Nelson de Souza Pinto - Mestrado

UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
AVALIAÇÃO DE EFEITOS DE UM EXTRATO DE “TRÊS BAILARINAS” EM
MODELOS EXPERIMENTAIS EM DIFERENTES NÍVEIS DE ORGANIZAÇÃO
BIOLÓGICA.
Natal, RN
2011
NELSON DE SOUZA PINTO
AVALIAÇÃO DE EFEITOS DE UM EXTRATO DE “TRÊS BAILARINAS” EM
MODELOS EXPERIMENTAIS EM DIFERENTES NÍVEIS DE ORGANIZAÇÃO
BIOLÓGICA.
Dissertação apresentada à Universidade Federal do
Rio Grande do Norte - UFRN, para a obtenção do
título de Mestre em Ciências da Saúde pelo Programa
de Pós- graduação em Ciências da Saúde.
Orientador: PROF. DR. MARIO BERNARDO-FILHO
Colaborador: PROF. DR. SEBASTIÃO DAVID SANTOS-FILHO
Natal, RN
2011
ii
UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
Coordenadora do Programa de Pós Graduação em Ciências da Saúde
Profa. Dra. Técia Maria de Oliveira Maranhão
Natal, RN
2011
iii
CATALOGAÇÃO NA FONTE
UERJ/REDE SIRIUS/BIBLIOTECA CB-A
P659
Pinto, Nelson de Souza.
Avaliação de efeitos de um extrato de três bailarinas em
modelos experimentais em diferentes níveis de organização
biológica / Nelson de Souza Pinto. - 2011.
57 f.
Orientador : Mario Bernardo-Filho.
Colaborador: Sebastião David Santos-Filho.
Dissertação (Mestrado) – Universidade Federal do Rio Grande
do Norte. Centro de Ciências da Saúde. Programa de Pósgraduação em Ciências da Saúde.
1. Drogas vegetais - Dissertação. 2. Eritrócitos - Dissertação.
3. Tecnécio–99m - Dissertação. 4. Escherichia coli - Dissertação.
I. Bernardo-Filho, Mario. II. Santos Filho, Sebastião David dos. III.
Universidade Federal do Rio Grande do Norte. Centro de Ciências
da Saúde. IV. Título.
CDU 615.32
NELSON DE SOUZA PINTO
AVALIAÇÃO DE EFEITOS DE UM EXTRATO DE “TRÊS BAILARINAS” EM
MODELOS EXPERIMENTAIS EM DIFERENTES NÍVEIS DE ORGANIZAÇÃO
BIOLÓGICA.
PRESIDENTE DA BANCA: Prof. Dr. Mario Bernardo-Filho (UERJ)
BANCA EXAMINADORA
Profa. Dra. Técia Maria de Oliveira Maranhão
Profa. Dra. Aurimery Gomes Chermont
SUPLENTES
Prof. Dr Aldo da Cunha Medeiros
Prof. Dr. Sebastião David dos Santos Filho
iv
DEDICATÓRIA
Dedico este trabalho:
A minha esposa, Alcy Batista da Costa, e aos meus filhos, Alan Costa de
Souza e Hugo Costa de Souza. Eles suportaram a minha ausência e vivenciaram
comigo todos os momentos de estresse e as dificuldades ocasionadas pela
sobrecarga de trabalho e das atividades pessoais acumuladas ao longo do período
de desenvolvimento do projeto de mestrado. Precisei muito da compreensão deles.
Os três são meus alicerces.
Aos meus seis irmãos, a todos os sobrinhos e sobrinhos netos. Juntos
formamos uma família maravilhosa.
In memoriam, de meu pai Nelson da Silva Pinto, minha mãe Odette de Souza
Pinto e minha irmã Neize Liliane de Souza Pinto, tenho certeza que estão
orgulhosos desse grande feito.
A meu orientador, Prof. Dr. Mario Bernardo Filho, por ter me acolhido.
A Deus por permitir que seu humilde servo tivesse alcançado mais essa
vitória.
v
AGRADECIMENTOS
A Deus que com sua infinita misericórdia proporcionou a esse filho a
realização de um grande sonho, que até então parecia impossível de ser realizado.
Agradeço ao Pai todo poderoso, pela minha vida, pela minha família e pelos meus
amigos.
Agradeço ao meu orientador Prof. Dr. Mario Bernardo-Filho, por me ensinar o
quão técnico, meticuloso, criterioso, dedicado e perseverante deve ser um
pesquisador.
Ao Programa de Pós-graduação em Ciências da Saúde da Universidade
Federal do Rio Grande do Norte por me proporcionar conhecimentos na área da
saúde e da educação, fato que me torna um ser humano e profissional melhor. Com
essas novas ferramentas adquiri um olhar mais apurado nos diferentes grupos de
atuação do meu dia-a-dia: ser humano, alunos e meus pacientes.
Aos funcionários da Secretaria do Programa de Pós-graduação em Ciências
da Saúde, em especial a Alana, que sempre foi atenciosa, me orientando com
relação aos prazos e as normas a serem cumpridas.
Aos amigos do Laboratório de Radiofarmácia Experimental do Departamento
de Biofísica e Biometria do Instituto Roberto Alcântara Gomes da Universidade do
Estado do Rio de Janeiro, Márcia, Fernanda, Claudia e Daniele, molas mestras do
laboratório. Aos alunos de iniciação científica: Eric e Marcela por fazerem parte
dessa grande equipe.
Ao Tecnólogo em Radiologia, especialista em radiologia veterinária, Daniel
Teixeira de Menezes, com a minha ausência na clínica veterinária, conduziu de
forma brilhante todas as atividades internas e externas, dessa forma permitiu que eu
vi
assistisse às aulas de todas as disciplinas, participasse das reuniões do laboratório
de radiofarmácia e desenvolvesse o projeto de mestrado. Minha eterna gratidão.
Ao grande amigo Prof. Dr. Sebastião David dos Santos Filho, sem ele esse
projeto não teria sido realizado. Sua amizade, paciência, dedicação e vontade de
ajudar ao próximo fazem dele um ser humano impar, exemplo a ser seguido.
vii
SUMÁRIO
Sumário.............................................................................................................viii
Lista de abreviações siglas e símbolos...............................................................x
Resumo…………….…………………………………........……………...................xi
1. Introdução........................................................................................................1
2. Revisão de literatura........................................................................................3
3. Artigos anexados.............................................................................................7
3.1. Artigo publicado............................................................................................8
3.2. Artigo submetido ........................................................................................21
4. Comentários, críticas e conclusões...............................................................42
5.Anexos (comitê de ética, carta de aceite)......................................................46
6. Referências...................................................................................................48
7. Abstract..........................................................................................................57
viii
LISTA DE ABREVIAÇÕES, SIGLAS E SÍMBOLOS.
BC
Blood Cell (célula sanguínea)
ber
base excision repair
reb
reparo de excisão de bases
C
célula, celular
°C
graus centigrados
cm
centímetro
CAPES
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
CNPq
Conselho Nacional de Desenvolvimento Científico e Tecnológico
UFC
unidades formadora de colônias
DNA
Acido desoxirribonucléico
E. coli
Escherichia coli
FAPERJ
Fundação de Amparo a Pesquisa do Rio de Janeiro
FI-C
fração insolúvel da célula
FS-C
fração solúvel da célula
FI-P
fração insolúvel do plasma
FS-P
fração solúvel do plasma
g
grama
h
hora
IBRAG
Instituto de Biologia Roberto Alcantara Gomes
kBq
quilobecquerel
LB
Luria Broth
LRE
Laboratório de Radiofarmácia Experimental
ix
µg
micrograma
µl
microlitro
mg
miligrama
ml
mililitro
min
minutos
mo
molibdênio
mSv
miliSievert
NaCl
cloreto de sódio
nm
nanômetro
P
plasma
PET
tomografia por emissão de positron
% ATI
porcentagem de radioatividade incorporada
PubMed
base de dados da “U.S. National Library of Medicine”
RBC
células vermelhas do sangue
RL
radical livre
rpm
rotações por minuto
SnCl2
cloreto estanoso
Sn
2+
íon estanoso
SPECT
tomografia computadorizada por emissão de fóton único
Na99mTcO4
pertecnetato de sódio
99m
tecnécio-99m
Tc
TB
Três bailarinas
TCA
ácido tricloroacético
x
RESUMO
Drogas naturais ou sintéticas podem ser capazes de alterar a sobrevivência de
culturas bacterianas, interferir na marcação de estruturas sanguíneas com tecnécio99m (99mTc) e alterar a morfologia das hemácias. De acordo com as instruções do
fabricante, a formula denominada de Três Bailarinas (TB) é sugerida para ser usada,
como bebida, por pessoas que desejam ajustar o peso sem dieta. Os ingredientes
dessa fórmula são a Cassia angustifolia e a Malva verticellate. Informações
cientificas sobre TB não foram encontradas no indexador PubMed, e esse fato tem
estimulado nossas investigações sobre seus efeitos biológicos. O objetivo deste
estudo foi avaliar, em diferentes modelos experimentais, o efeito de um extrato
aquoso de Três Bailarinas: (i) na sobrevivência de culturas de E. coli AB1157, ii)
efeito do SnCl2 em culturas bacterianas, iii) na marcação das hemácias e proteínas
plasmáticas e celulares com
99m
Tc e iv) na morfologia de hemácias de sangue de
ratos Wistar. Os resultados encontrados demonstram que o extrato de TB não
alterou a sobrevivência de cultura de E. coli AB1157 e aboliu o efeito letal do SnCl2
na sobrevivência dessa cultura bacteriana. Na marcação de estruturas sangüíneas
com
99m
99m
Tc o extrato de TB reduziu a percentagem de atividade (%ATI) referente ao
Tc no compartimento celular e nas proteínas plasmáticas, mas não alterou a
%ATI nas proteínas celulares. O extrato de TB não foi capaz de alterar a morfologia
das hemácias. Os modelos experimentais realizados mostram a importância dos
mesmos na avaliação de efeitos biológicos de agentes químicos, e contribui para um
melhor entendimento das propriedades do extrato de Três Bailarinas. Esse trabalho
abrange varias áreas do conhecimento, tais como: radiobiologia, botânica, fitoterapia
e hematologia.
Palavras-chaves: hemácias, tecnécio-99m, cloreto estanoso, Escherichia coli.
xi
1
1 INTRODUÇÃO
O consumo e o uso de plantas medicinais vêm crescendo em todo mundo ao
longo dos anos (1, 2, 3). Essas plantas são empregadas no tratamento de diversas
alterações metabólicas (4).
Em relação ao produto denominado “Três Bailarinas” (TB), composto por
Cassia angustifolia, da família Fabaceae e Malva verticellata, da família Malvaceae,
segundo as instruções do fabricante (Truong Giang Corp., South El Monte, CA
91733, USA), é sugerido que extrato desse produto ajuda a perder peso sem
necessidade de dieta .
Apesar da importância e uso das plantas medicinais, determinados efeitos
biológicos, que poderiam acarretar reações adversas e mesmo tóxicas, são
relatados o que estimula o uso de modelos experimentais que permitam uma melhor
compreensão dos mecanismos de ação desses produtos naturais (5, 6, 7).
Modelos experimentais empregando culturas bacterianas são utilizados para
avaliar a citotoxicidade e genotoxicidade de extratos vegetais (8, 9).
Estudos sobre o efeito de plantas medicinais na marcação de constituintes
sanguíneos com tecnécio-99m (99mTc) (10, 11, 12, 13 ) e na morfologia de hemácias
também têm sido realizados (14).
Na medicina nuclear, o cloreto estanoso (SnCl2) é freqüentemente usado
como um agente redutor na marcação de estruturas moleculares e celulares com
99m
Tc para obtenção de radiofármacos. Apesar de seu uso, diversos autores relatam
que o cloreto estanoso apresenta efeitos citotóxicos e genotóxicos (8, 15, 16).
Estudos com culturas bacterianas com diferentes capacidades do reparo
de lesões no DNA revelaram que essas lesões causadas pelo SnCl2 acarretavam
um efeito letal que parece ser mediado pela geração de radicais livres (17, 18, 19).
2
O efeito do SnCl2 também dependeria da presença de mecanismo de restauração
(18). Determinados extratos vegetais diminuem ou abolem essa ação do cloreto
estanoso (20, 21). Esse agente redutor também é capaz de promover a quebra de
DNA plasmidial (22, 23, 24, 25, 26).
Os mecanismos de ação de TB ainda não estão bem esclarecidos, e até essa
data não foi possível encontrar na literatura publicações de possíveis efeitos em
culturas bacterianas e na radiomarcação de constituintes sangüíneos empregado-se
o indexador PubMed (www.pubmed.com). Esse fato tem estimulado nossas
investigações sobre os efeitos biológicos de um extrato de TB em modelos
experimentais em diferentes níveis de organização biológica.
A hipótese dessa investigação é avaliar se o extrato de TB apresenta
propriedades redoxi. O presente trabalho tem como objetivo avaliar, em modelos
experimentais, o efeito de um extrato aquoso de Três Bailarinas (i) na sobrevivência
de culturas de E.coli AB1157 (selvagem), (ii) efeito do SnCl2 em culturas bacterianas,
(iii) na marcação dos constituintes sanguíneos com 99mTc, e (iv) na morfologia de
hemácias isoladas de ratos Wistar.
Os experimentos foram desenvolvidos no Laboratório de Radiofarmácia
Experimental do Departamento de Biofísica e Biometria do Instituto de Biologia
Roberto Alcantara Gomes da Universidade do Estado do Rio de Janeiro (UERJ) no
período de agosto de 2009 a julho de 2011 e teve aprovação da Comissão de Ética
para o Cuidado e Uso de Animais Experimentais (CEA/024/2009). O projeto foi
possível através de convênio firmado entre a UERJ e a Universidade Federal do Rio
Grande do Norte, sob a orientação do Professor Doutor Mario Bernardo Filho e na
vigência de auxílios concedidos pelo CNPq, CAPES, FAPERJ e UERJ.
3
2 REVISÃO DE LITERATURA
O consumo de produtos naturais vem aumentando ao longo dos tempos. É
desejável que plantas medicinais possam curar muitas doenças e que não tenham
toxicidade para o organismo (7, 27, 28, 30). A avaliação de forma mais cientifica dos
efeitos biológicos dos produtos de origem vegetal foi progressivamente realizada
através de diversas investigações ao longo dos anos (27, 28, 29).
Apesar da importância e uso de plantas medicinais, determinados efeitos
biológicos que podem acarretar reações adversas e mesmo tóxicas têm sido
descritos, o que estimula o desenvolvimento e implementação de estudos adicionais
(7, 30). Modelos experimentais de fácil utilização e baixo custo que permitem a
avaliação do efeito de extratos de produtos naturais ou drogas sintéticas têm sido
propostos e importantes resultados vêm sendo obtidos.
A determinação da sobrevivência de culturas bacterianas na presença de
extratos de plantas medicinais tem sido útil para a avaliação da possível
citotoxicidade de produtos naturais (8, 17, 26).
A ação de extratos de plantas medicinais no efeito de agente lesivo para
avaliar possível proteção de culturas bacterianas ou potencialização de danos
biológicos de agente agressor tem sido empregada (37). A avaliação de efeitos de
extratos vegetais na ação (potencializando, abolindo ou não alterando) do cloreto
estanoso (SnCl2) (18, 21, 37, 38) tem demonstrado a relevância desses estudos.
Autores têm descrito a ação tóxica e mesmo genotóxica associada com esse agente
redutor (9, 26, 29, 30, 39). O mecanismo de ação do cloreto estanoso parece
também estar associado com a geração de radicais livres (17), embora uma ação
direta sobre o sistema biológico não possa ser eliminada (30).
4
Com relação aos estudos com culturas bacterianas, pesquisadores relatam
potencial citotóxico e genotóxico do SnCl2 em culturas bacterianas de diferentes
cepas de Escherichia coli. Cepas bacterianas com mutação em único gene, cujo
produto, esteja envolvido com sistema de reparo do DNA são mais resistentes ao
tratamento com cloreto estanoso do que cepas com mais de uma mutação (8, 9, 19,
54, 55, 60).
Uma das características do íon estanoso é o seu possível transporte através
da membrana (41), isso poderia justificar seus efeitos tóxicos no interior da célula.
Aceptores de radicais livres (como a tiouréia, benzoato de sódio e dipiridil) ou
fitoterápicos (Cymbopogon citratus, Baccharis genistelloides, Maytenus ilicifolia e
Peumus boldus) aumentam a sobrevivência de culturas bacterianas, proficientes e
deficientes nos mecanismos de reparo de lesões induzidas no DNA, ao tratamento
com SnCl2. O efeito protetor destes fitoterápicos ocorreria devido à presença de
substâncias presentes nos extratos que atuariam como: (i) quelantes do íon
estanoso, protegendo as células bacterianas contra a oxidação e diminuindo a
geração de radicais livres, (ii) aceptores de radicais livres gerados pela oxidação do
SnCl2, e/ou (iii) substâncias que oxidariam o íon estanoso, reduzindo os efeitos do
SnCl2 (18).
A marcação de estruturas celulares ou moleculares de interesse biológico com
tecnécio-99m (99mTc), empregadas como radiofármacos (radiobiocomplexos) (28)
envolve a utilização do agente redutor cloreto estanoso (56). Constituintes
sanguíneos, como hemácias, leucócitos, plaquetas e proteínas plasmáticas podem
ser marcados com
99m
99m
Tc (44, 62). Entre as aplicações das hemácias marcadas com
Tc na medicina nuclear pode ser citada a avaliação do sistema cardiovascular
5
(45, 59), medida do fluxo sangüíneo em artérias periféricas (46), localização de
hemorragias gastrintestinais (47, 48) e avaliação da função excretora (49).
Um método de marcação de constituintes sangüíneos com
99m
Tc é utilizado
também como modelo experimental (27, 28, 29). Esse modelo foi proposto e
utilizado com sucesso para avaliação de efeitos biológicos de extratos de plantas
medicinais (10,11, 12, 13, 27,31, 32, 33, 34, 35, 36,). Um dos métodos de marcação
comumente empregado é a marcação in vitro. O sangue é isolado com
anticoagulante, incubado com o extrato de planta medicinal, posteriormente com o
íon estanoso e, após com íon pertecnetato.
Estudos sugerem que na marcação de hemácias com
99m
Tc, o ânion
pertecnetato difundi-se para o espaço intracelular através da proteína da banda-3
por troca com os íons cloreto e/ou bicarbonato (20, 40). A proteína da banda-3 é
uma proteína integral que constitui a principal molécula relacionada com o transporte
de anions das hemácias. Estes fatos indicam que o processo de marcação ocorre
em nível intracelular. O agente redutor, SnCl2, também parece ser transportado para
o interior da hemácia por um sistema de transporte específico, o canal de cálcio (41,
42).
O mecanismo que envolve a ligação
99m
Tc-hemácias não é completamente
entendido. Ainda assim, alguns autores sugerem que: (i) o íon estanho difunde-se
para o interior das células, ligando-se ao componente celular, (ii) o íon pertecnetato
se difunde livremente dentro e fora da célula, (iii) o íon pertecnetato, dentro da célula
na presença do Sn+2 é reduzido e se liga principalmente à cadeia-beta da
hemoglobina (43, 44).
As proteínas plasmáticas e celulares podem ser marcadas com
99m
Tc. Ao
administrar-se um radiofármaco a um paciente, uma fração do mesmo é sempre
6
encontrada ligada às proteínas plasmáticas (50, 51, 52). A marcação das proteínas
plasmáticas e celulares no procedimento de marcação de hemácias com
99m
Tc pode
ser avaliada através da precipitação das proteínas plasmáticas e celulares com
ácido tricloroacético (53).
Estudos da alteração qualitativa da forma das hemácias de sangue de ratos
Wistar realizados através de esfregaços de sangue sobre uma lâmina para
microscopia de luz, também têm relevância na avaliação do efeito de extratos de
plantas medicinais (57).
As hemácias são células que sofreram o processo de
extrusão de seu núcleo durante a diferenciação celular, o que facilita sua
visualização na microscopia óptica (análise qualitativa), visto que não apresenta
sistemas intracelulares de membranas (58).
O produto “Três Bailarinas” (TB), composto por Cassia angustifolia, da família
Fabaceae e Malva verticellata, da família Malvaceae, segundo as instruções do
fabricante (Truong Giang Corp., South El Monte, CA 91733, USA), ajuda a perder
peso sem necessidade de dieta. No entanto, os Efeitos biológicos do TB ainda não
estão bem definidos. No indexador PubMed (www.pubmed.com) não foram
encontradas informações cientificas sobre TB, e esse fato tem estimulado a presente
investigação sobre seus efeitos biológicos.
7
3 ARTIGOS ANEXADOS
3.1 ARTIGO PUBLICADO
a - Effects of an aqueous extract of Three Ballerina on the survival of
Escherichia coli AB1157 cultures and in the action of stannous chloride. Aceito
para publicação em 2011, no periódico “The Journal of Medicinal Plants Research”,
fator de impacto 0,879. Qualis internacional B.
3.2 MANUSCRITO SUBMETIDO
a- Evaluation of the in vitro effect of a Three Ballerina extract on the
labeling of blood constituents of rats with technetium-99m and on the
morpohology of the red blood cell. Submetido ao periódico “Russian
Journal of Plant Physiology”, fator de impacto 0,500. Qualis internacional B
8
3.1 ARTIGO PUBLICADO
Separata
Journal of Medicinal Plants Research
v. 5, suplemento 14, pp. 3256-3259, 2011.
Effects of an aqueous extract of Three Ballerina on the survival of
Escherichia coli AB1157 cultures and in the action of stannous chloride.
Pinto NS, Carmo FS, Diniz CL, Almeida DS, Pereira MO, Santos-Filho SD,
Medeiros AC, Bernardo-Filho M
Journal of Medicinal Plants Research Vol. 5(14), pp. 3256-3259, 18 July, 2011
Available online at http://www.academicjournals.org/JMPR
ISSN 1996-0875 ©2011 Academic Journals
Full Length Research Paper
Effects of an aqueous extract of Three Ballerina on the
survival of Escherichia coli AB1157 cultures and in the
action of stannous chloride
Pinto N. S.1, Carmo F. S.2, Diniz C. L.3, Almeida D. S.3, Pereira M. O.1, Santos-Filho S. D.3*,
Medeiros A. C.1 and Bernardo-Filho M.3,4
1
Programa de Pós-Graduação em Ciências da Saúde da Universidade Federal do Rio Grande do Norte, Natal, RN,
Brasil.
2
Programa de Pós-Graduação de Ciências Médicas da Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ,
Brasil.
3
Laboratório de Radiofarmácia Experimental, Departamento de Biofísica e Biometria, Instituto Roberto Alcantara
Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brasil.
4
Controladoria de Pesquisa, Instituto Nacional do Câncer, Rio de Janeiro, RJ, Brasil.
Accepted 1 April, 2011
The global interest in natural substances has increased. Three Ballerina (TB) is a natural formula that
has medicinal properties. Scientific information about this formula is not found in the PubMed and this
fact has stimulated investigations about its biological effects. An experimental model using a reducing
agent, stannous chloride (SnCl2), was carried out. Although, SnCl2 is used in nuclear medicine to obtain
technetium-99 m radiopharmaceuticals, it is capable to generate free radicals. The aim of this work was
to evaluate the biological effects of an aqueous extract of TB on the survival of Escherichia coli (E. coli)
AB1157 (wild type) cultures and to verify if this extract protect a E. coli culture against the action of
SnCl2. E. coli AB1157 cultures (exponential growth phase) were incubated with TB extract (23.4 mg/ml)
or 0.9% NaCl solution as a control. E. coli cultures were also incubated with SnCl2 (25 µg/ml) + TB
extract (23.4 mg/ml) or SnCl2 (25 µg/ml) alone. Aliquots from these treatments were spread onto Petri
dishes containing solidified LB medium. The colony-forming units (CFU) were counted after overnight
and the survival fractions calculated. Data reveal that the TB did not alter the survival of the E. coli
culture, however, it has protected the cells against the lethal effect of the SnCl2. In conclusion, the TB
extract did not present cytotoxic effects, but it appears to have redox action.
Key words: Escherichia coli, three ballerina, stannous chloride, cytotoxic effect.
INTRODUCTION
The global interest in natural substances has increased
over the world (Rotblatt and Ziment, 2002; Li et al., 2008;
Monbaliu et al., 2010) and, there is a growing interest in
the studies about various properties of medicinal and
dietary plants (Bahramikia and Yazdanparast, 2008). The
prevalence of obesity is growing worldwide and in the
United States, it is estimated that this prevalence has
reached almost 30% (Flegal et al., 2010). Due to the lack
*Corresponding author. E-mail: santos-filho@uerj.br Tel: +5521-28688332.
and limitation of weight loss medicines, herbal teas and
functional food ingredients have become important tools
in improving obesity-related parameters. Tea has been
consumed as a popular beverage worldwide for the last
thousands of years because of its health benefits and
pleasant aroma (Watson and Preedy, 2008). Green tea is
one of the most extensively studied plants for the
prevention of metabolic syndrome by stimulating fat
oxidation and increasing energy expenditure (Dulloo et
al., 1999). “3 Ballerina” Tea Dieters' drink is blended with
the premium natural herbs. Cassia angustifolia and Malva
verticellate are the vegetables of this formula. Following
the instructions of the manufacturer (Truong Giang Corp.),
Pinto et al.
“this special formula Dieters’ drink is all natural tea,
soothing and relaxing especially delightful for those
desiring to adjust weight, although this statement has not
been evaluated by the Food and Drug Administration”
(Soyuncu et al., 2008).
Different experimental models have been used to try to
get scientific information about the natural products
(Bahramikia and Yazdanparast, 2008; Li et al., 2008;
Lima et al., 2002). Investigations to evaluate the
citotoxicity of medicinal plants (Raphael et al., 2009;
Almeida et al., 2007) have been performed. The effects
of extracts of Baccharis genistellodes (Scheila et al.,
2002) in the action of a reducing agent, the stannous
chloride (SnCl2), have been assessed using Escherichia
coil (E. coli) cultures.
SnCl2 is used in nuclear medicine to obtain technetium99m
99m ( Tc) radiopharmaceuticals (Saha, 2010; Guedes
et al., 2006). However, some deleterious effects of this
substance have been described. In humans, it has been
reported that it is highly irritant to the mucous membrane
and skin, although it presents low systemic toxicity. In
animals, it can produce stimulation or depression of the
central nervous system. As for bacterial assays, SnCl2
appears to be capable of inducing and/or producing
injuries in deoxyribonucleic acid (DNA), being considered
as a potential genotoxic agent (Soares et al., 2004;
Guedes et al., 2006). These effects may be, at least in
part, attributed to free radicals (FR), generated during
SnCl2 treatment (Bernardo-Filho et al., 1994; Dantas et
al., 1999; Mattos et al., 1999).
As the scientific publications about “3 Ballerina” was
not found in the PubMed databank, (TB), we have
investigated the effects of an aqueous extract of TB on
the survival of E. coli AB1157 (wild type) cultures and if
this extract protect a E. coli culture against the action of
SnCl2.
MATERIALS AND METHODS
3257
Preparation of the E. coli culture to be used
The E. coli AB1157, a wild-type strain, proficient to repair damage
in the DNA, was used in this work and its characteristics are
reported in Howard-Flanders et al. (1964). From stock (in glycerol
50% v/v) a sample (50 ȝl) of the culture was grown on liquid LB
medium (5 ml, Luria and Burrous, 1957) at 37°C overnight on a
shaking water bath (reciprocal water bath shaker, model R76, New
Brunswick, USA) up to the stationary growth phase. A sample (200
ȝl) was taken from this culture and further incubated (20 ml, liquid
LB medium) under the same conditions, for 2 h to obtain an
exponential growth phase (exponential growth culture with
approximately 108 cells/ml). The cells were collected by
centrifugation, washed with 10 ml of saline and suspended again in
the same solution until they reached 108 cells/ml.
Effect of the TB extract on the survival of E. coli culture
Samples (0.8 ml) of the washed cultures (108 cells/ml) were
incubated on the shaking water bath with (1) 0.2 ml of saline, or (2)
0.1 ml of saline and 0.1 ml of the 3TB extract 23.4 mg/ml up to 60
min, at 37°C. During the assay, at 0 and 60 min, aliquots (0.1 ml)
were taken and diluted with saline and spread onto Petri dishes
containing solidified LB medium (1.5% agar). Colony-forming units
(CFU) formed after overnight incubation at 37°C were counted and
the survival fraction was calculated as described previously (Dantas
et al., 1996). The survival fraction was calculated dividing the
number of viable cells obtained per ml in each time of the treatment
(N) by the number of viable cells obtained per ml in zero time (No).
Effect of the TB extract on the action of the stannous chloride
in E. coli culture
Samples (0.8 ml) of the washed cultures (108 cells/ml) were
incubated on the shaking water bath with (1) 0.1 ml of SnCl2 (25
µg/ml) and 0.1 ml of saline, or (2) 0.1 ml of the TB extract 23.4
mg/ml and 0.1 ml of saline, or (3) 0.2 ml of saline as control, on
initial time and after 60 min, at 37°C. During the assay, at 0 and 60
min, aliquots (0.1 ml) were taken and diluted with saline and spread
onto Petri dishes containing solidified LB medium (1.5% agar). CFU
formed after overnight incubation at 37°C were counted and the
survival fraction was calculated as described previously (Dantas et
al., 1996). The survival fraction was calculated dividing the number
of viable cells obtained per ml in each time of the treatment (N) by
the number of viable cells obtained per ml in zero time (No).
Strategy in the PubMed
A search using the key words “Three Ballerina “or “3 Ballerina” was
performed in the PubMed (www.ncbi.nlm.nih.gov/sites/entrez) on
March 15th 2011.
Preparation of TB extract
As the commercial extract of Three Ballerina (dried powder),
(Truong Giang Corp. South El Monte, CA 91733) has only small
solubility, a solution with 2.34 g of Three Ballerina extract was
prepared with 100 ml of a hot (ebullition) 0.9% NaCl solution
(saline). The preparation was centrifuged (clinical centrifuge,
2000 rpm, 15 min) and the supernatant was isolated. Then, the
obtained solution was considered 23.4 mg/ml. Saline was used in
all the dilutions. All experiments were carried out during the period
of validity of this product. The quality control of the preparation of
this extract was controlled by the optical density of 0.93 ± 0.01
obtained at 490 nm that was used as a marker and the quality
control of each preparation of TB in the all experiments.
RESULTS
The search that has been done in the PubMed has
revealed that no items were found with the key words
“Three Ballerina” or “3 Ballerina”.
Results shown in Figure 1 reveal that the TB was not
capable to interfere on survival of the E. coli AB1157
culture up to 60 min with the treatment using a
concentration of 23.4 mg/ml.
Figure 2 shows that the strong lethal effect of the
stannous chloride has already described for different
authors (Bernardo-Filho et al., 1994; Almeida et al.,
2007). In addition, it is shown that the TB extract has a
protective effect against the treatment with the SnCl2,
abolishing the effect of the reducing agent in the
concentration used in the assay.
3258
J. Med. Plant. Res.
Survival fractions of E. coli AB1157
treated with 3 Ballerina extract
Time (min)
Figure 1. Absence of an effect of the TB extract on E. coli AB1157. Cells suspended in 0.9%
NaCl were treated with TB extract. Survival fractions of E. coli AB1157 strain treated for different
time of incubation in the presence or absence of the extract. The survival fractions were
determined. Ƈ, 0.9% NaCl; ů, extract. N, number of viable cells obtained per ml in each time of the
treatment; No, number of viable cells obtained per ml in zero time.
Survival fractions of E. coli AB1157 treated
with 3 Ballerina extract and SnCl2
Control
SnCl2
SnCl2 + Extract
Time (min)
Figure 2. Effect of the extract from TB on the inactivation induced by SnCl2 on E. coli AB1157. Cells
suspended in 0.9% NaCl were treated with SnCl. Survival fractions of E. coli AB1157 strain treated
with SnCl2 for different time of incubation in the presence or absence of the extract and with the
extract alone. The survival fractions were determined. Ƈ, 0.9% NaCl; Ŷ, SnCl2; Ÿ, SnCl2 + extract. N,
number of viable cells obtained per ml in each time of the treatment; No, number of viable cells
obtained per ml in zero time.
DISCUSSION
Due to the use of the medicinal plants, it is increasing
around the world, and given the limited scientific
information about the effect of many of them, it is highly
relevant to try to assess biological properties of extract of
natural products. Concerning to the Three Ballerina, there
are no publications in the PubMed. This fact stimulates
scientific investigations about this formula.
The extract of Three Ballerina was not capable in
interfering in the survival of an E. coli culture AB1157
(Figure 1), demonstrating an absence of cytotoxic effect
Pinto et al.
in the conditions used in the experiments. This fact has
already been demonstrated to another extracts, as
cauliflower (Lima et al., 2002).
Although the stannous chloride has been used in the
nuclear medicine (Saha, 2010), important deleterious
effects associated to this reducing agent has been
reported (Melo et al., 2001; Guedes et al., 2006; Souza
et al., 2009). Another important finding of our work was
demonstrated that the extract of Three Ballerina
abolished the lethal effect of the stannous chloride
(Figure 2). As the action of this reducing agent is related
with the generation of free radicals, we can suggest that
the TB extract has redox properties. The protective effect
in E. coli AB1157 induced by the extract of TB against the
inactivation produced by the treatment with SnCl2 was
also observed with Peumu boldus (Reiniger et al., 1999)
and with Cymbbopogon citratus, Maytenus ilicifolia and
Baccharis genistelloides (Melo et al., 2001).
In conclusion, the findings in this work show that the
chemical products present in extracts of TB, in the
concentration used, are not toxic to the E. coli AB1157
culture. Moreover, as the TB extract has probably redox
properties, it can prevent the generation of free radicals
or act as a scavenger. Additional studies should be
performed to try to elucidate the action mechanisms
involved in the effects of TB extract obtained in this work.
ACKNOWLEDGEMENTS
The authors are grateful to the following CNPq, CAPES
and UERJ for supporting this work.
REFERENCES
Almeida MC, Soares SF, Abreu PR, Jesus LM, Brito LC, Bernardo FM
(2007). Protective effect of an aqueous extract of Harpagophytum
procumbens up on Escherichia coli strains submitted to the lethal
action of stannous chloride. Cell. Mol. Biol., (Noisy-le-grand) 53
Suppl: OL923-7.
Bahramikia S, Yazdanparast R (2008). Antioxidant and free radical
scavenging activities of different fractions of Anethum graveolens
leaves using in vitro models. Pharmacol. Online, pp. 219-233.
Bernardo FM, Cunha MC, Valsa JO, Caldeira DAA, Silva FCP, Fonseca
AS (1994). Evaluation of potential genotoxic of stannous chloride:
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Food Chem. Toxicol., 32: 477-479.
Dantas FJS, Moraes MO, Carvalho EF, Valsa JO, Bernardo FM,
Caldeira DAA (1996). Lethality induced by stannous chloride on
Escherichia coli AB1157: Participation of reactive oxygen species.
Food Chem. Toxicol., 34: 959-962.
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Dantas FJS, Moraes MO, Mattos JCP, Bezerra RJAC, Carvalho EF,
Bernardo FM (1999). Stannous chloride mediates single strand
breaks in plasmid DNA through reactive oxygen species formation.
Toxicol. Lett., 110: 129-136.
Dulloo AG, Duret C, Rohrer D, Girardier L, Mensi N, Fathi M, Chantre P,
Vandermander J (1999). Efficacy of a green tea extract rich in
catechin polyphenols and caffeine in increasing 24-h energy
expenditure and fat oxidation in humans. Am. J. Clin. Nutr., 70: 10401045.
Flegal KM, Carrol MD, Ogden CL, Curtin LR (2010). Prevalence and
trends in obesity among US adults, 1999-2008. Ann. Pharmacother.,
44: 1141-1151.
Guedes AP, Cardoso VN, De MJCP, Dantas FJS, Matos VC, Silva JCF,
Bezerra RJAC, Caldeira DAA (2006). Cytotoxic and genotoxic effects
induced by stannous chloride associated to nuclear medicine kits.
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Howard-Flanders P, Simon E, Theriot T (1964). A locus that control
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Genetics, 53: 237-246.
Li J, Li Q, Feng T, Li K (2008). Aqueous extract of Solanum nigrum
inhibit growth of cervical carcinoma (U14) via modulating immune
response of tumor bearing mice and inducing apoptosis of tumor
cells. Fitoterapia, 79: 548-556.
Mattos DMM, Gomes LM, Freitas RS, Rodrigues PC, Paula EF,
Bernardo FM (1999). Model to evaluate the biological effect of natural
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Vincristine
action
on
the
biodistribution
of
radiopharmaceuticals in Balb/c female mice. J. Appl. Toxicol., 19:
251-254.
Melo SF, Soares SF, da Costa RF, da Silva CR, de Oliveira MB,
Bezerra RJ, Caldeira DAA, Bernardo FM (2001). Effect of the
Cymbopogon citratus, Maytenus ilicifolia and Baccharis genistelloides
extracts against the stannous chloride oxidative damage in
Escherichia coli. Mutat. Res., 496: 33-38.
Monbaliu S, Wu A, Zhang D, Peteghem CV, Saeger SD (2010).
Multimycotoxin UPLC-MS/MS for tea, herbal infusion and the derived
drinkable products. J. Agric. Food Chem., 58: 12664-12671.
Reiniger IW, Ribeiro DSC, Felzenszwalb I, De MJC, De OJF, Da SDFJ,
Bezerra RJ, Caldeira DAA, Bernardo FM (1999). Boldine action
against the stannous chloride effect. J. Ethnopharmacol., 68: 345348.
Rotblatt PM, Ziment I (2002). Evidence-Based Herbal Medicine. Hanley
and Belfus Philadelphia.
Saha GB (2010). Fundamentals of nuclear pharmacy. Sixth edition.
Springer: New York.
Soares SF, Brito LC, Souza DE, Almeida MC, Bernardo LC, Bernardo
FM (2004). Citotoxic effects of stannous salts and the action of
Maytenus ilicifolia, Baccharis genistelloides and Cymbopogon citratus
aqueous extracts. Braz. J. Biom. Eng., 20: 73-79.
Souza RS, Almeida MC, Manoel CV, Santos FSD, Fonseca AS,
Bernardo FM (2009). Biological effects of an aqueous extract of Salix
alba on the survival of Escherichia coli AB 1157 cultures submitted to
the action of stannous chloride. Biol. Res., 42: 199-203
Soyuncu S, Cete Y, Nokay AE (2008). Portal vein thrombosis related to
Cassia angustifolia. Clin. Toxicol., (Phila), 46: 774-777.
Watson RR, Preedy VR (2008). Botanical Medicine in Clinical Practice.
ND-Cabi Publishing, p. 395.
21
3.2 MANUSCRITO SUBMETIDO
Russian Journal of Plant Physiology, fator de impacto 0,5. Qualis Internacional
B
EVALUATION OF THE IN VITRO EFFECT OF A THREE BAILLERINA EXTRACT
ON
THE
LABELING
OF
BLOOD
CONSTITUENTS
OF
RATS
WITH
TECHNETIUM-99M AND ON THE MORPHOLOGY OF THE RED BLOOD CELL
Pinto NS1, Oliveira MP1, Carmo FS2, Diniz CL2, Missailidis S3, Santos-Filho SD2,
Medeiros AC1, Bernardo-Filho M2,4
1-Programa de Pós-Graduação em Ciências da Saúde, Universidade Federal do Rio
Grande do Norte, Natal, RN, Brasil; 2-Departamento de Biofísica e Biometria, Instituto
Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro,
RJ, Brasil; 3- The Open University, UK; 4-Coordenadoria de Pesquisa, Instituto
Nacional do Câncer, Rio de Janeiro, RJ, Brasil.
Corresponding author:
Sebastião David Santos-Filho
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, fundos, 4º andar
20551-030, Vila Isabel, Rio de Janeiro, RJ, Brasil
Phone/Fax: 55-21-2568-8332; Mobile: 55-21-8715-1371
E-mail: santos-filho@uerj.br or sdavidsfilho@gmail.com
22
Abstract
The aim of this work is to verify the effect of a TB extract on the labeling of blood
constituents with 99mTc and on the morphology of the red blood cells (RBC). Some
physical characteristics, as visible absorbance spectrum, electric conductivity, and
refractive index of this extract were also determined. Plasma (P) and blood cells (BC)
were labeling with 99mTc, and also isolated and precipitated with trichloroacetic acid
and soluble (SF) and insoluble fractions (IF) were separated. The %ATI in these
samples was calculated. The morphology of the treated RBC showed no shape’s
qualitative alterations. The TB extract was characterized with an absorbance peak at
490nm (0.93±0.01), electric conductivity of 1.35±0.04mSv/cm and refractive index of
2.21±0.15%BRIX. TB extract also decreased significantly (p<0.05) the radioactivity
distribution in cellular compartment of 96.97±1.30% to 88.48±7.13%, and in IF-P from
74.29±4.12 to 14.26±5.73%. In conclusion, some physical chemical parameters could
be suitable to characterize an extract of TB. Moreover, substances present in the TB
extract should probably have an effect on transport of the ions through the RBC
membrane and/or should have redoxi properties justifying the effect on the fixation of
the radioactivity on the plasma proteins.
Keywords: blood constituents, technetium-99m, red blood cell, Three Baillerina,
characterization of the extract
23
Introduction
Technetium-99m (99mTc) has been the most utilized radionuclide in the single
photon emission computed tomography (SPECT) (Harbert et al, 1996; Prech et al, 2006;
Rasilla et al, 2009; Schinkel et al, 2010) and it has also been used in basic research
(Burke et al, 2005; Pettersson et al, 2005; O'Connor et al, 2009; Gropler et al, 2010).
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) (Bernardo-Filho et al, 2005, Rasilla et al, 2009; Schinkel et al, 2010).
99mTc-labeled RBC scan is the nuclear study best suited for identifying slowbleeding sources as gastrointestinal bleeding (Karacalioglu et al, 2003; ManningDimmitt et al, 2005; Kiratli et al, 2009), as well as (i) in the determination of the left
ventricular function by measuring the ejection fractions (Leitha et al, 2001; Gropler et
al, 2010) and (ii) in the evaluation of wall motion abnormalities (Sampson, 1999; Prech
et al, 2006; Schinkel et al, 2010), 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
2+
stannous ion (Sn ) is usually used for this purpose (Harbert et al, 1996; Oliveira et al,
2003; Moreno et al, 2005; Prech et al, 2006). When whole blood is used for the labeling
of RBC with 99mTc, radioactivity is mainly found on RBC, however it is also bound on
plasma proteins (Saha, 2004, Bernardo-Filho et al, 1990). Authors have reported that
some herbal medicines are capable to alter the labeling of blood constituents with
technetium99m (O’Neill et al, 1998; Oliveira et al, 2000; Lima et al, 2002; Moreno et
al, 2002; Oliveira et al, 2002; Diré et al, 2004; Santos-Filho et al, 2004; Santos-Filho et
al, 2005; Misra et al, 2007).
24
Qualitative morphological analysis has been used as a method to evaluate if the
effects of drugs on this radiolabeling process could be related to changes of shape of
RBC (Benarroz et al, 2007).
The use of medicinal plants has grown in world (Rotblatt and Ziment, 2002;
Gullett et al, 2010), and the development and implementation of experimental models
(Júnior et al., 2005; Nakhai et al, 2007) are important to permit a better comprehension
of the action mechanisms of these natural products.
Three Baillerina (TB) is a formulae and experimental data suggest a great
pharmacologic potentiality of the substances that there are on it. The activities described
are laxative and against complement activity (Soyuncu et al., 2008; Tomoda et al.,
1992).
A useful physical chemical property to aid to characterize and to estimate the
purity or to determine the concentration of a substance or solution is the refractive index
(Castilho et al, 2006). Electric conductivity and the absorbance spectrum profile are
other physical parameters that could be measured and also used to characterize a
preparation of unknown composition, such as an extract of a medicinal plant (Néhémie
et al, 2007).
Publications about physical properties of TB extract were not found yet in
PubMed (www.pubmed.com). Moreover, since that the extract of TB can be used also
humans and several effects of this natural product are not well understood, the aim of
this work was to characterize some physical-chemical properties and to evaluate the
effect of TB aqueous extract on the labeling of blood constituents with 99mTc using an
in vitro experimental model, as well as to verify the consequences of this extract on the
morphology of the RBC.
Materials and Methods
25
Animals
The experiments were performed with rats maintained in a controlled
environment. The animals had free access to water and food and the ambient
temperature was kept at 25±2 °C. 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±15 g of weight). All the experimental
procedures have followed the Ethical Guidelines of the Instituto de Biologia Roberto
Alcantara
Gomes,
Universidade
do
Estado
do
Rio
de
Janeiro
(Protocol
CEA/024/2009).
Preparation of TB extract
As the commercial extract of Three Baillerina (dried powder, Astron Comercial
LTDA, São Paulo, Brasil, Lot 1563) has only small solubility, a solution with 2.34 g
of Three Baillerina extract was prepared with 100 ml of a hot (ebullition) 0.9% NaCl
(saline). The preparation was centrifuged (clinical centrifuge, 2000 rpm, 15 min) and
the supernatant was isolated. Then, the obtained solution was considered 23.4 mg/ml
(100%). Saline was used in all the dilutions. All experiments were carried out during the
period of validity of this product.
Spectrophotometry of TB extract
Absorbance spectrum (Spectrophotometer, 800M, Analyser Comércio e
Indústria Ltda., São Paulo, Brazil) was determined with the TB extract (23.4 mg/ml)
prepared as described above in the range of 400–700 nm. Saline solution was used as
the blank. The absorbance was measured at each interval of 10 nm. This value was
26
considered as the marker of the reproducibility of the conditions used to prepare all the
extracts utilized in the assays.
Electric conductivity of TB extract
Electric conductivity (mS/cm) of the TB extract was performed with a
conductivimeter (Marte Balanças e Aparelhos de Precisão Ltda., São Paulo). Saline
solution was used as the control.
Refractive index of TB extract
The refractive index (%BRIX) of TB extract was measured with a refractometer
(Ningbo Utech International Co. Ltd., Ningbo, People’s Republic of China) at room
temperature. Saline solution was also used as the control.
In vitro radiolabeling of blood constituents
Samples of 0.5 ml of whole blood were incubated with 100 µl of different
concentrations of diluted (0.9% NaCl) TB 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. All the tubes used in this experiment were
previously closed with a rubber cap and a syringe was used to reduce the air atmosphere
(vacuum) inside the vials. Then, 0.5 ml of a freshly prepared stannous chloride solution
(1.2 µg/ml), as SnCl2 (Sigma, USA) was added and the incubation continued for another
1 hour. After this period of time, 99mTc (0.1 ml with 370kBq), as sodium pertechnetate,
recently milked from a 99Mo/99mTc 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
27
(P) and BC cells were separated. Samples (20 µl) 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 (Oliveira et al,
2002; Oliveira et al, 2003).
Morphological evaluation of RBC
Preparations for morphological (microscopic) analyses were carried out with
blood samples treated in vitro with TB extract at different concentrations for 60 minutes
at room temperature or with saline solution as the control group. Blood smears were
prepared, dried, fixed, and staining by the May-Grünwald-Giemsa method (Barcia,
2007). After that, images of the RBCs were acquired (CANON, model Power Shot
SX200 IS) from blood smears to analyze the qualitative morphology by optical
microscopy (NIKON, model E 200, x 1000). Three independent researches with
expertise in analysis of blood smears have done a qualitative analysis of the RBC.
Analysis of the results
The data were analyzed using the GraphPad InStat (version 3.01 for Windows
95/NT, GraphPad Software, San Diego Ca, USA). Data from the analyses were tested
for any differences between treatments using one-way analysis of variance (ANOVA).
The means and standard errors of the means (mean ± SE) are also reported. Test with
the significance level being p<0.05.
28
Results
Physical chemical determinations, as, spectrum of absorbance, electric
conductivity and refractive index of the extract of TB were performed. Figure 1 shows
the spectrum of absorbance of the TB extract at higher concentration used (23.4mg/ml)
in the range of 400–700 nm. The data show an absorption peak of the extract
(0.93±0.01) at 490 nm. This value was considered as the marker of the reproducibility
of the conditions used to prepare the extracts used in the assays. The value of electric
conductivity (1.35±0.04mS/cm) of the extract at higher concentration was used as a
second marker of the reproducibility of the conditions used to prepare the extract. The
value of the refractive index (2.21±0.15%BRIX) of the extract at higher concentration
was used as a third marker of the reproducibility of the conditions used to prepare the
extract.
Table I shows the distribution of the radioactivity in the cellular and plasma
compartments isolated from whole blood treated with different concentrations of the TB
extract. The results indicate that there is a significant decrease (p<0.05) in the
distribution of the 99mTc in the cellular compartment from 96.97±1.30% to
88.48±7.13% in presence of TB extract.
Table II shows 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 TB extract. There is no alteration in the 99mTc fixation by the
cellular proteins (IF-BC) in presence of TB extract.
Table III shows 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 TB extract. There is a significant (P<0.05) decrease in the
29
radioactivity fixation in the plasma proteins (IF-P) in presence of TB extract from
74.29±4.12 to 14.26±5.73%.
Figures 2 and 3 show photomicrographs of smears from blood treated with
saline solution (control) and with TB extract at the higher concentration used
(23.4mg/ml), respectively. The qualitative morphological analysis suggests that the
treatment with TB extract does not induce important changes in the shape of RBC
observed under optical microscopy.
Discussion
To our knowledge, there is no description about physical chemical properties of
TB extracts. This fact has also stimulated this investigation, and a commercial TB was
used. This study shows that TB extract could be characterized by an absorbance
spectrum at 490 nm (Figure 1), an electric conductivity of 1.35±0.04mSv/ml and a
refraction index of 2.21±0.15%BRIX. These physical parameters could be used in other
studies to characterize the preparation conditions of an aqueous TB extract.
Nuclear medicine procedures as SPECT and PET have proven increasingly
effective imaging modalities in the study of several clinical disorders (Kumakura et al,
2004; Lodge et al, 2005; Palestro et al, 2005; Kiratli et al, 2009; Gropler et al, 2010).
Besides the disease, some authors have reported that these procedures could be altered
by medications that the patient is undergoing (Hesslewood and Leung, 1994). Natural
products could alter the biodistribution of radiobiocomplexes in a specific target or the
fixation of the radionuclide 99mTc to blood constituents (Sampson, 1999; Caprilles et
al, 2002; Moreno et al, 2002; Oliveira et al, 2002; Oliveira et al, 2003; Diré et al, 2004;
Fonseca et al, 2005). Other products are not capable to interfere with this labeling
process (Frydman et al, 2004; Fernandes et al, 2005). This procedure has been proposed
30
as an in vitro assay to verify some important properties, as chelating/redox activities or
interactions on cellular membrane, of products used daily by humans (Benarroz et al,
2007; Fonseca et al, 2007).
Although, there are data concerning to the interaction of diagnostic agents,
including radiobiocomplexes, with therapeutic drugs (synthetic and natural),
investigations about these interactions are worthwhile. Knowledge about drug–
radiopharmaceutical interactions could contribute to reduce the risk of misdiagnosis
and/or repetition of the examinations (Saha, 2004) that could contribute to increase the
radiation dose to patients and staff in nuclear medicine. The development and use of in
vitro
assays
may
provide
important
information
about
possible
drug–
radiopharmaceutical interactions (Fonseca et al, 2007).
The analysis of the data presented in Tables 1 and 3 indicates that the TB extract
could alter the distribution of 99mTc between P and cellular compartments or the
fixation of this radionuclide on plasma proteins. In general, the labeling of blood
constituents could decrease because of action of drugs by (a) alteration of the plasma
membrane structure or modifying the transport systems of stannous and pertechnetate
ions into cells, (b) direct oxidation or generation of free radicals that could oxidize the
stannous ion, (c) direct inhibition (chelating action) of the stannous and pertechnetate
ions, or (d) binding at same sites on the blood constituents.
Interactions involved with the ion transport systems could alter the transport of
stannous and pertechnetate ions, decreasing the labeling of RBC with 99mTc and
explain, in part, the data obtained (Table 1). In fact, other researchers have
demonstrated that drugs that interact with calcium channels (Gutfilen et al, 1992) and
band-3 protein (Callahan and Rabito, 1990) to alter the labeling of RBC with 99mTc.
Moreover, no modifications, at least under optical microscopy, of the shape of RBC
31
were not found when blood samples were incubated with TB extract (Figures 2 and 3),
suggesting that other mechanisms could be involved in the decrease of the labeling
efficiency of RBC with 99mTc (Table 1).
The redox properties of phenolic compounds present in medicinal plants have
been related to various mechanisms: free radical scavenging activity, transition metal
chelating, and singlet oxygen-quenching capacity (Shan et al, 2005). These substances
could act as chelators on stannous ions, decreasing the distribution of radioactivity in
blood cell compartment (Table 1) and the fixation of 99mTc on Plasma proteins (Table
3). In fact, experimental data have suggested antioxidant and pro-oxidant actions of
caffeine and its metabolites (Azam et al, 2003). Other data have suggested that caffeine
could alter the labeling of blood constituent at higher concentrations (Frydman et al,
2004). These findings and our morphological data from RBC could indicate that TB
extract could alter the labeling of blood constituents with 99mTc due to chelating/redox
properties of chemical compounds at the highest concentration of TB.
Although
scientific
information
about
the
pharmacokinetics
and
pharmacodynamics of the products present in TB is scarce, in general, the actions of
drugs has been shown to be depend on the plasma protein binding (Musteata et al,
2006). Despite previous data indicating that caffeine at low concentrations does not alter
the radiolabeling of plasma and cellular proteins (Frydman et al, 2004), other chemical
compounds in TB extract could decrease the fixation of 99mTc on these proteins (Table
3).
An unexpected finding is the no alteration of the labeling of the blood cell
proteins in presence of extract of TB (Table 2). Probably, the quantity of the TB that is
inside the RBC is not capable to interfere also on the fixation of the 99mTc on the blood
cell proteins.
32
Conclusion
In conclusion, our data show some physical chemical parameters that could be
suitable to characterize an extract of TB. Moreover, substances present in the TB extract
should probably have an effect on transport of the ions through the RBC 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. Moreover,
although our experiments were carried out with animals, it is suggested precaution in
the interpretation of the examinations that use labeled blood constituents in patients who
are using TB extract.
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39
Tables
Table I: Effect of TB extract on the distribution of the radioactivity in the cellular and
plasmatic compartments
TB concentrations (%)
0.00 (control)
6.25
12.50
25.00
50.00
100.00
Cellular
compartment
96.97±1.30
96.56±0.39
98.73±0.22
97.14±0.67
98.24±1.28
88.48±7.13
Plasma
compartment
3.03±1.30
3.43±0.39
1.26±0.22
2.85±0.67
1.75±1.28
11.51±7.13
Samples of heparinized blood were incubated with different concentrations of TB extract. A sample of
heparinized whole blood was incubated with saline solution (NaCl 0.9%) as control. Then, stannous
chloride (1.2µg/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 TB extract in the labeling of soluble (SF) and insoluble (IF) fractions
of the BC with 99mTc
TB concentrations (%)
0.00 (control)
6.25
12.50
25.00
50.00
100.00
IF-BC
93.45±7.64
96.49±1.69
98.26±0.72
95.45±4.67
97.66±0.99
96.43±1.95
SF-BC
6.54±7.64
3.50±1.69
1.73±0.72
4.54±4.67
2.33±0.99
3.56±1.95
Samples of heparinized blood were incubated with different concentrations of TB extract. A sample of
heparinized whole blood was incubated with saline solution (NaCl, 0.9%) as control. Then, stannous
chloride (1.2µg/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 TB extract in the labeling of soluble (SF) and insoluble (IF)
fractions of the plasma (P) with 99mTc
TB concentrations (%)
0.00 (control)
6.25
12.50
25.00
50.00
100.00
IF-P
74.29±4.12
72.11±0.84
71.38±0.23
8.64±7.59
9.58±8.95
14.26±5.73
SF-P
25.70±4.12
27.88±0.84
28.61±0.23
91.35±7.59
90.41±8.95
85.73±5.73
Samples of heparinized blood were incubated with different concentrations of TB extract. Sample of
heparinized whole blood was incubated with saline solution (NaCl, 0.9%) as control. Then, stannous
chloride (1.2µg/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.
40
Figures
Figure 1: Absorbance spectrum of TB extract. To prepare the extract, 2.34 g of dust was dissolved with
100ml of hot saline (0.9% NaCl). After 20 minutes, this preparation was centrifuged at 2000rpm for 15
minutes, and the extract obtained was considered to be 23.4mg/ml. The absorbance spectrum was
determined with the TB extract (23.4mg/ml) in the range of 400–700 nm at intervals of 10 nm. Saline
solution (0.9% NaCl) was used as the blank.
Figure 2: Photomicrograph of blood smear from blood samples treated with saline (control). Blood
samples from Wistar rats were incubated with saline (0.9% NaCl) for 1 hour. After that, blood smears
were prepared, dried, and stained by the May-Grünwald-Giemsa method. The slides were analyzed by
optical microscopy (X 1,000).
41
Figure 3: Photomicrograph of blood smear from blood samples treated with TB extract. Blood samples
from Wistar rats were incubated with TB extract (23.4mg/ml) for 1 hour. After that, blood smears were
prepared, dried, and stained by the May-Grünwald-Giemsa method. The slides were analyzed by optical
microscopy (X 1,000).
42
4 - COMENTÁRIOS, CRÍTICAS E CONCLUSÕES
A vivência e a paixão como médico veterinário e o interesse em clínica
radiológica estimulou a realização de uma dissertação de mestrado que envolvesse
o uso de fonte de radiação.
Com o objetivo de aprimorar os conhecimentos dos efeitos produzidos pelas
plantas medicinais em diferentes modelos experimentais apresentamos junto ao
Programa de Pós-Graduação em Ciências da Saúde o projeto de dissertação
intitulado “Avaliação de efeitos de um extrato de Três Bailarinas em modelos
experimentais em diferentes níveis de organização biológica”.
Além disso,
informações cientificas sobre as Três Bailarinas não foram encontradas no banco de
dados PubMed (http://www.ncbi.nlm.nih.gov/sites/entrez/).
Para o desenvolvimento do projeto usamos um extrato aquoso de Três Bailarinas
em modelos experimentais em diferentes níveis de organização biológica para
avaliação dos efeitos: (i) na sobrevivência de culturas de E.coli AB1157 (selvagem),
(ii) na avaliação de efeitos do extrato na ação do agente lesivo SnCl2 em culturas
bacterianas, (iii) na marcação dos constituintes sanguíneos com 99mTc, e (iv) na
morfologia de hemácias isoladas de ratos Wistar.
No inicio do projeto tivemos dificuldades em desenvolver e executar as diversas
técnicas empregadas no laboratório visto não estar familiarizado com instrumentos
de precisão. Porém, com persistência, vontade de aprender e a ajuda do orientador
e demais colegas do laboratório, conseguimos resultados preliminares que foram
apresentados no projeto de mestrado e encaminhados ao programa de PósGraduação em Ciências da Saúde, sendo então aprovado o meu ingresso no
43
mestrado. Os experimentos foram realizados de acordo com o estabelecido no
cronograma do projeto.
Os resultados obtidos com o modelo avaliando a sobrevivência de Escherichia
coli na presença ou ausência de cloreto estanoso propiciaram a elaboração de um
artigo intitulado “Effects of an aqueous extract of Three Ballerina on the survival of
Escherichia coli AB1157 cultures and in the action of stannous chloride”, que foi
aceito para publicação no periódico “The Journal of Medicinal Plants Research”, fator
de impacto 0,879. Qualis Internacional B.
Estudos em modelos experimentais que envolviam o efeito dos extratos na
marcação de constituintes sanguíneos com tecnécio-99m, e na morfologia das
hemácias foram realizados. Essas investigações feitas também com esfregaços
sanguíneos seguidos de captura das imagens, e a análise morfológica qualitativa
das imagens obtidas por microscópio óptico originaram mais um manuscrito
intitulado “Evaluation of the in vitro effect of a Three Ballerina extract on the labeling
of blood constituents of rats with technetium-99m and on the morpohology of the red
blood cell”, submetido ao periódico “Russian Journal of Plant Physiology”, fator de
impacto 0,500.
Ao concluirmos nosso estudo, verificamos que o extrato de Três Bailarinas não
apresentou citotoxicidade em cultura bacteriana e também não modificou a
morfologia da membrana eritrocitária. Mais ainda, os compostos químicos existentes
nesse extrato parecem ter um importante efeito protetor redoxi contra a ação de
agente lesivo (cloreto estanoso). Além disso, o extrato de Três Bailarinas alterou a
fixação do tecnécio-99m em determinados constituintes sanguíneos. Embora esses
resultados de marcação tenham sido obtidos com sangue de animais, esse fato
44
poderia justificar possíveis comprometimentos em exames de medicina nuclear
envolvendo a marcação desses constituintes sanguíneos.
O conhecimento dessa alteração de marcação de hemácias com tecnécio-99m
em ensaios com o extrato de Três Bailarinas é de importância para profissionais de
saúde que utilizam os exames da medicina nuclear para o diagnóstico e o
acompanhamento de diversas desordens, assim como os médicos veterinários em
seu campo de atuação. O médico veterinário é profissional de nível superior que
prescreve, ministra e supervisiona o tratamento com o objetivo de presenciar,
manter, desenvolver ou restaurar a integridade de um órgão, sistema ou função do
corpo de um animal, através de condutas clínicas determinadas. Para um bom êxito
do tratamento utilizado o médico veterinário necessita de exames complementares
que forneçam informações precisas para diagnóstico e acompanhamento da
evolução do tratamento de um paciente como os exames da medicina nuclear
utilizados na avaliação por imagem.
Através deste estudo acreditamos haver contribuído para que os profissionais da
área da saúde, e entre eles o médico veterinário, tenham novos parâmetros sobre os
efeitos das plantas medicinais estudadas sobre o organismo humano.
Esperamos que as informações levantadas por esta pesquisa possam servir de
ponto de partida para novos estudos sobre a avaliação de outros mecanismos de
ação no organismo vivo, favorecendo modos de ação em pacientes que estejam
utilizando produtos naturais e venham realizar exames de medicina nuclear.
Além disso, a vivência e experiência adquirida durante o Curso de Mestrado
propiciaram
amadurecimento
científico
e
profissional,
e
como
educador,
principalmente na busca do conhecimento comprovado cientificamente e digno de
crédito. Entretanto, a experiência vivenciada no Curso de Mestrado e a aquisição de
45
novos conhecimentos poderão ser relevantes para ações em projetos futuros junto a
grupos de pesquisa da Universidade do Estado do Rio de Janeiro, e no ingresso
futuro em programas de pós-graduação para o doutorado.
Por fim, os nossos objetivos em aprimorar os conhecimentos dos efeitos
produzidos pelo extrato de Três Bailarinas em modelos experimentais em diferentes
níveis de organização biológica apresentados junto ao Programa de Pós-Graduação
em Ciências da Saúde foram atingidos graças ao intercambio entre a Universidade
Federal do Rio Grande do Norte e a Universidade do Estado do Rio de Janeiro.
46
5 ANEXOS
ANEXO 1 Comitê de ética
47
ANEXO 2
Carta de aceite do manuscrito
---------- Forwarded message ---------From: JMPR Medicinal Plants Research <jmpr.acadjourn@gmail.com>
Date: Fri, 1 Apr 2011 09:52:34 +0100
Subject: Accepted for publication (JMPR-11-337 Pinto et al)
To: sdavidsfilho@gmail.com
Dear Sebastiao David Santos Filho ,
I am pleased to inform you that your manuscript; JMPR-11-337 Pinto et al has
been accepted for publication in the Journal of Medicinal Plants Research.
There are some corrections which we will make before the proof is sent to
you.
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57
ABSTRACT
Natural and synthetic drugs can modify the survival of bacterial cultures, interfere on
the labeling of blood constituents with technetium-99m (99mTc) and alter the
morphology of red blood cells (RBC). Following the instructions of the manufacturer,
the formula Three Ballerina (TB) is suggested to be used, as drink, for people
desiring to adjust weight without dietary. The ingredients of this formula are Cassia
angustifolia and Malva verticellate. As in the PubMed databank scientific publications
about TB were not found, we have investigated the effect of an aqueous extract of
TB (i) on the survival of Escherichia coli (E. coli) AB1157 (wild type) cultures, (ii) on
the protection of a E.coli culture against the action of stannous chloride (SnCl2), (iii)
on the labeling of the RBC, and plasma and blood cells proteins with 99mTc and iv) on
RBC morphology of Wistar rats. Data reveal that the TB did not alter the survival of
the E. coli culture, however, it has protected the cells against the lethal effect of the
SnCl2. TB extract also decreased the percentage of radioactivity (%ATI) in cellular
compartment and on the plasma proteins, but not altered the %ATI on the cells
proteins. The extract not was capable to alter the shape of the RBC. The performed
experimental models show the importance of them in the evaluation of biological
effects due to chemical agents, and these investigations were also useful to a better
understanding of the TB properties. This work included some knowledge areas, as
radiobiology, botany, phytotherapy and hematology.
Keywords: Red blood cells, technetium-99m, stannous chloride, Escherichia coli.