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Adalgisa Ieda Maiworm
UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
EFEITOS DE UM EXTRATO AQUOSO DE LANTANA CAMARA
(CAMBARÁ DE ESPINHO) NA MARCAÇÃO DE CONSTITUINTES SANGÜÍNEOS
COM TÉCNECIO-99m E NA MORFOLOGIA DE HEMÁCIAS DE RATOS WISTAR
Dissertação apresentada à Universidade Federal do Rio
Grande do Norte para a obtenção do título de Mestre em
Ciências da Saúde.
Natal
2007
Adalgisa Ieda Maiworm
EFEITOS DE UM EXTRATO AQUOSO DE LANTANA CAMARA
(CAMBARÁ DE ESPINHO) NA MARCAÇÃO DE CONSTITUINTES SANGÜÍNEOS
COM TÉCNECIO-99m E NA MORFOLOGIA DE HEMÁCIAS DE RATOS WISTAR
Orientador: Professor Doutor Mário Bernardo-Filho
Co-Orientador: Professor Doutor Aldo da Cunha Medeiros
Dissertação de mestrado apresentada ao Programa de
Pós-graduação em Ciências da Saúde, para obtenção do
título de Mestre em Ciências da Saúde.
Natal
2007
ii
UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
Prof. Dr. Aldo da Cunha Medeiros
Coordenador do Programa de Pós-graduação em Ciências da Saúde
Natal
2007
iii
Adalgisa Ieda Maiworm
EFEITOS DE UM EXTRATO AQUOSO DE LANTANA CAMARA
(CAMBARÁ DE ESPINHO) NA MARCAÇÃO DE CONSTITUINTES SANGÜÍNEOS
COM TÉCNECIO-99m E NA MORFOLOGIA DE HEMÁCIAS DE RATOS WISTAR
PRESIDENTE DA BANCA: Prof. Dr. Adenilson de Souza da Fonseca (UERJ)
BANCA EXAMINADORA
Prof. Dr. Adenilson de Souza da Fonseca (UERJ)
___________________________________________________________________
Profa. Dra. Ivonete Batista de Araújo (UFRN)
___________________________________________________________________
Prof. Dr. Luis Alberto Lira Soares (UNICAP)
Natal
2007
iv
DEDICATÓRIA
A Deus que em tantos momentos desesperadores carregou-me no colo, não
permitindo que eu me afastasse de meu propósito.
In memoriam, dedico este título ao meu pai Pedro Martiniano Maiworm que me
educou respeitando os limites do próximo, mostrando-me o quanto é valioso ser
justo e reto nas atitudes.
In vivo, dedico esta vitória aos meus filhos Erick e Bernardo Maiworm
Bromerschenckel que mesmo diante de todas as minhas limitações e ausências
estiveram sempre comigo, e a minha mãe Iedda Leonina Barillo Maiworm que me
ensinou a tolerância, a compreensão e a determinação, asseverando que sempre
vale a pena ir de encontro aos sonhos.
“O Homem deverá mover-se dentro do mundo
sem pretender suplantar o semelhante,
ou tirar-lhe seu lugar, pois cada um tem o seu,
que pode ampliar sem molestar a ninguém”
Carlos Bernardo González Pecotche
( Pensador argentino)
v
AGRADECIMENTOS
Aos amigos... que nunca me abandonaram...
Aos colegas de laboratório... por toda a paciência de ensinar as técnicas e ajudar-
me em tarefas, às vezes, tão árduas para minha formação religiosa...
Aos colaboradores na pesquisa:
Dr. Giuseppe Antonio Presta
Dr. Severo de Paoli
Dr. Sebastião David dos Santos-Filho e
Dra. Tania Giani...amigos das horas mais conturbadas, mas também de tantos
momentos inesquecíveis,... eternamente grata por todo o carinho e
companheirismo...
Não posso deixar de agradecer ao Prof. Doutor José Brandão Neto, pela acolhida no
Programa de Pós-Graduação em Ciências da Saúde da Universidade Federal do Rio
Grande do Norte e a amiga natalense Lucia de Fatima Amorim, que deu todo o
apoio necessário junto à secretaria do programa. À Profa. Dra Lavinia Brito que
ajudou-me no início desta jornada. Ao Botânico Ricardo Carneiro da Cunha Reis que
catalogou a exsicata de Lantana camara no Herbário RB - Jardim Botânico – Brasil.
Ao Prof. Mário Pereira dos Santos que ajudou na verificação estatística da pesquisa.
Ao Professor Doutor Adenilson de Souza da Fonseca que ajudou na finalização
desta dissertação....... só posso agradecer à todos vocês....
Obrigado de coração
Aos orientadores:
Professor Doutor Mário Bernardo Filho e Professor Doutor Aldo da Cunha Medeiros
por toda a compreensão, bem como toda a disposição de mostrar-me o caminho
correto para fazer ciência, meu muito obrigado.
vi
LISTA DE ABREVIAÇÕES 99mTc - Tecnécio-99m
C - Hemácias
P - Plasma
FS - C - Fração solúvel da célula
FI-C - Fração insolúvel da célula
FS-P - Fração solúvel do plasma
FI-P - Fração insolúvel do plasma
%ATI - Percentagem de radioatividade
%ATI – M - Percentagem de radioatividade mantida
99Mo - Molibidênio
Na99mTcO4 - Pertecnetato de sódio
kBq - quilobecquerel
SPECT - Single Photon Emission Computed Tomography
PET - Positron Emission Tomography
W1 - Washed 1
W2 - Washed 2
RBC - Red Blood Cell
OD - Optical density
keV - quiloeletronvolt
SOD – Superoxidismutase
vii
1) RESUMO
Extratos de plantas medicinais são utilizados amplamente em todo o mundo.
Entretanto, efeitos biológicos associados aos mesmos não têm sido investigados
exaustivamente. Através do uso de suas folhas e flores a Lantana camara (Cambará
de espinho) tem sido usado para tratamento de várias doenças na medicina popular.
O presente estudo teve como objetivo verificar o efeito do extrato desta planta na
marcação de constituintes sangüíneo com tecnécio 99m, bem como a morfologia de
hemácias de ratos Wistar. Os resultados mostraram que o extrato de Lantana
camara acarretou uma diminuição de fixação de radioatividade na fração insolúvel
do plasma. Este efeito não foi observado na célula e nem na fração insolúvel da
célula. O percentual de atividade teve diminuição em todas as concentrações
quando as células foram lavadas, o que não foi observado com o controle. O extrato
estudado aumentou a fragilidade osmótica das hemácias e modificou a morfologia
dessas células. Os efeitos do extrato de Lantana camara podem estar associados a
algumas propriedades farmacológicas de componentes químicos do extrato
estudado. O estudo teve caráter multidisciplinar com a participação das seguintes
áreas do conhecimento: Radiobiologia, Botânica, Fitoterapia e Hematologia.
Palavras-chave: constituintes sangüíneos, tecnécio-99m, fragilidade osmótica,
Lantana camara
viii
SUMÁRIO
Lista de abreviações……………………………………….....……………………..vi
Resumo……………………………………………………………………………....vii
Sumário……………………………………………………………………………….ix
1. Introdução........................................................................................................01
2. Revisão de literatura........................................................................................03
3. Artigos em publicações....................................................................................08
4. Comentários, críticas e conclusões.................................................................39
5. Anexos.............................................................................................................41
6. Referências......................................................................................................43
7. Abstract............................................................................................................49
ix
1
1. INTRODUÇÃO
O uso de produtos naturais vem crescendo ao longo dos anos. De
modo geral através da utilização desses produtos busca-se a cura para muitas doenças
e a identificação de substâncias menos tóxicas. Logo se torna de grande importância
uma avaliação dos efeitos biológicos de produtos de origem vegetal através de
modelos experimentais (1).
A distribuição, a fixação, a retenção e a eliminação de um
radiobiocomplexo (ou radifármaco) dependem de vários fatores, tais como metabolismo
tecidual, fluxo sanguíneo regional e sua ligação a constituintes do sangue. Essa
radiofarmacocinética, assim como o próprio processo de marcação de muitos
radiobiocomplexos, têm sido influenciadas por diferentes fatores. Um deles é a
interação de produtos naturais e sintéticos com os referidos radiobiocomplexos (2,3). A
marcação de constituintes sanguíneos com tecnécio-99m (99mTc), como as hemácias,
pode ser alterada em decorrência de modificações em nível de membrana celular (4,
5,6).
A capacidade do eritrócito de resistir à hemólise caracteriza uma
propriedade físico-química denominada fragilidade osmótica da membrana. O
comportamento da membrana do eritrócito é de particular interesse nos estudos de
substâncias que possam interferir na fragilidade da membrana e causar o risco de
hemólise (6, 7, 8).
O objetivo desse trabalho foi avaliar o efeito de um extrato aquoso
de Lantana camara na marcação de constituintes sanguíneos com 99mTc e na
morfologia de hemácias de ratos Wistar. Parte dos resultados obtidos, foram utilizados
no preparo de manuscritos que foram submetidos à periódicos indexados:
1
2
Evaluation of the in vitro effect of a Lantana camara extract on the labeling of
blood constituents with technetium-99m. Aceito para publicação no periódico “Acta
Physiologica Hungarica.
Osmotic and Morphological Effects on Red Blood Cell membrane:
Action of an aqueous extract of Lantana camara. Aceito para
publicação no periódico “Brazilian Journal of Pharmacognosy”
2. REVISÃO DE LITERATURA
A descoberta das radiações ionizantes e dos radionuclídeos
despertou o interesse nas suas aplicações na Biologia e nas Ciências Médicas pelo
seu valor como meio para auxiliar o diagnóstico e o tratamento de doenças. Como a
origem do fenômeno radioativo é nuclear, os nuclídeos que emitem radiação são
chamados mais apropriadamente de radionuclídeos (12,13, 14,15).
Os radionuclídeos podem ser utilizados em medicina nuclear como
fontes de radiação e como traçadores (16, 17). No primeiro caso, o material biológico
recebe apenas as radiações emitidas pelo radionuclídeo usado. No segundo o próprio
radionuclídeo está incorporado ao constituinte celular e molecular (radiofármaco ou
radiobiocomplexo) para ser utilizado no estudo de determinado fenômeno biológico
e/ou fisiológico (14,16,18). Dessa forma, os radiobiocomplexos podem ser empregados
para diagnóstico e terapia de doenças (16,17,19). A utilização em medicina nuclear é
interessante pelo fato das doses administradas em diagnóstico acarretarem, em geral,
2
3
baixas doses de radiação para o paciente e produzirem imagens de excelente
qualidade (20).
O radionuclídeo tecnécio-99m (99mTc) é um dos isótopos do
elemento tecnécio, que é classificado como um metal de transição do grupo VII. Ele
possui uma meia vida de 6 horas, uma emissão de radiação gama de 140 keV de
energia e apresenta estados de oxidação que variam de -1 a +7. É obtido através de
geradores 99Mo/99mTc na forma de pertecnetato de sódio (Na 99mTcO ) (21). 4
A redução do íon pertecnetato pode ser obtida através de diferentes
agentes químicos. O cloreto estanoso (SnCl2) é o mais amplamente utilizado para esta
finalidade, pois possui uma eficiência de redução do íon pertecnetato superior a de
outros agentes redutores, justificando sua preferência (17, 23).
Íons pertecnetato livres difundem através das membranas dos
capilares para o líquido intersticial, de onde são captados por diferentes órgãos como o
estômago (24), intestinos(25), glândulas salivares (26), tireóide (27), plexo coróide e
rins (22). Após a administração, apenas 30% da dose é excretada na urina em 24h. O
íon pertecnetato é facilmente absorvido pelo sistema digestivo após administração oral
ou após injeção intramuscular por processo de difusão simples (13, 12).
O sangue é um líquido contido em um compartimento fechado, o
aparelho circulatório (28,29). Esse é formado pelos glóbulos sanguíneos e pelo plasma,
parte líquida, na qual os primeiros estão suspensos (28, 29). Os glóbulos sanguíneos
são os eritrócitos (hemácias), as plaquetas e diversos tipos de leucócitos ou glóbulos
brancos. (28, 29, 30). O plasma é uma solução aquosa contendo muitas substâncias
em suspensão assim como sais inorgânicos, proteínas (albumina, imunoglobulinas, por
exemplo) e diversos outros compostos não protéicos (28, 29, 30).
3
4
As hemácias dos mamíferos são anucleadas e, em humanos, têm
forma de disco bicôncavo. A forma bicôncava proporciona grande superfície em relação
ao volume, o que facilita as trocas de gases. (28, 29, 30).
A composição interna da hemácia é mantida através de mecanismos
que requerem energia. A membrana que reveste a hemácia é semipermeável,
permitindo a passagem de água e eletrólitos. Quando colocadas em solução
hipertônica, as hemácias perdem água, tornado-se crenadas. Em solução
suficientemente hipotônica, as hemácias absorvem água, até que ocorra o equilíbrio
osmótico ou até que ocorra ruptura da membrana ou hemólise (28, 29, 30).
Existem muitas metodologias para marcação de hemácias com
99mTc (13,17,31), e esta é de grande relevância na medicina nuclear (22). Nos
métodos que empregam a marcação in vitro, o sangue isolado é incubado com o íon
estanoso e, posteriormente, com íon pertecnetato (12, 17, 32).
Hemácias marcadas com 99mTc também são utilizadas em
medicina nuclear para obtenção de imagens através do pool sanguíneo, para avaliação
do sistema cardiovascular, hemorragias gastrintestinais e volemia (33, 34 ).
6A hemácia tem cerca de 10 exemplares de proteína integral de
membrana, chamada de glicoproteína (4). A banda-3 além de desempenhar um papel
estrutural na fixação do citoesqueleto à membrana, ela constitui a principal proteína no
transporte de ânions das hemácias, estando envolvida na difusão de íons cloreto, que
atuam durante o transporte de O e CO2 2 (29). Estudos realizados demonstraram que
na marcação de hemácias com 99mTc, o íon pertecnetato atravessa o espaço
intracelular por um mecanismo de troca com os íons cloreto e/ou bicarbonato (35).
Estes fatos indicam que o processo de marcação ocorre em nível intracelular.
4
5
Somando-se a isso, o agente redutor, SnCl2, também parece ser transportado para o
interior da hemácia por um sistema de transporte específico, o canal de cálcio (32, 36).
Na marcação de hemácias com 99mTc utilizando SnCl2 também
ocorre a ligação desse radionuclídeo às proteínas plasmáticas dependendo da
concentração do agente redutor, do radiobiocomplexo utilizado e do tempo de
incubação, como pode ser observado através da técnica de precipitação com ácido
tricloroacético (37). Ao se administrar um radiobiocomplexo a um paciente, teremos
sempre uma fração do mesmo ligado às proteínas plasmáticas (17,38).
O uso de drogas é fundamental na obtenção de um resultado
terapêutico desejado no tratamento de doenças. Neste caso existe a possibilidade de
uma droga estar modificando os efeitos farmacológicos de outra droga administrada
concomitantemente, podendo modificar a resposta farmacológica esperada (18,39). Os
mecanismos de ação envolvendo os produtos sintéticos em geral estão bem
estabelecidos (39), entretanto não é o que acontece com muitos dos produtos naturais
usados na clínica médica (1).
A interação medicamentosa com radiofármacos pode ser devido à
possibilidade do medicamento (i) mudar a natureza química do radiobiocomplexo, (ii)
alterar o meio bioquímico do alvo ou do não alvo no qual o radiofármaco pode se
concentrar, (iii) favorecer ou dificultar a ligação do radiobiocomplexo às proteínas
plasmáticas e aos elementos sanguíneos e (v) em excesso pode, ainda, exercer seu
efeito terapêutico mimetizando sintomas (40).
A compreensão da interação medicamentosa é de suma importância
para profissionais de saúde que utilizam os exames da medicina nuclear para
tratamento e/ou diagnóstico de diversas doenças. A pesquisa básica trouxe novos
conhecimentos sobre as plantas e suas propriedades terapêuticas. O crescente
5
6
aumento do interesse da comunidade científica em estudar os medicamentos naturais
tem dado respaldo às drogas à base de ervas. Alguns autores têm descrito os efeitos
de plantas medicinais na marcação de hemácias com 99mTc (37, 41, 42).
A Lantana camara (Cambará de espinho), do gênero (Verbenaceae)
como descrito por Linnaeus em 1753, é uma planta arborizada com varias cores de
floração: vermelha, rosa, branca, amarela e violeta (9).
Alguns complexos de Lantana camara são tóxicos para ruminantes
de pequeno porte, e estes efeitos têm sido associados ao tipo e a espécie. Entretanto,
estudos recentes sobre a Lantana camara vêm demonstrando a atividade biológica
positiva desta planta. O Cambará de espinho é utilizado freqüentemente na medicina
popular, por possuir propriedades farmacológicas, tais como antipirético, antimicrobial,
antitumoral, antitrombótico, antiinflamatório e antimutagênicos ( 9,10,43,44, 45, 46). A
sua utilização é distinta para cada doença e/ou sintoma, para a bronquite tem sido
utilizado o chá da planta incluindo raízes, caule, flores e folhas. Folhas em infusão
como antipirético, vermífugo, anti-tumoral e analgésico para dores estomacais. Sob a
forma de chá em decocção para lepra e sarna. Como cataplasma para feridas e
sarampo (9,11).
3. MANUSCRITO ACEITO PARA PUBLICAÇÃO – I
Evaluation of the in vitro effect of a Lantana camara extract on the labeling of blood
constituents with technetium-99m.
Running title: L. camara effect on 99mTc-blood constituents.
6
7
Adalgisa I. Maiworm1,2 1, 2, S.D. Santos-Filho , G.A. Presta1,2, Tania S. Giani1,2, S.
Paoli1,2, M. Bernardo-Filho1,3
1Departamento de Biofísica e Biometria, Universidade do Estado do Rio de Janeiro,
Instituto de Biologia Roberto Alcantara Gomes, Rio de Janeiro, RJ, Brasil.
2Programa de Pós-Graduação em Ciências da Saúde, Universidade Federal do Rio
Grande do Norte, Natal, RN, Brasil.
3Instituto Nacional do Câncer, Coordenadoria de Pesquisa Básica, Rio de Janeiro, RJ,
Brasil.
Abstract
Blood constituents labeled with technetium-99m(99mTc) have been used in nuclear
medicine procedures and drugs are capable to interfere on this labeling. Lantana
camara(lantana) has medicinal properties and it has been used in folk medicine. The
aim is to verify the effect of a lantana extract on the labeling of blood constituents with
99m 99mTc. Blood was incubated with extract, stannous chloride and Tc, as sodium
pertechnetate. Plasma(P) and blood cells(BC) were isolated, also precipitated with
trichloroacetic acid and soluble(SF) and insoluble fractions(IF) were separated. The %
of radioactivity(%ATI) in these samples was calculated. Samples of labeled BC were
washed and the %ATI maintained(%ATI-M) in the BC was determined. The results
showed that lantana extract has significantly(p<0.05) decreased on the IF-P from
70.24 2.59 to 11.95±3.07. This effect was not observed in the BC and IF-BC. The BC-
%ATI-M was significantly(p<0.05) decreased in all concentrations tested when the BC
was washed. This fact was not observed in the control. Substances present on the
extract should have redoxi action decreasing the concentration of the stannous ion and
7
8
this condition could justify the effect on the IF-P. The results about the BC-%ATI-M
should indicate a possible effect on the transport of ions through the erythrocyte
membrane.
Keywords: blood constituents, technetium-99m, red blood cell, Lantana camara
2) 1-Introduction Technetium-99m (99mTc) has been the most utilized radionuclide in the single
photon emission computed tomography (SPECT) (11, 34) and it has also been used in
basic research (5, 33). Chemical compounds or cellular structures (red blood cells-RBC,
platelets and white blood cells) have been labeled with this radionuclide and used as
radiopharmaceutical (radiobiocomplex) (4).
99mTc-labeled RBC scan is the nuclear study best suited for identifying slow-
bleeding sources as gastrointestinal bleeding (14, 19), as well as (i) in the determination
of the left ventricular function by measuring the ejection fractions (16) and (ii) in the
evaluation of wall motion abnormalities (34, 39), in cardiovascular nuclear medicine.
The RBC has been labeled with 99mTc for in vitro, in vivo or in vivo/ in vitro techniques.
The labeled process with 99mTc, as sodium pertechnetate, depends on a reducing agent
and stannous ion (Sn+2) is usually used for this purpose (11, 24, 29, 34). When whole
blood is used on the labeling of RBC with 99mTc, radioactivity is mainly found on RBC,
however it is also bound on plasma proteins (3). Authors have reported that some
herbal medicines are capable to alter the labeling of blood constituents with technetium-
99m (8, 17, 21, 23, 26, 27, 28, 40, 41).
Lantana camara L. (Verbenaceae; lantana) is one of the most prevalent and
noxious weeds causing hepatotoxicity in grazing animals. Lantana poisoning causes
obstructive jaundice and within a few hours of browsing upon its foliage, animals go off-
feed and become severely constipated within 48 h (36). On the contrary, lantana plant
8
9
has been reported to possess a number of medicinal properties (10). Some metabolites
isolated from their leaves possess antitumor activity (42), antithrombin activity (26), anti-
inflammatory (25), antinociceptive and antipyretic activity (43) has tested the antifilarial
activity of a crude extract of stem portion of the lantana with the concentration of 1g/kg
for 5 days by oral route (21).
As the extract of Lantana camara can be used by the human beings and several
effects about this natural product are not well understood, the aim of this work was to
evaluate the effect of lantana aqueous extract on the labeling of blood constituents with
99mTc using an in vitro experimental model, as well as to verify the consequences of
several washings on the maintenance of the radioactivity on the labeled blood cells.
Seção 1.02
Seção 1.03 2-Material and Methods
The experiments were performed with rats that were maintained in a controlled
environment. The animals had free access to water and food and ambient temperature
was kept at 25 2ºC. The experiments were carried out without sacrificing the animals.
Heparinized whole blood was withdrawn by cardiac puncture from adult male Wistar
rats (9 animals, 3 months of age, 250±15g of weight). All the experimental procedures
have followed the Ethical Guidelines of the Institution (Protocol CEA/127/2006).
Lantana camara fresh leaves, collected in the local small forest of Petrópolis city,
Rio de Janeiro State, Brazil. It was identified by the Biologist Ricardo Carneiro da
Cunha Reis, Botanic Herbarium RB of the Jardim Botânico, Rio de Janeiro State, Brazil,
where a voucher specimen (4070081) was kept.
The extract of Lantana camara was prepared with 150 mg of a macerated of
leaves freshly collected that was added in 15mL of 0.9% NaCl at 100°C incubated by
10min. The preparation was filtered through paper (quality filter paper). In the tentative
9
10
to estimate the concentration of the extract, a difference between the filter paper after
the filtration (dried at 37 oC) and the filter paper with the extract before the filtration was
calculated. This difference (10 mg) was the quantity of the extract that was extracted in
15 mL and the filtered solution was considered to be 660 g/mL or 100%.
The tubes used in these experiments were previously closed with a rubber cap
and a syringe was used to reduce the air atmosphere (vacuum) inside the vials.
Samples of 0.5 ml were incubated with 100 l of different concentrations of diluted
aqueous (0.9% NaCl) lantana extract (6.25, 12.5, 25, 50 and 100 %) for 1 hour at room
temperature. A sample of heparinized whole blood was incubated with saline solution
(NaCl 0.9%) as control. Then, 0.5 ml of a freshly prepared stannous chloride solution
(1.2 g/ml), as SnCl2 (Sigma, USA) was added and the incubation continued for another
1 hour. After this period of time, Tc-99m (0.1 ml with 370 kBq), as sodium
pertechnetate, recently milked from a 99 99mMo/ Tc generator (Instituto de Pesquisas
Energéticas e Nucleares, Comissão Nacional de Energia Nuclear, Brasil), was added
and the incubation continued for another 10 min. These samples were centrifuged and
plasma (P) and BC cells were separated. Samples (20 l) of P and BC were also
precipitated with 1 ml of trichloroacetic acid 5% and soluble (SF) and insoluble fractions
(IF) were separated. The radioactivity in P, BC, IF-P, SF-P, IF-BC and SF-BC were
determined in a well counter (Automatic Gamma Counter, Packard Instrument Co,
USA). After that, the % of radioactivity (%ATI) was calculated as described elsewhere
(28, 29).
Samples of labeled BC were washed twice with saline solution and the %ATI
maintained (%ATI-M) in the BC was determined, dividing the radioactivity after each
washing by the initial radioactivity. In this assay, the labeled whole blood was
centrifuged (clinical centrifuge), the supernatant was discharged and an aliquot (20 l)
10
11
of the pellet (BC) was isolated. In the first washing, saline solution (1 mL) was added
and the BC was gently homogenized, centrifuged and 20 l of the pellet was isolated
again. These steps were repeated in the second washing.
Statistical analysis involved one-way ANOVA, followed by the Turkey-Kramer
Multiple Comparisons Test, with the significance level being P<0.05. InStat Graphpad
software was used to perform statistical analysis (GraphPad InStat version 3.01 for
Windows 95/NT, GraphPad Software, San Diego Ca, USA).
3-Results:
Table I shows the distribution of the radioactivity in the cellular and plasma
compartments isolated from whole blood treated with different concentrations of the
lantana extract. The analysis of the results indicates that there is not alteration in the
radioactivity distribution of the 99mTc on the cellular and plasma compartments in
presence of lantana extract.
The results in table II indicate the distribution of the radioactivity on the soluble
and insoluble fractions of the cellular compartment isolated from whole blood treated
with different concentrations of the lantana extract. There is not alteration in the
radiation fixation by the cellular proteins (IF-BC) in presence of lantana extract.
The analysis of the results shows in table III indicates the distribution of the
radioactivity in the soluble and insoluble fractions of the plasma compartment isolated
from whole blood treated with different concentrations of the lantana extract. There is a
significant (P<0.05) decrease in the radioactivity fixation in the plasma proteins (IF-P) in
presence of lantana extract from 70.24 2.59 to 11.95±3.07.
11
12
Table IV shows the radioactivity in the samples of labeled BC that were washed.
The results indicate that there was a significant (p<0.05) decreased in all concentrations
of the lantana extract, except in the control.
4-Discussion:
Nuclear medicine procedures as SPECT and PET have proven increasingly
effective imaging modalities in the study of several clinical disorders (15, 18, 31).
Besides the disease, some authors have reported that these procedures could be
altered by medications that the patient is undergoing. The natural products could be
altering the biodistribution of radiobiocomplexes in a specific target or the fixation of the
radionuclide 99mTc to blood constituents (6, 8, 9, 13, 23, 28, 29, 39).
The phytochemical analysis of the lantana leaf extract has earlier been shown to
contain flavonoids (32), terpenes (2) and their derivatives and pentacyclic triterpenoids
(1). These constituents may mediate the anti-diarrheal action of the lantana extract. A
verbascoside (12) isolated from Lantana camara has been shown to be an inhibitor of
protein kinase C. The role of this enzyme has been demonstrated in signal transduction,
inflammation and smooth muscle contraction (34, 38). Terpenes, flavonoids and
terpenoid derivatives may act by inhibiting release of autocoids and prostaglandins (7,
25) thereby inhibit the motility and secretion induced by neostigmine (20). Probably
some of these results seem to be associated with effects on the plasma membrane of
the cells of different tissues. In consequence our findings reported in tables I and II
could not be justified due to effects of the lantana extract on the erythrocyte membrane.
However, unexpected results about the %ATI-M of the BC (Table IV) would indicate a
possible effect on the lantana extract on the erythocyte membrane. As the washings of
the labeled blood cells have reduced the radioactivity on the BC compartment, it is
12
13
possible to speculate a blockage of the transport of the stannous ions through the
erythrocyte membrane. The pertechnetate ion could enter into the cell, but without the
stannous ion, the 99mTc could be not fixed in the hemoglobin and with the washings
the radioactivity would be washed out in the presence of the lantana extract as
demonstrated in Table IV. Although, studies that could permit to evaluate the transport
kinetics of chemical agents through the erythrocyte membrane, our findings with the
washings should be worthwhile and have permitted to verify that the washings effect
has increased when the highest concentrations of lantana were tested.
The extract of lantana leaves suppressed the emergence of leaf buds of water
hyacinth plant, and caused the decay of its leaves by foliar spraying. In addition, the
increase of SOD activity in water hyacinth leaves was in accordance with the
accumulation of H O2 2 and the increase in degree of membrane peroxidation, while the
activity of catalase, which might remove the excessive H O2 2 in water hyacinth leaves,
was inhibited by the treatment with lantana extract. At tissue level, high H O2 2
histochemical labeling was detected in guard cells after treatment with lantana extract.
This overproduction of H O2 2 could kill the leaf cells and cause leaf necrosis in the
treated plant. Therefore, the high toxicity of lantana leaf extract to water hyacinth might
be due to oxidative stress (44). If these phenomena could be occurring in the plasma of
the whole blood in presence of the lantana extract, as consequence would be the
oxidation of the stannous ions decreasing the fixation of the 99mTc on the plasma
proteins as indicated in table III.
The comparison of the concentrations of extract used in our experiments would
be in agreement with Misra et al (21). These authors have used 1g/kg and as it is
possible to consider a man with 60 kg and 5 000 mL of whole blood, the concentration
on the blood would be 12 mg/ml. In our experiments, considering the highest
13
14
concentration of lantana, we have used 100 l of an extract with 660 g/ml in a
preparation of 1.2 ml, and the final concentration of the extract was about 66 g/ml.
In conclusion, probably substances present in the lantana extract should have an
effect on transport of the ions through the erythrocyte membrane and/or should have
redoxi properties and the stannous ion would decrease and could justify the effect on
the fixation of the radioactivity on the plasma proteins. Although, the experiments were
carried out with animals, it is suggested precaution in the interpretation of the
examinations that use labeled blood constituents in the patients that are undergoing
Lantana camara extract. Furthermore, as the radioactivity from the labeled BC was
washed, it is necessary more attention due to this phenomenon.
5-References
1 - Ahmed ZF, Shoaib AM, Wassel GM, Sayyad SM: Phytochemical study of Lantana
camara L . Planta Med. 21, 282–288 (1972).
2 - Begum S, Wahab A, Siddiqui BS: Pentacyclic triterpenoids from the aerial parts of
Lantana camara. Chem. Pharm. Bull. 51,134–137 (2003).
3 - Bernardo-Filho M, Nogueira J, Sturm J, Boasquevisque E: Plasma proteins labelling
with 99mTechnetium. Braz. Arch. Biol. Technol. 33, 811-817 (1990).
14
15
4 - Bernardo-Filho M, Santos-Filho SD, Moura EG, Maiworm AI, Orlando MMC, Penas
ME, Cardoso VN, Bernardo LC, Brito LC: Drug interaction with radiopharmaceuticals: a
review. Braz. Arch. Biol. Technol. 48, 13-27 (2005).
5 - Burke IT, Boothman C, Lloyd JR, Mortimer RJ, Livens FR, Morris K: Effects of
progressive anoxia on the solubility of technetium in sediments. Environ. Sci. Technol.
39, 4109-4116 (2005).
6 - Caprilles PVSZ, Dias APM, Costa TEMM, Oliveira MBN, Faria MVC, Moura EG,
Abreu BAL, Bernardo-Filho M: Effect of eggplant (Solanum melongena) extract on the in
vitro labeling of blood elements with technetium-99m and on the biodistribution of
sodium pertechnetate in rats. Cell. Mol. Biol. 48, 771-776 (2002).
7 - Chitme HR, Chandra R, Kaushik S: Studies on anti-diarrhoeal activity of Calotropis
gigantea R.Br. in experimental animals. J. Pharm. Pharmaceut. Sci. 7, 70–75 (2004)
8 - Diré G, Gomes ML, Lima EAC, Jales RL, Faria MC, Bernardo-Filho M: Assessment
of a fruit extract (Sechium edule) on the labeling of blood elements with technetium-
99m. Afr. J. Biotechnol. 3, 484-488 (2004).
9 - Fonseca AS, Frydman JN, Santos R, Bernardo-Filho M: Influence of antipyretic
drugs on the labeling of blood elements with technetium-99m. Acta Biol. Hung. 56, 275-
282 (2005).
10 - Ghisalberti EL: Lantana camara Linn. (Review). Fitoterapia, 71, 467–485 (2000)
11 - Harbert JC, Eckelman WC, Neumann RD (1996): Nuclear Medicine Diagnosis and
therapy. Thieme Medical Publishers, New York.
12 - Herbert JM, Maffrand JP, Taoubi K, Augereau JM, Fouraste I, Gleye J:
Verbascoside isolated from Lantana camara, an inhibitor of protein kinase C. J. Nat.
Prod., 54, 1595–1600 (1991).
15
16
13 - Hesslewood S, Leung E: Drug interactions with radiopharmaceuticals. Eur. J. Nucl.
Med., 21, 348-356 (1994)
14 - Karacalioglu O, Ilgan S, Arslan N, Ozguven M: Uterine doughnut in early
proliferating phase: potential pitfall in gastrointestinal bleeding studies. Ann. Nucl. Med.
17, 685-687 (2003).
15 - Kumakura Y, Danielsen EH, Reilhac A, Gjedde A, Cumming P: Levodopa effect on
18-F fluorodopa influx to brain: normal volunteers and patients with Parkinson’s
disease. Acta Neurol. Scand. 110, 188–195 (2004).
16 - Leitha T, Gwechenberger M, Pruckmayer M, Staudenherz A, Bailer H, Kronik G:
Does motion analysis in postexercise gated sestamibi SPECT reflect rest left ventricular
motion even in severe coronary artery disease? Clin. Nucl. Med. 26, 694-700 (2001).
17 - Lima EAC, Diré G, Mattos DMM, Freiras RS, Gomes ML, Oliveira MBN, Faria
MVC, Jales RL, Bernardo-Filho M: Effect of an extract of cauliflower (leaf) on the
labeling of blood elements with technetium-99m and on the survival of Escherichia coli
AB1157 submitted to the treatment with stannous chloride. Food Chem. Toxicol. 40,
919-923 (2002).
18 - Lodge MA, Braess H, Mahmoud F, Suh J, Englar N, Geyser-Stoops S, Jenkins J,
Bacharach SL, Dilsizian V: Developments in nuclear cardiology: transition from single
photon emission computed tomography to positron emission tomography-computed
tomography. J. Invasive Cardiol. 17, 491-496 (2005).
19 - Manning-Dimmitt LL, Dimmitt SG, Wilson GR: Diagnosis of gastrointestinal
bleeding in adults. Am. Fam. Physician. 71, 1339-1346 (2005).
20 - McNicol A, Nickolaychuk BR: Inhibition of collagen-induced platelet activation by
arachidonyl trifluoromethyl ketone. Biochem. Pharmacol. 50, 1795-1802 (1999).
16
17
21 - Misra N, Sharma M, Raj K, Dangi A, Srivastava S, Misra-Bhattacharya S: Chemical
constituents and antifilarial activity of Lantana Câmara against human lymphatic filariid
Brugia malayi and rodent filariid Acanthocheilonema viteae maintained in rodent hosts.
Parasitol. Res. 100, 439-448 (2007)
22 - Moreno SRF, Rocha EK, Feliciano GD, Bernardo-Filho M, Farah MB, Freitas RS,
Gomes ML: Effect of Ginkgo biloba on the in vitro labeling of red blood cells and plasma
proteins with technetium-99m. J. Labelled Comp. Radiopharm. 44, 639-641 (2001).
23 - Moreno SRF, Diré, G, Freitas RS, Farah MB, Lima-Filho GL, Rocha EK, Jales RLC,
Bernardo-Filho M: Effect of Ginkgo biloba on the labeling of blood elements with
technetium-99m: in vitro study. Rev. Bras. Farmacognosia. 12, 62-63 (2002).
24 - Moreno SRF, Carvalho JJ, Nascimento AL, Pereira M, Rocha EK, Diré G, Arnobio
A, Caldas LQA, Bernardo-Filho M: Bioavailability of the sodium pertechnetate and
morphometry of organs isolated from Wistar rats: study of possible pharmacokinetic
interactions of a Ginkgo biloba extract. Braz. Arch. Biol.Technol. 48(special issue), 73-
78 (2005).
25 - Nikiema JB, Vanhaelen Fastre R, Vanhaelen M, Fontaine J, De Graef C, Heenen
M: Effects of anti-inflammatory triterpenes isolated from Leptadenia hastate latex on
keratinocyte proliferation. Phytother. Res. 15, 131–134 (2001).
26 - O' Neill MJ, Lewis JA, Noble HM, Holland S, Mansat C, Farthing JE, Foster G,
Noble D, Lane SJ, Sidebottom PJ, Lynn SM, Hayes MV, Dix CJ: Isolation of
translactone-containing triterpenes with thrombin inhibitory activities from the leaves of
Lantana camara. J. Nat. Prod. 61,1328–1331 (1998).
27 - Oliveira JF, Braga AC, de Oliveira MB, Avila AS, Caldeira-de-Araujo A, Cardoso
VN, Bezerra RJ, Bernardo-Filho M: Assessment of the effect of Maytenus ilicifolia
17
18
(espinheira santa) extract on the labeling of red blood cells and plasma proteins with
technetium-99m. J. Ethnopharmacol. 72,179-184 (2000).
28 - Oliveira JF, Avila AS, Braga ACS, Oliveira MBN, Boasquevisque EM, Jales RL,
Cardoso VN, Bernardo-Filho M: Effect of extract of medicinal plants on the labeling of
blood elements with technetium-99m and on the morphology of red blood cells: I – a
study with Paullinia cupana. Fitoterapia 73, 305-312 (2002).
29 - Oliveira JF, Santos-Filho SD, Catanho MTJA, Srivastava SC, Lima-Filho GL,
Bernardo Filho M: Effect of extract of medicinal plants on the labeling of blood elements
with technetium-99m and on the morphology of red blood cells (RBC): Toxicological
actions of roast coffee beans (Coffea arabica). Indian J. Nucl. Med. 18, 52-56 (2003).
30 - Oliveira JFF, Brito LC, Frydman JNG, Santos-Filho SD, Bernardo-Filho M: An
aqueous extract of Pfaffia sp. does not alter the labeling of blood constituents with
technetium-99m and the morphology of the red blood cells. Rev. Bras. Farmacognosia.
15,126-132 (2005).
31 - Palestro CJ, Tomas MB, Tronco GG: Radionuclide imaging of the parathyroid
glands. Semin. Nucl. Med. 35, 266-276 (2005).
32 - Pan WD, Mai LT, Li YJ, Xu XL, Yu DQ: Studies on the chemical constituents of the
leaves of Lantana camara. Yao Xue Xue Bao. 28, 35–39 (1993).
33 - Pettersson F, Vogt AM, Jonsson C, Mok BW, Shamaei-Tousi A, Bergstrom S, Chen
Q, Wahlgren M: Whole-body imaging of sequestration of Plasmodium falciparum In the
rat. Infect. Immun. 73, 7736-7746 (2005).
34 - Prech M, Grajek S, Marszalek A, Lesiak M, Jemielity M, Araszkiewicz A, Mularek-
Kubzdela T, Cieslinski A. Chronic infarct-related artery occlusion is associated with a
reduction in capillary density: Effects on infarct healing. Eur. J. Heart Fail. 8, 373-80
(2006).
18
19
35 - Rotblatt M, Ziment I (2002): Evidence-Based Herbal Medicine. Hanley & Belfus,
Philadelphia.
36 - Sagar L, Sehgal R, Ojha S: Evaluation of antimotility effect of Lantana camara L.
var. acuelata constituents on neostigmine induced gastrointestinal transit in mice. BMC
Complement Altern. Med. 5, 18 (2005).
37 - Saha GB (2004): Fundamentals in Nuclear Pharmacy. Springer-Verlag, New York.
38 - Sales ME, Sterin-Borda L, de Bracco MM, Borda ES: Tyrosine kinase regulatory
action on ileal muscarinic effects of IFN-gamma. J. Interferon Cytokine Res. 19, 375–
382 (1999).
39 - Sampson CB (1999): Textbook of radiopharmacy theory and practice. Gordon and
Breach, Amsterdam.
40 - Santos-Filho SD, Diré GL, Lima E, Oliveira MN, Bernardo-Filho M: Effect of Mentha
crispa (mint) extract on the labeling of blood elements with technetium-99m: A possible
evaluation of free radicals. J. Biol. Sci. 4, 266-270 (2004).
41 - Santos-Filho SD, Bernardo-Filho M: Effect of Hypericum perforatum extract on in
vitro labeling of blood elements with technetium-99m and on biodisponibility of sodium
pertechnetate in Wistar rats. Acta Cir. Bras. 20,121-125 (2005).
42 - Shashi BM, Niranjan PS, Subodh KR, Sharma OP: Potential Antitumor Agents
fromf Lantana camara: Structures of Flavonoid and Phenylpropanoid Glycosides.
Tetrahedron. 50, 9439–9446 (1994).
43 - Uzcategui B, Avila D, Heberto SR, Quintero L, Ortega J, Gonzalez YB: Anti-
inflammatory, antinociceptive and antipyretic effects of Lantana trifolia Linnaeus in
experimental animals. Invest. Clin. 45, 317-322 (2004).
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44 - Zheng HQ, Wei N, Wang LF, He P: Effects of Lantana camara Leaf Extract on the
Activity of Superoxide Dismutase and Accumulation of H(2)O(2) in Water Hyacinth Leaf.
Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao. 32,189-194 (2006).
TABLES
Table I - Effect of Lantana camara extract on the distribution of the radioactivity in the
cellular and plasmatic compartments.
Lantana camara
concentrations (%) Cellular compartment Plasma compartment
0.00 (control) 98.58±1.22 1.42±1.22
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6.25% 98.72±0.23 1.28±0.23
12.5% 98.12±1.26 1.88±1.26
25% 97.14±1.19 2.86±1.19
50% 96.55±1.17 3.45±1.17
100% 96.15±1.53 3.85±1.53
Samples of heparinized blood were incubated with different concentrations of Lantana
camara extract. A sample of heparinized whole blood was incubated with saline solution
(NaCl 0.9%) as control. Then, stannous chloride (1.2 g/ml) and 99mTc, as sodium
pertechnetate were added. The % ATI in Plasma and cellular compartments were
determined in a well counter and the percent of radioactivity was calculated.
Table II - Effect of Lantana camara extract in the labeling of soluble (SF) and insoluble
(IF) fractions of the BC with 99mTc.
Lantana camara
concentrations (%) IF-BC SF-BC
0.00 (control) 90.73±1.43 9.27±1.43
6.25% 89.04±3.81 10.96±3.81
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12.5% 88.75±1.33 11.25±1.33
25% 88.07±0.83 11.93±0.83
50% 87.32±1.34 12.68±1.34
100% 87.03±2.34 12.97±2.34
Samples of heparinized blood were incubated with different concentrations of Lantana
camara extract. A sample of heparinized whole blood was incubated with saline solution
(NaCl, 0.9%) as control. Then, stannous chloride (1.2 g/ml) and 99mTc, as sodium
pertechnetate were added. ATI% in IF-BC and SF-BC were determined in a well
counter and the % of radioactivity was calculated.
Table III - Effect of Lantana camara extract in the labeling of soluble (SF) and
99m insoluble (IF) fractions of the plasma (P) with Tc.
Lantana camara
concentrations (%) IF-P SF-P
0.00 (control) 29.76±2.5970.24 2.59
6.25% 23.43±0.88 76.57±0.88
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12.5% 16.88±2.92 83.11±2.92
25% 15.02±1.40 84.98±1.40
50% 12.12±1.82 87.88±1.82
100% 11.95±3.07 88.05±3.07
Samples of heparinized blood were incubated with different concentrations of Lantana
camara extract. Sample of heparinized whole blood was incubated with saline solution
(NaCl, 0.9%) as control. Then, stannous chloride (1.2 g/ml) and 99mTc, as sodium
pertechnetate were added. The %ATI in IF-P and SF-P were determined in a well
counter and the percent of radioactivity was calculated.
Table IV - Effect of Lantana camara extract on the %ATI maintained (%ATI-M)
in the samples of labeled BC that were washed.
Lantana camara
concentrations (%) BC W1 W2
0.00 (control) 98.58±1.22 95.23±2.12 93.62±3.21
6.25% 98.72±0.23 92.57±0.84 88.98±1.01
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12.5% 98.12±1.26 87.89±1.15 83.56±1.15
25% 97.14±1.19 91.32±1.09 87.93±1.01
50% 96.55±1.17 80.76±0.62 71.78±0.26
100% 96.15±1.53 79.97±1.16 66.89±1.10
Samples of blood treated with different concentrations of Lantana camara extract were
washed with saline solution. The %ATI maintained (%ATI-M) in each BC samples
washed was determined. W1 (washed 1); W2 (washed 2).
3. MANUSCRITO ACEITO PARA PUBLICAÇÃO – II
Osmotic and Morphological Effects on Red Blood Cell membrane:
Action of an aqueous extract of Lantana camara
Adalgisa I. Maiworm1,2*,Giuseppe A. Presta1,2,3, Sebastião D. Santos-Filho1,2, Severo de
Paoli1,2, Tânia S. Giani1,2 , Adenilson S. Fonseca2 1,3 and Mario Bernardo-Filho .
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1Universidade Federal do Rio Grande do Norte, Programa de Pós-Graduação em
Ciências da Saúde, Avenida General Gustavo de Farias s/n, CEP 59010-180, Natal,
Rio Grande do Norte, Brazil.
2* Universidade do Estado do Rio de Janeiro, Instituto de Biologia Roberto Alcântara
Gomes, Departamento de Biofísica e Biometria, Avenida 28 de Setembro, 87, CEP
20551-030, Rio de Janeiro, Brazil.
3Instituto Nacional do Câncer, Coordenadoria de Pesquisa, Praça Cruz Vermelha, 23,
CEP 20230-130, Rio de Janeiro, Brazil.
Abstract
The Lantana camara (cambara de espinho) leaves infusions are
used popularly in some countries to treat gastrointestinal diseases.
Osmotic fragility assay and morphometric analysis have been used to try
to verify the interaction of drugs with the membrane of red blood cells
(RBC). The aim of this work was to evaluate the effect of an aqueous
25
26
extract of Lantana camara on the osmotic fragility and on the morphology
of RBC. Blood samples were treated with extract of Lantana camara (10
mg/ml), osmotic fragility assay and morphological analysis were carried
out. In presence of the extract, the data obtained indicated (i) osmotic
fragility and a significant (p<0.05) increase of hemolysis and (ii)
modifications on the morphology of RBC. These effects of the Lantana
camara could be associated with some pharmacological properties of the
chemical compounds of this studied extract.
Keywords: Lantana camara, morphology, red blood cells,osmotic
fragility.
Introduction
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The Lantana camara (cambara de espinho) leaves infusions are used popularly in
some countries to treat gastrointestinal diseases (Sagar et al., 2005; Hernandes et al.,
2003). Lantana camara plant has been reported to possess a number of
pharmacological properties as antipyretic, antimicrobial, antimutagenic, antithrombin,
anti-inflamatory, antitumor and antinoceptive (Ahmed et al.,1972;Sashi et al., 1994,
Ghisalbert et al., 2000; Uzcategui et al., 2004; Zheng et al., 2006; Misra et al., 2007).
The chemical compounds present in Lantana camara extracts include mono and
sesquiterpenes, flavonoids, iridoid glycosides, furanonaphoquinones, sthsteroids and
phenyl ethanoid glicosides, triterpene and diterpenes.(Ghisalbert et al,. 2000).
The volume of the red blood cells (RBC) seems to be regulated by a direct action
of the sodium-potassium pump: it controls the solute concentration inside the cell,
thereby regulating the osmotic forces that can make a cell swell or shrink (Alberts et al.,
2002). The resistance of RBC to hemolysis characterizes what is called the osmotic
fragility (OF) of the cell membrane. The osmotic fragility is classically used as a in vitro
assay to evaluate the effects of natural and synthetic drugs on membrane (Didelon et
al., 2000). The “fragility curve” reflects the structural and geometrical changes in RBC.
Hemolysis results from a structural perturbation of the RBC and cytoskeleton caused by
high partition in the membrane (Cruz Silva et al.; Didelon et al., 2000).
RBC have been proposed as a prototypical cellular system regarding drug
mediated plasma membrane effects (Li et al., 1999). Different techniques have
demonstrated that therapeutic drugs can modify the structure and morphology of these
cells (Nwafor & Coakley, 1986; Scheiman & Elta, 1990; Li et al., 1999; Shacter &
Weitzman, 2002; Suwalsky et al., 2003; Hubner et al., 2005; Santos et al., 2005; Zhang
et al., 2005).
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The aim of this study was to investigate the effects of an aqueous extract of the
Lantana camara on the osmotic fragility and on the morphology of red blood cells.
Methods
Animals
The animals were maintained under environmental conditions (25±2°C, 12h of
light/dark cycle), water ad libitum and normal diet. Blood was withdrawn by cardiac
puncture with a heparinized syringe from adult male Wistar rats (n=6, 3-4 months,
245±35g). The experimental procedures have followed the Ethical Guidelines of the
Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de
Janeiro with the protocol number CEA/127/2006.
Identification and preparation of the Lantana camara extract
The leaves were collected in the forest of Petrópolis city, Rio de Janeiro State,
Brazil. The material collected was identified by the Biologist Ricardo Carneiro da Cunha
Reis, Botanic Herbarium RB of the Jardim Botânico, Rio de Janeiro State, Brazil, where
a voucher specimen (4070081) was kept. The extract of Lantana camara was prepared
with leaves triturated (150 mg, dried in ambient air) added in 15 ml of boiling 0.9% NaCl
during 10 minutes. The preparation was filtered through paper (quality filter paper) and
considered as 10 mg/ml. As reproductibility control of the extract was used the value of
the absorbance of a alíquot of extract at 480 nm (0.17±0.03) determined in a
spectrophotometer (Analyser 800M Analyser comércio e indústria LTDA, São Paulo)
a) The experimental procedure
The osmotic fragility evaluations of the RBC were performed with whole blood
samples incubated with Lantana camara extract (10 mg/ml) or with sodium chloride
solution (0.9% NaCl) as a control for 60 minutes at room temperature. Samples of these
whole blood (control and treated) were centrifuged (Clinical centrifuge, BIO ENG Ind e
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Com LTDA) and aliquots of RBC are separated. Briefly, RBC samples (100 l) treated
with Lantana camara extract or with saline solution (control) were gently mixed with
different hypotonic NaCl (from 0.12 to 0.9%) solutions according to Dacie (2001)
modified method (Dacie & Lewis, 2001). After 60 min, at room temperature, the
preparations were centrifuged (1500 rpm, 15 minutes). The supernatants were isolated
and the optical density (OD) to each NaCl concentration was in a spectrophotometer
(Analyser 800M Analyser comercio industria LTDA,São Paulo) at 540 nm. The OD of
each supernatant was compared with the OD of the 0.12% NaCl solution (100% of
lysis). The supernatant at 0.9% NaCl was considered the blank for the preparation,
because it has no hemolysis. According to curve tendency, three intervals were
determinates: interval I between 0.12 and 0.36% NaCl, interval II between 0.36 and
0.60% NaCl and interval III between 0.60 and 0.90% NaCl (Cavalcanti et al., 2003).
After measuring of osmotic fragility, “fragility curves” were drawn by plotting the
percentage of lysis or hemolysis (% hemolysis) for each tube (relative to 100%
hemolysis tube) and the corresponding concentration of NaCl. The experiments were
analyzed with paired t-test to verify potential differences between hypotonic and isotonic
phases (% concentrations of NaCl) versus relative hemolysis (% hemolysis).
Morphological Evaluation
Histological preparations were carried out with blood samples in vitro treated with
extract Lantana camara (10 mg/ml) during 60 min at room temperature, or with saline
solution as control group. Blood smears were prepared, dried, fixed and staining by
May-Grünwald-Giensa method (Barcia, 2007). After that, the images of the red blood
cells were acquired (Optronics, USA) from blood smears to qualitative morphology
analysis under optical microscopy (x1000, Olympus, BX model, Japan).
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Statistical analysis
The data of mean of hemolysis percentage in each interval in the fragility curve
were presented as means ± standard deviation. Paired t-test was used to compare the
intervals I, II and III between treated and control groups. A significance level at p<0.05
was adopted. InStat Graphpad software was used to perform statistical analysis
(GraphPad InStat version 3.01 for Windows 95/NT, GraphPad Software, San Diego,
USA).
Artigo II. Results
Figure 1 shows the osmotic fragility curves obtained after treatment with
an aquous extract of Lantana camara of RBC samples from Wistar rats. The
results indicate that the extract used alters the profile of osmotic fragility
curves when compared with the control group. The figure 2 presents the
mean of the hemolysis percentage after analysis of the three NaCl
concentrations intervals obtained of the osmotic curve of the figure 1.
These data confirm the results presented in Figure 1. The analysis of the
results showed a significant statistical increase (p<0.05) on osmotic fragility
of RBC incubated with Lantana camara extract in the interval II (0.36 to
0.60% NaCL) and in the interval III (0.60 to 0.90% NaCL). In the interval 3
(0.60 to 0.90% NaCL, that is related to the isotonic interval, the osmotic
fragility also increased significantly (p<0.05) in blood samples treated with
Lantana camara extract when compared to control group.
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The figures 3 and 4 represent photomicrographies of blood smears
from samples of blood treated with saline (control) and treated with the
extract of Lantana camara. The comparison between these figures
indicates that the extract is capable to induce alterations on the morphology
of the red blood cells.
Artigo III. Discussion
Authors have described that some drugs are capable of inducing alterations on
the shape and physiology of the red cells (Ammus et al,1989; Oliveira et al., 2005).
Different techniques have been used to evaluate the effects of the interaction between
drugs and plasma membrane (Li et al. 1999, Pompei et al. 2005). The osmotic fragility
assay is a classical, rapid, useful and easy technique that have permitted to obtain
relevant information about the interactions of natural and synthetic drugs with plasma
membrane (Khanna et al. 2002). Morphological analysis is another available method
that have permitted to evaluate the effects of natural products on membrane of red
blood cells (Oliveira et al. 2002, Oliveira et al. 2003).
The data obtained from osmotic fragility assay in this work indicated that extract
of Lantana camara could alter the membrane integrity at NaCl concentrations near to
physiologic level (Figures 1 and 2). In the same way, the morphological analysis of
blood smears suggested alteration on the shape of the red blood cells from whole blood
treated with Lantana camara (Figures 3 and 4). These alterations on the membrane
integrity could be related to components present in the aqueous extract of Lantana
camara capable of interact with membrane components and that could modify the
erythrocyte membrane ions transport or the osmotic transport balance.
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It was reported that compounds present in Lantana camara alter the function of
protein C (Herbert et al.1991). Other data suggested that an antifilarial (Misra et al.
2007) and antitumor (Shashi et al. 1994) effects to this natural product. Moreover,
serveral phrmacological properties have been associated with the studied extract.
Taken together, these findings could indicate an action of the chemical compounds of
the Lantana camara on membrane structure and they could be in agreement with the
results obtained in this work.
In conclusion, the aqueous extract of Lantana camara used could affect the
membrane integrity decreasing the osmotic resistance and altering the shape of red
blood cells. These findings could be related with some properties of the chemical
compounds of this studied extract.
Acknowledgements
This research was supported by Fundação de Amparo a Pesquisa do Estado do
Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq) and Universidade do Estado do Rio de Janeiro (UERJ).
i) References
Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson JD 2002. Molecular
Biology of The Cell. New York & London: Garland Publishing Inc.
Ahmed ZF, Shoaib AM, Wassel GM, Sayyad SM 1972. Phytochemical study of Lantana
camara L . Planta Med 21: 282–288.
Ammus S, Yunis A 1989.Drug-induced red cell dyscrasias. Blood Rev 3: 71-82 .
Barcia JJ 2007.The Giemsa stain: its history and applications. Int J surg patho 15: 292-
6.
Cavalcanti TC, Gregorini CG, Guimarães F, Rettori O, Vieira-Matos A N 2003. Changes
in red blood cell osmotic fragility induced by total plasma and plasma fractions obtained
32
33
from rats bearing progressive and regressive variants of the Walker 256 tumor. Braz
Med Biol Res 36: 887-895.
Cruz Silva MM, Madeira VMC, Almeida LM, Custódio JBA 2000. Hemolysis of human
erythrocytes induced by tamoxifen is related to disruption of membrane structure.
Biochim Biophys Acta 1464: 49-61.
Dacie J, Lewis SM 2001. Practical Haematology. London: Churchill Livingstone.
Didelon J, Mazeron P, Muller S, Stoltz JF 2000. Osmotic fragility of the erythrocyte
membrane: Characterization by modeling of the transmittance curve as a function of the
NaCl concentration. Biorheol 37: 409-416.
Fonseca AS, Frydman JN, Santos R, Bernardo-Filho M 2005. Influence of antipyretic
drugs on the labeling of blood elements with technetium-99m. Acta Biol Hung 56: 275-
282.
Ghisalberti EL 2000.Lantana camara Linn. Fitoterapia 71: 467–485.
Herbert JM, Maffrand JP, Taoubi K, Augereau JM, Fouraste I, Gleye J
1991.Verbascoside isolated from Lantana camara, an inhibitor of protein kinase C. J
Nat Prod 54: 1595–1600.
Hernandes T, Canales M, Avila JG, Duran A, Caballero J, Romo de Vivar A, Lira R
2003. Ethnobotany and antibacterial activity of some plants used in tradional medicine
of Zapotitlann de las Salinas.J Ethnopharmacol 88: 181-188.
Hubner Y, Hoettges KF, Kass G E, Ogin S L, Hughes MP 2005. Parallel measurements
of drug actions on Erythrocytes by dielectrophoresis, using a three-dimensional
electrode design. IEE Proc Nanobiotechnol 152: 150-154.
Junqueira LC, Carneiro J, 2004. Histologia Básica. Rio de Janeiro: Guanabara Koogan.
Li A, Seipelt H, Muller C, Shi Y, Artmann M 1999. Effects of salicylic acid derivatives on
red blood cell membranes. Pharmacol Toxicol 85: 206-211.
33
34
Khanna R, Chang SH, Andrabi S, Azam M, Kim A, Rivera A, Brugnara C, Low OS, Liu
SC,Chishti AH 2002. Headpiece domain of dematin is requires for the stability of the
erythrocyte membrane. Biochem 99: 6637-6642.
Mello FB, Jacobus D, de Carvalho KC, de Mello JR 2003. Effects of Lantana câmara
(Verbenaceae) on rat fertility. Vet Hum Toxicol 45:20-23.
Misra N, Sharma M, Raj K, Dangi A, Srivastava S, Misra-Bhattacharya S 2007.
Chemical constituents and antifilarial activity of Lantana Camara against human
lymphatic filariid Brugia malayi and rodent filariid Acanthocheilonema viteae maintained
in rodent hosts. Parasitol Res 100: 439-448.
Nwafor A, Coakley W T 1986. Charge-independent effects of drugs on erythrocyte
morphology. Biochem Pharmacol 35: 953-957.
Pompei LM, Carvalho FM, Ortiz SC, Motta MC, Cruz RJ, Melo NR 2005. Morphometric
evaluation of effects of two sex steroids on mammary gland of female rats. Maturitas 5:
370-379.
Oliveira JF, Avila AS, Braga ACS, Oliveira MBN, Boasquevisque EM, Jales RL,
Cardoso VN, Bernardo-Filho M 2002. Effect of extract of medicinal plants on the
labeling of blood elements with technetium-99m and on the morphology of red blood
cells: I – a study with Paullinia cupana. Fitoterapia 73: 305-312.
Oliveira JF, Santos-Filho SD, Catanho MTJA, Srivastava SC, Lima-Filho GL, Bernardo
Filho M 2003. Effect of extract of medicinal plants on the labeling of blood elements with
technetium-99m and on the morphology of red blood cells (RBC): Toxicological actions
of roast coffee beans (Coffea arabica). Indian J Nucl Med 18: 52-56.
Oliveira JFF, Brito LC, Frydman JNG, Santos-Filho SD, Bernardo-Filho M 2005 An
aqueous extract of Pfaffia sp. does not alter the labeling of blood constituents with
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35
technetium-99m and the morphology of the red blood cells. Rev Bras Farmacogn
15:126-132.
Sagar L, Sehgal R, Ojha S 2005. Evaluation of antimotility effect of Lantana camara L.
var. acuelata constituents on neostigmine induced gastrointestinal transit in mice. BMC
Complement Altern Med 5: 18-23.
Santos N C, Martins-Silva J, Saldanha C 2005. Gramicidin D and dithiothreitol effects
on erythrocyte exovesiculation. Cell Biochem Biophys 43: 419-430.
Shacter E, Weitzman SA 2002. Chronic inflammation and cancer. Oncology 16: 217-
226.
Shashi BM, Niranjan PS, Subodh KR, Sharma OP 1994. Potential Antitumor Agents
from Lantana camara: Structures of Flavonoid and Phenylpropanoid Glycosides.
Tetrahedron 50: 9439–9446.
Scheiman J M, Elta GH 1990. Gastroduodenal mucosal damage with salsalate versus
aspirin: results of experimental models and endoscopic studies in humans. Semin
Arthritis Rheum 20: 121-127.
Suwalsky M, Hernandez PL, Villena F,Sotomayor C P 2003. The anticancer drug
cytarabine does not interact with the human erythrocyte membrane. Z Naturforschung C
58: 885-890.
Uzcategui B, Avila D, Heberto SR, Quintero L, Ortega J, Gonzalez YB 2004. Anti-
inflammatory, antinociceptive and antipyretic effects of Lantana trifolia Linnaeus in
experimental animals. Invest Clin 45: 317-322.
Zhang X, Inukai T, Hirose K, Akahane K, Nemoto A, Takahashi K, Sato H, Kagami K,
Goi K, Sugita K, Nakazawa S 2005. Induction of impaired membrane phospholipid
asymmetry in mature erythrocytes after chemotherapy. Int J Hematol 82: 132-136.
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36
Zheng HQ, Wei N, Wang LF, He P 2006. Effects of Lantana camara Leaf Extract on the
Activity of Superoxide Dismutase and Accumulation of H(2)O(2) in Water Hyacinth Leaf.
Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao 32:189-194.
FIGURE 1
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
0,12 0,24 0,36 0,48 0,6 0,72 0,9
NaCl (%)
Hem
olys
is (%
)
ControlTreated
I II III
FIGURE 2
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37
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
I II III
Intervals
Mea
n of
Hem
olys
is
Control
Treated*
*
FIGURE 3
FIGURE 4
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(b) FIGURE LEGENDS
FIGURE 1: Osmotic fragility of blood samples treated or not treated with extract of Lantana camara. Blood samples were incubated with extract of Lantana camara or with sodium chloride solution (0.9% NaCl), as control. Aliquots of blood were gently mixed with NaCl at different concentrations, centrifuged, the supernatants were isolated and the optical densities (OD) were determined in a spectrophotometer at 540 nm. The hemolysis percentage was calculed and “fragility curves” were drawn plotting the percentage of hemolysis (% hemolysis) for beach NaCl concentration (relative to 100% hemolysis tube). ( ) control, ( ) treated with extract of Lantana camara.
FIGURE 2: Means of hemolysis of the blood samples treated or not treated with extract of Lantana camara. Three intervals were determinate: interval I (from 0.12 to 0.24%), interval II (from 0.24 to 0.48%), and interval III (from 0.48 to 0.9%) according the curve tendency. The means and standard deviations of each interval were determinate and the statistical analysis was performed. ( )control, ( ) treated with extract of Lantanacamara. (*) p<0.05.
FIGURE 3: Photomicrography of blood smears from blood samples in vitro treated with NaCl 0.9% solution (control group). Samples of whole blood from Wistar rats were treated with NaCl 0.9% solution during 60 minutes. Blood smears were prepared, dried, fixed and staining by May-Grünwald-Giensa method. The morphology of red blood cells was evaluated under optical microscopy (x 1000) after image capture.
FIGURE 4: Photomicrography of blood smears from blood samples in vitro treated with extract of Lantana camara. Samples of whole blood from Wistar rats were treated with extract of Lantana camara (10 mg/ml) during 60 minutes. Blood smears were prepared, dried, fixed and staining by May-Grünwald-Giensa method. The morphology of red blood cells was evaluated under optical microscopy (x 1000) after image capture.
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4. COMENTÁRIOS, CRÍTICAS E CONCLUSÕES
A experiência vivenciada no Curso de Mestrado foi de grande
importância para a aquisição de conhecimentos que podem contribuir para o
desenvolvimento de modelos experimentais em outras pesquisas, bem como avançar
nesse mesmo modelo, para a verificação de outros resultados como por exemplo, o
estudo de biodistribuição em ratos wistar tratados com este extrato,e experimentos em
cepas bacterianas.
Uma das maiores preocupações foi tentar evitar riscos, buscando
seguir rigorosamente as condições metodológicas, dessa forma permitindo a
reprodutibilidade integral de nossos estudos pela comunidade científica, objetivando
estimular a obtenção de outros resultados que tragam maiores contribuições ao meio
acadêmico.
A possibilidade de estudar este extrato vegetal, possibilitada pelo
Orientador, Professor Doutor Mario Bernardo-Filho com total apoio da Universidade
Federal do Rio Grande do Norte, permitiu a oportunidade de viver a experiência
inigualável e gratificante da pesquisa básica.
Como profissional da área da saúde (fisioterapeuta) tenho a
preocupação de que efeitos produzidos pela Lantana camara possam interferir nos
resultados de exames clínicos, especialmente os da medicina nuclear. A interação
medicamentosa pode modificar a resposta farmacológica. Dessa forma alterar a
distribuição normal dos radiotraçadores, podendo apresentar resultados imprecisos que
levariam à repetição de exames, expondo o paciente a doses de radiação
desnecessárias. Como fisioterapeuta, utilizo condutas com o objetivo de preservar,
manter, desenvolver ou restaurar a integridade de um órgão, sistema ou função do
corpo humano, através de terapias físicas e cinéticas. Desta forma o laudo de exames
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40
complementares, em especial os da medicina nuclear, corroboram o diagnóstico
cinético-funcional com informações precisas para o fechamento do diagnóstico
fisioterapêutico e permite uma conduta mais apropriada.
As dificuldades em ter plenos recursos para fazer pesquisa foram
supridos em parte pelo suporte dado por Setor de Medicina Nuclear, Laboratório de
Endocrinologia e Laboratório Central do Hospital Universitário Pedro Ernesto -
Universidade do Estado do Rio de Janeiro, os quais foram fundamentais para a
realização dessa dissertação, junto ao apoio da Fundação de Amparo a Pesquisa do
Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico
e Tecnológico (CNPq) e Instituto de Biologia Roberto Alcântara Gomes, através do
Laboratório de Radiofarmacia experimental. Sem a participação efetiva de todos essas
instituições e seus respectivos serviços não teria sido possível concluir esta
dissertação.
Os resultados obtidos permitiram a confecção de dois manuscritos
que foram submetidos e aceitos em revistas científicas. Gerando publicações que
estarão contribuindo para a pesquisa.
.
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5 . ANEXOS
Anexo I
Carta de aceite do manuscrito I
Acta Physiologica Hungarica A Periodical of the Hungarian Academy of Sciences
Managing Editor: Jen Bartha Editor-in-Chief: Emil MonosEditorial Office Semmelweis University Semmelweis University Institute of Human Physiology Institute of Physiology H-1446 Budapest, POB. 448. Hungary Editorial Correspondence Phone: (36-1) 210-6038 H-1445 Budapest, POB. 294. Hungary Fax: (36-1) 334-3162 Fax: (36-1) 266-7480 E-mail: [email protected] E-mail: [email protected]
Adalgisa I. Maiworm 12 July 2007 Universidade do Estado do Rio de Janeiro
Instituto de Biologia Roberto Alcantara Gomes Departamento de Biofísica e Biometria Laboratório de Radiofarmácia Experimental Av. 28 de setembro, 87 20551-030 - Rio de Janeiro – RJ
BRASIL
Dear Doctor Adalgisa I. Maiworm
I am pleased to inform you that your paper entitled
(38/6) Adalgisa I. Maiworm et al.: Evaluation of the in vitro effect of a Lantana camara
extract on the labeling of blood constituents with technetium-99m.
has been accepted for publication by the editorial staff of Acta Physiologica Hungarica.
Please send the employments of all authors and your postal address in english, too
Yours sincerely
J. Bartha, MD, PhD Managing Editor
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Anexo II
Carta de aceite do manuscrito II
Prezada Profa. Adalgisa Maiworm,
Informo que o manuscrito intitulado "Osmotic and morphological effects on red blood cellmembrane: Action of an aqueous extract of Lantana camara", de autoria de Adalgisa I. Maiworm, Giuseppe A. Presta, Sebastião D. Santos-Filho, Severo de Paoli, Tânia S. Giani, Adenilson S. Fonseca e Mário Bernardo-Filho, foi aceito para publicação na REVISTA BRASILEIRA DEFARMACOGNOSIA.
Atenciosamente,
José Maria Barbosa Filho Editor Chefe
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6. REFERÊNCIAS
1. Rotblatt M, Ziment I. Evidence-Based Herbal Medicine. Hanley & Belfus, Philadelphia
2002.
2. Hladik III WB, Saha GB, Study KT. Essentials of Nuclear Medicine Science. Williams
& Wilkins, Baltimore 1987.
3. Sampson CB. Textbook of radiopharmacy theory and practice. Gordon and Breach,
Amsterdam 1999.
4. Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson JD. Molecular Biology of
The Cell.Garland Publishing Inc, New York & London 2002.
5. Santos-Filho SD, Diré GL, Lima E, Oliveira MN, Bernardo-Filho M. Effect of Mentha
crispa (mint) extract on the labeling of blood elements with technetium-99m: A possible
evaluation of free radicals. Journal of Biology and Science 2004; 4, 266-270.
6. Moreno SRF, Carvalho JJ, Nascimento AL, Pereira M, Rocha EK, Diré G, Arnobio A,
Caldas LQA, Bernardo-Filho M. Bioavailability of the sodium pertechnetate and
morphometry of organs isolated from Wistar rats: study of possible pharmacokinetic
interactions of a Ginkgo biloba extract. Brazilian Archieves of Biology and Technology
2005; 48: 73 -78.
7. Didelon J, Mazeron P, Muller S, Stoltz JF. Osmotic fragility of the erythrocyte
membrane: Characterization by modeling of the transmittance curve as a function of the
NaCl concentration. Biorheology 2000; 37: 409 - 416.
8. Oliveira JFF, Brito LC, Frydman JNG, Santos-Filho SD, Bernardo-Filho M An
aqueous extract of Pfaffia sp. does not alter the labeling of blood constituents with
43
44
technetium-99m and the morphology of the red blood cells. Revista Brasileira de
Farmacognosia 2005; 15:126 -132.
9. Ghisalberti EL Lantana camara Linn. (Review). Fitoterapia, 2000; 71: 467–485
10. Mello FB, Jacobus D, de Carvalho KC, de Mello JR. Effects of Lantana camara
(Verbenaceae) on rat fertility. Veterinary and Human Toxicology 2003; 45:20-23.
11. Deena M J, Thoppil J E. Atividade antimicrobiana do óleo de Lantana Camara.
Fitoterapia 2000; 71:453 - 455.
12. Early PJ & Sodee DB. Principles and Practice of Nuclear Medicine. Mosby, London.
1995.
13. Owunwanne A, Patel M, Sadek S. The Handbook of RadioPharmaceuticals.
Chapman and Hall, London 1995.
14. Bushong SC. Radiologic Science for Technologists: Physics, Biology and
Protection. Mosby, Missouri 2001.
15. Thrall Jh, Jiessman HA. Medicina Nuclear. Guanabara Koogan, Rio de Janeiro
2003.
16. Perkins A, Frier M. Nuclear Medicine Pharmaceutical Research. Taylor & Francis,
London 1999.
17. Saha GB. Fundamentals in Nuclear Pharmacy. Springer-Verlag, New York 2004.
18. Bernardo-Filho M, Santos-Filho SD, Moura EG, Maiworm AI, Orlando MMC, Penas
ME, Cardoso VN, Bernardo LC, Brito LC. Drug interaction with radiopharmaceuticals: a
review. Brazilian Archieves of Biology and Technology 2005; 48: 13 - 27.
19. Van de Wiele C, Lahorte C, Oyen W, Boerman O, Goethals I, Slegers G, Dierckx
RA. Nuclear medicine imaging to predict response to radiotherapy: a review.
International Journal of Radiation Oncology, Biology, Physics. 2003; 551: 5 -15.
44
45
20. Propper DJ, de Bono J, Saleem A, Ellard S, Flanagan E, Paul J, Genesan TS,
Taibot DC, Aboagye EO, Price P, Harris A, Twelves C. Use of positron emission
tomography in pharmacokinetic studies to investigate therapeutic advantage in a phase
I study of 120-hour intravenous infusion XR5000. Journal of Clinical Oncology 2003; 21:
203 - 210.
21. Kowalsky RJ & Perry JR. Current Practice in Nuclear Medicine. Appleton e Lange.
Los Altos, California 1987.
22. Palestro CJ, Tomas MB, Tronco GG: Radionuclide imaging of the parathyroid
glands. Seminars Nuclear Medicine 2005; 35: 266 – 276.
23. Guedes AP, Cardoso VN, De Mattos JC, Dantas FJ, Matos VC, Silva JC, Bezerra
RJ, Caldeira-de-Araujo A.; Cytotoxic and genotoxic effects induced by stannous
chloride associated to nuclear medicine kits. Nuclear Medicine and Biology 2006; 33:
915 - 21.
24. Bennink RJ, van den Elzen BD, Kuiken SD, Boeckxstaens GE. Noninvasive
easurement of gastric accommodation by means of pertechnetate SPECT: limiting
radiation dose without losing image quality. Journal of Nuclear Medicine 2004; 45:147-
52.
25. Mehmet R, Mehmet A, Fuat AY, Nebil B A . Jejunoileal duplication diagnosed by Tc-
99m pertechnetate abdominal scintigraphy. Journal of Postgraduate Medicine 2005;
51:245 - 246.
26. Leibovitch I, Hoyama E, Limawararut V, Crompton J, Selva D. Novel technique to
control hypersecretion from a transplanted autologous submandibular salivary gland for
keratoconjunctivitis sicca. Cornea 2006; 25:1251 - 1253.
27. Goethals I, Mervillie K, De Winter O, Delrue L, Mekeirele K, Ham H. Mismatch of F-
18 fluorodeoxyglucose (FDG) positron emission tomography (PET) and Tc-99m
45
46
pertechnetate single photon emission computed tomography (SPECT) in a euthyroid
multinodular goiter. Clinical Nuclear Medicine. 2007; 32: 6 - 8.
28. Junqueira LC, Carneiro J. Histologia Básica. Guanabara Koogan, Rio de Janeiro
2004.
29. Berne RM, Levy MN, Koepper BM, Stanton BA. Fisiologia. Elsevier, Rio de Janeiro
2004.
30. Guyton AC & Hall JE. Tratado de Fisiologia Médica. Guanabara Koogan, Rio de
Janeiro 2006.
31. Arano Y. Recent advances in 99mTc radiopharmaceuticals. Annals of Nuclear
Medicine 2002; 16: 79 - 93.
32. Bernardo-Filho M, Nogueira J, Sturm J, Boasquevisque E. Plasma proteins labelling
with 99mTechnetium. Brazilian Archieves of Biology and Technology 1990; 33: 811-
817.
33. Manning-Dimmitt LL, Dimmitt SG, Wilson GR. Diagnosis of gastrointestinal bleeding
in adults. American Family Physician, 2005; 71: 1339 -1346.
34. Karacalioglu O, Ilgan S, Arslan N, Ozguven M. Uterine doughnut in early
proliferating phase: potential pitfall in gastrointestinal bleeding studies. Annals of
Nuclear Medicine 2003; 17: 685 – 687.
35. Callahan RJ & Rabito AC. Radiolabeling of erythrocytes with technetium-99m: role
of band-3 protein in the transport of pertechnetate across the cell membrane. Journal of
Nuclear Medicine 1990; 31: 2004 -2010.
36. Gutfilen B, Boasquevisque EM, Bernardo-Filho M. Calcium channel blockers:
interference on red blood cells and plasma proteins labeling with Tc-99m. Revista
Espanhola de Medicina Nuclear 1992; 11: 195 - 199.
46
47
37. Freitas RS, Moreno SR, Lima-Filho GL, Fonseca AS, Bernardo-Filho M. Effect of a
commercial extract of Paullinia cuPana (guarana) on the binding of 99mTc-DMSA on
blood constituents: An in vivo study. Applied Radiation and Isotopes 2007; 65: 528 -
533.
38. Harbert JC, Eckelman WC, Neumann RD. Nuclear Medicine Diagnosis and
therapy. Thieme Medical Publishers, New York 1996.
39. Hardman JG, Limbird LE, Molinoff PB. Goodman & Gilman’s: The Pahrmacological
basis of therapeutic. Mc Graw Hill, New York 2005.
40. Gomes ML, Oliveira MBN, Bernardo-Filho M. Drug interaction with
radiopharmaceuticals: Effect on the labeling of the red blood cell with technetium-99m
ando n the bioavailability of rafiopharmaceuticals. Brazilian Archieves of Biology and
Technology 2002; 45: 143 -149.
41. Santos-Filho SD, Bernardo-Filho M: Effect of Hypericum perforatum extract on in
vitro labeling of blood elements with technetium-99m and on biodisponibility of sodium
pertechnetate in Wistar rats. Acta Cirurgica Brasileira 2005; 20:121 -125.
42. Abreu PR, Almeida MC, Bernardo RM, Bernardo LC, Brito LC, Garcia EA, Fonseca
AS, Bernardo-Filho M. Guava extract (Psidium guajava) alters the labeling of blood
constituents with technetium-99m. Journal of Zhejiang University of Science B 2006; 7:
429 - 435.
43. Shashi BM, Niranjan PS, Subodh KR, Sharma OP. Potential Antitumor Agents fromf
Lantana camara: Structures of Flavonoid and Phenylpropanoid Glycosides. Tetrahedron
1994; 50: 9439 – 9446.
44. O' Neill MJ, Lewis JA, Noble HM, Holland S, Mansat C, Farthing JE, Foster G,
Noble D, Lane SJ, Sidebottom PJ, Lynn SM, Hayes MV, Dix CJ. Isolation of
47
48
translactone-containing triterpenes with thrombin inhibitory activities from the leaves of
Lantana camara . Journal of Natural Products 1998; 61:1328 – 1331.
45. Uzcategui B, Avila D, Heberto SR, Quintero L, Ortega J, Gonzalez YB. Anti-
inflammatory, antinociceptive and antipyretic effects of Lantana trifolia Linnaeus in
experimental animals. Investigation Clinical 2004; 45: 317 – 322.
46. Misra N, Sharma M, Raj K, Dangi A, Srivastava S, Misra-Bhattacharya S: Chemical
constituents and antifilarial activity of Lantana Camara against human lymphatic filariid
Brugia malayi and rodent filariid Acanthocheilonema viteae maintained in rodent hosts.
Parasitology of Research 2007; 100: 439 – 448.
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7. ABSTRACT
Medicinal plants have been studied and used in the world. Lantana camara has
medicinal properties and it has been used in folk medicine. The aim was to verify the
effect of a lantana extract on the labeling of blood constituents with 99mTc, and to
evaluate the effect of an aqueous extract of Lantana camara on the morphology of RBC
withdrawn from Wistar rats. The results showed that lantana extract has decreased the
fixation of radioactivity on the IF-P. This effect was not observed in the BC compartment
and in IF-BC. The BC-%ATI was decreased in all concentrations tested when the BC
was washed. The osmotic fragility assay and morphological analysis were carried out.
In presence of the extract, the data obtained indicated that (i) an increase of the
hemolysis and (ii) modifications on the morphology of RBC. These effects of the
Lantana camara could be associated with some pharmacological properties of the
chemical compounds of this studied extract.
Keywords: blood constituents, technetium-99m, osmotic fragility, Lantana camara
.
49
