J. Braz. Chem. Soc. vol.21 número10

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3’,8’’-Biisokaempferide, a Cytotoxic Bilavonoid and Other Chemical Constituents

of Nanuza plicata (V elloz iaceae)

Meri Emili F. Pinto,a_{Marcelo Sobral da Silva,*},a_{Elisabete Schindler,}b_{José Maria} Barbosa Filho,a_{Ramon dos Santos El- Bachá,}b_{Marianna V ieira S. C astello- Branco,}a

Maria de Fatima A g raa_{and Josean Fechine T avares}a

a_{Laboratório de Tecnologia Farmacêutica, Univers idade Federal da P araíba, CP 5 0 0 9 ,}

5 8 0 5 1 - 9 7 0 João P es s oa- P B, Brazil

b_{Laboratório de N euroq uímica e Biologia Celular ( LabN q ) , I ns tituto de Ciências da Saúde ( I CS) ,}

Univers idade Federal da Bahia, 4 0 1 1 0 - 9 0 2 Salvador- BA , Brazil

F oi isolado das f olh as de N anuza p licata um nov o b if lav onóide, ch am ado 3 ’,8’’-b iisocam p f erideo (1) , j untam ente com os conh ecidos com p ostos am entolav ona (2) , ácido

p atag ônico (3) , ( 4aR ,5 S,6 R ,8aR ) -5 -[ 2-( 2,5 -dih idro-5 -m etóx i-2-ox of uran-3 -il) etil] -3 ,4,4a,5 ,6 ,7 ,8, 8a-octah idro-5 ,6 ,8a-trim etilnaf taleno-1-ácido carb ox ílico (4) , caf eoilq uinato de m etila (5) , ácido

3 ,5 -di-caf eoilq uínico (6) e luteolina (7) . O s com p ostos 3, 4, 5 e 6 são relatados p ela p rim eira

v ez em Velloziaceae. A s estruturas dos com p ostos f oram elucidadas com b ase em m étodos esp ectroscóp icos, esp ecialm ente R M N e E M . A citotox icidade de 3 ’,8’’-b iisocam p f erideo f oi

estudada em células de g liob lastom a h um ano ( G L -15 ) . A concentração ef etiv a, q ue p roduziu m orte em 5 0% das células ap ós 7 2 h f oi 3 6 ,5 μm ol L-1_{. A lterações na m orf olog ia celular, incluindo}

retração e deg radação de citop lasm a, f oram ob serv adas q uando as células f oram tratadas com concentrações a p artir de 20 μm ol L-1_{de 3 ’,8’’-b iisocam p f erideo p or 7 2 h .}

A nov el b ilav onoid, nam ed 3 ’,8’’-b iisok aem p f eride (1) , along w ith th e k now n com p ounds

am entolav one (2) , p atag onic acid (3) , ( 4aR ,5 S,6 R ,8aR ) -5 -[ 2-( 2,5 -D ih y dro-5 -m eth ox y -2-oxof uran-3 -y l) eth y l] -uran-3 ,4,4a,5 ,6 ,7 ,8,8a-octah y dro-5 ,6 ,8a-trimeth y lnap h th alene-1-carb ox y lic acid (4) ,

5 -caf f eoy lq uinic acid m eth y l ester (5) , 3 ,5 -di-caf f eoy lq uinic acid (6) and luteolin (7) , w ere isolated

of th e leav es f rom N anuza p licata. T h e com p ounds 3, 4, 5 and 6 are rep orted f or th e irst tim e

in Velloziaceae. T h e structures of th e com p ounds w ere elucidated on th e b asis of sp ectroscop ic m eth ods, esp ecially NM R and M S analy ses. T h e cy totox icity of 3 ’,8’’-b iisok aem p f eride w as

studied in cultures of h um an g liob lastom a G L -15 cells. T h e ef f ectiv e concentration, w h ich k illed 5 0 % of cells af ter 7 2 h w as 3 6 .5 μm ol L-1_{. C h ang es in cellular m orp h olog y , including retraction}

and deg radation of cy top lasm , w ere ob serv ed w h en cells w ere treated w ith concentrations f rom 20 μm ol L-1_{of 3 ’,8’’-b iisok aem p f eride f or 7 2 h .}

K eyw ords: 3 ’,8’’-b iisok aem p f eride, N anuza p licata, cy totox icity , Velloziaceae

I ntroduction

Velloziaceae is a f am ily of m onocoty ledonous p lants containing ab out 27 0 sp ecies of th e trop ical

Brazilian lora occurring in rock y ields.1_{T h ese p lants}

liv e under conditions of h ig h solar irradiation and low

w ater av ailab ility .2_{Sp ecies of th is f am ily contain h ig h}

am ounts of diterp enes w ith a sk eleton of th e clerodane,2

cleisth antane, isop im arane,3_{k aurane,}4_{and lab dane}5_{ty p es,}

b esides lav onoids m onoisop reny lated, C-alk y lated and

b ilav onoids.6 -9_{T h e g enus}_{N anuza}_{com p rises th ree sp ecies,}

N . p licata, N . almeidae and N . luetzelburgii. N anuza

p licata ( M art.) L . B. Sm . & A y ensu ( Sy nony m y : V ellozia p licata and X erop hy ta p licata) , is k now n com m only as “canela d’em a”. I n earlier studies th e com p ounds

3 -g erany l-4-h y drox y b enzoate10-11_{and th e b if lav onoid}

am entolav one,12 _{w ere isolated f rom sp ecies collected in}

th e States of R io de J aneiro and M inas G erais, Brazil,

resp ectiv ely . I n th is w ork , w e rep ort th e isolation and

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3 ’,8’’-Biisok aem p f eride, a C y totox ic Bilav onoid and O th er C h em ical C onstituentsJ. Braz. Chem. Soc.

1820

b ilav onoids, 3 ’,8’’-b iisok aem p f eride (1) , a new natural

p roduct, and am entolav one (2) ,13 _{tw o diterp enes of th e}

clerodane ty p e, p atag onic acid (3)14_{and ( 4aR ,5 S,6 R ,8aR )}

5 [ 2( 2,5 dih y dro5 m eth ox y 2ox of uran3 y l) eth y l] -3 ,4,4a,5 ,6 ,7 ,8,8a-octah y dro-5 ,6 ,8a trim eth y lnap h th

alene-1-carb ox y lic acid (4) ,15_{tw o caf f eoy lq uinic acid deriv ates,}

5 -caf f eoy lq uinic acid m eth y l ester (5)16_{and 3 ,5}

-di-caf f eoy lq uinic acid (6)17 -18 _{and a lav one, luteolin (}₇₎19

( F ig ure 1) . T h e com p ounds 3, 4 , 5 and 6 are rep orted f or

th e irst tim e in Velloziaceae.

I nterest in th e p ossib le h ealth b eneits of lav onoids h as

increased b ecause som e h um an f eeding studies sup p ort a

p rotectiv e ef f ect of th eir consum p tion in cancer20_{and also}

ag ainst cy totox ic insults induced b y ox idativ e stress and

am y loid b sug g esting th eir th erap eutic p otential against

neurodeg enerativ e diseases.21_{T h e rep lacem ent of anim al}

tech niq ues w ith non-anim al ones is im p erativ e in saf e

tox icolog ical ev aluations of new com p ounds. M odels using

cultured cells to ev aluate th e cy totox icity of ch emicals

h av e th e adv antag e of b eing sim p le and q uick w ith a low

ev aluation cost.22_{T h e inv estig ation of new m olecules f or}

g liob lastom a treatm ent is relev ant b ecause desp ite current adv ances in th erap y , including surg ical resection f ollow ed b y radiation and ch em oth erap y , th e p rog nosis f or p atients

rem ains p oor.23_{I n th is p ap er, th e cy totox icity of}_{1 to h um an}

g liob lastom a G L -15 cells w as ev aluated in vitro.

R esults and D iscussion

T h e eth anol ex tract f rom N . p licata af f orded lav onoids,

clerodane diterp enes and deriv ativ es of ch lorog enic acid.

T h e new b ilav onoid (1 ) w as isolated in th e f orm of a

y ellow solid w ith m p 3 40-3 42º C , and th e H R -E SI -M S m ass

sp ectrum sh ow ed a m olecular ion p eak at 5 99.5 3 3 1 [ M +

H ] , com p atib le w ith th e m olecular f orm ula C_{3 2}H₂₃O₁₂. T h e

inf rared sp ectrum in K Br sh ow ed ab sorp tion at 3 3 5 0, 3 25 0

and 16 5 1 cm-1_{, ch aracteristic of O H and carb ony l g roup s,}

resp ectiv ely . T h e 13_{C NM R in C D}

3O D ( 5 00 M H z) sh ow ed

th e p resence of 3 2 sig nals w h ere 20 sig nals w ere attrib uted

to non-h y drog enated carb ons, 8 sig nals corresp onding to 10

m eth y nic carb ons and 2 to m eth ox y carb ons. T h e ch emical

sh if ts at dC 180.0 and 17 9.9 conirm th e ab sorp tion of

carb ony l g roup s. T h e sig nals at d_C 94.8 and 99.8, com p ared

w ith literature data19_{w ere attrib uted to C -8 and C -6 ,}

resp ectiv ely , of th e lav onoids A ring . L ik ew ise, the sig nals at

dC 121.9, 106 .8 and 101.4 w ere attrib uted to C -3 ’, C -8’’ and

C -6 ’’ consistent w ith b ilav onoids b ond C -3 ’-C -8’’, b ecause

w h en f usion is C -3 ’-C -6 ’’, th e ch em ical sh if ts f or th e sam e

carb ons are 116 .7 , 93 .9 and 103 .5 , resp ectiv ely . T he b ond

C -3 ’-C -8’’ w as conirm ed b y th e H M BC correlations of

th e sig nal at 6 .3 1 ( s, H -6 ’’) , and 7 .08 ( d, J 9.0, H -5 ’) w ith

106 .8 ( C -8’’) .

T h e ab sence of carb on sig nals at d_C 104.0 and 103 .4,

as in am entolav one (2) , and th e ch em ical sh if ts f or th e

m eth ox y carb ons at d_C 6 0.5 and 6 0.6 and com p arison

w ith th e ch em ical sh if ts of isok aem p f eride19_{sug g est th e}

insertion of th ese g roup s at C -3 and C -3 ’’. T h is notion w as

conirm ed b y th e H M BC sp ectrum th roug h th e correlations

of th e m eth ox y h y drog ens w ith th e carb ons at d_C 13 9.4

and 13 9.3 ( C -3 and C -3 ’’) . I t is f urth er sup p orted b y th e R O E SY sp ectrum th roug h th e correlations of th e m eth ox y

p rotons at C -3 and C -3 ’’ w ith th e h y drog ens H -6 ’ and

H -2’’’, resp ectiv ely . T h us, (1) dif f ers f rom am entolav one

b y h av ing p ositions C -3 and C -3 ’’ occup ied b y m eth ox y g roup s. By com p aring th e ch em ical sh if ts of m onom eric units of (1) w ith th e literature could b e ch aracterized

th em as units isok aem p f eride. T h eref ore, (1) is a new

b ilav onoid w ith union C -3 ’-C -8’’ of isok aem p f eride, nam ed

3 ’,8’’-b iisok aem p f eride. T h e data f or 1_{H ,}13_{C , H M QC and}

H M BC NM R are com p iled in T ab le 1. T h e k now n lav onoid

am entolav one (2) w as identiied b y sp ectral data analy sis

and com p arison w ith literature v alues.19 _{T h e structures}

of 3 and 4 w ere identiied b y NM R as p atag onic acid14

and ( 4aR ,5 S,6 R ,8aR ) -5 -[ 2-( 2,5 -dih y dro-5 -m eth ox y

-2-ox of uran-3 -y l) eth y l] -3 ,4,4a,5 ,6 ,7 ,8,8a-octah y dro-5 ,6

,8a-trim eth y lnap h th alene-1-carb ox y lic acid, resp ectiv ely , b oth

diterp enes of th e clerodane ty p e.15 ,24,25_{A ccording to th e}

literature14_{on sub stance}_{3 th e ch em ical sh if ts of C -2 is 27 .4}

and C -7 is 27 .2. H M BC sp ectrum sh ow ed a correlation of

th e sig nal at 0.85 ( d, J 6 .7 H z, C H₃-17 ) w ith th e sig nal at

27 .2, b eing th is sig nal assig ned to C -7 , and a correlation of th e sig nal at 6 .81 ( b r s H -3 ) w ith th e sig nal at 27 .4,

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Pinto et al. 1821 Vol. 21, No. 10, 2010

w h ich w as assig ned to C -2. T h us, 13_{C NM R data of}_{3 w ere}

uneq uiv ocally m ark ed.

T h e sp ectral data analy sis of 5 and 6 identiied th ese

com p ounds as 5 -caf f eoy lq uinic acid m eth y l ester26_{and 3 ,5}

-di-caf f eoy lq uinic acid,18_{resp ectiv ely , b oth rep orted f or th e irst}

tim e in th e f am ily Velloziaceae. L uteolin (7) w as identiied b y

sp ectral data analy sis and com p arison w ith literature v alues.19

T h e ef f ect of th e concentration of com p ound 1 on th e

induction of cy totox icity to h um an g liob lastom a G L -15

cells w as inv estig ated ( F ig ure 2) . I t w as f ound th at th e

m inim al cy totox ic concentration w as 20 μm ol L-1_as

com p ared to control g roup , k illing 46 .9% of cells af ter

7 2 h 3 ’,8’’-b iisok aem p f eride -induced cy totox icity w as

itted to eq uation 1:

V = 4.987 + { 115 .113 / [ 1 + 10 ( 1.991 log [ C ] - 2.919)_{] }} _{( 1)}

( R2_{= 0.926 2)}

in w h ich V corresp onds to cell v iab ility norm alized to

data m easured under control conditions, and [ C ] is th e

3 ’,8’’-b iisok aem p f eride concentration. T h e calculated

ef f ectiv e concentration, w h ich k illed 5 0% ( E C_{5 0}) of G L -15

cells af ter 7 2 h w as 3 6 .5 μm ol L-1_.

T h is is im p ortant to state th at th e sam e inv estig ation

carried out w ith am entolav one sh ow ed th at it does not

p ossess any antitum oral activ ity ag ainst G L -15 cells until

6 00 μm ol L-1 _{( data not sh ow n) . Since th e dif f erence b etw een}

th ese com p ounds sh ow n in f orm ulas 1 and 2 ( F ig ure 1) is

th e p resence of tw o m eth ox y carb ons, th ese g roup s enh ance

th e induction of cy totox icity to G L -15 cells.

T h e m orp h olog y of cells treated w ith 1.5 μm ol L-1

3 ’,8’’-b iisok aem p f eride w as not m odif ied af ter 7 2 h,

w h en com p ared to th e neg ativ e control g roup . C h ang es

in cellular m orp h olog y w ere ob serv ed w h en cells w ere

treated w ith 20 μm ol L-1_{3 ’,8’’-b iisok aem p f eride f or 7 2 h ,}

som e cells sh ow ed retraction and deg radation of cy top lasm ,

oth ers b ecam e round and b ig . F urth erm ore, som e cells

detach ed f rom p lates. C ells treated w ith 200 μm ol L-1

3 ’,8’’-b iisok aem p f eride f or 7 2 h p resented an alm ost

T ab le 1 . NM R data f or 1a

Positions H M QC H M BC

1_H 13_C

2 - 15 8.1

3 - 13 9.4

4 - 180.0

5 - 16 2.9

6 6 .14 ( d, J 2.0) 99.8 C -5 , C -7 , C -10

7 - 16 6 .2

8 6 .19 ( d, J 2.0) 94.8 C -7 , C -9, C -10

9 - 15 8.4

10 - 105 .7

1’ - 122.7

2’ 8.16 ( d, J 2.0) 13 4.6 C -2, C -4’, C -6 ’, C -8’’

3 ’ - 121.9

4’ - 16 1.1

5 ’ 7 .08 ( d, J 9.0) 118.4 C -1’ , C -3 ’ , C -4’

6 ’ 8.08 ( dd, J 9.0; 2.0) 13 0.4 C -4’

2’’ - 15 7 .2

3 ’’ - 13 9.3

4’’ - 17 9.9

5 ’’ - 16 2.1

6 ’’ 6 .3 1 ( s) 101.4 C -5 ’’, C -7 ’’, C -8’’

7 ’’ - 16 7 .5

8’’ - 106 .8

9’’ - 15 5 .6

10’’ - 105 .2

1’’’ - 122.6

2’’’ 7 .7 8 ( d, J 8.0) 13 1.3 C -2’’, C -4’’’

3 ’’’ 6 .6 5 ( d, J 8.0) 116 .3 C -1’’’, C -4’’’

4’’’ - 16 1.4

5 ’’’ 6 .6 5 ( d, J 8.0) 116 .3 C -4’’’

6 ’’’ 7 .7 8 ( d, J 8.0) 13 1.3 C -2’, C -4’’’

3 -O C H3 3 .7 3 ( s) 6 0.5 C -3

3 ’’-O C H₃ 3 .7 8 ( s) 6 0.6 C -3 ’’

5 -O H 12.7 4 ( s)b

-5 ’’-O H 12.86 ( s)b

-a_{D ata ob tained at 5 00 M H z, C D}

3O D ; J in H z and d in p p m ; b_Values

ob tained in D M SO -d₆.

F ig ure 2 . 3 ’,8’’-b iisok aem p f eride induced cy totox icity to h um an g liob lastom a G L -15 cells. G L -15 cells w ere ex p osed to 3 ’,8’’-b iiso-k aem p f eride 1.5 -200 μm ol L-1_{f or 7 2 h , at 3 7 °C , and 5 % C O}

2, in 96 -w ell

p lates ( n = 8 f or each concentration) . C ell v iab ility w as norm alized to data m easured under control conditions w ith out 3 ’,8’’-b iisok aem p f eride.

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1822

com p lete deg radation of cy top lasm , ch aracterizing cellular

death .

T em ozolom ide ( T M Z ) w as th e irst ch em oth erap eutic

ag ent ap p rov ed f or treatm ent of h ig h -g rade m alig nant

g liom as in m ore th an 20 y ears. I n p rim ary culture of

h um an g liob lastom a cells, dif f erences in T M Z cy totox icity

w as ob serv ed, th e indiv idualized E C_{5 0} af ter 7 2 h sh ow ed

v ariab ility b etw een p atients, 45 0-900 m m ol L-1_.27_{A lth oug h}

relativ ely uncom m on, m alig nant g liom as are associated

w ith disp rop ortionately h ig h m orb idity and m ortality , and

desp ite op tim al treatm ent, th e m edian surv iv al is only 12 to

15 m onth s f or p atients w ith g liob lastom a.28_{G liob lastom a}

stem cells disp lay strong cap ab ility of tum or’s resistance to

T M Z .29_{I n th is study , th e 3 ’,8’’-b iisok aem p f eride p resente}_d

an E C_{5 0} of 3 6 .5 μm ol L-1_{in G L -15 cells, a cy totox ic}

concentration b eneath th at f ound f or T M Z .27

E xperimental

G eneral p rocedures

M elting p oints h av e not b een corrected. I R sp ectra w ere

recorded on a BO M E M -M B 100 sp ectrop h otom eter. 1_H

( 5 00 M H z) and 13_{C ( 125 M H z) NM R sp ectra w ere recorded}

on a VA R I A N-Sy stem sp ectrom eter using D Md₆, SO

-C D₃O D or C D C l₃ w ith T M S as internal standard. H R E SI

m ass sp ectra w ere ob tained w ith a Brük er D altonics

U ltrO T O F -Q, Billerica, M A , sp ectrom eter using ( H₂O ,

A r) , 20 eV f or M S and 45 eV f or M S/ M S in p ositiv e m ode.

C olum n ch rom atog rap h y w ith silica g el ( M erck 0.06 3 -0.20

m m ) and Sep h adex L H -20 ( Sig m a, U SA ) ; silica g el F 25 4 G ( Vetec) w as used f or p rep arativ e T L C , silica g el p lates

PF_{25 4} 7 7 49 ( M erck ) w as used f or analy tical T L C ; w ith

v isualization under U V ( 25 4 and 3 6 6 nm ) , or ex p osure to iodine v ap or.

P lant material

T h e entire p lant of N anuza p licata ( M art.) L .B.Sm . &

A y ensu ( Velloziaceae) w as collected in M arch 2007 in th e sem i-arid north east reg ion of Brazil, city of Serra Branca

in Paraib a State. Vouch er sp ecim en ( A g ra et al.; 5 7 3 0)

is dep osited at th e H erb arium Prof . L auro Pires X av ier, U niv ersidade F ederal da Paraíb a, J oão Pessoa-PB, Brazil.

E x traction and is olation

T h e dry and p ulv erized p lant m aterial ( 2.0 k g ) w as ex tracted successiv ely w ith 95 % eth anol at room tem p erature. T h e solv ent w as rem ov ed under reduced

p ressure, ob taining th e crude eth anol ex tract ( 6 0 g) .

E ig h t g ram s of th is ex tract w ere sub m itted to g el

iltration th roug h Sep h adex L H 20, eluted w ith m eth anol

f ollow ed b y a m eth anol: ch lorof orm ( 1: 1) , resulting in

7 6 f ractions ( A 1-A 7 6 ) . T h e eluate f rom th e colum n

w as m onitored b y analy tical th in-lay er ch rom atog raph y

and sim ilar f ractions w ere com b ined. T h e g roup of

f ractions A 5 -A 7 ( 3 5 m g ) w as sub m itted to p rep arative

T L C w ith ch lorof orm : m eth anol ( 9: 1) , ob taining

1 ( 20 m g ) . T h e g roup of f ractions A 8-A 9 ( 25 m g )

w as recry stallized f rom acetone ob taining 2 ( 15 m g ) .

T h e f ractions A 10-A 13 ( 100 m g ) w ere ch rom atog rap h ed

on a silica g el colum n utilizing ch lorof orm : m eth anol as

th e m ob ile p h ase, w ith an increasing p olarity g radient,

ob taining 3 and 4 ( 10 m g ) , 5 ( 11 m g ) and 6 ( 12 m g ) ,

resp ectiv ely . T h e g roup of f ractions A 23 -A 28 w as

recry stallized f rom acetone, ob taining 7 ( 8 m g ) .

G lioblas toma cell cultures

E x p erim ents w ere carried out using h um an g liob lastom a

G L -15 cell line of clonal orig in, w h ich h as b een p rev iously

estab lish ed.3 0_{G L -15 cell cultures w ere p rep ared as}

describ ed p rev iously .3 1_{C ells w ere g row n in a h um idiied}

95 % air and 5 % C O₂ atm osp h ere at 3 7 ºC , and th e culture

m edium w as rep laced th ree tim es a w eek . A t th e tim e of

th e ex p erim ent, conluent cells w ere try p sinized and p lated

in a 96 -w ell p late, at a inal density of 3 × 104_{cells/ cm}2_.

E x p erim ents w ere initiated 7 2 h af ter p lating .

I n vitro evaluation of 3 ’,8 ’’- biis ok aemp f eride induced cy totox icity

3 ’,8’’-b iisok aem p f eride at concentrations b etw een

1.5 -200 m m ol L-1_{, and am entolav one at concentrations}

b etw een 3 -6 00 m m ol L-1_{w ere used to ex am ine its}

cy totox ic ef f ects. E ig h t rep licates w ere used f or each

dose in a 96 -w ell p late. C ells w ere ex p osed to 1 f or 7 2 h .

C ells v iab ility w as assessed using 3 -( 4,5 -dim eth y lth

iazol-2-y l) -2,5 -dip h eny ltetrazolium b rom ide ( M T T ; Sig m a,

St. L ouis, M O ) . M T T w as dissolv ed at a concentration

of 5 m g L-1_{in sterile p h osp h ate b uf f ered saline ( PBS) at}

room tem p erature, and th e solution w as f urth er sterilized b y p assing th roug h a 0.2 m m ilter and stored at 4 °C in th e dark . T h e inal concentration of M T T added to each

w ell w as 1 m g L-1_{. A f ter 2 h of incub ation at 3 7 ºC , a}

sam e v olum e of ly sis b uf f er w as added. L y sis b uf f er w as p rep ared as f ollow s: 20% ( m / v ) sodium dodecy l sulp h ate ( SD S) w as dissolv ed at 3 7 ºC in a solution of 5 0% ( v / v )

dim eth y lf orm am ide ( D M F ) and reag ent g rade w ater, p H 4.7 .

A f ter an ov ernig h t incub ation at room tem p erature, op tical

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Pinto et al. 1823 Vol. 21, No. 10, 2010

norm alized to data m easured under control conditions,

w ith out 1.

P has e contras t micros cop y

C ell m orp h olog y w as ev aluated b y p h ase contrast m icroscop y using an inv erted m icroscop e E clip se T S100 ( Nik on, T ok y o, J ap an) . Ph otog rap h s w ere tak en b y a C oolp ix 43 00 dig ital cam era ( Nik on) attach ed to th e

m icroscop e. Nik on View v ersion 6 .1.0 w as used to transf er

im ag es f rom th e cam era to a com p uter to b e edited. T h e only p rocess used to edit im ag es w as to transf orm color

p h otog rap h s into h alf tone p ictures. A ruler w ith tick s ev ery

10 m m ( O ly m p us, T ok y o, J ap an) w as p h otog rap h ed under th e sam e conditions. A new lay er containing th e ruler w as

added to p ictures using Ph oto I m p ression 4.0 ( A rcSof t,

F rem ont, U SA ) .

Statis tical analy s is

T h e data f or th e concentration-resp onse curv e w ere analy zed using one-w ay A NO VA and g roup s w ere com p ared b y Student-New m an-K euls test. T h e data w ere

itted using nonlinear reg ression p erf orm ed w ith G rap h Pad

Prism sof tw are ( San D ieg o, U SA ) .

3 ’,8 ’’- biis ok aemp f eride (1)

Y ellow am orp h ous p ow der; I R ( K Br) ν

m ax/ cm

-1_{: 3 3 5 0,}

16 5 1, 15 00, 13 5 7 , 1980; H R Mm/ z: 5 99.5 3 3 1 S-E SI

( M + H ) +_{, ( calc. f or C}

3 2H23O12 5 99.5 3 89) , f ound 46 4.3 6 6 2

( 100% ) ; 1_{H and}13_{C NM R sp ectral data: see T ab le 1.}

P atagonic acid (3)

W h ite am orp h ous p ow der; I R ( K Br) ν

m ax/ cm

-1_{: 3 43 3 ,}

295 8, 17 5 1, 16 7 8, 145 8, 126 1; 1_{H NM R ( C D C l}

3; 5 00M H z) :

d 1.7 0 ( m , 2H -1) , 1.45 ( m , 2H -2) , 6 .81 ( b r s, H -3 ) , 1.43 ( m ,

H -6 eq ) , 2.40 ( m , H -6 ax ) , 2.25 ( m , 2H -7 ) , 1.5 0 ( m , H -8) , 1.3 5 ( m , H -10) , 1.19 ( m , H -11eq ) , 2.40 ( m , H -11ax ) , 2,05 ( m , H -12eq ) , 2.20 ( m , H -12ax ) , 7 .06 ( b r s, H -14) , 4.7 9 ( b r s, 2H 15 ) , 0.85 ( d, 3 H 17 ) , 1.27 ( s, 3 H 19) , 0.7 5 ( s, 3 H

-20) ; NM R ( C D C l₃; 5 00M H z) : d 17 .4 ( C H₂-1) , 27 .4 ( C H₂

-2) , 140.1 ( C H -3 ) , 141.1 ( C -4) , 3 7 .4 ( C -5 ) , 3 6 .0 ( C H₂-6 ) ,

27 .2 ( C H₂-7 ) , 3 6 .3 ( C H -8) , 3 8.7 ( C -9) , 46 .7 ( C H -10) , 3 5 .7

( C H₂-11) , 19.0 ( C H₂-12) , 13 5 .0 ( C -13 ) , 143 .4 ( C H -14) ,

7 0.1 ( C H₂-15 ) , 17 4.3 ( C -16 ) , 15 .8 ( C H₃-17 ) , 17 4.2 ( C -18) ,

20.5 ( C H₃-19) , 18.1 ( C H₃-20) .

S upplementary I nformation

Sup p lem entary data are av ailab le f ree of ch arg e at h ttp : / / j b cs.sb q .org .b r, as PD F ile.

A cknow ledg ments

T h e auth ors are g ratef ul to C NPq , F A PE SB and C A PE S

f or sch olarsh ip s and inancial sup p ort, as w ell as to V. C . de

O . C osta ( U F PB) f or th e NM R sp ectra, and N. P. L op es of

F aculdade de C iências F arm acêuticas-U SP-R ib eirão Preto

f or th e H R -E SI M S.

R eferences

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2 2, 2017 .

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1 9 84, 3 9, 16 7 .

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3 6 5 .

18. M eira, M .; D av id, J . M .; D av id, J . P.; A raúj o, S. V.; R eg is, T . L .; G iulietti, A . M .; Queiróz, L . P.;Q uim. N ova

2008, 3 1, 7 5 1.

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Y ak ugak u Z as s hi 2 0 0 9 , 1 2 9 , 1113 . ( C oden: Y K K Z A J I SSN: 003 1-6 903 , A N 2009: 13 1486 9 C aPlus) .

23 . Valensin, S.; G h iron, C .; L am anna, C .; K rem er, A .; R ossi, M .; F erruzzi, P.; Niev o, M .; Bak k er, A .; BM C Cancer 2 0 0 9, 9, 196 .

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25 . A h m ad, V. U .; K h an, A .; F arooq , U .; K ousar, F .; K h an, S. S.; Naw az, S. A .; A b b asi, M . A .; C h oudh ary , M . I .; Chem. P harm.

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3 0. Bocch ini, V.; C asalone, R .; C ollini, P. ; R eb el, G .; L o C urto, F .; Cell Tis s ue R es . 1 9 9 1 , 2 6 5, 7 3.

3 1. Planch enault, T .; C osta, S. L .; F ag es, C .; R ich e, D .; C h

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Submitted: D ecember 1 8 , 2 0 0 9

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Su

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Inf

or

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*e-mail: marcelosobral@ltf.ufpb.br

3’,8’’-Biisokaempferide, a Cytotoxic Bilavonoid, and Other Chemical Constituents

of Nanuza plicata (V elloz iaceae)

Meri Emili F. Pinto,a_{Marcelo Sobral da Silva,*},a_{Elisabete Schindler,}b_{José Maria} Barbosa Filho,a_{Ramon dos Santos El- Bachá,}b_{Marianna V ieira S. C astello- Branco,}a

Maria de Fatima A g raa_{and Josean Fechine T avares}a

a_{Laboratório de Tecnologia Farmacêutica, Univers idade Federal da P araíba, CP 5 0 0 9 ,}

5 8 0 5 1 - 9 7 0 João P es s oa- P B, Brazil

b_{Laboratório de N euroq uímica e Biologia Celular ( LabN q ) , I ns tituto de Ciências da Saúde ( I CS) ,}

Univers idade Federal da Bahia, 4 0 1 1 0 - 9 0 2 Salvador- BA , Brazil

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3’,8’’-Biisokaempferide, a Cytotoxic Bilavonoid, and Other Chemical Constituents J. Braz. Chem. Soc.

2

Figure S2. 13_{C NMR spectrum (CD}

3OD, 125 MHz) of the compound 1 isolated of N anuza p licata.

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Pinto et al. 3 Vol. 21, No. 10, 2010

3OD, 125 MHz, dC 94-139) of the compound 1 isolated of N anuza p licata.

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4

Figure S6. 1_{H NMR spectrum (CD}

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Pinto et al. 5 Vol. 21, No. 10, 2010

3OD, 500 MHz, dH 6.1-7.1) of the compound 1 isolated of N anuza p licata.

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6

Figure S10. HMBC NMR experiment (CD3OD, 500×125 MHz, dC 158-168) of the compound 1 isolated of N anuza p licata.

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Pinto et al. 7 Vol. 21, No. 10, 2010

Figure S12. HMBC NMR experiment (CD3OD, 500 x 125 MHz, dC 138-140) of the compound 1 isolated of N anuza p licata.

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8

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Pinto et al. 9 Vol. 21, No. 10, 2010

3OD, 125 MHz, dC 95-135) of the compound 2 isolated of N anuza p licata.

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10

3OD, 500 MHz, dH 6.1-7.9) of the compound 2 isolated of N anuza p licata.

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Pinto et al. 11 Vol. 21, No. 10, 2010

Figure S20. 13_{C NMR spectrum (CDCl}

3, 125 MHzdC 102-174) of the compounds 3 and 4 isolated of N anuza p licata.

Figure S21. 13_{C NMR spectrum (CDCl}

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12

Figure S22. 1_{H NMR spectrum (CDCl}

3, 500 MHz) of the compounds 3 and 4 isolated of N anuza p licata.

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Pinto et al. 13 Vol. 21, No. 10, 2010

3, 500 MHz,dH 2.0-2.4) of the compounds 3 and 4 isolated of N anuza p licata.

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14

Figure S26. HMQC NMR experiment (CDCl₃, 500×125 MHz

,

dC 140-144) of the compounds 3 and 4 isolated of N anuza p licata.

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Pinto et al. 15 Vol. 21, No. 10, 2010

Figure S28. HMBC NMR experiment (CDCl3, 500 x 125 MHz, dC 100-145) of the compounds 3 and 4 isolated of N anuza p licata. * Correlation of

compounds 4.

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16

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Pinto et al. 17 Vol. 21, No. 10, 2010