teste0r2an rnraid)~ 5 Uheer thoncetrat ritin 0 p o (o r jI r41 Ic w c op o p a~dz - g n

501n) inthti nume of~ Hrtl t ~test e 12rLk ld4WhiC t0CTapie te n

difieret frum5testieHC11si [OtE(per mental~n he1muW~EIS Moppanr)~aged wamwh eF rado~ we muag

Tap EEC OFan nF de OspnoEFEQE CHaRo22NEHC SISTERgr oMATI 4000ZEXHA n GE 006m m Thes cocet l atI1 ia 11 Ae -I Ipfw Ill44

conctratiron de excasingesH~aesed all~ i Ii wh~ uh44 1s41howno MtAgnnesiv F~f~tl i wcanbednduedtbha

5 olte f f ntfr Tbt h in g di ca Lim 1 steMLtgnccm-r )FSagr de taiV eII neths ta

~ ofXamp

4 irohe oms 1 ste ca 1ltV 11 c eseha we ~ exchnge eid~db

andtC is aOt ll l if brtht~l~ey 6 conswsidere d C))

chfiromosme- amage- C hae b~ye~ 41 i4se1ogt

Thece 17~4111n s 1 t a li fo these ex e ime t eeY 901144weu

_COand ~ oh e I 414werp414141e7ns embryos a f 4 ~ th

1I 111411 ~I1~~1~414 1 1 1411 1141ltj I t 4 4111lt1 11 14 4~ ~ 14y

4-~

21 Evauationriof Citotoxici ty6of Tasp1 ne7 CI on V79AP4

~att n asp i ne -~C Io ~ ~ ed95f II l 4e tpi-

t~l~~UAnter~ adcr tiwuo d j

ar~fCO~ta fieamp L t c~pellst~descrr andor he of frmthe

pnu~rI-U~df~atnt L-5an

groni D4heCDr mdfi~ agep edL 5Lh~e mntedny mcw o th

24 hours r~aoh The reUl ts showed that concertrations of 176~ 000B and C) 14 L Im ~ 6f Taspine HCl were toxica for th1e el Is hi1

4 concentratins~ of C)2641 nd 0000 Oqml f Taspipe6-HdC were in~ocUOU~S for the cellis

Sb tCUS~d e thSi MUt~t rmntoJf-heitegrit f eI

S-mjembrane-and th ciiyo ntraeua aae5 me od- b f ae1o tYaerate

m~hodorRotan ad P (P~rocgt )a1~cadSLP USA 55 13-4~ 1966) ran Edidin IdjrjtI 13

Thlis technique Makes use of two dyes

The fir~ on~e a -nn ~lrn on f trec q compound~ -f curc that-6 coud t rav1erse thee I I i dlct ea~i

membaneOce nsid th cl I ine peifica stear (s I i ber atLek

~oneecon~d apoar f I1-2sceentsi~-T is dye etidium bromid~e~wh ich uP-onlexci tati on(wi th UV ligt fluIorsered This dy

Ci Ulonly enter theoeI whe the~r me-rni damaged~~Th cnetain fFursc~n ctt and et Id i Utn bromide14~Lsedwere 2 Ugr1a~d 5~bilWheI-ee~10y

ml 4nUgmiilerctvely e Ca~nlltrbaea twiththdy~s nd6~eredUnder U- gt in~ a~fILkoresceLt mlsectp~ 1 0h cel a b4~ed~Lrigh green6

fluorescencejunhealth 1 oed iIi1htgrPer1 freF5C ncampe anfagd ciidedcells ed~edf~oe~

- This~method wa also- sed inri order- toevaluate the ~ci otWlti

With the- oes described abv- th is con c en~rat ions5o asp btween 1761 and 0 4 4e V not ito nic while

Ce shy

on~~~~t-nIf)6~ ~ ~ n 6 nlad lwr wren t

weret+ne I r nt4t e cibe e -einthep m IJ

41~ ~

ME 4i

E2val at an of C0oait o f a n-HCl and Sanigre d

Grado on CHO cell1s

jCE~lI Hre-mp e resi tant t ig her conentrton or

23 Evlainof teeffect of Taspine HC1I on Sister

pe Choai Exhng~e- dfe age

SLuppl 6e~ted wh 5 eal bovine seru and005m~gertami~cin arindApCLbated in hL~Ii i a aos p h e eo C

~reedeTaspihe HM1 to afinal concentrationW C264 ~ M~Yof j1 -i~nI-0088 ugmi respectively A thr sto pl tes di d notI recei ye an~y Taspinej-ICl Iand ~remai ned as -control~ Simultaneou~sly with the tr tmnt die tweeamp sets~(oPaL

-~received~5Doodoirdn to ilicnetaino ltgM1 O nedi P~a t es hbrethen incubatedmt I

~~additional 22 hou~rs in a-hUnidifiedjaoshr of5 5 at -35r~a~7C (abdo2tt repli CatiVe-rQuL~WO nd

0~t the enid of the 22~ hours of InicLu3ajampion C61 mid~ o~a5 added ~toamptbe pl-ates (3final i-Y6f~ U andto ocneE- I- (pn I J

3 pc nLatdI~er f or and addit ion I twodhour s ftri at ubat i n i hao 31i n -fe e a~icai

and stain~ed wi~th tiplFllu iocromejP1lUZGe techni qie c)f per ry and Wol f f Nature 56-18 17il) and La tt (Pr 0c NAtI cad Sc i US7 3 3919l 7lt -K

7~The resul ts areshown in -Table 14 Wat we observei s that 2YTaspile~HCL-i at~ non~ citotoxic concentr~ations~ increases thea

~fre~ue~ncy of- sister c armat id exchageson V79O4cls

Fr thIest i th CHOD cel Is that4 ware~ growin expone ti al wer e seeded on 60 mmi tissue C1ture pt~ji dishe (3 1 clsA

4-bova ne serum nd 0m m o etm and - nCbted in4i a hUitmsid ee o COzand 5 ai t_7C wny or

~hours I ater-ns tofIa toe srlte a~4de s e-1 CtLo final c~ on- 6ntrat 9 a 6tg )1 a nIot he 4r se L olaes receivedSangrea de rad6o~at n1Zoi 3 to of 0 00 at rdin

set- f tde r CracoG i nd nad-a he-eAi cio1S~n1a

treamen th the set of ~r1a~espl recaivd

-MredIIuM3 Pla1tes whr thniChtdf or and add~i t i nLI21

- ~ 1~ 4 -d4

4 U ~~W10ri1I1

(abVQCW rp ribte vea qiar s Iofe

pl4ateE~ d U Aue c fr iionWerL ndad

4 staied ihth lyrcrm nc fnSC techof - I AniqL

gtapine1Cand did -a a L1amage SO7 337-31 177-

The~~~~~~~~~~ na reu~ 5adwa b ev tar h

PofiteTIALomtiES OF ha RAOAnDgtAP as inSGR~D the r-ntar

4L~~oth sinfcnlwpe sed the ~~ 4 4sr1n

eol~o-at rsIlp-hFrmthe nexperimesdescryibe ad e e cocV ta Taspin-HClt anFsc6 Sanred WeeksddntP-dC hO~on

cr i no~a Sytemhand the t0dw uedoensi wee fMI owe i i Iat

aLs CDhthl-Toheon dac~n~eribed bypurd usld 71

j~nO~s1 a I a TClaoa

Kl-Ten krianto A SN - eesCE use 1 s - 1 a d N t OILSIi~sce op~d

Thbmaledeany1phrb M~c etate moths~ ofri the~wre shcet1aed i re~js ig phas hair~ 6c usd Mceery honedUIof-th wereeap

p ppiand pfahe inweteator de adI~Di i triTheafer cahe nod ltamp~4were 30Pan(riasp e Leachr dxermnag~~(roup6o

compoud~ uonted as-anp-ltracexerininM tgtA ih bfrue t soIvPd

carcinogenicou~~W-nd 7n-12 ewt~ole- 0(-)shy-~~~h

x and i

n~~ (l I s

I Thegr o n tume~or roe r compound ~used wa 7 ra decGoylpup rbltreate~s- (TA and ocn a-o -tdNo

wasOLI 2~Ug -in ofL actn ctone- gt i- e

G-C resnt I No

Group---------------------------------------------------------------------------

GrOLIP~~~ 10nTo ~ 3 i 1 l-c~~ fI-D

p 5r I10 n D Ajn 1s c e~ e

42 twi cf wekl e ef

t v4 CL we e4

Io4M C)A~~cng C Or oup 0I~ of rTrAn oar3 MOi Q~~~~IaeoneHrF

GrUP1 Wau ~Our cotro o efomt ion of sp n r-fpr~mQ SGroup 2 i4as Ou coto fo asacrcngn cDrMnq

r-0 o WiuRm mno r~a n iLWwht~U~ ldshwasiry zec p~b 7snbu ltzgtwo~t our twi~o-tg raiognen r6~wtd ~

tr~satedDrlI3Awilhai ard TP~ op -as ~wo$king~5nue ot

ourNoy rou Sndangre de Gnoranai Group VWithr ~~ts r 4 t -1C - acng

r n~dLndPh~fP~P e as 11

atou 7hend b for acarcnoge

h or Tasp n

Tho e )02 prtfrpd c i 2nd aneioMet er a on~ mbe e 190 vr

trpeetned _~ s nTAa n trwihDB dcr a m deloe

the pio~mast rekmonhs afther thji niLia n fthe treimn (2a oted of 294ba5ieay 1 rso rlice trearte wi thur-~d oleir o

of ~~~ ~ ~ ms~ ~ tju ~ ~ ~ i~ pe-~t ~ deeipdppnssa~d itA

no es w r oA4tp --4 d an

0

7t

J

22

-Grossge~i ti nowere sever a Iex e -p

GVL hcw a amintiom ma 94jf rom4~

by- hyep theao derm ppij aei wich er e ted Fo theI nthemi sufce he Ml gisa~ dc or inveIS aymtr~

igt beae4ikr aa~hs L wit r-ccnLgt (te hyekrami

a t om o f c n a s io y t T e r mj i- b i I

mainn chngs Th Kn4--oran did pr4n pah~oi

FFSiieii a e ca c n gei or tino promoters-

f ia e n in ocotty tfe eX p rim6 D it IFi aIhc I-54 SiAL-4 mo th in or r toi4i be i co c u F i ce I- mn ii iF

i ~~ t4Ie~ ~ os4iL-ig ~ ~ hL ~ ~iie ~ ~ v 4~ ~ ~ g Oilt494hrta ~h ra ~ wi~j theknow ogn sA~rtd t 3 doelo tv tmrcari DB

on mo th ag

4 4 ~

4 ONai

4CONCLUSIONS

rSnr de Grado etracted f romp ICiton 7er

o extracts fr-oT1 th~ecore~ fri in ofth Crotor lechleri tree

Thr4ure idtein Or trc EITOit in the ieavesu of thebAci~t n I 1eriand the most bkidant one was ~

T~asp inewas h cti O 1teri za tp i ncn 1 L-p e s en1 i Sa~ngre4 de Gracdo etracted from Croto hl1ech14er

~h Lid healin process~ r I

1~~~fibrobasts atonceritrtions1 be1llow2)- nOgm1 ard i t di d e Ih1 PEr at ion~not uincrease ratE1Qf-11P

w fon nt~h ibit the contac i on61

hnn f orEsk i ni i a fibr 0b I as

6~mutagunicitY 1tud using the 4rnes 4tcst showe htSJrgshydeGad a wh le taspine chloydrate hadttrcco k

~~ ~no Jn~ta ernii activity I1

StUdies on e ef f eect4 - Ire derao andAsnt A-t oBancr adpCdnd

ychorhdrteortisister choai eklnp (SCE) showe t I4bsTaspine hlrydrate increased the nIumber of SC[ 3in

V79if4 ellbt no increase wa5 abservedinCOcls 10Uigthe tW-tg O skincriogee syste w e V

4 14he shown that~4nri Uher Sng a ir tppeIJ1 were carci nb~geniviardor -tumor promtr f te n0ya +iitrea t4ment 1

--- --- --- --- --- --- ---

0 11

FXTROTrONOOS9NJRF DE GRA1DO FRMDFgRE T RE P1)0tITUITSC DING -m RSTTRP

TREETPg~ OF] PH ~VOLiUMEK OF~ YILIL - XT I ON E--~ Ig11TV~ XTROCTION

6-- 91 -~agm ~ mJ-1 -- ~ 22 272

M- 5ql ar-To each of the sample wre addced tJ n - -qri

~~~~e alktalinized to p 9 wth amn~iui hydjroXidftt~eJ~h and extatede0hautv- wie~t~h choroform Each clooorm extractwa concentrated i n a rotavapor then dryed and -the~~shy

ht 0os theE 6k gaIkaIoidvr eaU d i napl- alI7 24 -065j-~9-- ~

C) Iml ofec f Lhe 5ape wer Alded Ijlo ae th as t e aI kalinized-to pH 92wtha

etract iasdred and hei redissolved i 1 mo-o lLlou and0 1e bobnamp t347lt was merLrdL~igaZi

Spe ro fotome er-jThextinction coefficient of taspine at 47 i

a si gni i can t di FF$ernce2 between thee gbFUPS anthegOUS-n

The col lact n datE wJas Dec mber~ 19 1901

-- ---Y -- ~4A shy- ~----

I A

4 AB 2 ~4

q 7

EXRCIN F AGE1 1ACFOM -ER RL RMIU

~4 1 4-112 lt 4 44 4

44 42 I-4lt C) 6

- ~~ ~ ~ ~ ~ ~~44-- -- -- ----4 -shy~~ ~~~4441533c~ de Grado was co lced fo t e te s nm rae hc

on ebr 1 15and 5 a d n M y 2 90 h I a m- dary 19

4tet4wa d -4 e44 de 11o etha To~4lt4I ml 4~44 each 4 44444 were ed of wa eof o ~ th sV ad I4~ l th

mixturwa al t 4lt4 yrxd -iUPH~ 9 IVizp and 4l~444 wi- 4 o c rfextr c~e l a~~iv 4 h 4aof 4ofri E4a4 h

r-~ lt4~4 444 ~ 44 ~44 4) gt44

Xh e~= 2 8c tr-ts 0 da gr7380frI~ aawas 1+C

wasra sign~ficat diiffrec betwee the to ~grup Plt 0

i 44 ~ f~cho ~ h ui 1 4 l V~n11~9 7 rW hnaIdiin 4 hdoidlt465 W~qamp

T

22

I~~_7

control (rD 11

SG 51 1 1 1 8

Tabpie tm11C () r 71 0 1

Obrevi itions WS = WUndbeakinsteghSIG =Sargre de Grado 2

STaspine-HICI = Taspirn chlrhydrate

Nce wepre anesthesized with vapors fdiethyl ete and a~ IcM A ci ion as made perpendi CIler _to the ais fsmfnmet-y f tea i anid thtwo4borders of the Wo~nd werestichdtgte tiscntr

g~tete ~ n~p~vry 12 hOrs the comfpol-tbeinQt~ted Ias~ap llied Ato the w~ound -For thi p~pGUQXn M2Ishy

mealt p m crop 1diwpe 4 ddrj1 Veins a WacLIndy h ant-h otosrrcie ny

~Fnt iii2which liheror6 wa dtdI1 horst ~ pib elte

f gone of the borders of t~he on aFtr 1-itinLh t hle4 g ret h other onet For moeJh tai _hi p in a 2MeaSLrable_ fore teoe hmred

t 1i anlYi S~ ohtps thUi ng ~ s sh2tt w d2itt ic aie

di erence betwepri Aher can Lrol grouip and tgtheSG-g roup (p0nO5 -n bewenr theQcntrol pU idLPtpi6Hl L(p C CI)i1

fr C an 22gn 12 2

1r

wo Ours a aae Ned i Wq Ed~-I~e i t 111- lato hes ) Q tta~ci5 t t i~ a n 1 A ho~iur5 3

boedl9od nedadwahecw~L~amp5 c -th ph It nopatt I 6 b f~e e Ii$5 I

4 t

-re4plce overEKItheOLAS~rf IOf Vr

4 0ug g-tsp the pljpe arvnd o ntamcin an th fI skfinvertd an-o erediu wa adedt pevn te lties f a0

wee l~t e cletadx ons of son nizt The ineHCI

for teath1primntsflrtairj~hpretaamp rfd

Itcwaseimprantc1~ ust-ir- ~in f t t4 0 iL( oe Z

In~ sdond oee w tded o the f feec t ciotaf p nei-111 O

sme ofrOr- exei-tsWl MIae L CO n c~ s ltInthis type of I r Or f iJ nqsarre e eowinrimniTs~a e 6~wa nwhat 047 auldv~iJ th k

ecrv nc~en tra -1 1oo rnm l of~ taspine-HCI~ e t-d sxncnpnriothe4 pmrrcentage o d-atrIiigh~yr f increased Iocnraln thimtseayoh~

1--J-h I shy

eSU~~~~~~ ~~prmns ~ ~ 5 b ad~ ~ aesonin 7 4s a

I114

r a pt i caruase h0

22 EFFECTI OF TASP I NE-HC IbON fTHE MIGRATIOW OF HUMAN FORESKIN FIBROBLASTS

The miral4o of fbolssto th~e W7Owund a _O

andL th~emtod We1olowd wasteoe eirbd by1 LiUr kR

Iine s Wmd 1acingr razor blade ered lr~t kIl pl te nd thenlbli

the right tho ell ~he twas pt amp1ediordpe t T r

tOhe rghtofth liA~ne Pl ats wer thn washd wth PDS in n ordpr~~~~~~~~~~~tc)~~~--t ceII e b m it t e IaII e ninated id d c

a]Og h in -Iero ells werearked~ wit th onto

the nube of c Isln tha crosLed~th I in a h all re- W~r ePCounfted under~~a Phase a t t

OUL o cells ~ wao e l -spine i-iC on the-mill t of the

frbl aststi th cl e L s of the p1t Th elOffil was obser at very Iow 1ocncentrati ons o taspine-1I~ Ta lII hw h eut o f an exper i men ts iIa t n descriIbeoli thprevol4 Table but~ here tJhe i -c i Lill Vwil~

migrtiol the f~lbroblasts at concientrations between A2and C

23 EFF~ECT OF TASPINE7HCI~ ON THE CONTRACTION OF LATTICES MADE OF HUMAN FORESKIN FIBROBLASTS~AND COLLAGENlt~

Won ontraction - isa -aohr~i mot atpl [t

~ci atri zatibprocess that~ we hav StL~ded using i s model sy4stem theEclntractiotn of cel4-colagn lttice described b Y

Deli-and colabaos(rc N ciUA712417

- 4 us

1979

11121 ~

Co 1f ri r Mo Ii ibb ario astt

ll C175r er2iWpr eP l p on eibobt e

b~ate l0 caIgIa5 dise i OlIs o dot t 2iL fct ) hG

r 5) no a u qn~fln 4 Kfcst

coptr I tvi Hank hNM fetald t~oine QImlr~~ shownCC)5m h

jn~P ti[ampI tio Eag 1ti sbaV1t 5 H)

5rLIM IM -~ Iyan f tapnAI1 So h s ia 7n71 k p n i e b h o t

7bt7-IampI a~r were7 added in 0 m4I77f M 7 1 Cinc

44gt7 74 744 n di ed d Am Ed gt a 5h77 J n i- r L tt b dia ef i re

444th d4~ 5 were~ me su i ti 4 e diffn e

The7l s o w i f r n 4ril4i~ f4-rLnn

1)c n atiel sn at L4o n -ach7ag on 4 two

dif4 re4 con4 -n 4to a i -H 444 s i I 4lt4( 14 Fi~ue Iand in~4 Asv pc01 -

744- )p ca~ b w ht44~~c 4

j 4

4l s4e 7 4 of7~7~ Laspill4 I 7 77 i4e _t4 ~ 2

tht 7i lhd4gi~~ca to 744g

the74~ 6- 6 0n -4 7 of f44s L ge I jhiIr -) t ce a on ori d_ - s7i7r

4ke C)44~~ I4447 ed7 th rj at w ic 4

I7t 4e- 4t aL ted 4 s 4 a-e an 774-4-474 atcon 74r o

~~ - a t47e 7 7 44ui n 747444 ~Ath 4 e7f h 4o Latt ce5~ by~

p 7n L h7-l c ~ 4

64 4 4 - j X

T~PN~I1SUDIES TE EFFE9T ICGS ON UTGEIC O S jE D 6RAORAN O

q--PO1TR POETALTE OFhAGE EGAD ADTS

FrTI o po eje u ed t i TEt o Am es Afe tiof~ MLAat0 isb~ on Rsth 77

-c -111 l 74

spheciticaitsin being ruse t fro ahitiin r Griq irOlntba to1 p trophy anwde ait~) r~t L~mwpby1~ T ~d~C tainod ThisoacorL-11a r h~u~a bee aiae rtedt-_to of carcinoges aSMtagL~ by F JAL b

tst ed in) the conentona animafr atns infi mutati fbraness

72- 5135-5139197

iThe m thodrLn seha We U~e~i~s r T 0T Jt)IA T 7 45 oratoyI~~ ofV n N Amefse~ate-a tst

st-a s~~o-interesting tcompoindt Ot t tSe iht ti knovm hi~t 1 Mhnq~L ~ hE varC~o gi opn f or

r) 4 aed n -reiin~p Tid su I sItIt~amp~gues - t- ( Darr Pi d~ r a sa ~tc~apu tio othL

-m tibLthe sm ic h5tris h pro nS1 s hte C b w th

The mleod Le sona e~ ~ -To 2 fmle)S o gra

e~t~I ti f ants r edtovmpai th

m0(w en r I i er on oe

frct ~~

n)te

wr aPtddwidla ia sdi-Va

mi nd w

r er

- dI-iea

Gand s t l

d on~tapn i imshyTheb~4lta

M-~iI fias s5 t n(~W20ch

r~~U~~f~ na-d ia W tr - rqii-d - S

~~nb 00 -eetat pel~ com~clspar ao

Thcqpoid Taspo d5cgml4 an tape -apne7 Cl wa diovd nwtS ndSedb ilrto

~~~~~t h r enrns r wspoe- Am Snred o s yS er-i 1 n e

h 101111-

6 K

14 V MMq ~~ tpE

Vef s ) in ib trv Io i th t hih r-o r nra io 5 Po

obev ta gSr- f de frado a s3IRd

canentraton ofrw 11~ C h~ tetr 14I0 an ~ 2Vj~)For ~an ~f v i hube1 re etat of t5d~ nf -L~ ti

e ospeint toethehee~ Weo~o a CIWbpntnejsu~ bpred no t Lathe concentctionc5 testedidttgenic_

concw~entatiorcn5e of~o ~a de Gradmuand~fTaspif-k oLh hSagre I iTs~~s he wradk e ncr Miadotnoit anr e rre

~teste0r2an rnraid)~ 5 Uheer thoncetrat ritin 0 p o (o r jI r41 Ic w c op o p a~dz - g n

501n) inthti nume of~ Hrtl t ~test e 12rLk ld4WhiC t0CTapie te n

difieret frum5testieHC11si [OtE(per mental~n he1muW~EIS Moppanr)~aged wamwh eF rado~ we muag

Tap EEC OFan nF de OspnoEFEQE CHaRo22NEHC SISTERgr oMATI 4000ZEXHA n GE 006m m Thes cocet l atI1 ia 11 Ae -I Ipfw Ill44

conctratiron de excasingesH~aesed all~ i Ii wh~ uh44 1s41howno MtAgnnesiv F~f~tl i wcanbednduedtbha

5 olte f f ntfr Tbt h in g di ca Lim 1 steMLtgnccm-r )FSagr de taiV eII neths ta

~ ofXamp

4 irohe oms 1 ste ca 1ltV 11 c eseha we ~ exchnge eid~db

andtC is aOt ll l if brtht~l~ey 6 conswsidere d C))

chfiromosme- amage- C hae b~ye~ 41 i4se1ogt

Thece 17~4111n s 1 t a li fo these ex e ime t eeY 901144weu

_COand ~ oh e I 414werp414141e7ns embryos a f 4 ~ th

1I 111411 ~I1~~1~414 1 1 1411 1141ltj I t 4 4111lt1 11 14 4~ ~ 14y

4-~

21 Evauationriof Citotoxici ty6of Tasp1 ne7 CI on V79AP4

~att n asp i ne -~C Io ~ ~ ed95f II l 4e tpi-

t~l~~UAnter~ adcr tiwuo d j

ar~fCO~ta fieamp L t c~pellst~descrr andor he of frmthe

pnu~rI-U~df~atnt L-5an

groni D4heCDr mdfi~ agep edL 5Lh~e mntedny mcw o th

24 hours r~aoh The reUl ts showed that concertrations of 176~ 000B and C) 14 L Im ~ 6f Taspine HCl were toxica for th1e el Is hi1

4 concentratins~ of C)2641 nd 0000 Oqml f Taspipe6-HdC were in~ocUOU~S for the cellis

Sb tCUS~d e thSi MUt~t rmntoJf-heitegrit f eI

S-mjembrane-and th ciiyo ntraeua aae5 me od- b f ae1o tYaerate

m~hodorRotan ad P (P~rocgt )a1~cadSLP USA 55 13-4~ 1966) ran Edidin IdjrjtI 13

Thlis technique Makes use of two dyes

The fir~ on~e a -nn ~lrn on f trec q compound~ -f curc that-6 coud t rav1erse thee I I i dlct ea~i

membaneOce nsid th cl I ine peifica stear (s I i ber atLek

~oneecon~d apoar f I1-2sceentsi~-T is dye etidium bromid~e~wh ich uP-onlexci tati on(wi th UV ligt fluIorsered This dy

Ci Ulonly enter theoeI whe the~r me-rni damaged~~Th cnetain fFursc~n ctt and et Id i Utn bromide14~Lsedwere 2 Ugr1a~d 5~bilWheI-ee~10y

ml 4nUgmiilerctvely e Ca~nlltrbaea twiththdy~s nd6~eredUnder U- gt in~ a~fILkoresceLt mlsectp~ 1 0h cel a b4~ed~Lrigh green6

fluorescencejunhealth 1 oed iIi1htgrPer1 freF5C ncampe anfagd ciidedcells ed~edf~oe~

- This~method wa also- sed inri order- toevaluate the ~ci otWlti

With the- oes described abv- th is con c en~rat ions5o asp btween 1761 and 0 4 4e V not ito nic while

Ce shy

on~~~~t-nIf)6~ ~ ~ n 6 nlad lwr wren t

weret+ne I r nt4t e cibe e -einthep m IJ

41~ ~

ME 4i

E2val at an of C0oait o f a n-HCl and Sanigre d

Grado on CHO cell1s

jCE~lI Hre-mp e resi tant t ig her conentrton or

23 Evlainof teeffect of Taspine HC1I on Sister

pe Choai Exhng~e- dfe age

SLuppl 6e~ted wh 5 eal bovine seru and005m~gertami~cin arindApCLbated in hL~Ii i a aos p h e eo C

~reedeTaspihe HM1 to afinal concentrationW C264 ~ M~Yof j1 -i~nI-0088 ugmi respectively A thr sto pl tes di d notI recei ye an~y Taspinej-ICl Iand ~remai ned as -control~ Simultaneou~sly with the tr tmnt die tweeamp sets~(oPaL

-~received~5Doodoirdn to ilicnetaino ltgM1 O nedi P~a t es hbrethen incubatedmt I

~~additional 22 hou~rs in a-hUnidifiedjaoshr of5 5 at -35r~a~7C (abdo2tt repli CatiVe-rQuL~WO nd

0~t the enid of the 22~ hours of InicLu3ajampion C61 mid~ o~a5 added ~toamptbe pl-ates (3final i-Y6f~ U andto ocneE- I- (pn I J

3 pc nLatdI~er f or and addit ion I twodhour s ftri at ubat i n i hao 31i n -fe e a~icai

and stain~ed wi~th tiplFllu iocromejP1lUZGe techni qie c)f per ry and Wol f f Nature 56-18 17il) and La tt (Pr 0c NAtI cad Sc i US7 3 3919l 7lt -K

7~The resul ts areshown in -Table 14 Wat we observei s that 2YTaspile~HCL-i at~ non~ citotoxic concentr~ations~ increases thea

~fre~ue~ncy of- sister c armat id exchageson V79O4cls

Fr thIest i th CHOD cel Is that4 ware~ growin expone ti al wer e seeded on 60 mmi tissue C1ture pt~ji dishe (3 1 clsA

4-bova ne serum nd 0m m o etm and - nCbted in4i a hUitmsid ee o COzand 5 ai t_7C wny or

~hours I ater-ns tofIa toe srlte a~4de s e-1 CtLo final c~ on- 6ntrat 9 a 6tg )1 a nIot he 4r se L olaes receivedSangrea de rad6o~at n1Zoi 3 to of 0 00 at rdin

set- f tde r CracoG i nd nad-a he-eAi cio1S~n1a

treamen th the set of ~r1a~espl recaivd

-MredIIuM3 Pla1tes whr thniChtdf or and add~i t i nLI21

- ~ 1~ 4 -d4

4 U ~~W10ri1I1

(abVQCW rp ribte vea qiar s Iofe

pl4ateE~ d U Aue c fr iionWerL ndad

4 staied ihth lyrcrm nc fnSC techof - I AniqL

gtapine1Cand did -a a L1amage SO7 337-31 177-

The~~~~~~~~~~ na reu~ 5adwa b ev tar h

PofiteTIALomtiES OF ha RAOAnDgtAP as inSGR~D the r-ntar

4L~~oth sinfcnlwpe sed the ~~ 4 4sr1n

eol~o-at rsIlp-hFrmthe nexperimesdescryibe ad e e cocV ta Taspin-HClt anFsc6 Sanred WeeksddntP-dC hO~on

cr i no~a Sytemhand the t0dw uedoensi wee fMI owe i i Iat

aLs CDhthl-Toheon dac~n~eribed bypurd usld 71

j~nO~s1 a I a TClaoa

Kl-Ten krianto A SN - eesCE use 1 s - 1 a d N t OILSIi~sce op~d

Thbmaledeany1phrb M~c etate moths~ ofri the~wre shcet1aed i re~js ig phas hair~ 6c usd Mceery honedUIof-th wereeap

p ppiand pfahe inweteator de adI~Di i triTheafer cahe nod ltamp~4were 30Pan(riasp e Leachr dxermnag~~(roup6o

compoud~ uonted as-anp-ltracexerininM tgtA ih bfrue t soIvPd

carcinogenicou~~W-nd 7n-12 ewt~ole- 0(-)shy-~~~h

x and i

n~~ (l I s

I Thegr o n tume~or roe r compound ~used wa 7 ra decGoylpup rbltreate~s- (TA and ocn a-o -tdNo

wasOLI 2~Ug -in ofL actn ctone- gt i- e

G-C resnt I No

Group---------------------------------------------------------------------------

GrOLIP~~~ 10nTo ~ 3 i 1 l-c~~ fI-D

p 5r I10 n D Ajn 1s c e~ e

42 twi cf wekl e ef

t v4 CL we e4

Io4M C)A~~cng C Or oup 0I~ of rTrAn oar3 MOi Q~~~~IaeoneHrF

GrUP1 Wau ~Our cotro o efomt ion of sp n r-fpr~mQ SGroup 2 i4as Ou coto fo asacrcngn cDrMnq

r-0 o WiuRm mno r~a n iLWwht~U~ ldshwasiry zec p~b 7snbu ltzgtwo~t our twi~o-tg raiognen r6~wtd ~

tr~satedDrlI3Awilhai ard TP~ op -as ~wo$king~5nue ot

ourNoy rou Sndangre de Gnoranai Group VWithr ~~ts r 4 t -1C - acng

r n~dLndPh~fP~P e as 11

atou 7hend b for acarcnoge

h or Tasp n

Tho e )02 prtfrpd c i 2nd aneioMet er a on~ mbe e 190 vr

trpeetned _~ s nTAa n trwihDB dcr a m deloe

the pio~mast rekmonhs afther thji niLia n fthe treimn (2a oted of 294ba5ieay 1 rso rlice trearte wi thur-~d oleir o

of ~~~ ~ ~ ms~ ~ tju ~ ~ ~ i~ pe-~t ~ deeipdppnssa~d itA

no es w r oA4tp --4 d an

0

7t

J

22

-Grossge~i ti nowere sever a Iex e -p

GVL hcw a amintiom ma 94jf rom4~

by- hyep theao derm ppij aei wich er e ted Fo theI nthemi sufce he Ml gisa~ dc or inveIS aymtr~

igt beae4ikr aa~hs L wit r-ccnLgt (te hyekrami

a t om o f c n a s io y t T e r mj i- b i I

mainn chngs Th Kn4--oran did pr4n pah~oi

FFSiieii a e ca c n gei or tino promoters-

f ia e n in ocotty tfe eX p rim6 D it IFi aIhc I-54 SiAL-4 mo th in or r toi4i be i co c u F i ce I- mn ii iF

i ~~ t4Ie~ ~ os4iL-ig ~ ~ hL ~ ~iie ~ ~ v 4~ ~ ~ g Oilt494hrta ~h ra ~ wi~j theknow ogn sA~rtd t 3 doelo tv tmrcari DB

on mo th ag

4 4 ~

4 ONai

4CONCLUSIONS

rSnr de Grado etracted f romp ICiton 7er

o extracts fr-oT1 th~ecore~ fri in ofth Crotor lechleri tree

Thr4ure idtein Or trc EITOit in the ieavesu of thebAci~t n I 1eriand the most bkidant one was ~

T~asp inewas h cti O 1teri za tp i ncn 1 L-p e s en1 i Sa~ngre4 de Gracdo etracted from Croto hl1ech14er

~h Lid healin process~ r I

1~~~fibrobasts atonceritrtions1 be1llow2)- nOgm1 ard i t di d e Ih1 PEr at ion~not uincrease ratE1Qf-11P

w fon nt~h ibit the contac i on61

hnn f orEsk i ni i a fibr 0b I as

6~mutagunicitY 1tud using the 4rnes 4tcst showe htSJrgshydeGad a wh le taspine chloydrate hadttrcco k

~~ ~no Jn~ta ernii activity I1

StUdies on e ef f eect4 - Ire derao andAsnt A-t oBancr adpCdnd

ychorhdrteortisister choai eklnp (SCE) showe t I4bsTaspine hlrydrate increased the nIumber of SC[ 3in

V79if4 ellbt no increase wa5 abservedinCOcls 10Uigthe tW-tg O skincriogee syste w e V

4 14he shown that~4nri Uher Sng a ir tppeIJ1 were carci nb~geniviardor -tumor promtr f te n0ya +iitrea t4ment 1

--- --- --- --- --- --- ---

0 11

FXTROTrONOOS9NJRF DE GRA1DO FRMDFgRE T RE P1)0tITUITSC DING -m RSTTRP

TREETPg~ OF] PH ~VOLiUMEK OF~ YILIL - XT I ON E--~ Ig11TV~ XTROCTION

6-- 91 -~agm ~ mJ-1 -- ~ 22 272

M- 5ql ar-To each of the sample wre addced tJ n - -qri

~~~~e alktalinized to p 9 wth amn~iui hydjroXidftt~eJ~h and extatede0hautv- wie~t~h choroform Each clooorm extractwa concentrated i n a rotavapor then dryed and -the~~shy

ht 0os theE 6k gaIkaIoidvr eaU d i napl- alI7 24 -065j-~9-- ~

C) Iml ofec f Lhe 5ape wer Alded Ijlo ae th as t e aI kalinized-to pH 92wtha

etract iasdred and hei redissolved i 1 mo-o lLlou and0 1e bobnamp t347lt was merLrdL~igaZi

Spe ro fotome er-jThextinction coefficient of taspine at 47 i

a si gni i can t di FF$ernce2 between thee gbFUPS anthegOUS-n

The col lact n datE wJas Dec mber~ 19 1901

-- ---Y -- ~4A shy- ~----

I A

4 AB 2 ~4

q 7

EXRCIN F AGE1 1ACFOM -ER RL RMIU

~4 1 4-112 lt 4 44 4

44 42 I-4lt C) 6

- ~~ ~ ~ ~ ~ ~~44-- -- -- ----4 -shy~~ ~~~4441533c~ de Grado was co lced fo t e te s nm rae hc

on ebr 1 15and 5 a d n M y 2 90 h I a m- dary 19

4tet4wa d -4 e44 de 11o etha To~4lt4I ml 4~44 each 4 44444 were ed of wa eof o ~ th sV ad I4~ l th

mixturwa al t 4lt4 yrxd -iUPH~ 9 IVizp and 4l~444 wi- 4 o c rfextr c~e l a~~iv 4 h 4aof 4ofri E4a4 h

r-~ lt4~4 444 ~ 44 ~44 4) gt44

Xh e~= 2 8c tr-ts 0 da gr7380frI~ aawas 1+C

wasra sign~ficat diiffrec betwee the to ~grup Plt 0

i 44 ~ f~cho ~ h ui 1 4 l V~n11~9 7 rW hnaIdiin 4 hdoidlt465 W~qamp

T

22

I~~_7

control (rD 11

SG 51 1 1 1 8

Tabpie tm11C () r 71 0 1

Obrevi itions WS = WUndbeakinsteghSIG =Sargre de Grado 2

STaspine-HICI = Taspirn chlrhydrate

Nce wepre anesthesized with vapors fdiethyl ete and a~ IcM A ci ion as made perpendi CIler _to the ais fsmfnmet-y f tea i anid thtwo4borders of the Wo~nd werestichdtgte tiscntr

g~tete ~ n~p~vry 12 hOrs the comfpol-tbeinQt~ted Ias~ap llied Ato the w~ound -For thi p~pGUQXn M2Ishy

mealt p m crop 1diwpe 4 ddrj1 Veins a WacLIndy h ant-h otosrrcie ny

~Fnt iii2which liheror6 wa dtdI1 horst ~ pib elte

f gone of the borders of t~he on aFtr 1-itinLh t hle4 g ret h other onet For moeJh tai _hi p in a 2MeaSLrable_ fore teoe hmred

t 1i anlYi S~ ohtps thUi ng ~ s sh2tt w d2itt ic aie

di erence betwepri Aher can Lrol grouip and tgtheSG-g roup (p0nO5 -n bewenr theQcntrol pU idLPtpi6Hl L(p C CI)i1

fr C an 22gn 12 2

I114

r a pt i caruase h0

22 EFFECTI OF TASP I NE-HC IbON fTHE MIGRATIOW OF HUMAN FORESKIN FIBROBLASTS

The miral4o of fbolssto th~e W7Owund a _O

andL th~emtod We1olowd wasteoe eirbd by1 LiUr kR

Iine s Wmd 1acingr razor blade ered lr~t kIl pl te nd thenlbli

the right tho ell ~he twas pt amp1ediordpe t T r

tOhe rghtofth liA~ne Pl ats wer thn washd wth PDS in n ordpr~~~~~~~~~~~tc)~~~--t ceII e b m it t e IaII e ninated id d c

a]Og h in -Iero ells werearked~ wit th onto

the nube of c Isln tha crosLed~th I in a h all re- W~r ePCounfted under~~a Phase a t t

OUL o cells ~ wao e l -spine i-iC on the-mill t of the

frbl aststi th cl e L s of the p1t Th elOffil was obser at very Iow 1ocncentrati ons o taspine-1I~ Ta lII hw h eut o f an exper i men ts iIa t n descriIbeoli thprevol4 Table but~ here tJhe i -c i Lill Vwil~

migrtiol the f~lbroblasts at concientrations between A2and C

23 EFF~ECT OF TASPINE7HCI~ ON THE CONTRACTION OF LATTICES MADE OF HUMAN FORESKIN FIBROBLASTS~AND COLLAGENlt~

Won ontraction - isa -aohr~i mot atpl [t

~ci atri zatibprocess that~ we hav StL~ded using i s model sy4stem theEclntractiotn of cel4-colagn lttice described b Y

Deli-and colabaos(rc N ciUA712417

- 4 us

1979

11121 ~

Co 1f ri r Mo Ii ibb ario astt

ll C175r er2iWpr eP l p on eibobt e

b~ate l0 caIgIa5 dise i OlIs o dot t 2iL fct ) hG

r 5) no a u qn~fln 4 Kfcst

coptr I tvi Hank hNM fetald t~oine QImlr~~ shownCC)5m h

jn~P ti[ampI tio Eag 1ti sbaV1t 5 H)

5rLIM IM -~ Iyan f tapnAI1 So h s ia 7n71 k p n i e b h o t

7bt7-IampI a~r were7 added in 0 m4I77f M 7 1 Cinc

44gt7 74 744 n di ed d Am Ed gt a 5h77 J n i- r L tt b dia ef i re

444th d4~ 5 were~ me su i ti 4 e diffn e

The7l s o w i f r n 4ril4i~ f4-rLnn

1)c n atiel sn at L4o n -ach7ag on 4 two

dif4 re4 con4 -n 4to a i -H 444 s i I 4lt4( 14 Fi~ue Iand in~4 Asv pc01 -

744- )p ca~ b w ht44~~c 4

j 4

4l s4e 7 4 of7~7~ Laspill4 I 7 77 i4e _t4 ~ 2

tht 7i lhd4gi~~ca to 744g

the74~ 6- 6 0n -4 7 of f44s L ge I jhiIr -) t ce a on ori d_ - s7i7r

4ke C)44~~ I4447 ed7 th rj at w ic 4

I7t 4e- 4t aL ted 4 s 4 a-e an 774-4-474 atcon 74r o

~~ - a t47e 7 7 44ui n 747444 ~Ath 4 e7f h 4o Latt ce5~ by~

p 7n L h7-l c ~ 4

64 4 4 - j X

T~PN~I1SUDIES TE EFFE9T ICGS ON UTGEIC O S jE D 6RAORAN O

q--PO1TR POETALTE OFhAGE EGAD ADTS

FrTI o po eje u ed t i TEt o Am es Afe tiof~ MLAat0 isb~ on Rsth 77

-c -111 l 74

spheciticaitsin being ruse t fro ahitiin r Griq irOlntba to1 p trophy anwde ait~) r~t L~mwpby1~ T ~d~C tainod ThisoacorL-11a r h~u~a bee aiae rtedt-_to of carcinoges aSMtagL~ by F JAL b

tst ed in) the conentona animafr atns infi mutati fbraness

72- 5135-5139197

iThe m thodrLn seha We U~e~i~s r T 0T Jt)IA T 7 45 oratoyI~~ ofV n N Amefse~ate-a tst

st-a s~~o-interesting tcompoindt Ot t tSe iht ti knovm hi~t 1 Mhnq~L ~ hE varC~o gi opn f or

r) 4 aed n -reiin~p Tid su I sItIt~amp~gues - t- ( Darr Pi d~ r a sa ~tc~apu tio othL

-m tibLthe sm ic h5tris h pro nS1 s hte C b w th

The mleod Le sona e~ ~ -To 2 fmle)S o gra

e~t~I ti f ants r edtovmpai th

m0(w en r I i er on oe

frct ~~

n)te

wr aPtddwidla ia sdi-Va

mi nd w

r er

- dI-iea

Gand s t l

d on~tapn i imshyTheb~4lta

M-~iI fias s5 t n(~W20ch

r~~U~~f~ na-d ia W tr - rqii-d - S

~~nb 00 -eetat pel~ com~clspar ao

Thcqpoid Taspo d5cgml4 an tape -apne7 Cl wa diovd nwtS ndSedb ilrto

~~~~~t h r enrns r wspoe- Am Snred o s yS er-i 1 n e

h 101111-

6 K

14 V MMq ~~ tpE

Vef s ) in ib trv Io i th t hih r-o r nra io 5 Po

obev ta gSr- f de frado a s3IRd

canentraton ofrw 11~ C h~ tetr 14I0 an ~ 2Vj~)For ~an ~f v i hube1 re etat of t5d~ nf -L~ ti

e ospeint toethehee~ Weo~o a CIWbpntnejsu~ bpred no t Lathe concentctionc5 testedidttgenic_

concw~entatiorcn5e of~o ~a de Gradmuand~fTaspif-k oLh hSagre I iTs~~s he wradk e ncr Miadotnoit anr e rre

~teste0r2an rnraid)~ 5 Uheer thoncetrat ritin 0 p o (o r jI r41 Ic w c op o p a~dz - g n

501n) inthti nume of~ Hrtl t ~test e 12rLk ld4WhiC t0CTapie te n

difieret frum5testieHC11si [OtE(per mental~n he1muW~EIS Moppanr)~aged wamwh eF rado~ we muag

Tap EEC OFan nF de OspnoEFEQE CHaRo22NEHC SISTERgr oMATI 4000ZEXHA n GE 006m m Thes cocet l atI1 ia 11 Ae -I Ipfw Ill44

conctratiron de excasingesH~aesed all~ i Ii wh~ uh44 1s41howno MtAgnnesiv F~f~tl i wcanbednduedtbha

5 olte f f ntfr Tbt h in g di ca Lim 1 steMLtgnccm-r )FSagr de taiV eII neths ta

~ ofXamp

4 irohe oms 1 ste ca 1ltV 11 c eseha we ~ exchnge eid~db

andtC is aOt ll l if brtht~l~ey 6 conswsidere d C))

chfiromosme- amage- C hae b~ye~ 41 i4se1ogt

Thece 17~4111n s 1 t a li fo these ex e ime t eeY 901144weu

_COand ~ oh e I 414werp414141e7ns embryos a f 4 ~ th

1I 111411 ~I1~~1~414 1 1 1411 1141ltj I t 4 4111lt1 11 14 4~ ~ 14y

4-~

21 Evauationriof Citotoxici ty6of Tasp1 ne7 CI on V79AP4

~att n asp i ne -~C Io ~ ~ ed95f II l 4e tpi-

t~l~~UAnter~ adcr tiwuo d j

ar~fCO~ta fieamp L t c~pellst~descrr andor he of frmthe

pnu~rI-U~df~atnt L-5an

groni D4heCDr mdfi~ agep edL 5Lh~e mntedny mcw o th

24 hours r~aoh The reUl ts showed that concertrations of 176~ 000B and C) 14 L Im ~ 6f Taspine HCl were toxica for th1e el Is hi1

4 concentratins~ of C)2641 nd 0000 Oqml f Taspipe6-HdC were in~ocUOU~S for the cellis

Sb tCUS~d e thSi MUt~t rmntoJf-heitegrit f eI

S-mjembrane-and th ciiyo ntraeua aae5 me od- b f ae1o tYaerate

m~hodorRotan ad P (P~rocgt )a1~cadSLP USA 55 13-4~ 1966) ran Edidin IdjrjtI 13

Thlis technique Makes use of two dyes

The fir~ on~e a -nn ~lrn on f trec q compound~ -f curc that-6 coud t rav1erse thee I I i dlct ea~i

membaneOce nsid th cl I ine peifica stear (s I i ber atLek

~oneecon~d apoar f I1-2sceentsi~-T is dye etidium bromid~e~wh ich uP-onlexci tati on(wi th UV ligt fluIorsered This dy

Ci Ulonly enter theoeI whe the~r me-rni damaged~~Th cnetain fFursc~n ctt and et Id i Utn bromide14~Lsedwere 2 Ugr1a~d 5~bilWheI-ee~10y

ml 4nUgmiilerctvely e Ca~nlltrbaea twiththdy~s nd6~eredUnder U- gt in~ a~fILkoresceLt mlsectp~ 1 0h cel a b4~ed~Lrigh green6

fluorescencejunhealth 1 oed iIi1htgrPer1 freF5C ncampe anfagd ciidedcells ed~edf~oe~

- This~method wa also- sed inri order- toevaluate the ~ci otWlti

With the- oes described abv- th is con c en~rat ions5o asp btween 1761 and 0 4 4e V not ito nic while

Ce shy

on~~~~t-nIf)6~ ~ ~ n 6 nlad lwr wren t

weret+ne I r nt4t e cibe e -einthep m IJ

41~ ~

ME 4i

E2val at an of C0oait o f a n-HCl and Sanigre d

Grado on CHO cell1s

jCE~lI Hre-mp e resi tant t ig her conentrton or

23 Evlainof teeffect of Taspine HC1I on Sister

pe Choai Exhng~e- dfe age

SLuppl 6e~ted wh 5 eal bovine seru and005m~gertami~cin arindApCLbated in hL~Ii i a aos p h e eo C

~reedeTaspihe HM1 to afinal concentrationW C264 ~ M~Yof j1 -i~nI-0088 ugmi respectively A thr sto pl tes di d notI recei ye an~y Taspinej-ICl Iand ~remai ned as -control~ Simultaneou~sly with the tr tmnt die tweeamp sets~(oPaL

-~received~5Doodoirdn to ilicnetaino ltgM1 O nedi P~a t es hbrethen incubatedmt I

~~additional 22 hou~rs in a-hUnidifiedjaoshr of5 5 at -35r~a~7C (abdo2tt repli CatiVe-rQuL~WO nd

0~t the enid of the 22~ hours of InicLu3ajampion C61 mid~ o~a5 added ~toamptbe pl-ates (3final i-Y6f~ U andto ocneE- I- (pn I J

3 pc nLatdI~er f or and addit ion I twodhour s ftri at ubat i n i hao 31i n -fe e a~icai

and stain~ed wi~th tiplFllu iocromejP1lUZGe techni qie c)f per ry and Wol f f Nature 56-18 17il) and La tt (Pr 0c NAtI cad Sc i US7 3 3919l 7lt -K

7~The resul ts areshown in -Table 14 Wat we observei s that 2YTaspile~HCL-i at~ non~ citotoxic concentr~ations~ increases thea

~fre~ue~ncy of- sister c armat id exchageson V79O4cls

Fr thIest i th CHOD cel Is that4 ware~ growin expone ti al wer e seeded on 60 mmi tissue C1ture pt~ji dishe (3 1 clsA

4-bova ne serum nd 0m m o etm and - nCbted in4i a hUitmsid ee o COzand 5 ai t_7C wny or

~hours I ater-ns tofIa toe srlte a~4de s e-1 CtLo final c~ on- 6ntrat 9 a 6tg )1 a nIot he 4r se L olaes receivedSangrea de rad6o~at n1Zoi 3 to of 0 00 at rdin

set- f tde r CracoG i nd nad-a he-eAi cio1S~n1a

treamen th the set of ~r1a~espl recaivd

-MredIIuM3 Pla1tes whr thniChtdf or and add~i t i nLI21

- ~ 1~ 4 -d4

4 U ~~W10ri1I1

(abVQCW rp ribte vea qiar s Iofe

pl4ateE~ d U Aue c fr iionWerL ndad

4 staied ihth lyrcrm nc fnSC techof - I AniqL

gtapine1Cand did -a a L1amage SO7 337-31 177-

The~~~~~~~~~~ na reu~ 5adwa b ev tar h

PofiteTIALomtiES OF ha RAOAnDgtAP as inSGR~D the r-ntar

4L~~oth sinfcnlwpe sed the ~~ 4 4sr1n

eol~o-at rsIlp-hFrmthe nexperimesdescryibe ad e e cocV ta Taspin-HClt anFsc6 Sanred WeeksddntP-dC hO~on

cr i no~a Sytemhand the t0dw uedoensi wee fMI owe i i Iat

aLs CDhthl-Toheon dac~n~eribed bypurd usld 71

j~nO~s1 a I a TClaoa

Kl-Ten krianto A SN - eesCE use 1 s - 1 a d N t OILSIi~sce op~d

Thbmaledeany1phrb M~c etate moths~ ofri the~wre shcet1aed i re~js ig phas hair~ 6c usd Mceery honedUIof-th wereeap

p ppiand pfahe inweteator de adI~Di i triTheafer cahe nod ltamp~4were 30Pan(riasp e Leachr dxermnag~~(roup6o

compoud~ uonted as-anp-ltracexerininM tgtA ih bfrue t soIvPd

carcinogenicou~~W-nd 7n-12 ewt~ole- 0(-)shy-~~~h

x and i

n~~ (l I s

I Thegr o n tume~or roe r compound ~used wa 7 ra decGoylpup rbltreate~s- (TA and ocn a-o -tdNo

wasOLI 2~Ug -in ofL actn ctone- gt i- e

G-C resnt I No

Group---------------------------------------------------------------------------

GrOLIP~~~ 10nTo ~ 3 i 1 l-c~~ fI-D

p 5r I10 n D Ajn 1s c e~ e

42 twi cf wekl e ef

t v4 CL we e4

Io4M C)A~~cng C Or oup 0I~ of rTrAn oar3 MOi Q~~~~IaeoneHrF

GrUP1 Wau ~Our cotro o efomt ion of sp n r-fpr~mQ SGroup 2 i4as Ou coto fo asacrcngn cDrMnq

r-0 o WiuRm mno r~a n iLWwht~U~ ldshwasiry zec p~b 7snbu ltzgtwo~t our twi~o-tg raiognen r6~wtd ~

tr~satedDrlI3Awilhai ard TP~ op -as ~wo$king~5nue ot

ourNoy rou Sndangre de Gnoranai Group VWithr ~~ts r 4 t -1C - acng

r n~dLndPh~fP~P e as 11

atou 7hend b for acarcnoge

h or Tasp n

Tho e )02 prtfrpd c i 2nd aneioMet er a on~ mbe e 190 vr

trpeetned _~ s nTAa n trwihDB dcr a m deloe

the pio~mast rekmonhs afther thji niLia n fthe treimn (2a oted of 294ba5ieay 1 rso rlice trearte wi thur-~d oleir o

of ~~~ ~ ~ ms~ ~ tju ~ ~ ~ i~ pe-~t ~ deeipdppnssa~d itA

no es w r oA4tp --4 d an

0

7t

J

22

-Grossge~i ti nowere sever a Iex e -p

GVL hcw a amintiom ma 94jf rom4~

by- hyep theao derm ppij aei wich er e ted Fo theI nthemi sufce he Ml gisa~ dc or inveIS aymtr~

igt beae4ikr aa~hs L wit r-ccnLgt (te hyekrami

a t om o f c n a s io y t T e r mj i- b i I

mainn chngs Th Kn4--oran did pr4n pah~oi

FFSiieii a e ca c n gei or tino promoters-

f ia e n in ocotty tfe eX p rim6 D it IFi aIhc I-54 SiAL-4 mo th in or r toi4i be i co c u F i ce I- mn ii iF

i ~~ t4Ie~ ~ os4iL-ig ~ ~ hL ~ ~iie ~ ~ v 4~ ~ ~ g Oilt494hrta ~h ra ~ wi~j theknow ogn sA~rtd t 3 doelo tv tmrcari DB

on mo th ag

4 4 ~

4 ONai

4CONCLUSIONS

rSnr de Grado etracted f romp ICiton 7er

o extracts fr-oT1 th~ecore~ fri in ofth Crotor lechleri tree

Thr4ure idtein Or trc EITOit in the ieavesu of thebAci~t n I 1eriand the most bkidant one was ~

T~asp inewas h cti O 1teri za tp i ncn 1 L-p e s en1 i Sa~ngre4 de Gracdo etracted from Croto hl1ech14er

~h Lid healin process~ r I

1~~~fibrobasts atonceritrtions1 be1llow2)- nOgm1 ard i t di d e Ih1 PEr at ion~not uincrease ratE1Qf-11P

w fon nt~h ibit the contac i on61

hnn f orEsk i ni i a fibr 0b I as

6~mutagunicitY 1tud using the 4rnes 4tcst showe htSJrgshydeGad a wh le taspine chloydrate hadttrcco k

~~ ~no Jn~ta ernii activity I1

StUdies on e ef f eect4 - Ire derao andAsnt A-t oBancr adpCdnd

ychorhdrteortisister choai eklnp (SCE) showe t I4bsTaspine hlrydrate increased the nIumber of SC[ 3in

V79if4 ellbt no increase wa5 abservedinCOcls 10Uigthe tW-tg O skincriogee syste w e V

4 14he shown that~4nri Uher Sng a ir tppeIJ1 were carci nb~geniviardor -tumor promtr f te n0ya +iitrea t4ment 1

--- --- --- --- --- --- ---

0 11

FXTROTrONOOS9NJRF DE GRA1DO FRMDFgRE T RE P1)0tITUITSC DING -m RSTTRP

TREETPg~ OF] PH ~VOLiUMEK OF~ YILIL - XT I ON E--~ Ig11TV~ XTROCTION

6-- 91 -~agm ~ mJ-1 -- ~ 22 272

M- 5ql ar-To each of the sample wre addced tJ n - -qri

~~~~e alktalinized to p 9 wth amn~iui hydjroXidftt~eJ~h and extatede0hautv- wie~t~h choroform Each clooorm extractwa concentrated i n a rotavapor then dryed and -the~~shy

ht 0os theE 6k gaIkaIoidvr eaU d i napl- alI7 24 -065j-~9-- ~

C) Iml ofec f Lhe 5ape wer Alded Ijlo ae th as t e aI kalinized-to pH 92wtha

etract iasdred and hei redissolved i 1 mo-o lLlou and0 1e bobnamp t347lt was merLrdL~igaZi

Spe ro fotome er-jThextinction coefficient of taspine at 47 i

a si gni i can t di FF$ernce2 between thee gbFUPS anthegOUS-n

The col lact n datE wJas Dec mber~ 19 1901

-- ---Y -- ~4A shy- ~----

I A

4 AB 2 ~4

q 7

EXRCIN F AGE1 1ACFOM -ER RL RMIU

~4 1 4-112 lt 4 44 4

44 42 I-4lt C) 6

- ~~ ~ ~ ~ ~ ~~44-- -- -- ----4 -shy~~ ~~~4441533c~ de Grado was co lced fo t e te s nm rae hc

on ebr 1 15and 5 a d n M y 2 90 h I a m- dary 19

4tet4wa d -4 e44 de 11o etha To~4lt4I ml 4~44 each 4 44444 were ed of wa eof o ~ th sV ad I4~ l th

mixturwa al t 4lt4 yrxd -iUPH~ 9 IVizp and 4l~444 wi- 4 o c rfextr c~e l a~~iv 4 h 4aof 4ofri E4a4 h

r-~ lt4~4 444 ~ 44 ~44 4) gt44

Xh e~= 2 8c tr-ts 0 da gr7380frI~ aawas 1+C

wasra sign~ficat diiffrec betwee the to ~grup Plt 0

i 44 ~ f~cho ~ h ui 1 4 l V~n11~9 7 rW hnaIdiin 4 hdoidlt465 W~qamp

T

22

I~~_7

control (rD 11

SG 51 1 1 1 8

Tabpie tm11C () r 71 0 1

Obrevi itions WS = WUndbeakinsteghSIG =Sargre de Grado 2

STaspine-HICI = Taspirn chlrhydrate

Nce wepre anesthesized with vapors fdiethyl ete and a~ IcM A ci ion as made perpendi CIler _to the ais fsmfnmet-y f tea i anid thtwo4borders of the Wo~nd werestichdtgte tiscntr

g~tete ~ n~p~vry 12 hOrs the comfpol-tbeinQt~ted Ias~ap llied Ato the w~ound -For thi p~pGUQXn M2Ishy

mealt p m crop 1diwpe 4 ddrj1 Veins a WacLIndy h ant-h otosrrcie ny

~Fnt iii2which liheror6 wa dtdI1 horst ~ pib elte

f gone of the borders of t~he on aFtr 1-itinLh t hle4 g ret h other onet For moeJh tai _hi p in a 2MeaSLrable_ fore teoe hmred

t 1i anlYi S~ ohtps thUi ng ~ s sh2tt w d2itt ic aie

di erence betwepri Aher can Lrol grouip and tgtheSG-g roup (p0nO5 -n bewenr theQcntrol pU idLPtpi6Hl L(p C CI)i1

fr C an 22gn 12 2

11121 ~

Co 1f ri r Mo Ii ibb ario astt

ll C175r er2iWpr eP l p on eibobt e

b~ate l0 caIgIa5 dise i OlIs o dot t 2iL fct ) hG

r 5) no a u qn~fln 4 Kfcst

coptr I tvi Hank hNM fetald t~oine QImlr~~ shownCC)5m h

jn~P ti[ampI tio Eag 1ti sbaV1t 5 H)

5rLIM IM -~ Iyan f tapnAI1 So h s ia 7n71 k p n i e b h o t

7bt7-IampI a~r were7 added in 0 m4I77f M 7 1 Cinc

44gt7 74 744 n di ed d Am Ed gt a 5h77 J n i- r L tt b dia ef i re

444th d4~ 5 were~ me su i ti 4 e diffn e

The7l s o w i f r n 4ril4i~ f4-rLnn

1)c n atiel sn at L4o n -ach7ag on 4 two

dif4 re4 con4 -n 4to a i -H 444 s i I 4lt4( 14 Fi~ue Iand in~4 Asv pc01 -

744- )p ca~ b w ht44~~c 4

j 4

4l s4e 7 4 of7~7~ Laspill4 I 7 77 i4e _t4 ~ 2

tht 7i lhd4gi~~ca to 744g

the74~ 6- 6 0n -4 7 of f44s L ge I jhiIr -) t ce a on ori d_ - s7i7r

4ke C)44~~ I4447 ed7 th rj at w ic 4

I7t 4e- 4t aL ted 4 s 4 a-e an 774-4-474 atcon 74r o

~~ - a t47e 7 7 44ui n 747444 ~Ath 4 e7f h 4o Latt ce5~ by~

p 7n L h7-l c ~ 4

64 4 4 - j X

T~PN~I1SUDIES TE EFFE9T ICGS ON UTGEIC O S jE D 6RAORAN O

q--PO1TR POETALTE OFhAGE EGAD ADTS

FrTI o po eje u ed t i TEt o Am es Afe tiof~ MLAat0 isb~ on Rsth 77

-c -111 l 74

spheciticaitsin being ruse t fro ahitiin r Griq irOlntba to1 p trophy anwde ait~) r~t L~mwpby1~ T ~d~C tainod ThisoacorL-11a r h~u~a bee aiae rtedt-_to of carcinoges aSMtagL~ by F JAL b

tst ed in) the conentona animafr atns infi mutati fbraness

72- 5135-5139197

iThe m thodrLn seha We U~e~i~s r T 0T Jt)IA T 7 45 oratoyI~~ ofV n N Amefse~ate-a tst

st-a s~~o-interesting tcompoindt Ot t tSe iht ti knovm hi~t 1 Mhnq~L ~ hE varC~o gi opn f or

r) 4 aed n -reiin~p Tid su I sItIt~amp~gues - t- ( Darr Pi d~ r a sa ~tc~apu tio othL

-m tibLthe sm ic h5tris h pro nS1 s hte C b w th

The mleod Le sona e~ ~ -To 2 fmle)S o gra

e~t~I ti f ants r edtovmpai th

m0(w en r I i er on oe

frct ~~

n)te

wr aPtddwidla ia sdi-Va

mi nd w

r er

- dI-iea

Gand s t l

d on~tapn i imshyTheb~4lta

M-~iI fias s5 t n(~W20ch

r~~U~~f~ na-d ia W tr - rqii-d - S

~~nb 00 -eetat pel~ com~clspar ao

Thcqpoid Taspo d5cgml4 an tape -apne7 Cl wa diovd nwtS ndSedb ilrto

~~~~~t h r enrns r wspoe- Am Snred o s yS er-i 1 n e

h 101111-

6 K

14 V MMq ~~ tpE

Vef s ) in ib trv Io i th t hih r-o r nra io 5 Po

obev ta gSr- f de frado a s3IRd

canentraton ofrw 11~ C h~ tetr 14I0 an ~ 2Vj~)For ~an ~f v i hube1 re etat of t5d~ nf -L~ ti

e ospeint toethehee~ Weo~o a CIWbpntnejsu~ bpred no t Lathe concentctionc5 testedidttgenic_

concw~entatiorcn5e of~o ~a de Gradmuand~fTaspif-k oLh hSagre I iTs~~s he wradk e ncr Miadotnoit anr e rre

~teste0r2an rnraid)~ 5 Uheer thoncetrat ritin 0 p o (o r jI r41 Ic w c op o p a~dz - g n

501n) inthti nume of~ Hrtl t ~test e 12rLk ld4WhiC t0CTapie te n

difieret frum5testieHC11si [OtE(per mental~n he1muW~EIS Moppanr)~aged wamwh eF rado~ we muag

Tap EEC OFan nF de OspnoEFEQE CHaRo22NEHC SISTERgr oMATI 4000ZEXHA n GE 006m m Thes cocet l atI1 ia 11 Ae -I Ipfw Ill44

conctratiron de excasingesH~aesed all~ i Ii wh~ uh44 1s41howno MtAgnnesiv F~f~tl i wcanbednduedtbha

5 olte f f ntfr Tbt h in g di ca Lim 1 steMLtgnccm-r )FSagr de taiV eII neths ta

~ ofXamp

4 irohe oms 1 ste ca 1ltV 11 c eseha we ~ exchnge eid~db

andtC is aOt ll l if brtht~l~ey 6 conswsidere d C))

chfiromosme- amage- C hae b~ye~ 41 i4se1ogt

Thece 17~4111n s 1 t a li fo these ex e ime t eeY 901144weu

_COand ~ oh e I 414werp414141e7ns embryos a f 4 ~ th

1I 111411 ~I1~~1~414 1 1 1411 1141ltj I t 4 4111lt1 11 14 4~ ~ 14y

4-~

21 Evauationriof Citotoxici ty6of Tasp1 ne7 CI on V79AP4

~att n asp i ne -~C Io ~ ~ ed95f II l 4e tpi-

t~l~~UAnter~ adcr tiwuo d j

ar~fCO~ta fieamp L t c~pellst~descrr andor he of frmthe

pnu~rI-U~df~atnt L-5an

groni D4heCDr mdfi~ agep edL 5Lh~e mntedny mcw o th

24 hours r~aoh The reUl ts showed that concertrations of 176~ 000B and C) 14 L Im ~ 6f Taspine HCl were toxica for th1e el Is hi1

4 concentratins~ of C)2641 nd 0000 Oqml f Taspipe6-HdC were in~ocUOU~S for the cellis

Sb tCUS~d e thSi MUt~t rmntoJf-heitegrit f eI

S-mjembrane-and th ciiyo ntraeua aae5 me od- b f ae1o tYaerate

m~hodorRotan ad P (P~rocgt )a1~cadSLP USA 55 13-4~ 1966) ran Edidin IdjrjtI 13

Thlis technique Makes use of two dyes

The fir~ on~e a -nn ~lrn on f trec q compound~ -f curc that-6 coud t rav1erse thee I I i dlct ea~i

membaneOce nsid th cl I ine peifica stear (s I i ber atLek

~oneecon~d apoar f I1-2sceentsi~-T is dye etidium bromid~e~wh ich uP-onlexci tati on(wi th UV ligt fluIorsered This dy

Ci Ulonly enter theoeI whe the~r me-rni damaged~~Th cnetain fFursc~n ctt and et Id i Utn bromide14~Lsedwere 2 Ugr1a~d 5~bilWheI-ee~10y

ml 4nUgmiilerctvely e Ca~nlltrbaea twiththdy~s nd6~eredUnder U- gt in~ a~fILkoresceLt mlsectp~ 1 0h cel a b4~ed~Lrigh green6

fluorescencejunhealth 1 oed iIi1htgrPer1 freF5C ncampe anfagd ciidedcells ed~edf~oe~

- This~method wa also- sed inri order- toevaluate the ~ci otWlti

With the- oes described abv- th is con c en~rat ions5o asp btween 1761 and 0 4 4e V not ito nic while

Ce shy

on~~~~t-nIf)6~ ~ ~ n 6 nlad lwr wren t

weret+ne I r nt4t e cibe e -einthep m IJ

41~ ~

ME 4i

E2val at an of C0oait o f a n-HCl and Sanigre d

Grado on CHO cell1s

jCE~lI Hre-mp e resi tant t ig her conentrton or

23 Evlainof teeffect of Taspine HC1I on Sister

pe Choai Exhng~e- dfe age

SLuppl 6e~ted wh 5 eal bovine seru and005m~gertami~cin arindApCLbated in hL~Ii i a aos p h e eo C

~reedeTaspihe HM1 to afinal concentrationW C264 ~ M~Yof j1 -i~nI-0088 ugmi respectively A thr sto pl tes di d notI recei ye an~y Taspinej-ICl Iand ~remai ned as -control~ Simultaneou~sly with the tr tmnt die tweeamp sets~(oPaL

-~received~5Doodoirdn to ilicnetaino ltgM1 O nedi P~a t es hbrethen incubatedmt I

~~additional 22 hou~rs in a-hUnidifiedjaoshr of5 5 at -35r~a~7C (abdo2tt repli CatiVe-rQuL~WO nd

0~t the enid of the 22~ hours of InicLu3ajampion C61 mid~ o~a5 added ~toamptbe pl-ates (3final i-Y6f~ U andto ocneE- I- (pn I J

3 pc nLatdI~er f or and addit ion I twodhour s ftri at ubat i n i hao 31i n -fe e a~icai

and stain~ed wi~th tiplFllu iocromejP1lUZGe techni qie c)f per ry and Wol f f Nature 56-18 17il) and La tt (Pr 0c NAtI cad Sc i US7 3 3919l 7lt -K

7~The resul ts areshown in -Table 14 Wat we observei s that 2YTaspile~HCL-i at~ non~ citotoxic concentr~ations~ increases thea

~fre~ue~ncy of- sister c armat id exchageson V79O4cls

Fr thIest i th CHOD cel Is that4 ware~ growin expone ti al wer e seeded on 60 mmi tissue C1ture pt~ji dishe (3 1 clsA

4-bova ne serum nd 0m m o etm and - nCbted in4i a hUitmsid ee o COzand 5 ai t_7C wny or

~hours I ater-ns tofIa toe srlte a~4de s e-1 CtLo final c~ on- 6ntrat 9 a 6tg )1 a nIot he 4r se L olaes receivedSangrea de rad6o~at n1Zoi 3 to of 0 00 at rdin

set- f tde r CracoG i nd nad-a he-eAi cio1S~n1a

treamen th the set of ~r1a~espl recaivd

-MredIIuM3 Pla1tes whr thniChtdf or and add~i t i nLI21

- ~ 1~ 4 -d4

4 U ~~W10ri1I1

(abVQCW rp ribte vea qiar s Iofe

pl4ateE~ d U Aue c fr iionWerL ndad

4 staied ihth lyrcrm nc fnSC techof - I AniqL

gtapine1Cand did -a a L1amage SO7 337-31 177-

The~~~~~~~~~~ na reu~ 5adwa b ev tar h

PofiteTIALomtiES OF ha RAOAnDgtAP as inSGR~D the r-ntar

4L~~oth sinfcnlwpe sed the ~~ 4 4sr1n

eol~o-at rsIlp-hFrmthe nexperimesdescryibe ad e e cocV ta Taspin-HClt anFsc6 Sanred WeeksddntP-dC hO~on

cr i no~a Sytemhand the t0dw uedoensi wee fMI owe i i Iat

aLs CDhthl-Toheon dac~n~eribed bypurd usld 71

j~nO~s1 a I a TClaoa

Kl-Ten krianto A SN - eesCE use 1 s - 1 a d N t OILSIi~sce op~d

Thbmaledeany1phrb M~c etate moths~ ofri the~wre shcet1aed i re~js ig phas hair~ 6c usd Mceery honedUIof-th wereeap

p ppiand pfahe inweteator de adI~Di i triTheafer cahe nod ltamp~4were 30Pan(riasp e Leachr dxermnag~~(roup6o

compoud~ uonted as-anp-ltracexerininM tgtA ih bfrue t soIvPd

carcinogenicou~~W-nd 7n-12 ewt~ole- 0(-)shy-~~~h

x and i

n~~ (l I s

I Thegr o n tume~or roe r compound ~used wa 7 ra decGoylpup rbltreate~s- (TA and ocn a-o -tdNo

wasOLI 2~Ug -in ofL actn ctone- gt i- e

G-C resnt I No

Group---------------------------------------------------------------------------

GrOLIP~~~ 10nTo ~ 3 i 1 l-c~~ fI-D

p 5r I10 n D Ajn 1s c e~ e

42 twi cf wekl e ef

t v4 CL we e4

Io4M C)A~~cng C Or oup 0I~ of rTrAn oar3 MOi Q~~~~IaeoneHrF

GrUP1 Wau ~Our cotro o efomt ion of sp n r-fpr~mQ SGroup 2 i4as Ou coto fo asacrcngn cDrMnq

r-0 o WiuRm mno r~a n iLWwht~U~ ldshwasiry zec p~b 7snbu ltzgtwo~t our twi~o-tg raiognen r6~wtd ~

tr~satedDrlI3Awilhai ard TP~ op -as ~wo$king~5nue ot

ourNoy rou Sndangre de Gnoranai Group VWithr ~~ts r 4 t -1C - acng

r n~dLndPh~fP~P e as 11

atou 7hend b for acarcnoge

h or Tasp n

Tho e )02 prtfrpd c i 2nd aneioMet er a on~ mbe e 190 vr

trpeetned _~ s nTAa n trwihDB dcr a m deloe

the pio~mast rekmonhs afther thji niLia n fthe treimn (2a oted of 294ba5ieay 1 rso rlice trearte wi thur-~d oleir o

of ~~~ ~ ~ ms~ ~ tju ~ ~ ~ i~ pe-~t ~ deeipdppnssa~d itA

no es w r oA4tp --4 d an

0

7t

J

22

-Grossge~i ti nowere sever a Iex e -p

GVL hcw a amintiom ma 94jf rom4~

by- hyep theao derm ppij aei wich er e ted Fo theI nthemi sufce he Ml gisa~ dc or inveIS aymtr~

igt beae4ikr aa~hs L wit r-ccnLgt (te hyekrami

a t om o f c n a s io y t T e r mj i- b i I

mainn chngs Th Kn4--oran did pr4n pah~oi

FFSiieii a e ca c n gei or tino promoters-

f ia e n in ocotty tfe eX p rim6 D it IFi aIhc I-54 SiAL-4 mo th in or r toi4i be i co c u F i ce I- mn ii iF

i ~~ t4Ie~ ~ os4iL-ig ~ ~ hL ~ ~iie ~ ~ v 4~ ~ ~ g Oilt494hrta ~h ra ~ wi~j theknow ogn sA~rtd t 3 doelo tv tmrcari DB

on mo th ag

4 4 ~

4 ONai

4CONCLUSIONS

rSnr de Grado etracted f romp ICiton 7er

o extracts fr-oT1 th~ecore~ fri in ofth Crotor lechleri tree

Thr4ure idtein Or trc EITOit in the ieavesu of thebAci~t n I 1eriand the most bkidant one was ~

T~asp inewas h cti O 1teri za tp i ncn 1 L-p e s en1 i Sa~ngre4 de Gracdo etracted from Croto hl1ech14er

~h Lid healin process~ r I

1~~~fibrobasts atonceritrtions1 be1llow2)- nOgm1 ard i t di d e Ih1 PEr at ion~not uincrease ratE1Qf-11P

w fon nt~h ibit the contac i on61

hnn f orEsk i ni i a fibr 0b I as

6~mutagunicitY 1tud using the 4rnes 4tcst showe htSJrgshydeGad a wh le taspine chloydrate hadttrcco k

~~ ~no Jn~ta ernii activity I1

StUdies on e ef f eect4 - Ire derao andAsnt A-t oBancr adpCdnd

ychorhdrteortisister choai eklnp (SCE) showe t I4bsTaspine hlrydrate increased the nIumber of SC[ 3in

V79if4 ellbt no increase wa5 abservedinCOcls 10Uigthe tW-tg O skincriogee syste w e V

4 14he shown that~4nri Uher Sng a ir tppeIJ1 were carci nb~geniviardor -tumor promtr f te n0ya +iitrea t4ment 1

--- --- --- --- --- --- ---

0 11

FXTROTrONOOS9NJRF DE GRA1DO FRMDFgRE T RE P1)0tITUITSC DING -m RSTTRP

TREETPg~ OF] PH ~VOLiUMEK OF~ YILIL - XT I ON E--~ Ig11TV~ XTROCTION

6-- 91 -~agm ~ mJ-1 -- ~ 22 272

M- 5ql ar-To each of the sample wre addced tJ n - -qri

~~~~e alktalinized to p 9 wth amn~iui hydjroXidftt~eJ~h and extatede0hautv- wie~t~h choroform Each clooorm extractwa concentrated i n a rotavapor then dryed and -the~~shy

ht 0os theE 6k gaIkaIoidvr eaU d i napl- alI7 24 -065j-~9-- ~

C) Iml ofec f Lhe 5ape wer Alded Ijlo ae th as t e aI kalinized-to pH 92wtha

etract iasdred and hei redissolved i 1 mo-o lLlou and0 1e bobnamp t347lt was merLrdL~igaZi

Spe ro fotome er-jThextinction coefficient of taspine at 47 i

a si gni i can t di FF$ernce2 between thee gbFUPS anthegOUS-n

The col lact n datE wJas Dec mber~ 19 1901

-- ---Y -- ~4A shy- ~----

I A

4 AB 2 ~4

q 7

EXRCIN F AGE1 1ACFOM -ER RL RMIU

~4 1 4-112 lt 4 44 4

44 42 I-4lt C) 6

- ~~ ~ ~ ~ ~ ~~44-- -- -- ----4 -shy~~ ~~~4441533c~ de Grado was co lced fo t e te s nm rae hc

on ebr 1 15and 5 a d n M y 2 90 h I a m- dary 19

4tet4wa d -4 e44 de 11o etha To~4lt4I ml 4~44 each 4 44444 were ed of wa eof o ~ th sV ad I4~ l th

mixturwa al t 4lt4 yrxd -iUPH~ 9 IVizp and 4l~444 wi- 4 o c rfextr c~e l a~~iv 4 h 4aof 4ofri E4a4 h

r-~ lt4~4 444 ~ 44 ~44 4) gt44

Xh e~= 2 8c tr-ts 0 da gr7380frI~ aawas 1+C

wasra sign~ficat diiffrec betwee the to ~grup Plt 0

i 44 ~ f~cho ~ h ui 1 4 l V~n11~9 7 rW hnaIdiin 4 hdoidlt465 W~qamp

T

22

I~~_7

control (rD 11

SG 51 1 1 1 8

Tabpie tm11C () r 71 0 1

Obrevi itions WS = WUndbeakinsteghSIG =Sargre de Grado 2

STaspine-HICI = Taspirn chlrhydrate

Nce wepre anesthesized with vapors fdiethyl ete and a~ IcM A ci ion as made perpendi CIler _to the ais fsmfnmet-y f tea i anid thtwo4borders of the Wo~nd werestichdtgte tiscntr

g~tete ~ n~p~vry 12 hOrs the comfpol-tbeinQt~ted Ias~ap llied Ato the w~ound -For thi p~pGUQXn M2Ishy

mealt p m crop 1diwpe 4 ddrj1 Veins a WacLIndy h ant-h otosrrcie ny

~Fnt iii2which liheror6 wa dtdI1 horst ~ pib elte

f gone of the borders of t~he on aFtr 1-itinLh t hle4 g ret h other onet For moeJh tai _hi p in a 2MeaSLrable_ fore teoe hmred

t 1i anlYi S~ ohtps thUi ng ~ s sh2tt w d2itt ic aie

di erence betwepri Aher can Lrol grouip and tgtheSG-g roup (p0nO5 -n bewenr theQcntrol pU idLPtpi6Hl L(p C CI)i1

fr C an 22gn 12 2

T~PN~I1SUDIES TE EFFE9T ICGS ON UTGEIC O S jE D 6RAORAN O

q--PO1TR POETALTE OFhAGE EGAD ADTS

FrTI o po eje u ed t i TEt o Am es Afe tiof~ MLAat0 isb~ on Rsth 77

-c -111 l 74

spheciticaitsin being ruse t fro ahitiin r Griq irOlntba to1 p trophy anwde ait~) r~t L~mwpby1~ T ~d~C tainod ThisoacorL-11a r h~u~a bee aiae rtedt-_to of carcinoges aSMtagL~ by F JAL b

tst ed in) the conentona animafr atns infi mutati fbraness

72- 5135-5139197

iThe m thodrLn seha We U~e~i~s r T 0T Jt)IA T 7 45 oratoyI~~ ofV n N Amefse~ate-a tst

st-a s~~o-interesting tcompoindt Ot t tSe iht ti knovm hi~t 1 Mhnq~L ~ hE varC~o gi opn f or

r) 4 aed n -reiin~p Tid su I sItIt~amp~gues - t- ( Darr Pi d~ r a sa ~tc~apu tio othL

-m tibLthe sm ic h5tris h pro nS1 s hte C b w th

The mleod Le sona e~ ~ -To 2 fmle)S o gra

e~t~I ti f ants r edtovmpai th

m0(w en r I i er on oe

frct ~~

n)te

wr aPtddwidla ia sdi-Va

mi nd w

r er

- dI-iea

Gand s t l

d on~tapn i imshyTheb~4lta

M-~iI fias s5 t n(~W20ch

r~~U~~f~ na-d ia W tr - rqii-d - S

~~nb 00 -eetat pel~ com~clspar ao

Thcqpoid Taspo d5cgml4 an tape -apne7 Cl wa diovd nwtS ndSedb ilrto

~~~~~t h r enrns r wspoe- Am Snred o s yS er-i 1 n e

h 101111-

6 K

14 V MMq ~~ tpE

Vef s ) in ib trv Io i th t hih r-o r nra io 5 Po

obev ta gSr- f de frado a s3IRd

canentraton ofrw 11~ C h~ tetr 14I0 an ~ 2Vj~)For ~an ~f v i hube1 re etat of t5d~ nf -L~ ti

e ospeint toethehee~ Weo~o a CIWbpntnejsu~ bpred no t Lathe concentctionc5 testedidttgenic_

concw~entatiorcn5e of~o ~a de Gradmuand~fTaspif-k oLh hSagre I iTs~~s he wradk e ncr Miadotnoit anr e rre

~teste0r2an rnraid)~ 5 Uheer thoncetrat ritin 0 p o (o r jI r41 Ic w c op o p a~dz - g n

501n) inthti nume of~ Hrtl t ~test e 12rLk ld4WhiC t0CTapie te n

difieret frum5testieHC11si [OtE(per mental~n he1muW~EIS Moppanr)~aged wamwh eF rado~ we muag

Tap EEC OFan nF de OspnoEFEQE CHaRo22NEHC SISTERgr oMATI 4000ZEXHA n GE 006m m Thes cocet l atI1 ia 11 Ae -I Ipfw Ill44

conctratiron de excasingesH~aesed all~ i Ii wh~ uh44 1s41howno MtAgnnesiv F~f~tl i wcanbednduedtbha

5 olte f f ntfr Tbt h in g di ca Lim 1 steMLtgnccm-r )FSagr de taiV eII neths ta

~ ofXamp

4 irohe oms 1 ste ca 1ltV 11 c eseha we ~ exchnge eid~db

andtC is aOt ll l if brtht~l~ey 6 conswsidere d C))

chfiromosme- amage- C hae b~ye~ 41 i4se1ogt

Thece 17~4111n s 1 t a li fo these ex e ime t eeY 901144weu

_COand ~ oh e I 414werp414141e7ns embryos a f 4 ~ th

1I 111411 ~I1~~1~414 1 1 1411 1141ltj I t 4 4111lt1 11 14 4~ ~ 14y

4-~

21 Evauationriof Citotoxici ty6of Tasp1 ne7 CI on V79AP4

~att n asp i ne -~C Io ~ ~ ed95f II l 4e tpi-

t~l~~UAnter~ adcr tiwuo d j

ar~fCO~ta fieamp L t c~pellst~descrr andor he of frmthe

pnu~rI-U~df~atnt L-5an

groni D4heCDr mdfi~ agep edL 5Lh~e mntedny mcw o th

24 hours r~aoh The reUl ts showed that concertrations of 176~ 000B and C) 14 L Im ~ 6f Taspine HCl were toxica for th1e el Is hi1

4 concentratins~ of C)2641 nd 0000 Oqml f Taspipe6-HdC were in~ocUOU~S for the cellis

Sb tCUS~d e thSi MUt~t rmntoJf-heitegrit f eI

S-mjembrane-and th ciiyo ntraeua aae5 me od- b f ae1o tYaerate

m~hodorRotan ad P (P~rocgt )a1~cadSLP USA 55 13-4~ 1966) ran Edidin IdjrjtI 13

Thlis technique Makes use of two dyes

The fir~ on~e a -nn ~lrn on f trec q compound~ -f curc that-6 coud t rav1erse thee I I i dlct ea~i

membaneOce nsid th cl I ine peifica stear (s I i ber atLek

~oneecon~d apoar f I1-2sceentsi~-T is dye etidium bromid~e~wh ich uP-onlexci tati on(wi th UV ligt fluIorsered This dy

Ci Ulonly enter theoeI whe the~r me-rni damaged~~Th cnetain fFursc~n ctt and et Id i Utn bromide14~Lsedwere 2 Ugr1a~d 5~bilWheI-ee~10y

ml 4nUgmiilerctvely e Ca~nlltrbaea twiththdy~s nd6~eredUnder U- gt in~ a~fILkoresceLt mlsectp~ 1 0h cel a b4~ed~Lrigh green6

fluorescencejunhealth 1 oed iIi1htgrPer1 freF5C ncampe anfagd ciidedcells ed~edf~oe~

- This~method wa also- sed inri order- toevaluate the ~ci otWlti

With the- oes described abv- th is con c en~rat ions5o asp btween 1761 and 0 4 4e V not ito nic while

Ce shy

on~~~~t-nIf)6~ ~ ~ n 6 nlad lwr wren t

weret+ne I r nt4t e cibe e -einthep m IJ

41~ ~

ME 4i

E2val at an of C0oait o f a n-HCl and Sanigre d

Grado on CHO cell1s

jCE~lI Hre-mp e resi tant t ig her conentrton or

23 Evlainof teeffect of Taspine HC1I on Sister

pe Choai Exhng~e- dfe age

SLuppl 6e~ted wh 5 eal bovine seru and005m~gertami~cin arindApCLbated in hL~Ii i a aos p h e eo C

~reedeTaspihe HM1 to afinal concentrationW C264 ~ M~Yof j1 -i~nI-0088 ugmi respectively A thr sto pl tes di d notI recei ye an~y Taspinej-ICl Iand ~remai ned as -control~ Simultaneou~sly with the tr tmnt die tweeamp sets~(oPaL

-~received~5Doodoirdn to ilicnetaino ltgM1 O nedi P~a t es hbrethen incubatedmt I

~~additional 22 hou~rs in a-hUnidifiedjaoshr of5 5 at -35r~a~7C (abdo2tt repli CatiVe-rQuL~WO nd

0~t the enid of the 22~ hours of InicLu3ajampion C61 mid~ o~a5 added ~toamptbe pl-ates (3final i-Y6f~ U andto ocneE- I- (pn I J

3 pc nLatdI~er f or and addit ion I twodhour s ftri at ubat i n i hao 31i n -fe e a~icai

and stain~ed wi~th tiplFllu iocromejP1lUZGe techni qie c)f per ry and Wol f f Nature 56-18 17il) and La tt (Pr 0c NAtI cad Sc i US7 3 3919l 7lt -K

7~The resul ts areshown in -Table 14 Wat we observei s that 2YTaspile~HCL-i at~ non~ citotoxic concentr~ations~ increases thea

~fre~ue~ncy of- sister c armat id exchageson V79O4cls

Fr thIest i th CHOD cel Is that4 ware~ growin expone ti al wer e seeded on 60 mmi tissue C1ture pt~ji dishe (3 1 clsA

4-bova ne serum nd 0m m o etm and - nCbted in4i a hUitmsid ee o COzand 5 ai t_7C wny or

~hours I ater-ns tofIa toe srlte a~4de s e-1 CtLo final c~ on- 6ntrat 9 a 6tg )1 a nIot he 4r se L olaes receivedSangrea de rad6o~at n1Zoi 3 to of 0 00 at rdin

set- f tde r CracoG i nd nad-a he-eAi cio1S~n1a

treamen th the set of ~r1a~espl recaivd

-MredIIuM3 Pla1tes whr thniChtdf or and add~i t i nLI21

- ~ 1~ 4 -d4

4 U ~~W10ri1I1

(abVQCW rp ribte vea qiar s Iofe

pl4ateE~ d U Aue c fr iionWerL ndad

4 staied ihth lyrcrm nc fnSC techof - I AniqL

gtapine1Cand did -a a L1amage SO7 337-31 177-

The~~~~~~~~~~ na reu~ 5adwa b ev tar h

PofiteTIALomtiES OF ha RAOAnDgtAP as inSGR~D the r-ntar

4L~~oth sinfcnlwpe sed the ~~ 4 4sr1n

eol~o-at rsIlp-hFrmthe nexperimesdescryibe ad e e cocV ta Taspin-HClt anFsc6 Sanred WeeksddntP-dC hO~on

cr i no~a Sytemhand the t0dw uedoensi wee fMI owe i i Iat

aLs CDhthl-Toheon dac~n~eribed bypurd usld 71

j~nO~s1 a I a TClaoa

Kl-Ten krianto A SN - eesCE use 1 s - 1 a d N t OILSIi~sce op~d

Thbmaledeany1phrb M~c etate moths~ ofri the~wre shcet1aed i re~js ig phas hair~ 6c usd Mceery honedUIof-th wereeap

p ppiand pfahe inweteator de adI~Di i triTheafer cahe nod ltamp~4were 30Pan(riasp e Leachr dxermnag~~(roup6o

compoud~ uonted as-anp-ltracexerininM tgtA ih bfrue t soIvPd

carcinogenicou~~W-nd 7n-12 ewt~ole- 0(-)shy-~~~h

x and i

n~~ (l I s

I Thegr o n tume~or roe r compound ~used wa 7 ra decGoylpup rbltreate~s- (TA and ocn a-o -tdNo

wasOLI 2~Ug -in ofL actn ctone- gt i- e

G-C resnt I No

Group---------------------------------------------------------------------------

GrOLIP~~~ 10nTo ~ 3 i 1 l-c~~ fI-D

p 5r I10 n D Ajn 1s c e~ e

42 twi cf wekl e ef

t v4 CL we e4

Io4M C)A~~cng C Or oup 0I~ of rTrAn oar3 MOi Q~~~~IaeoneHrF

GrUP1 Wau ~Our cotro o efomt ion of sp n r-fpr~mQ SGroup 2 i4as Ou coto fo asacrcngn cDrMnq

r-0 o WiuRm mno r~a n iLWwht~U~ ldshwasiry zec p~b 7snbu ltzgtwo~t our twi~o-tg raiognen r6~wtd ~

tr~satedDrlI3Awilhai ard TP~ op -as ~wo$king~5nue ot

ourNoy rou Sndangre de Gnoranai Group VWithr ~~ts r 4 t -1C - acng

r n~dLndPh~fP~P e as 11

atou 7hend b for acarcnoge

h or Tasp n

Tho e )02 prtfrpd c i 2nd aneioMet er a on~ mbe e 190 vr

trpeetned _~ s nTAa n trwihDB dcr a m deloe

the pio~mast rekmonhs afther thji niLia n fthe treimn (2a oted of 294ba5ieay 1 rso rlice trearte wi thur-~d oleir o

of ~~~ ~ ~ ms~ ~ tju ~ ~ ~ i~ pe-~t ~ deeipdppnssa~d itA

no es w r oA4tp --4 d an

0

7t

J

22

-Grossge~i ti nowere sever a Iex e -p

GVL hcw a amintiom ma 94jf rom4~

by- hyep theao derm ppij aei wich er e ted Fo theI nthemi sufce he Ml gisa~ dc or inveIS aymtr~

igt beae4ikr aa~hs L wit r-ccnLgt (te hyekrami

a t om o f c n a s io y t T e r mj i- b i I

mainn chngs Th Kn4--oran did pr4n pah~oi

FFSiieii a e ca c n gei or tino promoters-

f ia e n in ocotty tfe eX p rim6 D it IFi aIhc I-54 SiAL-4 mo th in or r toi4i be i co c u F i ce I- mn ii iF

i ~~ t4Ie~ ~ os4iL-ig ~ ~ hL ~ ~iie ~ ~ v 4~ ~ ~ g Oilt494hrta ~h ra ~ wi~j theknow ogn sA~rtd t 3 doelo tv tmrcari DB

on mo th ag

4 4 ~

4 ONai

4CONCLUSIONS

rSnr de Grado etracted f romp ICiton 7er

o extracts fr-oT1 th~ecore~ fri in ofth Crotor lechleri tree

Thr4ure idtein Or trc EITOit in the ieavesu of thebAci~t n I 1eriand the most bkidant one was ~

T~asp inewas h cti O 1teri za tp i ncn 1 L-p e s en1 i Sa~ngre4 de Gracdo etracted from Croto hl1ech14er

~h Lid healin process~ r I

1~~~fibrobasts atonceritrtions1 be1llow2)- nOgm1 ard i t di d e Ih1 PEr at ion~not uincrease ratE1Qf-11P

w fon nt~h ibit the contac i on61

hnn f orEsk i ni i a fibr 0b I as

6~mutagunicitY 1tud using the 4rnes 4tcst showe htSJrgshydeGad a wh le taspine chloydrate hadttrcco k

~~ ~no Jn~ta ernii activity I1

StUdies on e ef f eect4 - Ire derao andAsnt A-t oBancr adpCdnd

ychorhdrteortisister choai eklnp (SCE) showe t I4bsTaspine hlrydrate increased the nIumber of SC[ 3in

V79if4 ellbt no increase wa5 abservedinCOcls 10Uigthe tW-tg O skincriogee syste w e V

4 14he shown that~4nri Uher Sng a ir tppeIJ1 were carci nb~geniviardor -tumor promtr f te n0ya +iitrea t4ment 1

--- --- --- --- --- --- ---

0 11

FXTROTrONOOS9NJRF DE GRA1DO FRMDFgRE T RE P1)0tITUITSC DING -m RSTTRP

TREETPg~ OF] PH ~VOLiUMEK OF~ YILIL - XT I ON E--~ Ig11TV~ XTROCTION

6-- 91 -~agm ~ mJ-1 -- ~ 22 272

M- 5ql ar-To each of the sample wre addced tJ n - -qri

~~~~e alktalinized to p 9 wth amn~iui hydjroXidftt~eJ~h and extatede0hautv- wie~t~h choroform Each clooorm extractwa concentrated i n a rotavapor then dryed and -the~~shy

ht 0os theE 6k gaIkaIoidvr eaU d i napl- alI7 24 -065j-~9-- ~

C) Iml ofec f Lhe 5ape wer Alded Ijlo ae th as t e aI kalinized-to pH 92wtha

etract iasdred and hei redissolved i 1 mo-o lLlou and0 1e bobnamp t347lt was merLrdL~igaZi

Spe ro fotome er-jThextinction coefficient of taspine at 47 i

a si gni i can t di FF$ernce2 between thee gbFUPS anthegOUS-n

The col lact n datE wJas Dec mber~ 19 1901

-- ---Y -- ~4A shy- ~----

I A

4 AB 2 ~4

q 7

EXRCIN F AGE1 1ACFOM -ER RL RMIU

~4 1 4-112 lt 4 44 4

44 42 I-4lt C) 6

- ~~ ~ ~ ~ ~ ~~44-- -- -- ----4 -shy~~ ~~~4441533c~ de Grado was co lced fo t e te s nm rae hc

on ebr 1 15and 5 a d n M y 2 90 h I a m- dary 19

4tet4wa d -4 e44 de 11o etha To~4lt4I ml 4~44 each 4 44444 were ed of wa eof o ~ th sV ad I4~ l th

mixturwa al t 4lt4 yrxd -iUPH~ 9 IVizp and 4l~444 wi- 4 o c rfextr c~e l a~~iv 4 h 4aof 4ofri E4a4 h

r-~ lt4~4 444 ~ 44 ~44 4) gt44

Xh e~= 2 8c tr-ts 0 da gr7380frI~ aawas 1+C

wasra sign~ficat diiffrec betwee the to ~grup Plt 0

i 44 ~ f~cho ~ h ui 1 4 l V~n11~9 7 rW hnaIdiin 4 hdoidlt465 W~qamp

T

22

I~~_7

control (rD 11

SG 51 1 1 1 8

Tabpie tm11C () r 71 0 1

Obrevi itions WS = WUndbeakinsteghSIG =Sargre de Grado 2

STaspine-HICI = Taspirn chlrhydrate

Nce wepre anesthesized with vapors fdiethyl ete and a~ IcM A ci ion as made perpendi CIler _to the ais fsmfnmet-y f tea i anid thtwo4borders of the Wo~nd werestichdtgte tiscntr

g~tete ~ n~p~vry 12 hOrs the comfpol-tbeinQt~ted Ias~ap llied Ato the w~ound -For thi p~pGUQXn M2Ishy

mealt p m crop 1diwpe 4 ddrj1 Veins a WacLIndy h ant-h otosrrcie ny

~Fnt iii2which liheror6 wa dtdI1 horst ~ pib elte

f gone of the borders of t~he on aFtr 1-itinLh t hle4 g ret h other onet For moeJh tai _hi p in a 2MeaSLrable_ fore teoe hmred

t 1i anlYi S~ ohtps thUi ng ~ s sh2tt w d2itt ic aie

di erence betwepri Aher can Lrol grouip and tgtheSG-g roup (p0nO5 -n bewenr theQcntrol pU idLPtpi6Hl L(p C CI)i1

fr C an 22gn 12 2

h 101111-

6 K

14 V MMq ~~ tpE

Vef s ) in ib trv Io i th t hih r-o r nra io 5 Po

obev ta gSr- f de frado a s3IRd

canentraton ofrw 11~ C h~ tetr 14I0 an ~ 2Vj~)For ~an ~f v i hube1 re etat of t5d~ nf -L~ ti

e ospeint toethehee~ Weo~o a CIWbpntnejsu~ bpred no t Lathe concentctionc5 testedidttgenic_

concw~entatiorcn5e of~o ~a de Gradmuand~fTaspif-k oLh hSagre I iTs~~s he wradk e ncr Miadotnoit anr e rre

~teste0r2an rnraid)~ 5 Uheer thoncetrat ritin 0 p o (o r jI r41 Ic w c op o p a~dz - g n

501n) inthti nume of~ Hrtl t ~test e 12rLk ld4WhiC t0CTapie te n

difieret frum5testieHC11si [OtE(per mental~n he1muW~EIS Moppanr)~aged wamwh eF rado~ we muag

Tap EEC OFan nF de OspnoEFEQE CHaRo22NEHC SISTERgr oMATI 4000ZEXHA n GE 006m m Thes cocet l atI1 ia 11 Ae -I Ipfw Ill44

conctratiron de excasingesH~aesed all~ i Ii wh~ uh44 1s41howno MtAgnnesiv F~f~tl i wcanbednduedtbha

5 olte f f ntfr Tbt h in g di ca Lim 1 steMLtgnccm-r )FSagr de taiV eII neths ta

~ ofXamp

4 irohe oms 1 ste ca 1ltV 11 c eseha we ~ exchnge eid~db

andtC is aOt ll l if brtht~l~ey 6 conswsidere d C))

chfiromosme- amage- C hae b~ye~ 41 i4se1ogt

Thece 17~4111n s 1 t a li fo these ex e ime t eeY 901144weu

_COand ~ oh e I 414werp414141e7ns embryos a f 4 ~ th

1I 111411 ~I1~~1~414 1 1 1411 1141ltj I t 4 4111lt1 11 14 4~ ~ 14y

4-~

21 Evauationriof Citotoxici ty6of Tasp1 ne7 CI on V79AP4

~att n asp i ne -~C Io ~ ~ ed95f II l 4e tpi-

t~l~~UAnter~ adcr tiwuo d j

ar~fCO~ta fieamp L t c~pellst~descrr andor he of frmthe

pnu~rI-U~df~atnt L-5an

groni D4heCDr mdfi~ agep edL 5Lh~e mntedny mcw o th

24 hours r~aoh The reUl ts showed that concertrations of 176~ 000B and C) 14 L Im ~ 6f Taspine HCl were toxica for th1e el Is hi1

4 concentratins~ of C)2641 nd 0000 Oqml f Taspipe6-HdC were in~ocUOU~S for the cellis

Sb tCUS~d e thSi MUt~t rmntoJf-heitegrit f eI

S-mjembrane-and th ciiyo ntraeua aae5 me od- b f ae1o tYaerate

m~hodorRotan ad P (P~rocgt )a1~cadSLP USA 55 13-4~ 1966) ran Edidin IdjrjtI 13

Thlis technique Makes use of two dyes

The fir~ on~e a -nn ~lrn on f trec q compound~ -f curc that-6 coud t rav1erse thee I I i dlct ea~i

membaneOce nsid th cl I ine peifica stear (s I i ber atLek

~oneecon~d apoar f I1-2sceentsi~-T is dye etidium bromid~e~wh ich uP-onlexci tati on(wi th UV ligt fluIorsered This dy

Ci Ulonly enter theoeI whe the~r me-rni damaged~~Th cnetain fFursc~n ctt and et Id i Utn bromide14~Lsedwere 2 Ugr1a~d 5~bilWheI-ee~10y

ml 4nUgmiilerctvely e Ca~nlltrbaea twiththdy~s nd6~eredUnder U- gt in~ a~fILkoresceLt mlsectp~ 1 0h cel a b4~ed~Lrigh green6

fluorescencejunhealth 1 oed iIi1htgrPer1 freF5C ncampe anfagd ciidedcells ed~edf~oe~

- This~method wa also- sed inri order- toevaluate the ~ci otWlti

With the- oes described abv- th is con c en~rat ions5o asp btween 1761 and 0 4 4e V not ito nic while

Ce shy

on~~~~t-nIf)6~ ~ ~ n 6 nlad lwr wren t

weret+ne I r nt4t e cibe e -einthep m IJ

41~ ~

ME 4i

E2val at an of C0oait o f a n-HCl and Sanigre d

Grado on CHO cell1s

jCE~lI Hre-mp e resi tant t ig her conentrton or

23 Evlainof teeffect of Taspine HC1I on Sister

pe Choai Exhng~e- dfe age

SLuppl 6e~ted wh 5 eal bovine seru and005m~gertami~cin arindApCLbated in hL~Ii i a aos p h e eo C

~reedeTaspihe HM1 to afinal concentrationW C264 ~ M~Yof j1 -i~nI-0088 ugmi respectively A thr sto pl tes di d notI recei ye an~y Taspinej-ICl Iand ~remai ned as -control~ Simultaneou~sly with the tr tmnt die tweeamp sets~(oPaL

-~received~5Doodoirdn to ilicnetaino ltgM1 O nedi P~a t es hbrethen incubatedmt I

~~additional 22 hou~rs in a-hUnidifiedjaoshr of5 5 at -35r~a~7C (abdo2tt repli CatiVe-rQuL~WO nd

0~t the enid of the 22~ hours of InicLu3ajampion C61 mid~ o~a5 added ~toamptbe pl-ates (3final i-Y6f~ U andto ocneE- I- (pn I J

3 pc nLatdI~er f or and addit ion I twodhour s ftri at ubat i n i hao 31i n -fe e a~icai

and stain~ed wi~th tiplFllu iocromejP1lUZGe techni qie c)f per ry and Wol f f Nature 56-18 17il) and La tt (Pr 0c NAtI cad Sc i US7 3 3919l 7lt -K

7~The resul ts areshown in -Table 14 Wat we observei s that 2YTaspile~HCL-i at~ non~ citotoxic concentr~ations~ increases thea

~fre~ue~ncy of- sister c armat id exchageson V79O4cls

Fr thIest i th CHOD cel Is that4 ware~ growin expone ti al wer e seeded on 60 mmi tissue C1ture pt~ji dishe (3 1 clsA

4-bova ne serum nd 0m m o etm and - nCbted in4i a hUitmsid ee o COzand 5 ai t_7C wny or

~hours I ater-ns tofIa toe srlte a~4de s e-1 CtLo final c~ on- 6ntrat 9 a 6tg )1 a nIot he 4r se L olaes receivedSangrea de rad6o~at n1Zoi 3 to of 0 00 at rdin

set- f tde r CracoG i nd nad-a he-eAi cio1S~n1a

treamen th the set of ~r1a~espl recaivd

-MredIIuM3 Pla1tes whr thniChtdf or and add~i t i nLI21

- ~ 1~ 4 -d4

4 U ~~W10ri1I1

(abVQCW rp ribte vea qiar s Iofe

pl4ateE~ d U Aue c fr iionWerL ndad

4 staied ihth lyrcrm nc fnSC techof - I AniqL

gtapine1Cand did -a a L1amage SO7 337-31 177-

The~~~~~~~~~~ na reu~ 5adwa b ev tar h

PofiteTIALomtiES OF ha RAOAnDgtAP as inSGR~D the r-ntar

4L~~oth sinfcnlwpe sed the ~~ 4 4sr1n

eol~o-at rsIlp-hFrmthe nexperimesdescryibe ad e e cocV ta Taspin-HClt anFsc6 Sanred WeeksddntP-dC hO~on

cr i no~a Sytemhand the t0dw uedoensi wee fMI owe i i Iat

aLs CDhthl-Toheon dac~n~eribed bypurd usld 71

j~nO~s1 a I a TClaoa

Kl-Ten krianto A SN - eesCE use 1 s - 1 a d N t OILSIi~sce op~d

Thbmaledeany1phrb M~c etate moths~ ofri the~wre shcet1aed i re~js ig phas hair~ 6c usd Mceery honedUIof-th wereeap

p ppiand pfahe inweteator de adI~Di i triTheafer cahe nod ltamp~4were 30Pan(riasp e Leachr dxermnag~~(roup6o

compoud~ uonted as-anp-ltracexerininM tgtA ih bfrue t soIvPd

carcinogenicou~~W-nd 7n-12 ewt~ole- 0(-)shy-~~~h

x and i

n~~ (l I s

I Thegr o n tume~or roe r compound ~used wa 7 ra decGoylpup rbltreate~s- (TA and ocn a-o -tdNo

wasOLI 2~Ug -in ofL actn ctone- gt i- e

G-C resnt I No

Group---------------------------------------------------------------------------

GrOLIP~~~ 10nTo ~ 3 i 1 l-c~~ fI-D

p 5r I10 n D Ajn 1s c e~ e

42 twi cf wekl e ef

t v4 CL we e4

Io4M C)A~~cng C Or oup 0I~ of rTrAn oar3 MOi Q~~~~IaeoneHrF

GrUP1 Wau ~Our cotro o efomt ion of sp n r-fpr~mQ SGroup 2 i4as Ou coto fo asacrcngn cDrMnq

r-0 o WiuRm mno r~a n iLWwht~U~ ldshwasiry zec p~b 7snbu ltzgtwo~t our twi~o-tg raiognen r6~wtd ~

tr~satedDrlI3Awilhai ard TP~ op -as ~wo$king~5nue ot

ourNoy rou Sndangre de Gnoranai Group VWithr ~~ts r 4 t -1C - acng

r n~dLndPh~fP~P e as 11

atou 7hend b for acarcnoge

h or Tasp n

Tho e )02 prtfrpd c i 2nd aneioMet er a on~ mbe e 190 vr

trpeetned _~ s nTAa n trwihDB dcr a m deloe

the pio~mast rekmonhs afther thji niLia n fthe treimn (2a oted of 294ba5ieay 1 rso rlice trearte wi thur-~d oleir o

of ~~~ ~ ~ ms~ ~ tju ~ ~ ~ i~ pe-~t ~ deeipdppnssa~d itA

no es w r oA4tp --4 d an

0

7t

J

22

-Grossge~i ti nowere sever a Iex e -p

GVL hcw a amintiom ma 94jf rom4~

by- hyep theao derm ppij aei wich er e ted Fo theI nthemi sufce he Ml gisa~ dc or inveIS aymtr~

igt beae4ikr aa~hs L wit r-ccnLgt (te hyekrami

a t om o f c n a s io y t T e r mj i- b i I

mainn chngs Th Kn4--oran did pr4n pah~oi

FFSiieii a e ca c n gei or tino promoters-

f ia e n in ocotty tfe eX p rim6 D it IFi aIhc I-54 SiAL-4 mo th in or r toi4i be i co c u F i ce I- mn ii iF

i ~~ t4Ie~ ~ os4iL-ig ~ ~ hL ~ ~iie ~ ~ v 4~ ~ ~ g Oilt494hrta ~h ra ~ wi~j theknow ogn sA~rtd t 3 doelo tv tmrcari DB

on mo th ag

4 4 ~

4 ONai

4CONCLUSIONS

rSnr de Grado etracted f romp ICiton 7er

o extracts fr-oT1 th~ecore~ fri in ofth Crotor lechleri tree

Thr4ure idtein Or trc EITOit in the ieavesu of thebAci~t n I 1eriand the most bkidant one was ~

T~asp inewas h cti O 1teri za tp i ncn 1 L-p e s en1 i Sa~ngre4 de Gracdo etracted from Croto hl1ech14er

~h Lid healin process~ r I

1~~~fibrobasts atonceritrtions1 be1llow2)- nOgm1 ard i t di d e Ih1 PEr at ion~not uincrease ratE1Qf-11P

w fon nt~h ibit the contac i on61

hnn f orEsk i ni i a fibr 0b I as

6~mutagunicitY 1tud using the 4rnes 4tcst showe htSJrgshydeGad a wh le taspine chloydrate hadttrcco k

~~ ~no Jn~ta ernii activity I1

StUdies on e ef f eect4 - Ire derao andAsnt A-t oBancr adpCdnd

ychorhdrteortisister choai eklnp (SCE) showe t I4bsTaspine hlrydrate increased the nIumber of SC[ 3in

V79if4 ellbt no increase wa5 abservedinCOcls 10Uigthe tW-tg O skincriogee syste w e V

4 14he shown that~4nri Uher Sng a ir tppeIJ1 were carci nb~geniviardor -tumor promtr f te n0ya +iitrea t4ment 1

--- --- --- --- --- --- ---

0 11

FXTROTrONOOS9NJRF DE GRA1DO FRMDFgRE T RE P1)0tITUITSC DING -m RSTTRP

TREETPg~ OF] PH ~VOLiUMEK OF~ YILIL - XT I ON E--~ Ig11TV~ XTROCTION

6-- 91 -~agm ~ mJ-1 -- ~ 22 272

M- 5ql ar-To each of the sample wre addced tJ n - -qri

~~~~e alktalinized to p 9 wth amn~iui hydjroXidftt~eJ~h and extatede0hautv- wie~t~h choroform Each clooorm extractwa concentrated i n a rotavapor then dryed and -the~~shy

ht 0os theE 6k gaIkaIoidvr eaU d i napl- alI7 24 -065j-~9-- ~

C) Iml ofec f Lhe 5ape wer Alded Ijlo ae th as t e aI kalinized-to pH 92wtha

etract iasdred and hei redissolved i 1 mo-o lLlou and0 1e bobnamp t347lt was merLrdL~igaZi

Spe ro fotome er-jThextinction coefficient of taspine at 47 i

a si gni i can t di FF$ernce2 between thee gbFUPS anthegOUS-n

The col lact n datE wJas Dec mber~ 19 1901

-- ---Y -- ~4A shy- ~----

I A

4 AB 2 ~4

q 7

EXRCIN F AGE1 1ACFOM -ER RL RMIU

~4 1 4-112 lt 4 44 4

44 42 I-4lt C) 6

- ~~ ~ ~ ~ ~ ~~44-- -- -- ----4 -shy~~ ~~~4441533c~ de Grado was co lced fo t e te s nm rae hc

on ebr 1 15and 5 a d n M y 2 90 h I a m- dary 19

4tet4wa d -4 e44 de 11o etha To~4lt4I ml 4~44 each 4 44444 were ed of wa eof o ~ th sV ad I4~ l th

mixturwa al t 4lt4 yrxd -iUPH~ 9 IVizp and 4l~444 wi- 4 o c rfextr c~e l a~~iv 4 h 4aof 4ofri E4a4 h

r-~ lt4~4 444 ~ 44 ~44 4) gt44

Xh e~= 2 8c tr-ts 0 da gr7380frI~ aawas 1+C

wasra sign~ficat diiffrec betwee the to ~grup Plt 0

i 44 ~ f~cho ~ h ui 1 4 l V~n11~9 7 rW hnaIdiin 4 hdoidlt465 W~qamp

T

22

I~~_7

control (rD 11

SG 51 1 1 1 8

Tabpie tm11C () r 71 0 1

Obrevi itions WS = WUndbeakinsteghSIG =Sargre de Grado 2

STaspine-HICI = Taspirn chlrhydrate

Nce wepre anesthesized with vapors fdiethyl ete and a~ IcM A ci ion as made perpendi CIler _to the ais fsmfnmet-y f tea i anid thtwo4borders of the Wo~nd werestichdtgte tiscntr

g~tete ~ n~p~vry 12 hOrs the comfpol-tbeinQt~ted Ias~ap llied Ato the w~ound -For thi p~pGUQXn M2Ishy

mealt p m crop 1diwpe 4 ddrj1 Veins a WacLIndy h ant-h otosrrcie ny

~Fnt iii2which liheror6 wa dtdI1 horst ~ pib elte

f gone of the borders of t~he on aFtr 1-itinLh t hle4 g ret h other onet For moeJh tai _hi p in a 2MeaSLrable_ fore teoe hmred

t 1i anlYi S~ ohtps thUi ng ~ s sh2tt w d2itt ic aie

di erence betwepri Aher can Lrol grouip and tgtheSG-g roup (p0nO5 -n bewenr theQcntrol pU idLPtpi6Hl L(p C CI)i1

fr C an 22gn 12 2

21 Evauationriof Citotoxici ty6of Tasp1 ne7 CI on V79AP4

~att n asp i ne -~C Io ~ ~ ed95f II l 4e tpi-

t~l~~UAnter~ adcr tiwuo d j

ar~fCO~ta fieamp L t c~pellst~descrr andor he of frmthe

pnu~rI-U~df~atnt L-5an

groni D4heCDr mdfi~ agep edL 5Lh~e mntedny mcw o th

24 hours r~aoh The reUl ts showed that concertrations of 176~ 000B and C) 14 L Im ~ 6f Taspine HCl were toxica for th1e el Is hi1

4 concentratins~ of C)2641 nd 0000 Oqml f Taspipe6-HdC were in~ocUOU~S for the cellis

Sb tCUS~d e thSi MUt~t rmntoJf-heitegrit f eI

S-mjembrane-and th ciiyo ntraeua aae5 me od- b f ae1o tYaerate

m~hodorRotan ad P (P~rocgt )a1~cadSLP USA 55 13-4~ 1966) ran Edidin IdjrjtI 13

Thlis technique Makes use of two dyes

The fir~ on~e a -nn ~lrn on f trec q compound~ -f curc that-6 coud t rav1erse thee I I i dlct ea~i

membaneOce nsid th cl I ine peifica stear (s I i ber atLek

~oneecon~d apoar f I1-2sceentsi~-T is dye etidium bromid~e~wh ich uP-onlexci tati on(wi th UV ligt fluIorsered This dy

Ci Ulonly enter theoeI whe the~r me-rni damaged~~Th cnetain fFursc~n ctt and et Id i Utn bromide14~Lsedwere 2 Ugr1a~d 5~bilWheI-ee~10y

ml 4nUgmiilerctvely e Ca~nlltrbaea twiththdy~s nd6~eredUnder U- gt in~ a~fILkoresceLt mlsectp~ 1 0h cel a b4~ed~Lrigh green6

fluorescencejunhealth 1 oed iIi1htgrPer1 freF5C ncampe anfagd ciidedcells ed~edf~oe~

- This~method wa also- sed inri order- toevaluate the ~ci otWlti

With the- oes described abv- th is con c en~rat ions5o asp btween 1761 and 0 4 4e V not ito nic while

Ce shy

on~~~~t-nIf)6~ ~ ~ n 6 nlad lwr wren t

weret+ne I r nt4t e cibe e -einthep m IJ

41~ ~

ME 4i

E2val at an of C0oait o f a n-HCl and Sanigre d

Grado on CHO cell1s

jCE~lI Hre-mp e resi tant t ig her conentrton or

23 Evlainof teeffect of Taspine HC1I on Sister

pe Choai Exhng~e- dfe age

SLuppl 6e~ted wh 5 eal bovine seru and005m~gertami~cin arindApCLbated in hL~Ii i a aos p h e eo C

~reedeTaspihe HM1 to afinal concentrationW C264 ~ M~Yof j1 -i~nI-0088 ugmi respectively A thr sto pl tes di d notI recei ye an~y Taspinej-ICl Iand ~remai ned as -control~ Simultaneou~sly with the tr tmnt die tweeamp sets~(oPaL

-~received~5Doodoirdn to ilicnetaino ltgM1 O nedi P~a t es hbrethen incubatedmt I

~~additional 22 hou~rs in a-hUnidifiedjaoshr of5 5 at -35r~a~7C (abdo2tt repli CatiVe-rQuL~WO nd

0~t the enid of the 22~ hours of InicLu3ajampion C61 mid~ o~a5 added ~toamptbe pl-ates (3final i-Y6f~ U andto ocneE- I- (pn I J

3 pc nLatdI~er f or and addit ion I twodhour s ftri at ubat i n i hao 31i n -fe e a~icai

and stain~ed wi~th tiplFllu iocromejP1lUZGe techni qie c)f per ry and Wol f f Nature 56-18 17il) and La tt (Pr 0c NAtI cad Sc i US7 3 3919l 7lt -K

7~The resul ts areshown in -Table 14 Wat we observei s that 2YTaspile~HCL-i at~ non~ citotoxic concentr~ations~ increases thea

~fre~ue~ncy of- sister c armat id exchageson V79O4cls

Fr thIest i th CHOD cel Is that4 ware~ growin expone ti al wer e seeded on 60 mmi tissue C1ture pt~ji dishe (3 1 clsA

4-bova ne serum nd 0m m o etm and - nCbted in4i a hUitmsid ee o COzand 5 ai t_7C wny or

~hours I ater-ns tofIa toe srlte a~4de s e-1 CtLo final c~ on- 6ntrat 9 a 6tg )1 a nIot he 4r se L olaes receivedSangrea de rad6o~at n1Zoi 3 to of 0 00 at rdin

set- f tde r CracoG i nd nad-a he-eAi cio1S~n1a

treamen th the set of ~r1a~espl recaivd

-MredIIuM3 Pla1tes whr thniChtdf or and add~i t i nLI21

- ~ 1~ 4 -d4

4 U ~~W10ri1I1

(abVQCW rp ribte vea qiar s Iofe

pl4ateE~ d U Aue c fr iionWerL ndad

4 staied ihth lyrcrm nc fnSC techof - I AniqL

gtapine1Cand did -a a L1amage SO7 337-31 177-

The~~~~~~~~~~ na reu~ 5adwa b ev tar h

PofiteTIALomtiES OF ha RAOAnDgtAP as inSGR~D the r-ntar

4L~~oth sinfcnlwpe sed the ~~ 4 4sr1n

eol~o-at rsIlp-hFrmthe nexperimesdescryibe ad e e cocV ta Taspin-HClt anFsc6 Sanred WeeksddntP-dC hO~on

cr i no~a Sytemhand the t0dw uedoensi wee fMI owe i i Iat

aLs CDhthl-Toheon dac~n~eribed bypurd usld 71

j~nO~s1 a I a TClaoa

Kl-Ten krianto A SN - eesCE use 1 s - 1 a d N t OILSIi~sce op~d

Thbmaledeany1phrb M~c etate moths~ ofri the~wre shcet1aed i re~js ig phas hair~ 6c usd Mceery honedUIof-th wereeap

p ppiand pfahe inweteator de adI~Di i triTheafer cahe nod ltamp~4were 30Pan(riasp e Leachr dxermnag~~(roup6o

compoud~ uonted as-anp-ltracexerininM tgtA ih bfrue t soIvPd

carcinogenicou~~W-nd 7n-12 ewt~ole- 0(-)shy-~~~h

x and i

n~~ (l I s

I Thegr o n tume~or roe r compound ~used wa 7 ra decGoylpup rbltreate~s- (TA and ocn a-o -tdNo

wasOLI 2~Ug -in ofL actn ctone- gt i- e

G-C resnt I No

Group---------------------------------------------------------------------------

GrOLIP~~~ 10nTo ~ 3 i 1 l-c~~ fI-D

p 5r I10 n D Ajn 1s c e~ e

42 twi cf wekl e ef

t v4 CL we e4

Io4M C)A~~cng C Or oup 0I~ of rTrAn oar3 MOi Q~~~~IaeoneHrF

GrUP1 Wau ~Our cotro o efomt ion of sp n r-fpr~mQ SGroup 2 i4as Ou coto fo asacrcngn cDrMnq

r-0 o WiuRm mno r~a n iLWwht~U~ ldshwasiry zec p~b 7snbu ltzgtwo~t our twi~o-tg raiognen r6~wtd ~

tr~satedDrlI3Awilhai ard TP~ op -as ~wo$king~5nue ot

ourNoy rou Sndangre de Gnoranai Group VWithr ~~ts r 4 t -1C - acng

r n~dLndPh~fP~P e as 11

atou 7hend b for acarcnoge

h or Tasp n

Tho e )02 prtfrpd c i 2nd aneioMet er a on~ mbe e 190 vr

trpeetned _~ s nTAa n trwihDB dcr a m deloe

the pio~mast rekmonhs afther thji niLia n fthe treimn (2a oted of 294ba5ieay 1 rso rlice trearte wi thur-~d oleir o

of ~~~ ~ ~ ms~ ~ tju ~ ~ ~ i~ pe-~t ~ deeipdppnssa~d itA

no es w r oA4tp --4 d an

0

7t

J

22

-Grossge~i ti nowere sever a Iex e -p

GVL hcw a amintiom ma 94jf rom4~

by- hyep theao derm ppij aei wich er e ted Fo theI nthemi sufce he Ml gisa~ dc or inveIS aymtr~

igt beae4ikr aa~hs L wit r-ccnLgt (te hyekrami

a t om o f c n a s io y t T e r mj i- b i I

mainn chngs Th Kn4--oran did pr4n pah~oi

FFSiieii a e ca c n gei or tino promoters-

f ia e n in ocotty tfe eX p rim6 D it IFi aIhc I-54 SiAL-4 mo th in or r toi4i be i co c u F i ce I- mn ii iF

i ~~ t4Ie~ ~ os4iL-ig ~ ~ hL ~ ~iie ~ ~ v 4~ ~ ~ g Oilt494hrta ~h ra ~ wi~j theknow ogn sA~rtd t 3 doelo tv tmrcari DB

on mo th ag

4 4 ~

4 ONai

4CONCLUSIONS

rSnr de Grado etracted f romp ICiton 7er

o extracts fr-oT1 th~ecore~ fri in ofth Crotor lechleri tree

Thr4ure idtein Or trc EITOit in the ieavesu of thebAci~t n I 1eriand the most bkidant one was ~

T~asp inewas h cti O 1teri za tp i ncn 1 L-p e s en1 i Sa~ngre4 de Gracdo etracted from Croto hl1ech14er

~h Lid healin process~ r I

1~~~fibrobasts atonceritrtions1 be1llow2)- nOgm1 ard i t di d e Ih1 PEr at ion~not uincrease ratE1Qf-11P

w fon nt~h ibit the contac i on61

hnn f orEsk i ni i a fibr 0b I as

6~mutagunicitY 1tud using the 4rnes 4tcst showe htSJrgshydeGad a wh le taspine chloydrate hadttrcco k

~~ ~no Jn~ta ernii activity I1

StUdies on e ef f eect4 - Ire derao andAsnt A-t oBancr adpCdnd

ychorhdrteortisister choai eklnp (SCE) showe t I4bsTaspine hlrydrate increased the nIumber of SC[ 3in

V79if4 ellbt no increase wa5 abservedinCOcls 10Uigthe tW-tg O skincriogee syste w e V

4 14he shown that~4nri Uher Sng a ir tppeIJ1 were carci nb~geniviardor -tumor promtr f te n0ya +iitrea t4ment 1

--- --- --- --- --- --- ---

0 11

FXTROTrONOOS9NJRF DE GRA1DO FRMDFgRE T RE P1)0tITUITSC DING -m RSTTRP

TREETPg~ OF] PH ~VOLiUMEK OF~ YILIL - XT I ON E--~ Ig11TV~ XTROCTION

6-- 91 -~agm ~ mJ-1 -- ~ 22 272

M- 5ql ar-To each of the sample wre addced tJ n - -qri

~~~~e alktalinized to p 9 wth amn~iui hydjroXidftt~eJ~h and extatede0hautv- wie~t~h choroform Each clooorm extractwa concentrated i n a rotavapor then dryed and -the~~shy

ht 0os theE 6k gaIkaIoidvr eaU d i napl- alI7 24 -065j-~9-- ~

C) Iml ofec f Lhe 5ape wer Alded Ijlo ae th as t e aI kalinized-to pH 92wtha

etract iasdred and hei redissolved i 1 mo-o lLlou and0 1e bobnamp t347lt was merLrdL~igaZi

Spe ro fotome er-jThextinction coefficient of taspine at 47 i

a si gni i can t di FF$ernce2 between thee gbFUPS anthegOUS-n

The col lact n datE wJas Dec mber~ 19 1901

-- ---Y -- ~4A shy- ~----

I A

4 AB 2 ~4

q 7

EXRCIN F AGE1 1ACFOM -ER RL RMIU

~4 1 4-112 lt 4 44 4

44 42 I-4lt C) 6

- ~~ ~ ~ ~ ~ ~~44-- -- -- ----4 -shy~~ ~~~4441533c~ de Grado was co lced fo t e te s nm rae hc

on ebr 1 15and 5 a d n M y 2 90 h I a m- dary 19

4tet4wa d -4 e44 de 11o etha To~4lt4I ml 4~44 each 4 44444 were ed of wa eof o ~ th sV ad I4~ l th

mixturwa al t 4lt4 yrxd -iUPH~ 9 IVizp and 4l~444 wi- 4 o c rfextr c~e l a~~iv 4 h 4aof 4ofri E4a4 h

r-~ lt4~4 444 ~ 44 ~44 4) gt44

Xh e~= 2 8c tr-ts 0 da gr7380frI~ aawas 1+C

wasra sign~ficat diiffrec betwee the to ~grup Plt 0

i 44 ~ f~cho ~ h ui 1 4 l V~n11~9 7 rW hnaIdiin 4 hdoidlt465 W~qamp

T

22

I~~_7

control (rD 11

SG 51 1 1 1 8

Tabpie tm11C () r 71 0 1

Obrevi itions WS = WUndbeakinsteghSIG =Sargre de Grado 2

STaspine-HICI = Taspirn chlrhydrate

Nce wepre anesthesized with vapors fdiethyl ete and a~ IcM A ci ion as made perpendi CIler _to the ais fsmfnmet-y f tea i anid thtwo4borders of the Wo~nd werestichdtgte tiscntr

g~tete ~ n~p~vry 12 hOrs the comfpol-tbeinQt~ted Ias~ap llied Ato the w~ound -For thi p~pGUQXn M2Ishy

mealt p m crop 1diwpe 4 ddrj1 Veins a WacLIndy h ant-h otosrrcie ny

~Fnt iii2which liheror6 wa dtdI1 horst ~ pib elte

f gone of the borders of t~he on aFtr 1-itinLh t hle4 g ret h other onet For moeJh tai _hi p in a 2MeaSLrable_ fore teoe hmred

t 1i anlYi S~ ohtps thUi ng ~ s sh2tt w d2itt ic aie

di erence betwepri Aher can Lrol grouip and tgtheSG-g roup (p0nO5 -n bewenr theQcntrol pU idLPtpi6Hl L(p C CI)i1

fr C an 22gn 12 2

ME 4i

E2val at an of C0oait o f a n-HCl and Sanigre d

Grado on CHO cell1s

jCE~lI Hre-mp e resi tant t ig her conentrton or

23 Evlainof teeffect of Taspine HC1I on Sister

pe Choai Exhng~e- dfe age

SLuppl 6e~ted wh 5 eal bovine seru and005m~gertami~cin arindApCLbated in hL~Ii i a aos p h e eo C

~reedeTaspihe HM1 to afinal concentrationW C264 ~ M~Yof j1 -i~nI-0088 ugmi respectively A thr sto pl tes di d notI recei ye an~y Taspinej-ICl Iand ~remai ned as -control~ Simultaneou~sly with the tr tmnt die tweeamp sets~(oPaL

-~received~5Doodoirdn to ilicnetaino ltgM1 O nedi P~a t es hbrethen incubatedmt I

~~additional 22 hou~rs in a-hUnidifiedjaoshr of5 5 at -35r~a~7C (abdo2tt repli CatiVe-rQuL~WO nd

0~t the enid of the 22~ hours of InicLu3ajampion C61 mid~ o~a5 added ~toamptbe pl-ates (3final i-Y6f~ U andto ocneE- I- (pn I J

3 pc nLatdI~er f or and addit ion I twodhour s ftri at ubat i n i hao 31i n -fe e a~icai

and stain~ed wi~th tiplFllu iocromejP1lUZGe techni qie c)f per ry and Wol f f Nature 56-18 17il) and La tt (Pr 0c NAtI cad Sc i US7 3 3919l 7lt -K

7~The resul ts areshown in -Table 14 Wat we observei s that 2YTaspile~HCL-i at~ non~ citotoxic concentr~ations~ increases thea

~fre~ue~ncy of- sister c armat id exchageson V79O4cls

Fr thIest i th CHOD cel Is that4 ware~ growin expone ti al wer e seeded on 60 mmi tissue C1ture pt~ji dishe (3 1 clsA

4-bova ne serum nd 0m m o etm and - nCbted in4i a hUitmsid ee o COzand 5 ai t_7C wny or

~hours I ater-ns tofIa toe srlte a~4de s e-1 CtLo final c~ on- 6ntrat 9 a 6tg )1 a nIot he 4r se L olaes receivedSangrea de rad6o~at n1Zoi 3 to of 0 00 at rdin

set- f tde r CracoG i nd nad-a he-eAi cio1S~n1a

treamen th the set of ~r1a~espl recaivd

-MredIIuM3 Pla1tes whr thniChtdf or and add~i t i nLI21

- ~ 1~ 4 -d4

4 U ~~W10ri1I1

(abVQCW rp ribte vea qiar s Iofe

pl4ateE~ d U Aue c fr iionWerL ndad

4 staied ihth lyrcrm nc fnSC techof - I AniqL

gtapine1Cand did -a a L1amage SO7 337-31 177-

The~~~~~~~~~~ na reu~ 5adwa b ev tar h

PofiteTIALomtiES OF ha RAOAnDgtAP as inSGR~D the r-ntar

4L~~oth sinfcnlwpe sed the ~~ 4 4sr1n

eol~o-at rsIlp-hFrmthe nexperimesdescryibe ad e e cocV ta Taspin-HClt anFsc6 Sanred WeeksddntP-dC hO~on

cr i no~a Sytemhand the t0dw uedoensi wee fMI owe i i Iat

aLs CDhthl-Toheon dac~n~eribed bypurd usld 71

j~nO~s1 a I a TClaoa

Kl-Ten krianto A SN - eesCE use 1 s - 1 a d N t OILSIi~sce op~d

Thbmaledeany1phrb M~c etate moths~ ofri the~wre shcet1aed i re~js ig phas hair~ 6c usd Mceery honedUIof-th wereeap

p ppiand pfahe inweteator de adI~Di i triTheafer cahe nod ltamp~4were 30Pan(riasp e Leachr dxermnag~~(roup6o

compoud~ uonted as-anp-ltracexerininM tgtA ih bfrue t soIvPd

carcinogenicou~~W-nd 7n-12 ewt~ole- 0(-)shy-~~~h

x and i

n~~ (l I s

I Thegr o n tume~or roe r compound ~used wa 7 ra decGoylpup rbltreate~s- (TA and ocn a-o -tdNo

wasOLI 2~Ug -in ofL actn ctone- gt i- e

G-C resnt I No

Group---------------------------------------------------------------------------

GrOLIP~~~ 10nTo ~ 3 i 1 l-c~~ fI-D

p 5r I10 n D Ajn 1s c e~ e

42 twi cf wekl e ef

t v4 CL we e4

Io4M C)A~~cng C Or oup 0I~ of rTrAn oar3 MOi Q~~~~IaeoneHrF

GrUP1 Wau ~Our cotro o efomt ion of sp n r-fpr~mQ SGroup 2 i4as Ou coto fo asacrcngn cDrMnq

r-0 o WiuRm mno r~a n iLWwht~U~ ldshwasiry zec p~b 7snbu ltzgtwo~t our twi~o-tg raiognen r6~wtd ~

tr~satedDrlI3Awilhai ard TP~ op -as ~wo$king~5nue ot

ourNoy rou Sndangre de Gnoranai Group VWithr ~~ts r 4 t -1C - acng

r n~dLndPh~fP~P e as 11

atou 7hend b for acarcnoge

h or Tasp n

Tho e )02 prtfrpd c i 2nd aneioMet er a on~ mbe e 190 vr

trpeetned _~ s nTAa n trwihDB dcr a m deloe

the pio~mast rekmonhs afther thji niLia n fthe treimn (2a oted of 294ba5ieay 1 rso rlice trearte wi thur-~d oleir o

of ~~~ ~ ~ ms~ ~ tju ~ ~ ~ i~ pe-~t ~ deeipdppnssa~d itA

no es w r oA4tp --4 d an

0

7t

J

22

-Grossge~i ti nowere sever a Iex e -p

GVL hcw a amintiom ma 94jf rom4~

by- hyep theao derm ppij aei wich er e ted Fo theI nthemi sufce he Ml gisa~ dc or inveIS aymtr~

igt beae4ikr aa~hs L wit r-ccnLgt (te hyekrami

a t om o f c n a s io y t T e r mj i- b i I

mainn chngs Th Kn4--oran did pr4n pah~oi

FFSiieii a e ca c n gei or tino promoters-

f ia e n in ocotty tfe eX p rim6 D it IFi aIhc I-54 SiAL-4 mo th in or r toi4i be i co c u F i ce I- mn ii iF

i ~~ t4Ie~ ~ os4iL-ig ~ ~ hL ~ ~iie ~ ~ v 4~ ~ ~ g Oilt494hrta ~h ra ~ wi~j theknow ogn sA~rtd t 3 doelo tv tmrcari DB

on mo th ag

4 4 ~

4 ONai

4CONCLUSIONS

rSnr de Grado etracted f romp ICiton 7er

o extracts fr-oT1 th~ecore~ fri in ofth Crotor lechleri tree

Thr4ure idtein Or trc EITOit in the ieavesu of thebAci~t n I 1eriand the most bkidant one was ~

T~asp inewas h cti O 1teri za tp i ncn 1 L-p e s en1 i Sa~ngre4 de Gracdo etracted from Croto hl1ech14er

~h Lid healin process~ r I

1~~~fibrobasts atonceritrtions1 be1llow2)- nOgm1 ard i t di d e Ih1 PEr at ion~not uincrease ratE1Qf-11P

w fon nt~h ibit the contac i on61

hnn f orEsk i ni i a fibr 0b I as

6~mutagunicitY 1tud using the 4rnes 4tcst showe htSJrgshydeGad a wh le taspine chloydrate hadttrcco k

~~ ~no Jn~ta ernii activity I1

StUdies on e ef f eect4 - Ire derao andAsnt A-t oBancr adpCdnd

ychorhdrteortisister choai eklnp (SCE) showe t I4bsTaspine hlrydrate increased the nIumber of SC[ 3in

V79if4 ellbt no increase wa5 abservedinCOcls 10Uigthe tW-tg O skincriogee syste w e V

4 14he shown that~4nri Uher Sng a ir tppeIJ1 were carci nb~geniviardor -tumor promtr f te n0ya +iitrea t4ment 1

--- --- --- --- --- --- ---

0 11

FXTROTrONOOS9NJRF DE GRA1DO FRMDFgRE T RE P1)0tITUITSC DING -m RSTTRP

TREETPg~ OF] PH ~VOLiUMEK OF~ YILIL - XT I ON E--~ Ig11TV~ XTROCTION

6-- 91 -~agm ~ mJ-1 -- ~ 22 272

M- 5ql ar-To each of the sample wre addced tJ n - -qri

~~~~e alktalinized to p 9 wth amn~iui hydjroXidftt~eJ~h and extatede0hautv- wie~t~h choroform Each clooorm extractwa concentrated i n a rotavapor then dryed and -the~~shy

ht 0os theE 6k gaIkaIoidvr eaU d i napl- alI7 24 -065j-~9-- ~

C) Iml ofec f Lhe 5ape wer Alded Ijlo ae th as t e aI kalinized-to pH 92wtha

etract iasdred and hei redissolved i 1 mo-o lLlou and0 1e bobnamp t347lt was merLrdL~igaZi

Spe ro fotome er-jThextinction coefficient of taspine at 47 i

a si gni i can t di FF$ernce2 between thee gbFUPS anthegOUS-n

The col lact n datE wJas Dec mber~ 19 1901

-- ---Y -- ~4A shy- ~----

I A

4 AB 2 ~4

q 7

EXRCIN F AGE1 1ACFOM -ER RL RMIU

~4 1 4-112 lt 4 44 4

44 42 I-4lt C) 6

- ~~ ~ ~ ~ ~ ~~44-- -- -- ----4 -shy~~ ~~~4441533c~ de Grado was co lced fo t e te s nm rae hc

on ebr 1 15and 5 a d n M y 2 90 h I a m- dary 19

4tet4wa d -4 e44 de 11o etha To~4lt4I ml 4~44 each 4 44444 were ed of wa eof o ~ th sV ad I4~ l th

mixturwa al t 4lt4 yrxd -iUPH~ 9 IVizp and 4l~444 wi- 4 o c rfextr c~e l a~~iv 4 h 4aof 4ofri E4a4 h

r-~ lt4~4 444 ~ 44 ~44 4) gt44

Xh e~= 2 8c tr-ts 0 da gr7380frI~ aawas 1+C

wasra sign~ficat diiffrec betwee the to ~grup Plt 0

i 44 ~ f~cho ~ h ui 1 4 l V~n11~9 7 rW hnaIdiin 4 hdoidlt465 W~qamp

T

22

I~~_7

control (rD 11

SG 51 1 1 1 8

Tabpie tm11C () r 71 0 1

Obrevi itions WS = WUndbeakinsteghSIG =Sargre de Grado 2

STaspine-HICI = Taspirn chlrhydrate

Nce wepre anesthesized with vapors fdiethyl ete and a~ IcM A ci ion as made perpendi CIler _to the ais fsmfnmet-y f tea i anid thtwo4borders of the Wo~nd werestichdtgte tiscntr

g~tete ~ n~p~vry 12 hOrs the comfpol-tbeinQt~ted Ias~ap llied Ato the w~ound -For thi p~pGUQXn M2Ishy

mealt p m crop 1diwpe 4 ddrj1 Veins a WacLIndy h ant-h otosrrcie ny

~Fnt iii2which liheror6 wa dtdI1 horst ~ pib elte

f gone of the borders of t~he on aFtr 1-itinLh t hle4 g ret h other onet For moeJh tai _hi p in a 2MeaSLrable_ fore teoe hmred

t 1i anlYi S~ ohtps thUi ng ~ s sh2tt w d2itt ic aie

di erence betwepri Aher can Lrol grouip and tgtheSG-g roup (p0nO5 -n bewenr theQcntrol pU idLPtpi6Hl L(p C CI)i1

fr C an 22gn 12 2

4 U ~~W10ri1I1

(abVQCW rp ribte vea qiar s Iofe

pl4ateE~ d U Aue c fr iionWerL ndad

4 staied ihth lyrcrm nc fnSC techof - I AniqL

gtapine1Cand did -a a L1amage SO7 337-31 177-

The~~~~~~~~~~ na reu~ 5adwa b ev tar h

PofiteTIALomtiES OF ha RAOAnDgtAP as inSGR~D the r-ntar

4L~~oth sinfcnlwpe sed the ~~ 4 4sr1n

eol~o-at rsIlp-hFrmthe nexperimesdescryibe ad e e cocV ta Taspin-HClt anFsc6 Sanred WeeksddntP-dC hO~on

cr i no~a Sytemhand the t0dw uedoensi wee fMI owe i i Iat

aLs CDhthl-Toheon dac~n~eribed bypurd usld 71

j~nO~s1 a I a TClaoa

Kl-Ten krianto A SN - eesCE use 1 s - 1 a d N t OILSIi~sce op~d

Thbmaledeany1phrb M~c etate moths~ ofri the~wre shcet1aed i re~js ig phas hair~ 6c usd Mceery honedUIof-th wereeap

p ppiand pfahe inweteator de adI~Di i triTheafer cahe nod ltamp~4were 30Pan(riasp e Leachr dxermnag~~(roup6o

compoud~ uonted as-anp-ltracexerininM tgtA ih bfrue t soIvPd

carcinogenicou~~W-nd 7n-12 ewt~ole- 0(-)shy-~~~h

x and i

n~~ (l I s

I Thegr o n tume~or roe r compound ~used wa 7 ra decGoylpup rbltreate~s- (TA and ocn a-o -tdNo

wasOLI 2~Ug -in ofL actn ctone- gt i- e

G-C resnt I No

Group---------------------------------------------------------------------------

GrOLIP~~~ 10nTo ~ 3 i 1 l-c~~ fI-D

p 5r I10 n D Ajn 1s c e~ e

42 twi cf wekl e ef

t v4 CL we e4

Io4M C)A~~cng C Or oup 0I~ of rTrAn oar3 MOi Q~~~~IaeoneHrF

GrUP1 Wau ~Our cotro o efomt ion of sp n r-fpr~mQ SGroup 2 i4as Ou coto fo asacrcngn cDrMnq

r-0 o WiuRm mno r~a n iLWwht~U~ ldshwasiry zec p~b 7snbu ltzgtwo~t our twi~o-tg raiognen r6~wtd ~

tr~satedDrlI3Awilhai ard TP~ op -as ~wo$king~5nue ot

ourNoy rou Sndangre de Gnoranai Group VWithr ~~ts r 4 t -1C - acng

r n~dLndPh~fP~P e as 11

atou 7hend b for acarcnoge

h or Tasp n

Tho e )02 prtfrpd c i 2nd aneioMet er a on~ mbe e 190 vr

trpeetned _~ s nTAa n trwihDB dcr a m deloe

the pio~mast rekmonhs afther thji niLia n fthe treimn (2a oted of 294ba5ieay 1 rso rlice trearte wi thur-~d oleir o

of ~~~ ~ ~ ms~ ~ tju ~ ~ ~ i~ pe-~t ~ deeipdppnssa~d itA

no es w r oA4tp --4 d an

0

7t

J

22

-Grossge~i ti nowere sever a Iex e -p

GVL hcw a amintiom ma 94jf rom4~

by- hyep theao derm ppij aei wich er e ted Fo theI nthemi sufce he Ml gisa~ dc or inveIS aymtr~

igt beae4ikr aa~hs L wit r-ccnLgt (te hyekrami

a t om o f c n a s io y t T e r mj i- b i I

mainn chngs Th Kn4--oran did pr4n pah~oi

FFSiieii a e ca c n gei or tino promoters-

f ia e n in ocotty tfe eX p rim6 D it IFi aIhc I-54 SiAL-4 mo th in or r toi4i be i co c u F i ce I- mn ii iF

i ~~ t4Ie~ ~ os4iL-ig ~ ~ hL ~ ~iie ~ ~ v 4~ ~ ~ g Oilt494hrta ~h ra ~ wi~j theknow ogn sA~rtd t 3 doelo tv tmrcari DB

on mo th ag

4 4 ~

4 ONai

4CONCLUSIONS

rSnr de Grado etracted f romp ICiton 7er

o extracts fr-oT1 th~ecore~ fri in ofth Crotor lechleri tree

Thr4ure idtein Or trc EITOit in the ieavesu of thebAci~t n I 1eriand the most bkidant one was ~

T~asp inewas h cti O 1teri za tp i ncn 1 L-p e s en1 i Sa~ngre4 de Gracdo etracted from Croto hl1ech14er

~h Lid healin process~ r I

1~~~fibrobasts atonceritrtions1 be1llow2)- nOgm1 ard i t di d e Ih1 PEr at ion~not uincrease ratE1Qf-11P

w fon nt~h ibit the contac i on61

hnn f orEsk i ni i a fibr 0b I as

6~mutagunicitY 1tud using the 4rnes 4tcst showe htSJrgshydeGad a wh le taspine chloydrate hadttrcco k

~~ ~no Jn~ta ernii activity I1

StUdies on e ef f eect4 - Ire derao andAsnt A-t oBancr adpCdnd

ychorhdrteortisister choai eklnp (SCE) showe t I4bsTaspine hlrydrate increased the nIumber of SC[ 3in

V79if4 ellbt no increase wa5 abservedinCOcls 10Uigthe tW-tg O skincriogee syste w e V

4 14he shown that~4nri Uher Sng a ir tppeIJ1 were carci nb~geniviardor -tumor promtr f te n0ya +iitrea t4ment 1

--- --- --- --- --- --- ---

0 11

FXTROTrONOOS9NJRF DE GRA1DO FRMDFgRE T RE P1)0tITUITSC DING -m RSTTRP

TREETPg~ OF] PH ~VOLiUMEK OF~ YILIL - XT I ON E--~ Ig11TV~ XTROCTION

6-- 91 -~agm ~ mJ-1 -- ~ 22 272

M- 5ql ar-To each of the sample wre addced tJ n - -qri

~~~~e alktalinized to p 9 wth amn~iui hydjroXidftt~eJ~h and extatede0hautv- wie~t~h choroform Each clooorm extractwa concentrated i n a rotavapor then dryed and -the~~shy

ht 0os theE 6k gaIkaIoidvr eaU d i napl- alI7 24 -065j-~9-- ~

C) Iml ofec f Lhe 5ape wer Alded Ijlo ae th as t e aI kalinized-to pH 92wtha

etract iasdred and hei redissolved i 1 mo-o lLlou and0 1e bobnamp t347lt was merLrdL~igaZi

Spe ro fotome er-jThextinction coefficient of taspine at 47 i

a si gni i can t di FF$ernce2 between thee gbFUPS anthegOUS-n

The col lact n datE wJas Dec mber~ 19 1901

-- ---Y -- ~4A shy- ~----

I A

4 AB 2 ~4

q 7

EXRCIN F AGE1 1ACFOM -ER RL RMIU

~4 1 4-112 lt 4 44 4

44 42 I-4lt C) 6

- ~~ ~ ~ ~ ~ ~~44-- -- -- ----4 -shy~~ ~~~4441533c~ de Grado was co lced fo t e te s nm rae hc

on ebr 1 15and 5 a d n M y 2 90 h I a m- dary 19

4tet4wa d -4 e44 de 11o etha To~4lt4I ml 4~44 each 4 44444 were ed of wa eof o ~ th sV ad I4~ l th

mixturwa al t 4lt4 yrxd -iUPH~ 9 IVizp and 4l~444 wi- 4 o c rfextr c~e l a~~iv 4 h 4aof 4ofri E4a4 h

r-~ lt4~4 444 ~ 44 ~44 4) gt44

Xh e~= 2 8c tr-ts 0 da gr7380frI~ aawas 1+C

wasra sign~ficat diiffrec betwee the to ~grup Plt 0

i 44 ~ f~cho ~ h ui 1 4 l V~n11~9 7 rW hnaIdiin 4 hdoidlt465 W~qamp

T

22

I~~_7

control (rD 11

SG 51 1 1 1 8

Tabpie tm11C () r 71 0 1

Obrevi itions WS = WUndbeakinsteghSIG =Sargre de Grado 2

STaspine-HICI = Taspirn chlrhydrate

Nce wepre anesthesized with vapors fdiethyl ete and a~ IcM A ci ion as made perpendi CIler _to the ais fsmfnmet-y f tea i anid thtwo4borders of the Wo~nd werestichdtgte tiscntr

g~tete ~ n~p~vry 12 hOrs the comfpol-tbeinQt~ted Ias~ap llied Ato the w~ound -For thi p~pGUQXn M2Ishy

mealt p m crop 1diwpe 4 ddrj1 Veins a WacLIndy h ant-h otosrrcie ny

~Fnt iii2which liheror6 wa dtdI1 horst ~ pib elte

f gone of the borders of t~he on aFtr 1-itinLh t hle4 g ret h other onet For moeJh tai _hi p in a 2MeaSLrable_ fore teoe hmred

t 1i anlYi S~ ohtps thUi ng ~ s sh2tt w d2itt ic aie

di erence betwepri Aher can Lrol grouip and tgtheSG-g roup (p0nO5 -n bewenr theQcntrol pU idLPtpi6Hl L(p C CI)i1

fr C an 22gn 12 2

p 5r I10 n D Ajn 1s c e~ e

42 twi cf wekl e ef

t v4 CL we e4

Io4M C)A~~cng C Or oup 0I~ of rTrAn oar3 MOi Q~~~~IaeoneHrF

GrUP1 Wau ~Our cotro o efomt ion of sp n r-fpr~mQ SGroup 2 i4as Ou coto fo asacrcngn cDrMnq

r-0 o WiuRm mno r~a n iLWwht~U~ ldshwasiry zec p~b 7snbu ltzgtwo~t our twi~o-tg raiognen r6~wtd ~

tr~satedDrlI3Awilhai ard TP~ op -as ~wo$king~5nue ot

ourNoy rou Sndangre de Gnoranai Group VWithr ~~ts r 4 t -1C - acng

r n~dLndPh~fP~P e as 11

atou 7hend b for acarcnoge

h or Tasp n

Tho e )02 prtfrpd c i 2nd aneioMet er a on~ mbe e 190 vr

trpeetned _~ s nTAa n trwihDB dcr a m deloe

the pio~mast rekmonhs afther thji niLia n fthe treimn (2a oted of 294ba5ieay 1 rso rlice trearte wi thur-~d oleir o

of ~~~ ~ ~ ms~ ~ tju ~ ~ ~ i~ pe-~t ~ deeipdppnssa~d itA

no es w r oA4tp --4 d an

0

7t

J

22

-Grossge~i ti nowere sever a Iex e -p

GVL hcw a amintiom ma 94jf rom4~

by- hyep theao derm ppij aei wich er e ted Fo theI nthemi sufce he Ml gisa~ dc or inveIS aymtr~

igt beae4ikr aa~hs L wit r-ccnLgt (te hyekrami

a t om o f c n a s io y t T e r mj i- b i I

mainn chngs Th Kn4--oran did pr4n pah~oi

FFSiieii a e ca c n gei or tino promoters-

f ia e n in ocotty tfe eX p rim6 D it IFi aIhc I-54 SiAL-4 mo th in or r toi4i be i co c u F i ce I- mn ii iF

i ~~ t4Ie~ ~ os4iL-ig ~ ~ hL ~ ~iie ~ ~ v 4~ ~ ~ g Oilt494hrta ~h ra ~ wi~j theknow ogn sA~rtd t 3 doelo tv tmrcari DB

on mo th ag

4 4 ~

4 ONai

4CONCLUSIONS

rSnr de Grado etracted f romp ICiton 7er

o extracts fr-oT1 th~ecore~ fri in ofth Crotor lechleri tree

Thr4ure idtein Or trc EITOit in the ieavesu of thebAci~t n I 1eriand the most bkidant one was ~

T~asp inewas h cti O 1teri za tp i ncn 1 L-p e s en1 i Sa~ngre4 de Gracdo etracted from Croto hl1ech14er

~h Lid healin process~ r I

1~~~fibrobasts atonceritrtions1 be1llow2)- nOgm1 ard i t di d e Ih1 PEr at ion~not uincrease ratE1Qf-11P

w fon nt~h ibit the contac i on61

hnn f orEsk i ni i a fibr 0b I as

6~mutagunicitY 1tud using the 4rnes 4tcst showe htSJrgshydeGad a wh le taspine chloydrate hadttrcco k

~~ ~no Jn~ta ernii activity I1

StUdies on e ef f eect4 - Ire derao andAsnt A-t oBancr adpCdnd

ychorhdrteortisister choai eklnp (SCE) showe t I4bsTaspine hlrydrate increased the nIumber of SC[ 3in

V79if4 ellbt no increase wa5 abservedinCOcls 10Uigthe tW-tg O skincriogee syste w e V

4 14he shown that~4nri Uher Sng a ir tppeIJ1 were carci nb~geniviardor -tumor promtr f te n0ya +iitrea t4ment 1

--- --- --- --- --- --- ---

0 11

FXTROTrONOOS9NJRF DE GRA1DO FRMDFgRE T RE P1)0tITUITSC DING -m RSTTRP

TREETPg~ OF] PH ~VOLiUMEK OF~ YILIL - XT I ON E--~ Ig11TV~ XTROCTION

6-- 91 -~agm ~ mJ-1 -- ~ 22 272

M- 5ql ar-To each of the sample wre addced tJ n - -qri

~~~~e alktalinized to p 9 wth amn~iui hydjroXidftt~eJ~h and extatede0hautv- wie~t~h choroform Each clooorm extractwa concentrated i n a rotavapor then dryed and -the~~shy

ht 0os theE 6k gaIkaIoidvr eaU d i napl- alI7 24 -065j-~9-- ~

C) Iml ofec f Lhe 5ape wer Alded Ijlo ae th as t e aI kalinized-to pH 92wtha

etract iasdred and hei redissolved i 1 mo-o lLlou and0 1e bobnamp t347lt was merLrdL~igaZi

Spe ro fotome er-jThextinction coefficient of taspine at 47 i

a si gni i can t di FF$ernce2 between thee gbFUPS anthegOUS-n

The col lact n datE wJas Dec mber~ 19 1901

-- ---Y -- ~4A shy- ~----

I A

4 AB 2 ~4

q 7

EXRCIN F AGE1 1ACFOM -ER RL RMIU

~4 1 4-112 lt 4 44 4

44 42 I-4lt C) 6

- ~~ ~ ~ ~ ~ ~~44-- -- -- ----4 -shy~~ ~~~4441533c~ de Grado was co lced fo t e te s nm rae hc

on ebr 1 15and 5 a d n M y 2 90 h I a m- dary 19

4tet4wa d -4 e44 de 11o etha To~4lt4I ml 4~44 each 4 44444 were ed of wa eof o ~ th sV ad I4~ l th

mixturwa al t 4lt4 yrxd -iUPH~ 9 IVizp and 4l~444 wi- 4 o c rfextr c~e l a~~iv 4 h 4aof 4ofri E4a4 h

r-~ lt4~4 444 ~ 44 ~44 4) gt44

Xh e~= 2 8c tr-ts 0 da gr7380frI~ aawas 1+C

wasra sign~ficat diiffrec betwee the to ~grup Plt 0

i 44 ~ f~cho ~ h ui 1 4 l V~n11~9 7 rW hnaIdiin 4 hdoidlt465 W~qamp

T

22

I~~_7

control (rD 11

SG 51 1 1 1 8

Tabpie tm11C () r 71 0 1

Obrevi itions WS = WUndbeakinsteghSIG =Sargre de Grado 2

STaspine-HICI = Taspirn chlrhydrate

Nce wepre anesthesized with vapors fdiethyl ete and a~ IcM A ci ion as made perpendi CIler _to the ais fsmfnmet-y f tea i anid thtwo4borders of the Wo~nd werestichdtgte tiscntr

g~tete ~ n~p~vry 12 hOrs the comfpol-tbeinQt~ted Ias~ap llied Ato the w~ound -For thi p~pGUQXn M2Ishy

mealt p m crop 1diwpe 4 ddrj1 Veins a WacLIndy h ant-h otosrrcie ny

~Fnt iii2which liheror6 wa dtdI1 horst ~ pib elte

f gone of the borders of t~he on aFtr 1-itinLh t hle4 g ret h other onet For moeJh tai _hi p in a 2MeaSLrable_ fore teoe hmred

t 1i anlYi S~ ohtps thUi ng ~ s sh2tt w d2itt ic aie

di erence betwepri Aher can Lrol grouip and tgtheSG-g roup (p0nO5 -n bewenr theQcntrol pU idLPtpi6Hl L(p C CI)i1

fr C an 22gn 12 2

0

7t

J

22

-Grossge~i ti nowere sever a Iex e -p

GVL hcw a amintiom ma 94jf rom4~

by- hyep theao derm ppij aei wich er e ted Fo theI nthemi sufce he Ml gisa~ dc or inveIS aymtr~

igt beae4ikr aa~hs L wit r-ccnLgt (te hyekrami

a t om o f c n a s io y t T e r mj i- b i I

mainn chngs Th Kn4--oran did pr4n pah~oi

FFSiieii a e ca c n gei or tino promoters-

f ia e n in ocotty tfe eX p rim6 D it IFi aIhc I-54 SiAL-4 mo th in or r toi4i be i co c u F i ce I- mn ii iF

i ~~ t4Ie~ ~ os4iL-ig ~ ~ hL ~ ~iie ~ ~ v 4~ ~ ~ g Oilt494hrta ~h ra ~ wi~j theknow ogn sA~rtd t 3 doelo tv tmrcari DB

on mo th ag

4 4 ~

4 ONai

4CONCLUSIONS

rSnr de Grado etracted f romp ICiton 7er

o extracts fr-oT1 th~ecore~ fri in ofth Crotor lechleri tree

Thr4ure idtein Or trc EITOit in the ieavesu of thebAci~t n I 1eriand the most bkidant one was ~

T~asp inewas h cti O 1teri za tp i ncn 1 L-p e s en1 i Sa~ngre4 de Gracdo etracted from Croto hl1ech14er

~h Lid healin process~ r I

1~~~fibrobasts atonceritrtions1 be1llow2)- nOgm1 ard i t di d e Ih1 PEr at ion~not uincrease ratE1Qf-11P

w fon nt~h ibit the contac i on61

hnn f orEsk i ni i a fibr 0b I as

6~mutagunicitY 1tud using the 4rnes 4tcst showe htSJrgshydeGad a wh le taspine chloydrate hadttrcco k

~~ ~no Jn~ta ernii activity I1

StUdies on e ef f eect4 - Ire derao andAsnt A-t oBancr adpCdnd

ychorhdrteortisister choai eklnp (SCE) showe t I4bsTaspine hlrydrate increased the nIumber of SC[ 3in

V79if4 ellbt no increase wa5 abservedinCOcls 10Uigthe tW-tg O skincriogee syste w e V

4 14he shown that~4nri Uher Sng a ir tppeIJ1 were carci nb~geniviardor -tumor promtr f te n0ya +iitrea t4ment 1

--- --- --- --- --- --- ---

0 11

FXTROTrONOOS9NJRF DE GRA1DO FRMDFgRE T RE P1)0tITUITSC DING -m RSTTRP

TREETPg~ OF] PH ~VOLiUMEK OF~ YILIL - XT I ON E--~ Ig11TV~ XTROCTION

6-- 91 -~agm ~ mJ-1 -- ~ 22 272

M- 5ql ar-To each of the sample wre addced tJ n - -qri

~~~~e alktalinized to p 9 wth amn~iui hydjroXidftt~eJ~h and extatede0hautv- wie~t~h choroform Each clooorm extractwa concentrated i n a rotavapor then dryed and -the~~shy

ht 0os theE 6k gaIkaIoidvr eaU d i napl- alI7 24 -065j-~9-- ~

C) Iml ofec f Lhe 5ape wer Alded Ijlo ae th as t e aI kalinized-to pH 92wtha

etract iasdred and hei redissolved i 1 mo-o lLlou and0 1e bobnamp t347lt was merLrdL~igaZi

Spe ro fotome er-jThextinction coefficient of taspine at 47 i

a si gni i can t di FF$ernce2 between thee gbFUPS anthegOUS-n

The col lact n datE wJas Dec mber~ 19 1901

-- ---Y -- ~4A shy- ~----

I A

4 AB 2 ~4

q 7

EXRCIN F AGE1 1ACFOM -ER RL RMIU

~4 1 4-112 lt 4 44 4

44 42 I-4lt C) 6

- ~~ ~ ~ ~ ~ ~~44-- -- -- ----4 -shy~~ ~~~4441533c~ de Grado was co lced fo t e te s nm rae hc

on ebr 1 15and 5 a d n M y 2 90 h I a m- dary 19

4tet4wa d -4 e44 de 11o etha To~4lt4I ml 4~44 each 4 44444 were ed of wa eof o ~ th sV ad I4~ l th

mixturwa al t 4lt4 yrxd -iUPH~ 9 IVizp and 4l~444 wi- 4 o c rfextr c~e l a~~iv 4 h 4aof 4ofri E4a4 h

r-~ lt4~4 444 ~ 44 ~44 4) gt44

Xh e~= 2 8c tr-ts 0 da gr7380frI~ aawas 1+C

wasra sign~ficat diiffrec betwee the to ~grup Plt 0

i 44 ~ f~cho ~ h ui 1 4 l V~n11~9 7 rW hnaIdiin 4 hdoidlt465 W~qamp

T

22

I~~_7

control (rD 11

SG 51 1 1 1 8

Tabpie tm11C () r 71 0 1

Obrevi itions WS = WUndbeakinsteghSIG =Sargre de Grado 2

STaspine-HICI = Taspirn chlrhydrate

Nce wepre anesthesized with vapors fdiethyl ete and a~ IcM A ci ion as made perpendi CIler _to the ais fsmfnmet-y f tea i anid thtwo4borders of the Wo~nd werestichdtgte tiscntr

g~tete ~ n~p~vry 12 hOrs the comfpol-tbeinQt~ted Ias~ap llied Ato the w~ound -For thi p~pGUQXn M2Ishy

mealt p m crop 1diwpe 4 ddrj1 Veins a WacLIndy h ant-h otosrrcie ny

~Fnt iii2which liheror6 wa dtdI1 horst ~ pib elte

f gone of the borders of t~he on aFtr 1-itinLh t hle4 g ret h other onet For moeJh tai _hi p in a 2MeaSLrable_ fore teoe hmred

t 1i anlYi S~ ohtps thUi ng ~ s sh2tt w d2itt ic aie

di erence betwepri Aher can Lrol grouip and tgtheSG-g roup (p0nO5 -n bewenr theQcntrol pU idLPtpi6Hl L(p C CI)i1

fr C an 22gn 12 2

4 ONai

4CONCLUSIONS

rSnr de Grado etracted f romp ICiton 7er

o extracts fr-oT1 th~ecore~ fri in ofth Crotor lechleri tree

Thr4ure idtein Or trc EITOit in the ieavesu of thebAci~t n I 1eriand the most bkidant one was ~

T~asp inewas h cti O 1teri za tp i ncn 1 L-p e s en1 i Sa~ngre4 de Gracdo etracted from Croto hl1ech14er

~h Lid healin process~ r I

1~~~fibrobasts atonceritrtions1 be1llow2)- nOgm1 ard i t di d e Ih1 PEr at ion~not uincrease ratE1Qf-11P

w fon nt~h ibit the contac i on61

hnn f orEsk i ni i a fibr 0b I as

6~mutagunicitY 1tud using the 4rnes 4tcst showe htSJrgshydeGad a wh le taspine chloydrate hadttrcco k

~~ ~no Jn~ta ernii activity I1

StUdies on e ef f eect4 - Ire derao andAsnt A-t oBancr adpCdnd

ychorhdrteortisister choai eklnp (SCE) showe t I4bsTaspine hlrydrate increased the nIumber of SC[ 3in

V79if4 ellbt no increase wa5 abservedinCOcls 10Uigthe tW-tg O skincriogee syste w e V

4 14he shown that~4nri Uher Sng a ir tppeIJ1 were carci nb~geniviardor -tumor promtr f te n0ya +iitrea t4ment 1

--- --- --- --- --- --- ---

0 11

FXTROTrONOOS9NJRF DE GRA1DO FRMDFgRE T RE P1)0tITUITSC DING -m RSTTRP

TREETPg~ OF] PH ~VOLiUMEK OF~ YILIL - XT I ON E--~ Ig11TV~ XTROCTION

6-- 91 -~agm ~ mJ-1 -- ~ 22 272

M- 5ql ar-To each of the sample wre addced tJ n - -qri

~~~~e alktalinized to p 9 wth amn~iui hydjroXidftt~eJ~h and extatede0hautv- wie~t~h choroform Each clooorm extractwa concentrated i n a rotavapor then dryed and -the~~shy

ht 0os theE 6k gaIkaIoidvr eaU d i napl- alI7 24 -065j-~9-- ~

C) Iml ofec f Lhe 5ape wer Alded Ijlo ae th as t e aI kalinized-to pH 92wtha

etract iasdred and hei redissolved i 1 mo-o lLlou and0 1e bobnamp t347lt was merLrdL~igaZi

Spe ro fotome er-jThextinction coefficient of taspine at 47 i

a si gni i can t di FF$ernce2 between thee gbFUPS anthegOUS-n

The col lact n datE wJas Dec mber~ 19 1901

-- ---Y -- ~4A shy- ~----

I A

4 AB 2 ~4

q 7

EXRCIN F AGE1 1ACFOM -ER RL RMIU

~4 1 4-112 lt 4 44 4

44 42 I-4lt C) 6

- ~~ ~ ~ ~ ~ ~~44-- -- -- ----4 -shy~~ ~~~4441533c~ de Grado was co lced fo t e te s nm rae hc

on ebr 1 15and 5 a d n M y 2 90 h I a m- dary 19

4tet4wa d -4 e44 de 11o etha To~4lt4I ml 4~44 each 4 44444 were ed of wa eof o ~ th sV ad I4~ l th

mixturwa al t 4lt4 yrxd -iUPH~ 9 IVizp and 4l~444 wi- 4 o c rfextr c~e l a~~iv 4 h 4aof 4ofri E4a4 h

r-~ lt4~4 444 ~ 44 ~44 4) gt44

Xh e~= 2 8c tr-ts 0 da gr7380frI~ aawas 1+C

wasra sign~ficat diiffrec betwee the to ~grup Plt 0

i 44 ~ f~cho ~ h ui 1 4 l V~n11~9 7 rW hnaIdiin 4 hdoidlt465 W~qamp

T

22

I~~_7

control (rD 11

SG 51 1 1 1 8

Tabpie tm11C () r 71 0 1

Obrevi itions WS = WUndbeakinsteghSIG =Sargre de Grado 2

STaspine-HICI = Taspirn chlrhydrate

Nce wepre anesthesized with vapors fdiethyl ete and a~ IcM A ci ion as made perpendi CIler _to the ais fsmfnmet-y f tea i anid thtwo4borders of the Wo~nd werestichdtgte tiscntr

g~tete ~ n~p~vry 12 hOrs the comfpol-tbeinQt~ted Ias~ap llied Ato the w~ound -For thi p~pGUQXn M2Ishy

mealt p m crop 1diwpe 4 ddrj1 Veins a WacLIndy h ant-h otosrrcie ny

~Fnt iii2which liheror6 wa dtdI1 horst ~ pib elte

f gone of the borders of t~he on aFtr 1-itinLh t hle4 g ret h other onet For moeJh tai _hi p in a 2MeaSLrable_ fore teoe hmred

t 1i anlYi S~ ohtps thUi ng ~ s sh2tt w d2itt ic aie

di erence betwepri Aher can Lrol grouip and tgtheSG-g roup (p0nO5 -n bewenr theQcntrol pU idLPtpi6Hl L(p C CI)i1

fr C an 22gn 12 2

--- --- --- --- --- --- ---

0 11

FXTROTrONOOS9NJRF DE GRA1DO FRMDFgRE T RE P1)0tITUITSC DING -m RSTTRP

TREETPg~ OF] PH ~VOLiUMEK OF~ YILIL - XT I ON E--~ Ig11TV~ XTROCTION

6-- 91 -~agm ~ mJ-1 -- ~ 22 272

M- 5ql ar-To each of the sample wre addced tJ n - -qri

~~~~e alktalinized to p 9 wth amn~iui hydjroXidftt~eJ~h and extatede0hautv- wie~t~h choroform Each clooorm extractwa concentrated i n a rotavapor then dryed and -the~~shy

ht 0os theE 6k gaIkaIoidvr eaU d i napl- alI7 24 -065j-~9-- ~

C) Iml ofec f Lhe 5ape wer Alded Ijlo ae th as t e aI kalinized-to pH 92wtha

etract iasdred and hei redissolved i 1 mo-o lLlou and0 1e bobnamp t347lt was merLrdL~igaZi

Spe ro fotome er-jThextinction coefficient of taspine at 47 i

a si gni i can t di FF$ernce2 between thee gbFUPS anthegOUS-n

The col lact n datE wJas Dec mber~ 19 1901

-- ---Y -- ~4A shy- ~----

I A

4 AB 2 ~4

q 7

EXRCIN F AGE1 1ACFOM -ER RL RMIU

~4 1 4-112 lt 4 44 4

44 42 I-4lt C) 6

- ~~ ~ ~ ~ ~ ~~44-- -- -- ----4 -shy~~ ~~~4441533c~ de Grado was co lced fo t e te s nm rae hc

on ebr 1 15and 5 a d n M y 2 90 h I a m- dary 19

4tet4wa d -4 e44 de 11o etha To~4lt4I ml 4~44 each 4 44444 were ed of wa eof o ~ th sV ad I4~ l th

mixturwa al t 4lt4 yrxd -iUPH~ 9 IVizp and 4l~444 wi- 4 o c rfextr c~e l a~~iv 4 h 4aof 4ofri E4a4 h

r-~ lt4~4 444 ~ 44 ~44 4) gt44

Xh e~= 2 8c tr-ts 0 da gr7380frI~ aawas 1+C

wasra sign~ficat diiffrec betwee the to ~grup Plt 0

i 44 ~ f~cho ~ h ui 1 4 l V~n11~9 7 rW hnaIdiin 4 hdoidlt465 W~qamp

T

22

I~~_7

control (rD 11

SG 51 1 1 1 8

Tabpie tm11C () r 71 0 1

Obrevi itions WS = WUndbeakinsteghSIG =Sargre de Grado 2

STaspine-HICI = Taspirn chlrhydrate

Nce wepre anesthesized with vapors fdiethyl ete and a~ IcM A ci ion as made perpendi CIler _to the ais fsmfnmet-y f tea i anid thtwo4borders of the Wo~nd werestichdtgte tiscntr

g~tete ~ n~p~vry 12 hOrs the comfpol-tbeinQt~ted Ias~ap llied Ato the w~ound -For thi p~pGUQXn M2Ishy

mealt p m crop 1diwpe 4 ddrj1 Veins a WacLIndy h ant-h otosrrcie ny

~Fnt iii2which liheror6 wa dtdI1 horst ~ pib elte

f gone of the borders of t~he on aFtr 1-itinLh t hle4 g ret h other onet For moeJh tai _hi p in a 2MeaSLrable_ fore teoe hmred

t 1i anlYi S~ ohtps thUi ng ~ s sh2tt w d2itt ic aie

di erence betwepri Aher can Lrol grouip and tgtheSG-g roup (p0nO5 -n bewenr theQcntrol pU idLPtpi6Hl L(p C CI)i1

fr C an 22gn 12 2

I A

4 AB 2 ~4

q 7

EXRCIN F AGE1 1ACFOM -ER RL RMIU

~4 1 4-112 lt 4 44 4

44 42 I-4lt C) 6

- ~~ ~ ~ ~ ~ ~~44-- -- -- ----4 -shy~~ ~~~4441533c~ de Grado was co lced fo t e te s nm rae hc

on ebr 1 15and 5 a d n M y 2 90 h I a m- dary 19

4tet4wa d -4 e44 de 11o etha To~4lt4I ml 4~44 each 4 44444 were ed of wa eof o ~ th sV ad I4~ l th

mixturwa al t 4lt4 yrxd -iUPH~ 9 IVizp and 4l~444 wi- 4 o c rfextr c~e l a~~iv 4 h 4aof 4ofri E4a4 h

r-~ lt4~4 444 ~ 44 ~44 4) gt44

Xh e~= 2 8c tr-ts 0 da gr7380frI~ aawas 1+C

wasra sign~ficat diiffrec betwee the to ~grup Plt 0

i 44 ~ f~cho ~ h ui 1 4 l V~n11~9 7 rW hnaIdiin 4 hdoidlt465 W~qamp

T

22

I~~_7

control (rD 11

SG 51 1 1 1 8

Tabpie tm11C () r 71 0 1

Obrevi itions WS = WUndbeakinsteghSIG =Sargre de Grado 2

STaspine-HICI = Taspirn chlrhydrate

Nce wepre anesthesized with vapors fdiethyl ete and a~ IcM A ci ion as made perpendi CIler _to the ais fsmfnmet-y f tea i anid thtwo4borders of the Wo~nd werestichdtgte tiscntr

g~tete ~ n~p~vry 12 hOrs the comfpol-tbeinQt~ted Ias~ap llied Ato the w~ound -For thi p~pGUQXn M2Ishy

mealt p m crop 1diwpe 4 ddrj1 Veins a WacLIndy h ant-h otosrrcie ny

~Fnt iii2which liheror6 wa dtdI1 horst ~ pib elte

f gone of the borders of t~he on aFtr 1-itinLh t hle4 g ret h other onet For moeJh tai _hi p in a 2MeaSLrable_ fore teoe hmred

t 1i anlYi S~ ohtps thUi ng ~ s sh2tt w d2itt ic aie

di erence betwepri Aher can Lrol grouip and tgtheSG-g roup (p0nO5 -n bewenr theQcntrol pU idLPtpi6Hl L(p C CI)i1

fr C an 22gn 12 2

T

22

I~~_7

control (rD 11

SG 51 1 1 1 8

Tabpie tm11C () r 71 0 1

Obrevi itions WS = WUndbeakinsteghSIG =Sargre de Grado 2

STaspine-HICI = Taspirn chlrhydrate

Nce wepre anesthesized with vapors fdiethyl ete and a~ IcM A ci ion as made perpendi CIler _to the ais fsmfnmet-y f tea i anid thtwo4borders of the Wo~nd werestichdtgte tiscntr

g~tete ~ n~p~vry 12 hOrs the comfpol-tbeinQt~ted Ias~ap llied Ato the w~ound -For thi p~pGUQXn M2Ishy

mealt p m crop 1diwpe 4 ddrj1 Veins a WacLIndy h ant-h otosrrcie ny

~Fnt iii2which liheror6 wa dtdI1 horst ~ pib elte

f gone of the borders of t~he on aFtr 1-itinLh t hle4 g ret h other onet For moeJh tai _hi p in a 2MeaSLrable_ fore teoe hmred

t 1i anlYi S~ ohtps thUi ng ~ s sh2tt w d2itt ic aie

di erence betwepri Aher can Lrol grouip and tgtheSG-g roup (p0nO5 -n bewenr theQcntrol pU idLPtpi6Hl L(p C CI)i1

fr C an 22gn 12 2

DOSE-REPONS CU TI IE Cv IY0 7 N FETO S

0u 002 s 54slt p 7 6p 12 5 gt

o oQc 5 9 870 + 6 csss39 9

00o- 1V5 66 2 7 6 115

Taspis s~Ss5S neHQTsieclrhvydrate 5

gtat tLica Anal~ysis Usiriqthe Stidentr-t- tt showedshy

e(ltr~di f-Ypruce with the control was signifctv (P~h~lt00 )

Fo th ehd TeablIe 3and text In this~experimnt vie use~ 5

group e pe each V i

P ~ s2 ~S55535 fs 5

5 5

~

5s ho r 5w

11 10

204

Contro 12 21 4 + 13 y

Ta e I 2 S 6 A 1 6 (6p H

-TsieHI 24 24 G7

Co to I VI I31+

Coto 10 10-9~

con~645620 vF 4~39 5

Coolt 164 14E39 C)yA

Taspi ne HC 9~w~6 92- +

Taspine-~ 12 74 7+

Co5n-4~r o1144Z 1 19 C4N~ - M2~42295 A

Control 16813 w I5 2269f ~~

aspinbampHCI~ Tai5p i e r-hIotrfyVrate v-WD _wotynd_ breakingl 5t t -shy

=~The diFference with~ the conLrol wa signi ficative(A5ggtvL Thebe d rf-fen wi Lhtecnrlwssgi ativ e (Plt1(_)r-je 4 Iw

the7 hFor44 me d e Tb e and text I thi cx p- rer -4

0 6 mgml 14~

11 10

204

Contro 12 21 4 + 13 y

Ta e I 2 S 6 A 1 6 (6p H

-TsieHI 24 24 G7

Co to I VI I31+

Coto 10 10-9~

con~645620 vF 4~39 5

Coolt 164 14E39 C)yA

Taspi ne HC 9~w~6 92- +

Taspine-~ 12 74 7+

Co5n-4~r o1144Z 1 19 C4N~ - M2~42295 A

Control 16813 w I5 2269f ~~

aspinbampHCI~ Tai5p i e r-hIotrfyVrate v-WD _wotynd_ breakingl 5t t -shy

=~The diFference with~ the conLrol wa signi ficative(A5ggtvL Thebe d rf-fen wi Lhtecnrlwssgi ativ e (Plt1(_)r-je 4 Iw

the7 hFor44 me d e Tb e and text I thi cx p- rer -4

0 6 mgml 14~

47

FX~ tB A

-FORESKIN

IN -A

ONQ -THE FIROLA4IG

11

PROIFE

HiA rs

AT0CON OF R CETL

isoc

100~yz

12 -70

12 7(C

17

5

15

00

0

c1

25 C0~nd95~i~

126270( ~At12 hn

a~ s

I~cC) a 2

146 C)()(too~p oLsDt 0p)

e

711

d io t hi-inin

oroski 1

E

f brbstswer

- 0

i

fe

oc~lat

alTb

d

v

o 3

o e

M G

~ f

I

I(M

SURC4

e e C

--- -- -- - - - - -- - - - - - - - - - - - - - - - - - -- - -

L 44 Iv

t F~ROL TBL -7944fU

ngm 4e 9 No I s9 ~ Dead No CI9 ea

20 391 ()()0 2~5 O4)C)C

~ 99

99~99~9 9994

5C)2 9 3409

4

~ 9 o099 2A42 91C 0 C

99 ~~ k~ ~1 9 6 304j~o J o~ ~ o4492

10--)For sk n f b ob asti we~ E -9 9 35 diss

uOg olr dise wer incubaed at 37C a fra dif9 e

ulae mm fo CUI fL-tr

~atm1o pdeonear 95 e~t Weeport ttal IS p diff tJ~~h~ anirb At hors h r -eltn

pecetg of deadj -ce11i caleCUlated by he~tr ypan b lue excj usi on

Eah oint repre tsthe avrg outo hee lts

4919 ~ lt 9 4 9~

9~~4~94~~9 ~ J 999A

9~999999999 1~ 990

9 99 9 19 9 q- 799

--- -- -- - - - - -- - - - - - - - - - - - - - - - - - -- - -

L 44 Iv

t F~ROL TBL -7944fU

ngm 4e 9 No I s9 ~ Dead No CI9 ea

20 391 ()()0 2~5 O4)C)C

~ 99

99~99~9 9994

5C)2 9 3409

4

~ 9 o099 2A42 91C 0 C

99 ~~ k~ ~1 9 6 304j~o J o~ ~ o4492

10--)For sk n f b ob asti we~ E -9 9 35 diss

uOg olr dise wer incubaed at 37C a fra dif9 e

ulae mm fo CUI fL-tr

~atm1o pdeonear 95 e~t Weeport ttal IS p diff tJ~~h~ anirb At hors h r -eltn

pecetg of deadj -ce11i caleCUlated by he~tr ypan b lue excj usi on

Eah oint repre tsthe avrg outo hee lts

4919 ~ lt 9 4 9~

9~~4~94~~9 ~ J 999A

9~999999999 1~ 990

9 99 9 19 9 q- 799

0F EFECO 0S 1INE IiC1I ON THE PR9OLIFERATION OF SUBCONFLUENT

HUMAN FORE~fIN FI8FRODLASTS ~ cUTRE~S

J

5~ 01610Q

74 3 31 4

16 1)DOc t5 16 217 o(

ce

tmsh o 0~eof ~ ~ a~ i ~r AtP 18hustedifrn

600 hors16aopjr L 1n220I -set f Sve oletdb

r

on a Jin d OU-7 0in ed i In~ he-o yt - -t33 We~lepot hrete tt z I umbe ofcelIs er plat S we I s

purej~ag c- dedCl1 yte IAC~aetya P IU

method

Tenme ofcs- s h- at 1 or q 0 C )da

or fSirb ~1wi~ ~~~~t ni p~e Ea-r-h po nt epeens aerge c~n o tre p aLes n~6

he

o

--- - - --- --------------

T

7

ngnil

7 705 1

2 986 1 20~ t5 192 1879 4

- - - - - - - ---- - -- 771

c io it g M M 1 1 0 e u plusmnn~1 m M l ep es 50 dighel gen~tmin The dishes were nFbted at 37 n ~m atmospherei o Co and-95gtair-Dnrce3t he cedlI rih- cnflOc

(3700cel7 s late the nujmber of m to- peri 1at~e Was COUnted t h and~ e mdi cagedi4our ) a

c o nta ng if fferent c o ncentraion s of t a spin c~-7 ume mi-Slt a als counted 24- 48 7_2di96 h6Url lter

Th following formula was applied inore to ci termin the um6ber

-Where Mt Mi os -~~a

MCMtOM5Counfted (In10 differenptarea5 of th aiteFi~ oh ~ aet p oF plate

Ac Jr ea co nte (Oe of the I10 d~i ffieet arE cou nt ed Y Achpoint~ repreqents th e avrg count of thiree plates

2--V - 44 7 Y tL -- X4 a

4

IB~L ST S - P A4

T4-I E-HAS 2f Hours 18 1 )urs Or

W 214~ +2 67_4- 23 7 0 3 + 7 9

44 2 2217 2 03 263~+ 06 2 +44~ 6

0 1b6 2 2 2 17+ 00 3Rt57+ 50 - - - - - -- - - - - - - -- - - - -- - - - ---- --- - -- ~4 4~4 ~ -~~i-7 4- ---- ----shy4

1- For es 1 -+444 brbI s s e edno4 ae t 0 M ti5LIc ltr

4 ni al4- ed 4 H sen74

i e a 4MHbov4n serum )MM4 -4 L- S- -n 4mam

The Sl We inCbtdat7 i a~uiife - 4toperpo

1 r4ncd 3di~~h ~ espitorntdnn nEa I asrih3sa~ ~ishy

lineForeNa by laamptqr- i tod~the~ SuLrfac P bladeime

of4-3 thedsepIa Ari ~tbymo nt-374-4Cfr az or d 4 rdsthPrig

the5clles tI order 4to -ciea areaamp t hi- ofWas pu9ie n i C rih the I ne PI Ateswere tten4aheai th PBS1i1n orderto 0 i mi nate al I thec 1ll4debries and media w~H60iM e nf M ss i tenilwi~

- o 9~1e~ wi th Hanks salts ~ 1-e~50e Oi i6M gen t avcan)4 ni~dh 4the ci fferamp64 E cofncertrati ons ~o f -tasp inE-HC 1 was d ed at ie r e

4then screened and the areas -aiong thrt line cJ e I I s w Ce- 2 mnarked with the puin of 4a4 n-ie bistur Plte ereth~n4plIac Levi~E~

at3 in a hUfnlif Ied f -Y a~h 444 L~n4pecified ontha Tab1e theruabto cl 3cdse th line along the fnarked Preas Were Counte~d unde cntas 94-44

For eah oin 30J mm 4were read on4 PAch ofthree-p]ates_

The- data viaGsu~bjected ~to staistca anlyi usir the - The dif e nc vath the c~ontrol-was ipg i eatEie- (I i Ot )~The difference withi te contr6 wa9s i tiv0l 0 j ~The di4terence withthe control w~ vc(Rlt0C Th ifeecewt coto Plt 0 C)0 tthe4 wasgriicativ

Z 44

pshy

4c C tS c

4 474-14 20 0 + 4 9

~ 4 6C4 3 10r 1 121 46 1

VIA -414 4- - -~ - - - - -4 -shy

ce t rNN1gtn4 f -IICIaonta daco1 elbvns~r o~44

4tde L r-t tet +Y 4444-Q~ m~ contro iv

iffer ~ e444 -i th-4h 4Y (PltC4 con~ tri ol was signii rit4

The dl~-444(f e e~t - - wi 44c4oin44tr4o4I was4 si n f c t v (P 0 lt

The ~Ji4 444thfere4c 4i coro was i ve p4I~4 lt4~444 o4

-The4 74pf~erenc wi44 tll Ake 4244 was s444f4 a lt

744~4 4 ~ R4~4gt4shy

~3444442~4-4 ~ 4gt4F~6441-S~3614 t444 I-~ J444

TABLE 12

+ I I l

il IDll

1E NI I HI 4-

dcIlK MI X

u)b I)IJ OND Ir6 I 1II[ - IC I ]11 1- I i II ITIt

IIIIILk IIAtllVLF

INI LY LIO OF 1IL- If I1 IV3 1010-I IA I U01SI lt577

II I1]) 1[101=

S P 0

1 L_

IU1 7

2192

r 70] I 20

(I 7

24l-02C

10(I-15 M

29 1

Is- L-

23 (1

0

I I U

419 t

270

27

114 112 9U

I1 (t

024

44

20 42

-2

is 12

17

14 21 21

110

) i175 U t 1lII)

+

l 4Jq I Ii

4 122I

14tl

2B 23 a)

84 81 96

e)(

165 1l 1[T5

101

26 CU

24

4 J+

10 1(

C

21 111 19

14

A131FV IAIIUNK 5 P E

0Ii

Spunrtrub(I

Efirr Ionpo-L]Ler

reversion Hvplected

r e er in obs ved

Fur~ Lhq su ~4 IIIdn ILa

T

A~~~oSS P

S G~CSciae

0Q23

4

4Y

004) ~

5rvr9a5pnafeu

I 42

4 9~7 4A3j U

47p~t~ 60

ee

72d~c 46 21 10

0 00--47 4 7 4 4 37 - 45 2 It 2744-4

0 1144 7 4~~ 2714

0 0450 72 444 24~4 444 -527K Z9 2 15

4x

--- - - --- --------------

T

7

ngnil

7 705 1

2 986 1 20~ t5 192 1879 4

- - - - - - - ---- - -- 771

c io it g M M 1 1 0 e u plusmnn~1 m M l ep es 50 dighel gen~tmin The dishes were nFbted at 37 n ~m atmospherei o Co and-95gtair-Dnrce3t he cedlI rih- cnflOc

(3700cel7 s late the nujmber of m to- peri 1at~e Was COUnted t h and~ e mdi cagedi4our ) a

c o nta ng if fferent c o ncentraion s of t a spin c~-7 ume mi-Slt a als counted 24- 48 7_2di96 h6Url lter

Th following formula was applied inore to ci termin the um6ber

-Where Mt Mi os -~~a

MCMtOM5Counfted (In10 differenptarea5 of th aiteFi~ oh ~ aet p oF plate

Ac Jr ea co nte (Oe of the I10 d~i ffieet arE cou nt ed Y Achpoint~ repreqents th e avrg count of thiree plates

2--V - 44 7 Y tL -- X4 a

4

IB~L ST S - P A4

T4-I E-HAS 2f Hours 18 1 )urs Or

W 214~ +2 67_4- 23 7 0 3 + 7 9

44 2 2217 2 03 263~+ 06 2 +44~ 6

0 1b6 2 2 2 17+ 00 3Rt57+ 50 - - - - - -- - - - - - - -- - - - -- - - - ---- --- - -- ~4 4~4 ~ -~~i-7 4- ---- ----shy4

1- For es 1 -+444 brbI s s e edno4 ae t 0 M ti5LIc ltr

4 ni al4- ed 4 H sen74

i e a 4MHbov4n serum )MM4 -4 L- S- -n 4mam

The Sl We inCbtdat7 i a~uiife - 4toperpo

1 r4ncd 3di~~h ~ espitorntdnn nEa I asrih3sa~ ~ishy

lineForeNa by laamptqr- i tod~the~ SuLrfac P bladeime

of4-3 thedsepIa Ari ~tbymo nt-374-4Cfr az or d 4 rdsthPrig

the5clles tI order 4to -ciea areaamp t hi- ofWas pu9ie n i C rih the I ne PI Ateswere tten4aheai th PBS1i1n orderto 0 i mi nate al I thec 1ll4debries and media w~H60iM e nf M ss i tenilwi~

- o 9~1e~ wi th Hanks salts ~ 1-e~50e Oi i6M gen t avcan)4 ni~dh 4the ci fferamp64 E cofncertrati ons ~o f -tasp inE-HC 1 was d ed at ie r e

4then screened and the areas -aiong thrt line cJ e I I s w Ce- 2 mnarked with the puin of 4a4 n-ie bistur Plte ereth~n4plIac Levi~E~

at3 in a hUfnlif Ied f -Y a~h 444 L~n4pecified ontha Tab1e theruabto cl 3cdse th line along the fnarked Preas Were Counte~d unde cntas 94-44

For eah oin 30J mm 4were read on4 PAch ofthree-p]ates_

The- data viaGsu~bjected ~to staistca anlyi usir the - The dif e nc vath the c~ontrol-was ipg i eatEie- (I i Ot )~The difference withi te contr6 wa9s i tiv0l 0 j ~The di4terence withthe control w~ vc(Rlt0C Th ifeecewt coto Plt 0 C)0 tthe4 wasgriicativ

Z 44

pshy

4c C tS c

4 474-14 20 0 + 4 9

~ 4 6C4 3 10r 1 121 46 1

VIA -414 4- - -~ - - - - -4 -shy

ce t rNN1gtn4 f -IICIaonta daco1 elbvns~r o~44

4tde L r-t tet +Y 4444-Q~ m~ contro iv

iffer ~ e444 -i th-4h 4Y (PltC4 con~ tri ol was signii rit4

The dl~-444(f e e~t - - wi 44c4oin44tr4o4I was4 si n f c t v (P 0 lt

The ~Ji4 444thfere4c 4i coro was i ve p4I~4 lt4~444 o4

-The4 74pf~erenc wi44 tll Ake 4244 was s444f4 a lt

744~4 4 ~ R4~4gt4shy

~3444442~4-4 ~ 4gt4F~6441-S~3614 t444 I-~ J444

TABLE 12

+ I I l

il IDll

1E NI I HI 4-

dcIlK MI X

u)b I)IJ OND Ir6 I 1II[ - IC I ]11 1- I i II ITIt

IIIIILk IIAtllVLF

INI LY LIO OF 1IL- If I1 IV3 1010-I IA I U01SI lt577

II I1]) 1[101=

S P 0

1 L_

IU1 7

2192

r 70] I 20

(I 7

24l-02C

10(I-15 M

29 1

Is- L-

23 (1

0

I I U

419 t

270

27

114 112 9U

I1 (t

024

44

20 42

-2

is 12

17

14 21 21

110

) i175 U t 1lII)

+

l 4Jq I Ii

4 122I

14tl

2B 23 a)

84 81 96

e)(

165 1l 1[T5

101

26 CU

24

4 J+

10 1(

C

21 111 19

14

A131FV IAIIUNK 5 P E

0Ii

Spunrtrub(I

Efirr Ionpo-L]Ler

reversion Hvplected

r e er in obs ved

Fur~ Lhq su ~4 IIIdn ILa

T

A~~~oSS P

S G~CSciae

0Q23

4

4Y

004) ~

5rvr9a5pnafeu

I 42

4 9~7 4A3j U

47p~t~ 60

ee

72d~c 46 21 10

0 00--47 4 7 4 4 37 - 45 2 It 2744-4

0 1144 7 4~~ 2714

0 0450 72 444 24~4 444 -527K Z9 2 15

4x

4

IB~L ST S - P A4

T4-I E-HAS 2f Hours 18 1 )urs Or

W 214~ +2 67_4- 23 7 0 3 + 7 9

44 2 2217 2 03 263~+ 06 2 +44~ 6

0 1b6 2 2 2 17+ 00 3Rt57+ 50 - - - - - -- - - - - - - -- - - - -- - - - ---- --- - -- ~4 4~4 ~ -~~i-7 4- ---- ----shy4

1- For es 1 -+444 brbI s s e edno4 ae t 0 M ti5LIc ltr

4 ni al4- ed 4 H sen74

i e a 4MHbov4n serum )MM4 -4 L- S- -n 4mam

The Sl We inCbtdat7 i a~uiife - 4toperpo

1 r4ncd 3di~~h ~ espitorntdnn nEa I asrih3sa~ ~ishy

lineForeNa by laamptqr- i tod~the~ SuLrfac P bladeime

of4-3 thedsepIa Ari ~tbymo nt-374-4Cfr az or d 4 rdsthPrig

the5clles tI order 4to -ciea areaamp t hi- ofWas pu9ie n i C rih the I ne PI Ateswere tten4aheai th PBS1i1n orderto 0 i mi nate al I thec 1ll4debries and media w~H60iM e nf M ss i tenilwi~

- o 9~1e~ wi th Hanks salts ~ 1-e~50e Oi i6M gen t avcan)4 ni~dh 4the ci fferamp64 E cofncertrati ons ~o f -tasp inE-HC 1 was d ed at ie r e

4then screened and the areas -aiong thrt line cJ e I I s w Ce- 2 mnarked with the puin of 4a4 n-ie bistur Plte ereth~n4plIac Levi~E~

at3 in a hUfnlif Ied f -Y a~h 444 L~n4pecified ontha Tab1e theruabto cl 3cdse th line along the fnarked Preas Were Counte~d unde cntas 94-44

For eah oin 30J mm 4were read on4 PAch ofthree-p]ates_

The- data viaGsu~bjected ~to staistca anlyi usir the - The dif e nc vath the c~ontrol-was ipg i eatEie- (I i Ot )~The difference withi te contr6 wa9s i tiv0l 0 j ~The di4terence withthe control w~ vc(Rlt0C Th ifeecewt coto Plt 0 C)0 tthe4 wasgriicativ

Z 44

pshy

4c C tS c

4 474-14 20 0 + 4 9

~ 4 6C4 3 10r 1 121 46 1

VIA -414 4- - -~ - - - - -4 -shy

ce t rNN1gtn4 f -IICIaonta daco1 elbvns~r o~44

4tde L r-t tet +Y 4444-Q~ m~ contro iv

iffer ~ e444 -i th-4h 4Y (PltC4 con~ tri ol was signii rit4

The dl~-444(f e e~t - - wi 44c4oin44tr4o4I was4 si n f c t v (P 0 lt

The ~Ji4 444thfere4c 4i coro was i ve p4I~4 lt4~444 o4

-The4 74pf~erenc wi44 tll Ake 4244 was s444f4 a lt

744~4 4 ~ R4~4gt4shy

~3444442~4-4 ~ 4gt4F~6441-S~3614 t444 I-~ J444

TABLE 12

+ I I l

il IDll

1E NI I HI 4-

dcIlK MI X

u)b I)IJ OND Ir6 I 1II[ - IC I ]11 1- I i II ITIt

IIIIILk IIAtllVLF

INI LY LIO OF 1IL- If I1 IV3 1010-I IA I U01SI lt577

II I1]) 1[101=

S P 0

1 L_

IU1 7

2192

r 70] I 20

(I 7

24l-02C

10(I-15 M

29 1

Is- L-

23 (1

0

I I U

419 t

270

27

114 112 9U

I1 (t

024

44

20 42

-2

is 12

17

14 21 21

110

) i175 U t 1lII)

+

l 4Jq I Ii

4 122I

14tl

2B 23 a)

84 81 96

e)(

165 1l 1[T5

101

26 CU

24

4 J+

10 1(

C

21 111 19

14

A131FV IAIIUNK 5 P E

0Ii

Spunrtrub(I

Efirr Ionpo-L]Ler

reversion Hvplected

r e er in obs ved

Fur~ Lhq su ~4 IIIdn ILa

T

A~~~oSS P

S G~CSciae

0Q23

4

4Y

004) ~

5rvr9a5pnafeu

I 42

4 9~7 4A3j U

47p~t~ 60

ee

72d~c 46 21 10

0 00--47 4 7 4 4 37 - 45 2 It 2744-4

0 1144 7 4~~ 2714

0 0450 72 444 24~4 444 -527K Z9 2 15

4x

Z 44

pshy

4c C tS c

4 474-14 20 0 + 4 9

~ 4 6C4 3 10r 1 121 46 1

VIA -414 4- - -~ - - - - -4 -shy

ce t rNN1gtn4 f -IICIaonta daco1 elbvns~r o~44

4tde L r-t tet +Y 4444-Q~ m~ contro iv

iffer ~ e444 -i th-4h 4Y (PltC4 con~ tri ol was signii rit4

The dl~-444(f e e~t - - wi 44c4oin44tr4o4I was4 si n f c t v (P 0 lt

The ~Ji4 444thfere4c 4i coro was i ve p4I~4 lt4~444 o4

-The4 74pf~erenc wi44 tll Ake 4244 was s444f4 a lt

744~4 4 ~ R4~4gt4shy

~3444442~4-4 ~ 4gt4F~6441-S~3614 t444 I-~ J444

TABLE 12

+ I I l

il IDll

1E NI I HI 4-

dcIlK MI X

u)b I)IJ OND Ir6 I 1II[ - IC I ]11 1- I i II ITIt

IIIIILk IIAtllVLF

INI LY LIO OF 1IL- If I1 IV3 1010-I IA I U01SI lt577

II I1]) 1[101=

S P 0

1 L_

IU1 7

2192

r 70] I 20

(I 7

24l-02C

10(I-15 M

29 1

Is- L-

23 (1

0

I I U

419 t

270

27

114 112 9U

I1 (t

024

44

20 42

-2

is 12

17

14 21 21

110

) i175 U t 1lII)

+

l 4Jq I Ii

4 122I

14tl

2B 23 a)

84 81 96

e)(

165 1l 1[T5

101

26 CU

24

4 J+

10 1(

C

21 111 19

14

A131FV IAIIUNK 5 P E

0Ii

Spunrtrub(I

Efirr Ionpo-L]Ler

reversion Hvplected

r e er in obs ved

Fur~ Lhq su ~4 IIIdn ILa

T

A~~~oSS P

S G~CSciae

0Q23

4

4Y

004) ~

5rvr9a5pnafeu

I 42

4 9~7 4A3j U

47p~t~ 60

ee

72d~c 46 21 10

0 00--47 4 7 4 4 37 - 45 2 It 2744-4

0 1144 7 4~~ 2714

0 0450 72 444 24~4 444 -527K Z9 2 15

4x

TABLE 12

+ I I l

il IDll

1E NI I HI 4-

dcIlK MI X

u)b I)IJ OND Ir6 I 1II[ - IC I ]11 1- I i II ITIt

IIIIILk IIAtllVLF

INI LY LIO OF 1IL- If I1 IV3 1010-I IA I U01SI lt577

II I1]) 1[101=

S P 0

1 L_

IU1 7

2192

r 70] I 20

(I 7

24l-02C

10(I-15 M

29 1

Is- L-

23 (1

0

I I U

419 t

270

27

114 112 9U

I1 (t

024

44

20 42

-2

is 12

17

14 21 21

110

) i175 U t 1lII)

+

l 4Jq I Ii

4 122I

14tl

2B 23 a)

84 81 96

e)(

165 1l 1[T5

101

26 CU

24

4 J+

10 1(

C

21 111 19

14

A131FV IAIIUNK 5 P E

0Ii

Spunrtrub(I

Efirr Ionpo-L]Ler

reversion Hvplected

r e er in obs ved

Fur~ Lhq su ~4 IIIdn ILa

T

A~~~oSS P

S G~CSciae

0Q23

4

4Y

004) ~

5rvr9a5pnafeu

I 42

4 9~7 4A3j U

47p~t~ 60

ee

72d~c 46 21 10

0 00--47 4 7 4 4 37 - 45 2 It 2744-4

0 1144 7 4~~ 2714

0 0450 72 444 24~4 444 -527K Z9 2 15

4x

T

A~~~oSS P

S G~CSciae

0Q23

4

4Y

004) ~

5rvr9a5pnafeu

I 42

4 9~7 4A3j U

47p~t~ 60

ee

72d~c 46 21 10

0 00--47 4 7 4 4 37 - 45 2 It 2744-4

0 1144 7 4~~ 2714

0 0450 72 444 24~4 444 -527K Z9 2 15

4x

EFFECT OF TsSP INE HCI ONTH-ERQEC Or SISTER ciHROMwTi) EX)CHANG TET -R SCEMETHAFHOSE4

Cotr 72 1 6l

0n26 g11~

SCE tstechrmatd xcha ]e

~ ~~ ~10 1~~~ eio V7KAcell

1) ~~C)

E di

d -i n io 6 Jn1PtS

LW C~t re

lb dih2

ule c s r dii- Ei e

and 005 mgnil of geptami c iri Li i ncpLi~t d i n a- d 0idi 5COil 91ai r at4-37 C rl 24i hourS 4~irn d1 Pent concqntrations f

s~lt~n~oUn1 iuwere added-Lcthe correpond~ing pl Si MLItaneulyw h 5 booeiid ea a f i nal conetrati on ofugmLf edUA2 hor If 6 c1evidtoRa J ialconcetratio 0

0 5 JM IOf Med i ur mis acded tothe plIates and i hcubated 4f7or 2 Q~SThne1-Cal S 1Aand YF stained W1

i he L-501 t of-~ twiD Bparate exrmnt n a talof- 7mlt~etaphases were readAniamp cha4ofthe tramngDIU

jSiviif i-ance o-f the differences were caclae icodn t0t th ~StLdenL t-Iest Th i~rrc uven contr~ol4 and the taseic 41m~gatQ68 u gmi grop (plt005) aKdI-beitweenthe c~o1o an -pin6

SHCl 2a 26 ug61l Lgroup i ni intcp~I~ K ~~~ II2gr l 1 (Plt 4 1 47 4 1 I 47

144 4 4 l47I~44lKKI~lK K 1447)

1 4 K Kfl 24 7 147

-77

CFFECrT OF TASFPINE-HC1 AND 6IONGRE DE GADONIEtSAD FRCIUENCY O I~GST 1F CHOMTI ECHNG

T A ME SCEa M-aa - I shyE--aT-aI--I -a- aP

C o 6 tr ) 1 1a- 2 21 5

-- - - - - - -- - - - ----- - - - - -a--a- -- - - - a-

ia- I 0~ a-aa-a- a-te a- a 1fet a b i Zn serumi--a ae a-a-a-a-ashy

gecbt~ ~ 1 ~ ~ ~n~a tnopeeo~ ~ aaaaa C029 a-a---n ~ ~ nahfidifie

Sagr d Sraon re G-md r---i1 --- 2aaa aa- shya-i the8aa-or esodigp shy

d tocarg - - L athe-plt gtand n a

S CanCe-o h fa~a-aaaciOa-a-a wrea-c At-afI0i --i(~vlrnrri nq t -ao 6 a a-denttri~ tes i~bt4i -

a~reI~Ur ea-Jaideda-pa--a-tetw the~ea~cntul aled tfh ts 1p00 a aX d a -tie i-~--aaaa ---- aaa a aoraOsL sieI-tat-~a f--a1-mtp~ aWr~aed~~ KJa

a-~ ja 0-gn-- i - a naata ----- aaa --- a a -- - )

tre-atm eaa--a-Ct g-O- IPS-aa-a-a - a~ -i -a-- aa--aa4( (a aa- -~amp -a-aP a-a--A

K3 amp

TAL 6

~ ~ApLA4A 3 1 EP I _ I E TTEPT

aTO-TO cmioGN rOE FIA CAR I

None ~

3 ~ DfDAi C

~~IACETONE ~ 30lt

5 ~DMI3~i+TP

6 DMDU-W +6GG C

EiFr- )ATIDNS JJMDA 771 D iacen

Pd Carcinogenicampdo of DMB(- (AI of 10Ci p61)DMDP=In ucrdoi~ofD11IA (I of tk)no

E3(3Sangred Grd 0am f5 (1t~ 4 e

rhe experiment began on December 12 94n h nimber ofni ccp per

p 5jingorfpafb-rite toh c) e 1 19E5 o r(U

Ac odng 0a~op~y Micedied o rons not related L totet FAP iL0ma~s starte~dto appear on Novembr 2190t Anraveragc-oi 7

papL LVLomaS OnUSP3 A~ ~~

Spap iIOM~k tar-ted tn appear onn~pt-il Ist 1905 An aveae of 7

For the fnethodo1Qgy-46P textK 7- ~

2~IP~ ~amp II

I I 3 k 1 4

T-)

Sarngre de Gradn

Co)nenL-atwd NH401(1l 1 1 to1

irownish Red Viscous MLLas

tr art J with CI (-l

hl oral orm I i oct I in

lowi d I vacou at 40 L

I WaJsihi d t h r-oo~m I p rmperatur (0 G ) I mothoniol anl-d i tp rp (

Dark Orate esi n Wh i clIlI ( lI ol I I t )

I r Lt f( J i O i oil ri t iTAI

Fi I Vrotu

Iteavie r crst Ls ir o sn rID

bottom of tzhe- ftasl

Csolid l (Whi t ti (e i Wiite mn(1)j-Lp C5rJ[

F crst lis kr ykia ized in Ac n iMlethanollrnrnrp

Yell ow crys-als White -mmrpols sol iid

FIUiJEd 1 Extraction and Iur firat in rncdure of the Al alrcili From Sanm jrp( de rad o

Y A7

44

- 7 _ - shy

91 9A 49 9 9

- I9 resin Ye lo 9 I

19~~~~~ 9 d ~

~ -- -- 9 ~- 499-9~A-- j - 9999-- 9 -~ -- --

D99999999 t9 ve tbq8 Or clnd r en

9999--------- -- -- --

FIUE2 hn aer hoaorpyo th jkal $ulampan Rei btie d rn teE c9 ip4 ng

~e99lt ~a4 99 ~ 99adoA fV) nSjwa per999~ore aGe C ~AAS4L

9 w t9 I 61U I i q 9h 6~99A an al -othc9o nm

9 g99 tj99 t W9v e--1UV ig L of 2 ni4 71A7 ogW

9

9 erf av~~ nex eah pt

- -

4 42

--- -- --22 - - -- - - - - - - - - - -- shy

722

122 222) 24

2 20 44 8

5~0 5 ~ 7

2

29

- ~ ~~ ~ ~ ~ ~- - - - - ~ 2 -2 2- ~ - -- 2 - - - - - - - - - - - - -

2222ZjI~D2 c 292i t~2i2J 22 t- o D22r222 ( v4L reE gE

(2222

F22 I29322~2~

~~VJ~ 2

2~ ~

22$22-Thin La rz a h

raclonEut2 -2222222222e

gt2 22222i212 2

2 Y 222~ 22

h Sys eyT s i2 ii a the2~ 666t 0 a42

e44 r s2 ih Ad22lt d2 ac ti

el vi E22 22kI2

of Rep

Dry2 Column2

22222 F 5

n2 e A

2)Mth M~2h ~2

at

22222 o

2

2rmog

c2

~2 2

2~en

2~2

2and2

22222 2 2 222 22 224222~ I2 2 4

44 4

ZI

C--IshyE~j44

a-4

A I- ~

~~~--4---4~~

~-

v-

~

-

~ 4k ~ 4

4

- -443-3-4 --

~~~ ~~~~~ FIUR E i

-~A 34603334

nPteno

~ - - N 4 -- - --3 -4 ---~- - -

K14---h CueA a

-$

i

-- -4

iXtO

- ~~ D _

~ ~ 4- 4-4Ai

e4tr

va

-2 4

L-i-n

upgtV to-~~

a p

~ hours

~ r

7 on~

4i

Ilk-~ 7 Ehtoi 4

7

to

IGURE 5 Ulrvoe pcr of the Chlor ydrate of thei

ProyIr-yided by Dr N~orman R~ -Fansorth (t5D)~~4 ~othSpectra Were- rt~n Wt~7~y i

Dot in W a -e

44~~ -~ - w~A

3

--

---

Ideni N

r U2

J4

f 0

5lt

46~

v~i ~

II

I K~-r~L pound

KJJzI--U

lt

1Ju

n 0 U

)

L7~tI ~1

~17 ~t K

--

0

~L

77PI if

-

~izLjplusmn1101~B

~

4

ifn

IGR 5

I

IIn

It I

FIGURE

6 UV

spectrum of

the H

ydrochloride of

the White

Amorphus Solid

from Sangre

do Grado i

nH20

41 40000 30

JA L3aS VRaLUICY Pp

7310 650411 iii1788 a1919 3847 4001 11351 73S4 1030 S51978 7974 404 71 7144 1087 7933403019 7 73

349055 43833463 318600 4L 3374 30835 3-154384 386349 3407

3400 33026 9 1083447 57 34 1 463444 371933 395

3432 217747 3721 170 9 2 4 68 5 3-65 2722 209388 823755 302510

2532 2345 489373 2367

3907 4T9436 3r 3022 Ir3845353661 3-299 004

3816 0000 0000 369 3299 - 0041 3793 - 30235 shy 00252

18ITLRAL- 4 8SITY

1441044 333441040 3 413470 339040 3490140 135310141 79

138

117438 313 48 bull9 5333411 14373743 1- 3938

348803 44400I300VV6 3113714lu

944453 1060393838 95040

1161735 15392

144704 13443

15- 0114 1 7921066 44176

9238012786720 7234502699

345992 44 48 3464991 448224 42576 84-392

3651793 431404 8474 49152 99-056 31776

go

C

1 E

4

04 amp209

N

-0

] 1

FIGJ1M 7 gR -ect of the Yellow Crystals obtained during the purification procedure of Sangre de Grado

-77

CFFECrT OF TASFPINE-HC1 AND 6IONGRE DE GADONIEtSAD FRCIUENCY O I~GST 1F CHOMTI ECHNG

T A ME SCEa M-aa - I shyE--aT-aI--I -a- aP

C o 6 tr ) 1 1a- 2 21 5

-- - - - - - -- - - - ----- - - - - -a--a- -- - - - a-

ia- I 0~ a-aa-a- a-te a- a 1fet a b i Zn serumi--a ae a-a-a-a-ashy

gecbt~ ~ 1 ~ ~ ~n~a tnopeeo~ ~ aaaaa C029 a-a---n ~ ~ nahfidifie

Sagr d Sraon re G-md r---i1 --- 2aaa aa- shya-i the8aa-or esodigp shy

d tocarg - - L athe-plt gtand n a

S CanCe-o h fa~a-aaaciOa-a-a wrea-c At-afI0i --i(~vlrnrri nq t -ao 6 a a-denttri~ tes i~bt4i -

a~reI~Ur ea-Jaideda-pa--a-tetw the~ea~cntul aled tfh ts 1p00 a aX d a -tie i-~--aaaa ---- aaa a aoraOsL sieI-tat-~a f--a1-mtp~ aWr~aed~~ KJa

a-~ ja 0-gn-- i - a naata ----- aaa --- a a -- - )

tre-atm eaa--a-Ct g-O- IPS-aa-a-a - a~ -i -a-- aa--aa4( (a aa- -~amp -a-aP a-a--A

K3 amp

TAL 6

~ ~ApLA4A 3 1 EP I _ I E TTEPT

aTO-TO cmioGN rOE FIA CAR I

None ~

3 ~ DfDAi C

~~IACETONE ~ 30lt

5 ~DMI3~i+TP

6 DMDU-W +6GG C

EiFr- )ATIDNS JJMDA 771 D iacen

Pd Carcinogenicampdo of DMB(- (AI of 10Ci p61)DMDP=In ucrdoi~ofD11IA (I of tk)no

E3(3Sangred Grd 0am f5 (1t~ 4 e

rhe experiment began on December 12 94n h nimber ofni ccp per

p 5jingorfpafb-rite toh c) e 1 19E5 o r(U

Ac odng 0a~op~y Micedied o rons not related L totet FAP iL0ma~s starte~dto appear on Novembr 2190t Anraveragc-oi 7

papL LVLomaS OnUSP3 A~ ~~

Spap iIOM~k tar-ted tn appear onn~pt-il Ist 1905 An aveae of 7

For the fnethodo1Qgy-46P textK 7- ~

2~IP~ ~amp II

I I 3 k 1 4

T-)

Sarngre de Gradn

Co)nenL-atwd NH401(1l 1 1 to1

irownish Red Viscous MLLas

tr art J with CI (-l

hl oral orm I i oct I in

lowi d I vacou at 40 L

I WaJsihi d t h r-oo~m I p rmperatur (0 G ) I mothoniol anl-d i tp rp (

Dark Orate esi n Wh i clIlI ( lI ol I I t )

I r Lt f( J i O i oil ri t iTAI

Fi I Vrotu

Iteavie r crst Ls ir o sn rID

bottom of tzhe- ftasl

Csolid l (Whi t ti (e i Wiite mn(1)j-Lp C5rJ[

F crst lis kr ykia ized in Ac n iMlethanollrnrnrp

Yell ow crys-als White -mmrpols sol iid

FIUiJEd 1 Extraction and Iur firat in rncdure of the Al alrcili From Sanm jrp( de rad o

Y A7

44

- 7 _ - shy

91 9A 49 9 9

- I9 resin Ye lo 9 I

19~~~~~ 9 d ~

~ -- -- 9 ~- 499-9~A-- j - 9999-- 9 -~ -- --

D99999999 t9 ve tbq8 Or clnd r en

9999--------- -- -- --

FIUE2 hn aer hoaorpyo th jkal $ulampan Rei btie d rn teE c9 ip4 ng

~e99lt ~a4 99 ~ 99adoA fV) nSjwa per999~ore aGe C ~AAS4L

9 w t9 I 61U I i q 9h 6~99A an al -othc9o nm

9 g99 tj99 t W9v e--1UV ig L of 2 ni4 71A7 ogW

9

9 erf av~~ nex eah pt

- -

4 42

--- -- --22 - - -- - - - - - - - - - -- shy

722

122 222) 24

2 20 44 8

5~0 5 ~ 7

2

29

- ~ ~~ ~ ~ ~ ~- - - - - ~ 2 -2 2- ~ - -- 2 - - - - - - - - - - - - -

2222ZjI~D2 c 292i t~2i2J 22 t- o D22r222 ( v4L reE gE

(2222

F22 I29322~2~

~~VJ~ 2

2~ ~

22$22-Thin La rz a h

raclonEut2 -2222222222e

gt2 22222i212 2

2 Y 222~ 22

h Sys eyT s i2 ii a the2~ 666t 0 a42

e44 r s2 ih Ad22lt d2 ac ti

el vi E22 22kI2

of Rep

Dry2 Column2

22222 F 5

n2 e A

2)Mth M~2h ~2

at

22222 o

2

2rmog

c2

~2 2

2~en

2~2

2and2

22222 2 2 222 22 224222~ I2 2 4

44 4

ZI

C--IshyE~j44

a-4

A I- ~

~~~--4---4~~

~-

v-

~

-

~ 4k ~ 4

4

- -443-3-4 --

~~~ ~~~~~ FIUR E i

-~A 34603334

nPteno

~ - - N 4 -- - --3 -4 ---~- - -

K14---h CueA a

-$

i

-- -4

iXtO

- ~~ D _

~ ~ 4- 4-4Ai

e4tr

va

-2 4

L-i-n

upgtV to-~~

a p

~ hours

~ r

7 on~

4i

Ilk-~ 7 Ehtoi 4

7

to

IGURE 5 Ulrvoe pcr of the Chlor ydrate of thei

ProyIr-yided by Dr N~orman R~ -Fansorth (t5D)~~4 ~othSpectra Were- rt~n Wt~7~y i

Dot in W a -e

44~~ -~ - w~A

3

--

---

Ideni N

r U2

J4

f 0

5lt

46~

v~i ~

II

I K~-r~L pound

KJJzI--U

lt

1Ju

n 0 U

)

L7~tI ~1

~17 ~t K

--

0

~L

77PI if

-

~izLjplusmn1101~B

~

4

ifn

IGR 5

I

IIn

It I

FIGURE

6 UV

spectrum of

the H

ydrochloride of

the White

Amorphus Solid

from Sangre

do Grado i

nH20

41 40000 30

JA L3aS VRaLUICY Pp

7310 650411 iii1788 a1919 3847 4001 11351 73S4 1030 S51978 7974 404 71 7144 1087 7933403019 7 73

349055 43833463 318600 4L 3374 30835 3-154384 386349 3407

3400 33026 9 1083447 57 34 1 463444 371933 395

3432 217747 3721 170 9 2 4 68 5 3-65 2722 209388 823755 302510

2532 2345 489373 2367

3907 4T9436 3r 3022 Ir3845353661 3-299 004

3816 0000 0000 369 3299 - 0041 3793 - 30235 shy 00252

18ITLRAL- 4 8SITY

1441044 333441040 3 413470 339040 3490140 135310141 79

138

117438 313 48 bull9 5333411 14373743 1- 3938

348803 44400I300VV6 3113714lu

944453 1060393838 95040

1161735 15392

144704 13443

15- 0114 1 7921066 44176

9238012786720 7234502699

345992 44 48 3464991 448224 42576 84-392

3651793 431404 8474 49152 99-056 31776

go

C

1 E

4

04 amp209

N

-0

] 1

FIGJ1M 7 gR -ect of the Yellow Crystals obtained during the purification procedure of Sangre de Grado

K3 amp

TAL 6

~ ~ApLA4A 3 1 EP I _ I E TTEPT

aTO-TO cmioGN rOE FIA CAR I

None ~

3 ~ DfDAi C

~~IACETONE ~ 30lt

5 ~DMI3~i+TP

6 DMDU-W +6GG C

EiFr- )ATIDNS JJMDA 771 D iacen

Pd Carcinogenicampdo of DMB(- (AI of 10Ci p61)DMDP=In ucrdoi~ofD11IA (I of tk)no

E3(3Sangred Grd 0am f5 (1t~ 4 e

rhe experiment began on December 12 94n h nimber ofni ccp per

p 5jingorfpafb-rite toh c) e 1 19E5 o r(U

Ac odng 0a~op~y Micedied o rons not related L totet FAP iL0ma~s starte~dto appear on Novembr 2190t Anraveragc-oi 7

papL LVLomaS OnUSP3 A~ ~~

Spap iIOM~k tar-ted tn appear onn~pt-il Ist 1905 An aveae of 7

For the fnethodo1Qgy-46P textK 7- ~

2~IP~ ~amp II

I I 3 k 1 4

T-)

Sarngre de Gradn

Co)nenL-atwd NH401(1l 1 1 to1

irownish Red Viscous MLLas

tr art J with CI (-l

hl oral orm I i oct I in

lowi d I vacou at 40 L

I WaJsihi d t h r-oo~m I p rmperatur (0 G ) I mothoniol anl-d i tp rp (

Dark Orate esi n Wh i clIlI ( lI ol I I t )

I r Lt f( J i O i oil ri t iTAI

Fi I Vrotu

Iteavie r crst Ls ir o sn rID

bottom of tzhe- ftasl

Csolid l (Whi t ti (e i Wiite mn(1)j-Lp C5rJ[

F crst lis kr ykia ized in Ac n iMlethanollrnrnrp

Yell ow crys-als White -mmrpols sol iid

FIUiJEd 1 Extraction and Iur firat in rncdure of the Al alrcili From Sanm jrp( de rad o

Y A7

44

- 7 _ - shy

91 9A 49 9 9

- I9 resin Ye lo 9 I

19~~~~~ 9 d ~

~ -- -- 9 ~- 499-9~A-- j - 9999-- 9 -~ -- --

D99999999 t9 ve tbq8 Or clnd r en

9999--------- -- -- --

FIUE2 hn aer hoaorpyo th jkal $ulampan Rei btie d rn teE c9 ip4 ng

~e99lt ~a4 99 ~ 99adoA fV) nSjwa per999~ore aGe C ~AAS4L

9 w t9 I 61U I i q 9h 6~99A an al -othc9o nm

9 g99 tj99 t W9v e--1UV ig L of 2 ni4 71A7 ogW

9

9 erf av~~ nex eah pt

- -

4 42

--- -- --22 - - -- - - - - - - - - - -- shy

722

122 222) 24

2 20 44 8

5~0 5 ~ 7

2

29

- ~ ~~ ~ ~ ~ ~- - - - - ~ 2 -2 2- ~ - -- 2 - - - - - - - - - - - - -

2222ZjI~D2 c 292i t~2i2J 22 t- o D22r222 ( v4L reE gE

(2222

F22 I29322~2~

~~VJ~ 2

2~ ~

22$22-Thin La rz a h

raclonEut2 -2222222222e

gt2 22222i212 2

2 Y 222~ 22

h Sys eyT s i2 ii a the2~ 666t 0 a42

e44 r s2 ih Ad22lt d2 ac ti

el vi E22 22kI2

of Rep

Dry2 Column2

22222 F 5

n2 e A

2)Mth M~2h ~2

at

22222 o

2

2rmog

c2

~2 2

2~en

2~2

2and2

22222 2 2 222 22 224222~ I2 2 4

44 4

ZI

C--IshyE~j44

a-4

A I- ~

~~~--4---4~~

~-

v-

~

-

~ 4k ~ 4

4

- -443-3-4 --

~~~ ~~~~~ FIUR E i

-~A 34603334

nPteno

~ - - N 4 -- - --3 -4 ---~- - -

K14---h CueA a

-$

i

-- -4

iXtO

- ~~ D _

~ ~ 4- 4-4Ai

e4tr

va

-2 4

L-i-n

upgtV to-~~

a p

~ hours

~ r

7 on~

4i

Ilk-~ 7 Ehtoi 4

7

to

IGURE 5 Ulrvoe pcr of the Chlor ydrate of thei

ProyIr-yided by Dr N~orman R~ -Fansorth (t5D)~~4 ~othSpectra Were- rt~n Wt~7~y i

Dot in W a -e

44~~ -~ - w~A

3

--

---

Ideni N

r U2

J4

f 0

5lt

46~

v~i ~

II

I K~-r~L pound

KJJzI--U

lt

1Ju

n 0 U

)

L7~tI ~1

~17 ~t K

--

0

~L

77PI if

-

~izLjplusmn1101~B

~

4

ifn

IGR 5

I

IIn

It I

FIGURE

6 UV

spectrum of

the H

ydrochloride of

the White

Amorphus Solid

from Sangre

do Grado i

nH20

41 40000 30

JA L3aS VRaLUICY Pp

7310 650411 iii1788 a1919 3847 4001 11351 73S4 1030 S51978 7974 404 71 7144 1087 7933403019 7 73

349055 43833463 318600 4L 3374 30835 3-154384 386349 3407

3400 33026 9 1083447 57 34 1 463444 371933 395

3432 217747 3721 170 9 2 4 68 5 3-65 2722 209388 823755 302510

2532 2345 489373 2367

3907 4T9436 3r 3022 Ir3845353661 3-299 004

3816 0000 0000 369 3299 - 0041 3793 - 30235 shy 00252

18ITLRAL- 4 8SITY

1441044 333441040 3 413470 339040 3490140 135310141 79

138

117438 313 48 bull9 5333411 14373743 1- 3938

348803 44400I300VV6 3113714lu

944453 1060393838 95040

1161735 15392

144704 13443

15- 0114 1 7921066 44176

9238012786720 7234502699

345992 44 48 3464991 448224 42576 84-392

3651793 431404 8474 49152 99-056 31776

go

C

1 E

4

04 amp209

N

-0

] 1

FIGJ1M 7 gR -ect of the Yellow Crystals obtained during the purification procedure of Sangre de Grado

T-)

Sarngre de Gradn

Co)nenL-atwd NH401(1l 1 1 to1

irownish Red Viscous MLLas

tr art J with CI (-l

hl oral orm I i oct I in

lowi d I vacou at 40 L

I WaJsihi d t h r-oo~m I p rmperatur (0 G ) I mothoniol anl-d i tp rp (

Dark Orate esi n Wh i clIlI ( lI ol I I t )

I r Lt f( J i O i oil ri t iTAI

Fi I Vrotu

Iteavie r crst Ls ir o sn rID

bottom of tzhe- ftasl

Csolid l (Whi t ti (e i Wiite mn(1)j-Lp C5rJ[

F crst lis kr ykia ized in Ac n iMlethanollrnrnrp

Yell ow crys-als White -mmrpols sol iid

FIUiJEd 1 Extraction and Iur firat in rncdure of the Al alrcili From Sanm jrp( de rad o

Y A7

44

- 7 _ - shy

91 9A 49 9 9

- I9 resin Ye lo 9 I

19~~~~~ 9 d ~

~ -- -- 9 ~- 499-9~A-- j - 9999-- 9 -~ -- --

D99999999 t9 ve tbq8 Or clnd r en

9999--------- -- -- --

FIUE2 hn aer hoaorpyo th jkal $ulampan Rei btie d rn teE c9 ip4 ng

~e99lt ~a4 99 ~ 99adoA fV) nSjwa per999~ore aGe C ~AAS4L

9 w t9 I 61U I i q 9h 6~99A an al -othc9o nm

9 g99 tj99 t W9v e--1UV ig L of 2 ni4 71A7 ogW

9

9 erf av~~ nex eah pt

- -

4 42

--- -- --22 - - -- - - - - - - - - - -- shy

722

122 222) 24

2 20 44 8

5~0 5 ~ 7

2

29

- ~ ~~ ~ ~ ~ ~- - - - - ~ 2 -2 2- ~ - -- 2 - - - - - - - - - - - - -

2222ZjI~D2 c 292i t~2i2J 22 t- o D22r222 ( v4L reE gE

(2222

F22 I29322~2~

~~VJ~ 2

2~ ~

22$22-Thin La rz a h

raclonEut2 -2222222222e

gt2 22222i212 2

2 Y 222~ 22

h Sys eyT s i2 ii a the2~ 666t 0 a42

e44 r s2 ih Ad22lt d2 ac ti

el vi E22 22kI2

of Rep

Dry2 Column2

22222 F 5

n2 e A

2)Mth M~2h ~2

at

22222 o

2

2rmog

c2

~2 2

2~en

2~2

2and2

22222 2 2 222 22 224222~ I2 2 4

44 4

ZI

C--IshyE~j44

a-4

A I- ~

~~~--4---4~~

~-

v-

~

-

~ 4k ~ 4

4

- -443-3-4 --

~~~ ~~~~~ FIUR E i

-~A 34603334

nPteno

~ - - N 4 -- - --3 -4 ---~- - -

K14---h CueA a

-$

i

-- -4

iXtO

- ~~ D _

~ ~ 4- 4-4Ai

e4tr

va

-2 4

L-i-n

upgtV to-~~

a p

~ hours

~ r

7 on~

4i

Ilk-~ 7 Ehtoi 4

7

to

IGURE 5 Ulrvoe pcr of the Chlor ydrate of thei

ProyIr-yided by Dr N~orman R~ -Fansorth (t5D)~~4 ~othSpectra Were- rt~n Wt~7~y i

Dot in W a -e

44~~ -~ - w~A

3

--

---

Ideni N

r U2

J4

f 0

5lt

46~

v~i ~

II

I K~-r~L pound

KJJzI--U

lt

1Ju

n 0 U

)

L7~tI ~1

~17 ~t K

--

0

~L

77PI if

-

~izLjplusmn1101~B

~

4

ifn

IGR 5

I

IIn

It I

FIGURE

6 UV

spectrum of

the H

ydrochloride of

the White

Amorphus Solid

from Sangre

do Grado i

nH20

41 40000 30

JA L3aS VRaLUICY Pp

7310 650411 iii1788 a1919 3847 4001 11351 73S4 1030 S51978 7974 404 71 7144 1087 7933403019 7 73

349055 43833463 318600 4L 3374 30835 3-154384 386349 3407

3400 33026 9 1083447 57 34 1 463444 371933 395

3432 217747 3721 170 9 2 4 68 5 3-65 2722 209388 823755 302510

2532 2345 489373 2367

3907 4T9436 3r 3022 Ir3845353661 3-299 004

3816 0000 0000 369 3299 - 0041 3793 - 30235 shy 00252

18ITLRAL- 4 8SITY

1441044 333441040 3 413470 339040 3490140 135310141 79

138

117438 313 48 bull9 5333411 14373743 1- 3938

348803 44400I300VV6 3113714lu

944453 1060393838 95040

1161735 15392

144704 13443

15- 0114 1 7921066 44176

9238012786720 7234502699

345992 44 48 3464991 448224 42576 84-392

3651793 431404 8474 49152 99-056 31776

go

C

1 E

4

04 amp209

N

-0

] 1

FIGJ1M 7 gR -ect of the Yellow Crystals obtained during the purification procedure of Sangre de Grado

Y A7

44

- 7 _ - shy

91 9A 49 9 9

- I9 resin Ye lo 9 I

19~~~~~ 9 d ~

~ -- -- 9 ~- 499-9~A-- j - 9999-- 9 -~ -- --

D99999999 t9 ve tbq8 Or clnd r en

9999--------- -- -- --

FIUE2 hn aer hoaorpyo th jkal $ulampan Rei btie d rn teE c9 ip4 ng

~e99lt ~a4 99 ~ 99adoA fV) nSjwa per999~ore aGe C ~AAS4L

9 w t9 I 61U I i q 9h 6~99A an al -othc9o nm

9 g99 tj99 t W9v e--1UV ig L of 2 ni4 71A7 ogW

9

9 erf av~~ nex eah pt

- -

4 42

--- -- --22 - - -- - - - - - - - - - -- shy

722

122 222) 24

2 20 44 8

5~0 5 ~ 7

2

29

- ~ ~~ ~ ~ ~ ~- - - - - ~ 2 -2 2- ~ - -- 2 - - - - - - - - - - - - -

2222ZjI~D2 c 292i t~2i2J 22 t- o D22r222 ( v4L reE gE

(2222

F22 I29322~2~

~~VJ~ 2

2~ ~

22$22-Thin La rz a h

raclonEut2 -2222222222e

gt2 22222i212 2

2 Y 222~ 22

h Sys eyT s i2 ii a the2~ 666t 0 a42

e44 r s2 ih Ad22lt d2 ac ti

el vi E22 22kI2

of Rep

Dry2 Column2

22222 F 5

n2 e A

2)Mth M~2h ~2

at

22222 o

2

2rmog

c2

~2 2

2~en

2~2

2and2

22222 2 2 222 22 224222~ I2 2 4

44 4

ZI

C--IshyE~j44

a-4

A I- ~

~~~--4---4~~

~-

v-

~

-

~ 4k ~ 4

4

- -443-3-4 --

~~~ ~~~~~ FIUR E i

-~A 34603334

nPteno

~ - - N 4 -- - --3 -4 ---~- - -

K14---h CueA a

-$

i

-- -4

iXtO

- ~~ D _

~ ~ 4- 4-4Ai

e4tr

va

-2 4

L-i-n

upgtV to-~~

a p

~ hours

~ r

7 on~

4i

Ilk-~ 7 Ehtoi 4

7

to

IGURE 5 Ulrvoe pcr of the Chlor ydrate of thei

ProyIr-yided by Dr N~orman R~ -Fansorth (t5D)~~4 ~othSpectra Were- rt~n Wt~7~y i

Dot in W a -e

44~~ -~ - w~A

3

--

---

Ideni N

r U2

J4

f 0

5lt

46~

v~i ~

II

I K~-r~L pound

KJJzI--U

lt

1Ju

n 0 U

)

L7~tI ~1

~17 ~t K

--

0

~L

77PI if

-

~izLjplusmn1101~B

~

4

ifn

IGR 5

I

IIn

It I

FIGURE

6 UV

spectrum of

the H

ydrochloride of

the White

Amorphus Solid

from Sangre

do Grado i

nH20

41 40000 30

JA L3aS VRaLUICY Pp

7310 650411 iii1788 a1919 3847 4001 11351 73S4 1030 S51978 7974 404 71 7144 1087 7933403019 7 73

349055 43833463 318600 4L 3374 30835 3-154384 386349 3407

3400 33026 9 1083447 57 34 1 463444 371933 395

3432 217747 3721 170 9 2 4 68 5 3-65 2722 209388 823755 302510

2532 2345 489373 2367

3907 4T9436 3r 3022 Ir3845353661 3-299 004

3816 0000 0000 369 3299 - 0041 3793 - 30235 shy 00252

18ITLRAL- 4 8SITY

1441044 333441040 3 413470 339040 3490140 135310141 79

138

117438 313 48 bull9 5333411 14373743 1- 3938

348803 44400I300VV6 3113714lu

944453 1060393838 95040

1161735 15392

144704 13443

15- 0114 1 7921066 44176

9238012786720 7234502699

345992 44 48 3464991 448224 42576 84-392

3651793 431404 8474 49152 99-056 31776

go

C

1 E

4

04 amp209

N

-0

] 1

FIGJ1M 7 gR -ect of the Yellow Crystals obtained during the purification procedure of Sangre de Grado

- -

4 42

--- -- --22 - - -- - - - - - - - - - -- shy

722

122 222) 24

2 20 44 8

5~0 5 ~ 7

2

29

- ~ ~~ ~ ~ ~ ~- - - - - ~ 2 -2 2- ~ - -- 2 - - - - - - - - - - - - -

2222ZjI~D2 c 292i t~2i2J 22 t- o D22r222 ( v4L reE gE

(2222

F22 I29322~2~

~~VJ~ 2

2~ ~

22$22-Thin La rz a h

raclonEut2 -2222222222e

gt2 22222i212 2

2 Y 222~ 22

h Sys eyT s i2 ii a the2~ 666t 0 a42

e44 r s2 ih Ad22lt d2 ac ti

el vi E22 22kI2

of Rep

Dry2 Column2

22222 F 5

n2 e A

2)Mth M~2h ~2

at

22222 o

2

2rmog

c2

~2 2

2~en

2~2

2and2

22222 2 2 222 22 224222~ I2 2 4

44 4

ZI

C--IshyE~j44

a-4

A I- ~

~~~--4---4~~

~-

v-

~

-

~ 4k ~ 4

4

- -443-3-4 --

~~~ ~~~~~ FIUR E i

-~A 34603334

nPteno

~ - - N 4 -- - --3 -4 ---~- - -

K14---h CueA a

-$

i

-- -4

iXtO

- ~~ D _

~ ~ 4- 4-4Ai

e4tr

va

-2 4

L-i-n

upgtV to-~~

a p

~ hours

~ r

7 on~

4i

Ilk-~ 7 Ehtoi 4

7

to

IGURE 5 Ulrvoe pcr of the Chlor ydrate of thei

ProyIr-yided by Dr N~orman R~ -Fansorth (t5D)~~4 ~othSpectra Were- rt~n Wt~7~y i

Dot in W a -e

44~~ -~ - w~A

3

--

---

Ideni N

r U2

J4

f 0

5lt

46~

v~i ~

II

I K~-r~L pound

KJJzI--U

lt

1Ju

n 0 U

)

L7~tI ~1

~17 ~t K

--

0

~L

77PI if

-

~izLjplusmn1101~B

~

4

ifn

IGR 5

I

IIn

It I

FIGURE

6 UV

spectrum of

the H

ydrochloride of

the White

Amorphus Solid

from Sangre

do Grado i

nH20

41 40000 30

JA L3aS VRaLUICY Pp

7310 650411 iii1788 a1919 3847 4001 11351 73S4 1030 S51978 7974 404 71 7144 1087 7933403019 7 73

349055 43833463 318600 4L 3374 30835 3-154384 386349 3407

3400 33026 9 1083447 57 34 1 463444 371933 395

3432 217747 3721 170 9 2 4 68 5 3-65 2722 209388 823755 302510

2532 2345 489373 2367

3907 4T9436 3r 3022 Ir3845353661 3-299 004

3816 0000 0000 369 3299 - 0041 3793 - 30235 shy 00252

18ITLRAL- 4 8SITY

1441044 333441040 3 413470 339040 3490140 135310141 79

138

117438 313 48 bull9 5333411 14373743 1- 3938

348803 44400I300VV6 3113714lu

944453 1060393838 95040

1161735 15392

144704 13443

15- 0114 1 7921066 44176

9238012786720 7234502699

345992 44 48 3464991 448224 42576 84-392

3651793 431404 8474 49152 99-056 31776

go

C

1 E

4

04 amp209

N

-0

] 1

FIGJ1M 7 gR -ect of the Yellow Crystals obtained during the purification procedure of Sangre de Grado

44 4

ZI

C--IshyE~j44

a-4

A I- ~

~~~--4---4~~

~-

v-

~

-

~ 4k ~ 4

4

- -443-3-4 --

~~~ ~~~~~ FIUR E i

-~A 34603334

nPteno

~ - - N 4 -- - --3 -4 ---~- - -

K14---h CueA a

-$

i

-- -4

iXtO

- ~~ D _

~ ~ 4- 4-4Ai

e4tr

va

-2 4

L-i-n

upgtV to-~~

a p

~ hours

~ r

7 on~

4i

Ilk-~ 7 Ehtoi 4

7

to

IGURE 5 Ulrvoe pcr of the Chlor ydrate of thei

ProyIr-yided by Dr N~orman R~ -Fansorth (t5D)~~4 ~othSpectra Were- rt~n Wt~7~y i

Dot in W a -e

44~~ -~ - w~A

3

--

---

Ideni N

r U2

J4

f 0

5lt

46~

v~i ~

II

I K~-r~L pound

KJJzI--U

lt

1Ju

n 0 U

)

L7~tI ~1

~17 ~t K

--

0

~L

77PI if

-

~izLjplusmn1101~B

~

4

ifn

IGR 5

I

IIn

It I

FIGURE

6 UV

spectrum of

the H

ydrochloride of

the White

Amorphus Solid

from Sangre

do Grado i

nH20

41 40000 30

JA L3aS VRaLUICY Pp

7310 650411 iii1788 a1919 3847 4001 11351 73S4 1030 S51978 7974 404 71 7144 1087 7933403019 7 73

349055 43833463 318600 4L 3374 30835 3-154384 386349 3407

3400 33026 9 1083447 57 34 1 463444 371933 395

3432 217747 3721 170 9 2 4 68 5 3-65 2722 209388 823755 302510

2532 2345 489373 2367

3907 4T9436 3r 3022 Ir3845353661 3-299 004

3816 0000 0000 369 3299 - 0041 3793 - 30235 shy 00252

18ITLRAL- 4 8SITY

1441044 333441040 3 413470 339040 3490140 135310141 79

138

117438 313 48 bull9 5333411 14373743 1- 3938

348803 44400I300VV6 3113714lu

944453 1060393838 95040

1161735 15392

144704 13443

15- 0114 1 7921066 44176

9238012786720 7234502699

345992 44 48 3464991 448224 42576 84-392

3651793 431404 8474 49152 99-056 31776

go

C

1 E

4

04 amp209

N

-0

] 1

FIGJ1M 7 gR -ect of the Yellow Crystals obtained during the purification procedure of Sangre de Grado

7

to

IGURE 5 Ulrvoe pcr of the Chlor ydrate of thei

ProyIr-yided by Dr N~orman R~ -Fansorth (t5D)~~4 ~othSpectra Were- rt~n Wt~7~y i

Dot in W a -e

44~~ -~ - w~A

3

--

---

Ideni N

r U2

J4

f 0

5lt

46~

v~i ~

II

I K~-r~L pound

KJJzI--U

lt

1Ju

n 0 U

)

L7~tI ~1

~17 ~t K

--

0

~L

77PI if

-

~izLjplusmn1101~B

~

4

ifn

IGR 5

I

IIn

It I

FIGURE

6 UV

spectrum of

the H

ydrochloride of

the White

Amorphus Solid

from Sangre

do Grado i

nH20

41 40000 30

JA L3aS VRaLUICY Pp

7310 650411 iii1788 a1919 3847 4001 11351 73S4 1030 S51978 7974 404 71 7144 1087 7933403019 7 73

349055 43833463 318600 4L 3374 30835 3-154384 386349 3407

3400 33026 9 1083447 57 34 1 463444 371933 395

3432 217747 3721 170 9 2 4 68 5 3-65 2722 209388 823755 302510

2532 2345 489373 2367

3907 4T9436 3r 3022 Ir3845353661 3-299 004

3816 0000 0000 369 3299 - 0041 3793 - 30235 shy 00252

18ITLRAL- 4 8SITY

1441044 333441040 3 413470 339040 3490140 135310141 79

138

117438 313 48 bull9 5333411 14373743 1- 3938

348803 44400I300VV6 3113714lu

944453 1060393838 95040

1161735 15392

144704 13443

15- 0114 1 7921066 44176

9238012786720 7234502699

345992 44 48 3464991 448224 42576 84-392

3651793 431404 8474 49152 99-056 31776

go

C

1 E

4

04 amp209

N

-0

] 1

FIGJ1M 7 gR -ect of the Yellow Crystals obtained during the purification procedure of Sangre de Grado

--

---

Ideni N

r U2

J4

f 0

5lt

46~

v~i ~

II

I K~-r~L pound

KJJzI--U

lt

1Ju

n 0 U

)

L7~tI ~1

~17 ~t K

--

0

~L

77PI if

-

~izLjplusmn1101~B

~

4

ifn

IGR 5

I

IIn

It I

FIGURE

6 UV

spectrum of

the H

ydrochloride of

the White

Amorphus Solid

from Sangre

do Grado i

nH20

41 40000 30

JA L3aS VRaLUICY Pp

7310 650411 iii1788 a1919 3847 4001 11351 73S4 1030 S51978 7974 404 71 7144 1087 7933403019 7 73

349055 43833463 318600 4L 3374 30835 3-154384 386349 3407

3400 33026 9 1083447 57 34 1 463444 371933 395

3432 217747 3721 170 9 2 4 68 5 3-65 2722 209388 823755 302510

2532 2345 489373 2367

3907 4T9436 3r 3022 Ir3845353661 3-299 004

3816 0000 0000 369 3299 - 0041 3793 - 30235 shy 00252

18ITLRAL- 4 8SITY

1441044 333441040 3 413470 339040 3490140 135310141 79

138

117438 313 48 bull9 5333411 14373743 1- 3938

348803 44400I300VV6 3113714lu

944453 1060393838 95040

1161735 15392

144704 13443

15- 0114 1 7921066 44176

9238012786720 7234502699

345992 44 48 3464991 448224 42576 84-392

3651793 431404 8474 49152 99-056 31776

go

C

1 E

4

04 amp209

N

-0

] 1

FIGJ1M 7 gR -ect of the Yellow Crystals obtained during the purification procedure of Sangre de Grado

ifn

IGR 5

I

IIn

It I

FIGURE

6 UV

spectrum of

the H

ydrochloride of

the White

Amorphus Solid

from Sangre

do Grado i

nH20

41 40000 30

JA L3aS VRaLUICY Pp

7310 650411 iii1788 a1919 3847 4001 11351 73S4 1030 S51978 7974 404 71 7144 1087 7933403019 7 73

349055 43833463 318600 4L 3374 30835 3-154384 386349 3407

3400 33026 9 1083447 57 34 1 463444 371933 395

3432 217747 3721 170 9 2 4 68 5 3-65 2722 209388 823755 302510

2532 2345 489373 2367

3907 4T9436 3r 3022 Ir3845353661 3-299 004

3816 0000 0000 369 3299 - 0041 3793 - 30235 shy 00252

18ITLRAL- 4 8SITY

1441044 333441040 3 413470 339040 3490140 135310141 79

138

117438 313 48 bull9 5333411 14373743 1- 3938

348803 44400I300VV6 3113714lu

944453 1060393838 95040

1161735 15392

144704 13443

15- 0114 1 7921066 44176

9238012786720 7234502699

345992 44 48 3464991 448224 42576 84-392

3651793 431404 8474 49152 99-056 31776

go

C

1 E

4

04 amp209

N

-0

] 1

FIGJ1M 7 gR -ect of the Yellow Crystals obtained during the purification procedure of Sangre de Grado

I

IIn

It I

FIGURE

6 UV

spectrum of

the H

ydrochloride of

the White

Amorphus Solid

from Sangre

do Grado i

nH20

41 40000 30

JA L3aS VRaLUICY Pp

7310 650411 iii1788 a1919 3847 4001 11351 73S4 1030 S51978 7974 404 71 7144 1087 7933403019 7 73

349055 43833463 318600 4L 3374 30835 3-154384 386349 3407

3400 33026 9 1083447 57 34 1 463444 371933 395

3432 217747 3721 170 9 2 4 68 5 3-65 2722 209388 823755 302510

2532 2345 489373 2367

3907 4T9436 3r 3022 Ir3845353661 3-299 004

3816 0000 0000 369 3299 - 0041 3793 - 30235 shy 00252

18ITLRAL- 4 8SITY

1441044 333441040 3 413470 339040 3490140 135310141 79

138

117438 313 48 bull9 5333411 14373743 1- 3938

348803 44400I300VV6 3113714lu

944453 1060393838 95040

1161735 15392

144704 13443

15- 0114 1 7921066 44176

9238012786720 7234502699

345992 44 48 3464991 448224 42576 84-392

3651793 431404 8474 49152 99-056 31776

go

C

1 E

4

04 amp209

N

-0

] 1

FIGJ1M 7 gR -ect of the Yellow Crystals obtained during the purification procedure of Sangre de Grado

41 40000 30

JA L3aS VRaLUICY Pp

7310 650411 iii1788 a1919 3847 4001 11351 73S4 1030 S51978 7974 404 71 7144 1087 7933403019 7 73

349055 43833463 318600 4L 3374 30835 3-154384 386349 3407

3400 33026 9 1083447 57 34 1 463444 371933 395

3432 217747 3721 170 9 2 4 68 5 3-65 2722 209388 823755 302510

2532 2345 489373 2367

3907 4T9436 3r 3022 Ir3845353661 3-299 004

3816 0000 0000 369 3299 - 0041 3793 - 30235 shy 00252

18ITLRAL- 4 8SITY

1441044 333441040 3 413470 339040 3490140 135310141 79

138

117438 313 48 bull9 5333411 14373743 1- 3938

348803 44400I300VV6 3113714lu

944453 1060393838 95040

1161735 15392

144704 13443

15- 0114 1 7921066 44176

9238012786720 7234502699

345992 44 48 3464991 448224 42576 84-392

3651793 431404 8474 49152 99-056 31776

go

C

1 E

4

04 amp209

N

-0

] 1

FIGJ1M 7 gR -ect of the Yellow Crystals obtained during the purification procedure of Sangre de Grado

a rciatLIrI-vc t Diori j edI tc r

tC I t tltro

l iTN 10[1r1c l 1 9

Lthyl attP a-f Li Ih oi- m V

VV

(qUOIiS phatse

Lx t r ac rI

0U~L1 Eit~racttion aind PuLrt fi~catl P-rOCF-dLre f or the Leaves of thp Croton chleri

A

-deg

C

FIGURE 9- Thin layer chromatography of the chloroform

extract frcn the leaves of the Croton lechshy

ieri

TLC was perfo_led on silica gel 60 HF 254 using as

eluent a mixture of Chloroform Methanol (Oil)

Spots A B and C with rfs of 063 051 and 029

respectively were positive to the Dragendorff reashy

gent

Fluorescence at 254 nm

Fluorescence at 366 nm (---------------

Dragerdorff positive (

II

-I1 -

_- __-t

FL1 Utvietpetuofak oIdBfe teea sofC tnlchein

Ethanol

qp- RI--shy

i-RE Ifrared SpeCtru of alkaloid B from the leaves of Croton lechleri in KBr

LA dITY 0 EO YR LQU

1 EdT 920 606 172IC Y 7577 594720 114432 932 60333 7 544 4557 504 629 760 NSS pp 9I LATI RAL

1031 58175e 7271 1631456 6942176191 546567 632 485656 132384 1229 535209 441776 6727 95040 1240 535-759 6697 4351424 5351129 1375 506096 6326 5577760 091392 1409 49861 6232 4299136 7541762260 311444 3593 14262 144 32230 c i -0 23 422309 2 93 4Us 165 6176300666 37583 2191-4246 - - 372- 152 202 496 2490 260856 3260 3562 6S0 230 752 Pj25 7 24231 3 03 4127-520 51 728 2730 205069 2676240 1260G 196 666 S- c 2749 203-90 2545 277246 1544002774 19 8391 2479 16212|6 226- 160 C)2755 15972 2449 1651232 2532446 2631 165654 2323 1469725 Is512 O

5 15515 2290 317024 234060327 131528 1644 6584500 1076394 J565 24417 0305 430976 1176323660 3519 0043 106C 160 1665283676 0000 0000 13163-136 4079 3w 3691 - 3299 0041 301 565 116632 il

te

FIGJE 12- NM Sectnzn of alkaloid B frcm the leaves of croton lechieri in CD Cl3

A

-deg

C

FIGURE 9- Thin layer chromatography of the chloroform

extract frcn the leaves of the Croton lechshy

ieri

TLC was perfo_led on silica gel 60 HF 254 using as

eluent a mixture of Chloroform Methanol (Oil)

Spots A B and C with rfs of 063 051 and 029

respectively were positive to the Dragendorff reashy

gent

Fluorescence at 254 nm

Fluorescence at 366 nm (---------------

Dragerdorff positive (

II

-I1 -

_- __-t

FL1 Utvietpetuofak oIdBfe teea sofC tnlchein

Ethanol

qp- RI--shy

i-RE Ifrared SpeCtru of alkaloid B from the leaves of Croton lechleri in KBr

LA dITY 0 EO YR LQU

1 EdT 920 606 172IC Y 7577 594720 114432 932 60333 7 544 4557 504 629 760 NSS pp 9I LATI RAL

1031 58175e 7271 1631456 6942176191 546567 632 485656 132384 1229 535209 441776 6727 95040 1240 535-759 6697 4351424 5351129 1375 506096 6326 5577760 091392 1409 49861 6232 4299136 7541762260 311444 3593 14262 144 32230 c i -0 23 422309 2 93 4Us 165 6176300666 37583 2191-4246 - - 372- 152 202 496 2490 260856 3260 3562 6S0 230 752 Pj25 7 24231 3 03 4127-520 51 728 2730 205069 2676240 1260G 196 666 S- c 2749 203-90 2545 277246 1544002774 19 8391 2479 16212|6 226- 160 C)2755 15972 2449 1651232 2532446 2631 165654 2323 1469725 Is512 O

5 15515 2290 317024 234060327 131528 1644 6584500 1076394 J565 24417 0305 430976 1176323660 3519 0043 106C 160 1665283676 0000 0000 13163-136 4079 3w 3691 - 3299 0041 301 565 116632 il

te

FIGJE 12- NM Sectnzn of alkaloid B frcm the leaves of croton lechieri in CD Cl3

II

-I1 -

_- __-t

FL1 Utvietpetuofak oIdBfe teea sofC tnlchein

Ethanol

qp- RI--shy

i-RE Ifrared SpeCtru of alkaloid B from the leaves of Croton lechleri in KBr

LA dITY 0 EO YR LQU

1 EdT 920 606 172IC Y 7577 594720 114432 932 60333 7 544 4557 504 629 760 NSS pp 9I LATI RAL

1031 58175e 7271 1631456 6942176191 546567 632 485656 132384 1229 535209 441776 6727 95040 1240 535-759 6697 4351424 5351129 1375 506096 6326 5577760 091392 1409 49861 6232 4299136 7541762260 311444 3593 14262 144 32230 c i -0 23 422309 2 93 4Us 165 6176300666 37583 2191-4246 - - 372- 152 202 496 2490 260856 3260 3562 6S0 230 752 Pj25 7 24231 3 03 4127-520 51 728 2730 205069 2676240 1260G 196 666 S- c 2749 203-90 2545 277246 1544002774 19 8391 2479 16212|6 226- 160 C)2755 15972 2449 1651232 2532446 2631 165654 2323 1469725 Is512 O

5 15515 2290 317024 234060327 131528 1644 6584500 1076394 J565 24417 0305 430976 1176323660 3519 0043 106C 160 1665283676 0000 0000 13163-136 4079 3w 3691 - 3299 0041 301 565 116632 il

te

FIGJE 12- NM Sectnzn of alkaloid B frcm the leaves of croton lechieri in CD Cl3

qp- RI--shy

i-RE Ifrared SpeCtru of alkaloid B from the leaves of Croton lechleri in KBr

LA dITY 0 EO YR LQU

1 EdT 920 606 172IC Y 7577 594720 114432 932 60333 7 544 4557 504 629 760 NSS pp 9I LATI RAL

1031 58175e 7271 1631456 6942176191 546567 632 485656 132384 1229 535209 441776 6727 95040 1240 535-759 6697 4351424 5351129 1375 506096 6326 5577760 091392 1409 49861 6232 4299136 7541762260 311444 3593 14262 144 32230 c i -0 23 422309 2 93 4Us 165 6176300666 37583 2191-4246 - - 372- 152 202 496 2490 260856 3260 3562 6S0 230 752 Pj25 7 24231 3 03 4127-520 51 728 2730 205069 2676240 1260G 196 666 S- c 2749 203-90 2545 277246 1544002774 19 8391 2479 16212|6 226- 160 C)2755 15972 2449 1651232 2532446 2631 165654 2323 1469725 Is512 O

5 15515 2290 317024 234060327 131528 1644 6584500 1076394 J565 24417 0305 430976 1176323660 3519 0043 106C 160 1665283676 0000 0000 13163-136 4079 3w 3691 - 3299 0041 301 565 116632 il

te

FIGJE 12- NM Sectnzn of alkaloid B frcm the leaves of croton lechieri in CD Cl3

LA dITY 0 EO YR LQU

1 EdT 920 606 172IC Y 7577 594720 114432 932 60333 7 544 4557 504 629 760 NSS pp 9I LATI RAL

1031 58175e 7271 1631456 6942176191 546567 632 485656 132384 1229 535209 441776 6727 95040 1240 535-759 6697 4351424 5351129 1375 506096 6326 5577760 091392 1409 49861 6232 4299136 7541762260 311444 3593 14262 144 32230 c i -0 23 422309 2 93 4Us 165 6176300666 37583 2191-4246 - - 372- 152 202 496 2490 260856 3260 3562 6S0 230 752 Pj25 7 24231 3 03 4127-520 51 728 2730 205069 2676240 1260G 196 666 S- c 2749 203-90 2545 277246 1544002774 19 8391 2479 16212|6 226- 160 C)2755 15972 2449 1651232 2532446 2631 165654 2323 1469725 Is512 O

5 15515 2290 317024 234060327 131528 1644 6584500 1076394 J565 24417 0305 430976 1176323660 3519 0043 106C 160 1665283676 0000 0000 13163-136 4079 3w 3691 - 3299 0041 301 565 116632 il

te

FIGJE 12- NM Sectnzn of alkaloid B frcm the leaves of croton lechieri in CD Cl3

11

Cr )tori I ict iIerer

F I (i EI I-U 3r r t1m c3 f a 1 1oicl 1 f ronn the I eaves of

S MASSIRA Male 441) OTAL 45IJNI- 4Z60464

16 ATI 1IM1 -303 TOTOIUD- J26219 ieisip 6f563

6 2 103 Ito

242 it

2

11L

szI aus n-iAU4 m~r 52TM1 Ii0US 2ti Dvi XSI

PIL RE MEL E

43 33 ~ 122 30 221 256 321~ 4 45 523 23 IO 23 21 4

SI 491 12274 1992 1 2542 1241

265~4

55 41 2 1 V ~ 4~ 1

56~ 23~~~~~~39 62 29 1 5 2 233lt 4 594 20

5311 42 24 56gt 24 2 2 46 2 4 5 41 197I35 22 35

141~ 4 1 39~ 539j 213 162 40 2659 ~ 4 53 - 46I 1) 9 263 112 2 60

4- w 6 1 1342 245 1 1 1 311~ 11 1242 6 15 t05-9- 26 to~- 42~ 4141 44 208 269 It~- I6 201 45 1444 ~ 211

14 237 141 Zr25 6 27 12f2146 04 4 ~ ~ 174~k245 569 216 2

41 4 4 156 532 2pj11

1 ~ 5 41I5t4 2 l 6~ ~ 2 7 2 34~ 1lt5V 1 5 g ~ ~ 41 4 146 4 2091 91 2 2~l14

65 2s 26Is 2534~ 275 298 i4ik 4 73 79 15 21 25 20 66 1

1105 11 41 2217 4 2921lt 1 9 5 126 18 39 0 5494-

of 263 155 5 -22 1 1 4-~221 36A2 217 653 -----(1 - 3 16~gt72

163

26 3451ll4 21 31A 23612 11 1gt 16 22 3

4 Ca 111 23 224 16 29at 4 2~ 16 423 1~ 2 232 12354-44 295 4 7~

559111 253 2321 344 644 29 -- I 69 -26 11 93 W lt 791 233 34 1 7-4

4444~le 06 44 4235-- 23 31112 21 - 3t 1--10146 1 11 1 239 4440

62 9654 7 9 239 0 31

A~ ~ e 42lt 126 2~~2 24 4 l1 f324 26i444gt4~41 12- 44~3lie f 465 179~ 2 42 5a1 - 324 - 2 41 --4lshy

101l 10 2 4 3 1 28 6 144 ~ l~ 44 1264l t92 242 4 )329 2-

2C4 9 14-21 246 333 4~

li 1 62 24 135 2417 2

)P41111

4

1

- - - - ---

T

~~-~ - ---shy

w~

D

00

-1 50~ -shy V

n C

0 2 4 T 2C 10TIME (

A2o inth

GUR 14 Kn tcso ii rsnc r j h

Co tr l witoi tapi e H

V C~~ ~ 7

-- 911~~

4o4

E I

R ~ I~4j

4 m

20 -i

-iK -j ----- - ----- ----- - - - -- - ----- ----------shy

Vz I -r T ME H

0~- 100 2()

f-fctof hlrhySdrae on oFIGRE 5 aspne the Contra3cap -

Ion -- l I sQLAn c ai nd 20 f~ r o~ gn rLn a tr~Inasp in E -Idc-I V~C v eIk

- t l ni1 otntro mp1(Oi 52 ngml CO 0 Coto 1fie witot e did no co it-amprct all

09C -- ~ tt~ Y ~ DEah rr s nde oA e era pacsoftf -th O I4 t

U-7

E I

n~

Ra~

O~

M~

I I I1 1

FIGRE6

-1 - -II -I1-3(-2-j-Y--

ffct of Taspine Chlorhy~drate oni thetContraction

Forh6 -general methodology n~e

T~e cohCncerations oC prWne-HAested werea

-aa1ttfceu jih(OLt cel1Isdi~d 1 t cotat t-I

Eac -s~de to thet~cr-aerage1 three platI Devila ation s tar shownbL-c Lie 1 _they wee very sm

(They wer e I es thranL+ 5of eac ViaIl e)1

Ink aiaa J

S MASSIRA Male 441) OTAL 45IJNI- 4Z60464

16 ATI 1IM1 -303 TOTOIUD- J26219 ieisip 6f563

6 2 103 Ito

242 it

2

11L

szI aus n-iAU4 m~r 52TM1 Ii0US 2ti Dvi XSI

PIL RE MEL E

43 33 ~ 122 30 221 256 321~ 4 45 523 23 IO 23 21 4

SI 491 12274 1992 1 2542 1241

265~4

55 41 2 1 V ~ 4~ 1

56~ 23~~~~~~39 62 29 1 5 2 233lt 4 594 20

5311 42 24 56gt 24 2 2 46 2 4 5 41 197I35 22 35

141~ 4 1 39~ 539j 213 162 40 2659 ~ 4 53 - 46I 1) 9 263 112 2 60

4- w 6 1 1342 245 1 1 1 311~ 11 1242 6 15 t05-9- 26 to~- 42~ 4141 44 208 269 It~- I6 201 45 1444 ~ 211

14 237 141 Zr25 6 27 12f2146 04 4 ~ ~ 174~k245 569 216 2

41 4 4 156 532 2pj11

1 ~ 5 41I5t4 2 l 6~ ~ 2 7 2 34~ 1lt5V 1 5 g ~ ~ 41 4 146 4 2091 91 2 2~l14

65 2s 26Is 2534~ 275 298 i4ik 4 73 79 15 21 25 20 66 1

1105 11 41 2217 4 2921lt 1 9 5 126 18 39 0 5494-

of 263 155 5 -22 1 1 4-~221 36A2 217 653 -----(1 - 3 16~gt72

163

26 3451ll4 21 31A 23612 11 1gt 16 22 3

4 Ca 111 23 224 16 29at 4 2~ 16 423 1~ 2 232 12354-44 295 4 7~

559111 253 2321 344 644 29 -- I 69 -26 11 93 W lt 791 233 34 1 7-4

4444~le 06 44 4235-- 23 31112 21 - 3t 1--10146 1 11 1 239 4440

62 9654 7 9 239 0 31

A~ ~ e 42lt 126 2~~2 24 4 l1 f324 26i444gt4~41 12- 44~3lie f 465 179~ 2 42 5a1 - 324 - 2 41 --4lshy

101l 10 2 4 3 1 28 6 144 ~ l~ 44 1264l t92 242 4 )329 2-

2C4 9 14-21 246 333 4~

li 1 62 24 135 2417 2

)P41111

4

1

- - - - ---

T

~~-~ - ---shy

w~

D

00

-1 50~ -shy V

n C

0 2 4 T 2C 10TIME (

A2o inth

GUR 14 Kn tcso ii rsnc r j h

Co tr l witoi tapi e H

V C~~ ~ 7

-- 911~~

4o4

E I

R ~ I~4j

4 m

20 -i

-iK -j ----- - ----- ----- - - - -- - ----- ----------shy

Vz I -r T ME H

0~- 100 2()

f-fctof hlrhySdrae on oFIGRE 5 aspne the Contra3cap -

Ion -- l I sQLAn c ai nd 20 f~ r o~ gn rLn a tr~Inasp in E -Idc-I V~C v eIk

- t l ni1 otntro mp1(Oi 52 ngml CO 0 Coto 1fie witot e did no co it-amprct all

09C -- ~ tt~ Y ~ DEah rr s nde oA e era pacsoftf -th O I4 t

U-7

E I

n~

Ra~

O~

M~

I I I1 1

FIGRE6

-1 - -II -I1-3(-2-j-Y--

ffct of Taspine Chlorhy~drate oni thetContraction

Forh6 -general methodology n~e

T~e cohCncerations oC prWne-HAested werea

-aa1ttfceu jih(OLt cel1Isdi~d 1 t cotat t-I

Eac -s~de to thet~cr-aerage1 three platI Devila ation s tar shownbL-c Lie 1 _they wee very sm

(They wer e I es thranL+ 5of eac ViaIl e)1

Ink aiaa J

- - - - ---

T

~~-~ - ---shy

w~

D

00

-1 50~ -shy V

n C

0 2 4 T 2C 10TIME (

A2o inth

GUR 14 Kn tcso ii rsnc r j h

Co tr l witoi tapi e H

V C~~ ~ 7

-- 911~~

4o4

E I

R ~ I~4j

4 m

20 -i

-iK -j ----- - ----- ----- - - - -- - ----- ----------shy

Vz I -r T ME H

0~- 100 2()

f-fctof hlrhySdrae on oFIGRE 5 aspne the Contra3cap -

Ion -- l I sQLAn c ai nd 20 f~ r o~ gn rLn a tr~Inasp in E -Idc-I V~C v eIk

- t l ni1 otntro mp1(Oi 52 ngml CO 0 Coto 1fie witot e did no co it-amprct all

09C -- ~ tt~ Y ~ DEah rr s nde oA e era pacsoftf -th O I4 t

U-7

E I

n~

Ra~

O~

M~

I I I1 1

FIGRE6

-1 - -II -I1-3(-2-j-Y--

ffct of Taspine Chlorhy~drate oni thetContraction

Forh6 -general methodology n~e

T~e cohCncerations oC prWne-HAested werea

-aa1ttfceu jih(OLt cel1Isdi~d 1 t cotat t-I

Eac -s~de to thet~cr-aerage1 three platI Devila ation s tar shownbL-c Lie 1 _they wee very sm

(They wer e I es thranL+ 5of eac ViaIl e)1

Ink aiaa J

-- 911~~

4o4

E I

R ~ I~4j

4 m

20 -i

-iK -j ----- - ----- ----- - - - -- - ----- ----------shy

Vz I -r T ME H

0~- 100 2()

f-fctof hlrhySdrae on oFIGRE 5 aspne the Contra3cap -

Ion -- l I sQLAn c ai nd 20 f~ r o~ gn rLn a tr~Inasp in E -Idc-I V~C v eIk

- t l ni1 otntro mp1(Oi 52 ngml CO 0 Coto 1fie witot e did no co it-amprct all

09C -- ~ tt~ Y ~ DEah rr s nde oA e era pacsoftf -th O I4 t

U-7

E I

n~

Ra~

O~

M~

I I I1 1

FIGRE6

-1 - -II -I1-3(-2-j-Y--

ffct of Taspine Chlorhy~drate oni thetContraction

Forh6 -general methodology n~e

T~e cohCncerations oC prWne-HAested werea

-aa1ttfceu jih(OLt cel1Isdi~d 1 t cotat t-I

Eac -s~de to thet~cr-aerage1 three platI Devila ation s tar shownbL-c Lie 1 _they wee very sm

(They wer e I es thranL+ 5of eac ViaIl e)1

Ink aiaa J

U-7

E I

n~

Ra~

O~

M~

I I I1 1

FIGRE6

-1 - -II -I1-3(-2-j-Y--

ffct of Taspine Chlorhy~drate oni thetContraction

Forh6 -general methodology n~e

T~e cohCncerations oC prWne-HAested werea

-aa1ttfceu jih(OLt cel1Isdi~d 1 t cotat t-I

Eac -s~de to thet~cr-aerage1 three platI Devila ation s tar shownbL-c Lie 1 _they wee very sm

(They wer e I es thranL+ 5of eac ViaIl e)1

Ink aiaa J