hypericum perforatum (hipÉrico) e de mentha … · o objetivo deste estudo foi comparar o efeito...
TRANSCRIPT
UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
COMPARAÇÃO DE EFEITOS DOS EXTRATOS DE
HYPERICUM PERFORATUM (HIPÉRICO) E DE MENTHA CRISPA (HORTELÃ)
EM DIFERENTES MODELOS EXPERIMENTAIS.
SEBASTIÃO DAVID DOS SANTOS FILHO
Natal, RN
2007
COMPARAÇÃO DE EFEITOS DOS EXTRATOS DE
HYPERICUM PERFORATUM (HIPÉRICO) E DE MENTHA CRISPA (HORTELÃ) EM
DIFERENTES MODELOS EXPERIMENTAIS.
SEBASTIÃO DAVID DOS SANTOS FILHO
Tese apresentada à Universidade Federal do Rio
Grande do Norte, para a obtenção do título de
Doutor em Ciências da Saúde pelo Programa de
Pós-Graduação em Ciências da Saúde.
Orientador: Prof. Dr. Mário Bernardo Filho
Co-orientador: Prof. Dr. Aldo da Cunha Medeiros
Natal, RN
2007
ii
CATALOGAÇÃO NA FONTE
S237 Santos Filho, Sebastião David dos.Comparação de efeitos dos extratos de Hypericum perforatum
(Hipérico) e de Mentha crispa (Hortelã) em diferentes modelosexperiemtais. / Sebastião David dos Santos Filho. – 2007.
x, 81f. : il.
Orientador : Mario Bernardo-Filho.Co-orientador : Aldo da Cunha Medeiros.
Tese (doutorado) – Universidade Federal do Rio Grande doNorte, Centro de Ciências da Saúde.
1. Plantas medicinais - Teses. 2. Radiobiologia - Teses .3.Tecnécio - Teses. 4. Eritrócitos - Teses. 5. Plasma sangüíneo - Teses.6. Proteínas - Teses. I. Bernardo-Filho, Mario. II. Medeiros, Aldo daCunha. III. Universidade Federal do Rio Grande do Norte.Centro deCiências da Saúde. IV. Título.
CDU 633.88
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, RN
2007
iii
SEBASTIÃO DAVID DOS SANTOS FILHO
COMPARAÇÃO DE EFEITOS DOS EXTRATOS DE
HYPERICUM PERFORATUM (HIPÉRICO) E DE MENTHA CRISPA (HORTELÃ) EM
DIFERENTES MODELOS EXPERIMENTAIS.
PRESIDENTE DA BANCA: Prof. Dr. Mario Bernardo Filho (UERJ)
BANCA EXAMINADORA
Prof. Dr. Mario Bernardo Filho (UERJ)
Prof. Dr. José Brandão Neto (UFRN)
Prof. Dr. Maria Teresa Jansem de Almeida Catanho (UFPE)
Prof. Dr. Eryvaldo Sócrates Tabosa do Egito (UFRN)
Prof. Dr. Adriana Augusto de Resende (UFRN)
Prof. Dr. Eveline Pipolo Milan (UFRN – suplente)
2007
iv
DEDICATÓRIA
In memoriam, dedico este título ao meu pai Sebastião David dos Santos que me
agraciou com seu amor, paciência e determinação, para que seu filho pudesse
alcançar objetivos que ele não alcançou.
À minha mãe Thereza Girão dos Santos, meus filhos Fabiana, Mariana, Luiz
Eduardo e Elisa que foram fonte de energia quando precisei para poder atingir este
sonho.
À minha esposa e companheira de todas as horas, alegres e tristes, Maristela
Mascarenhas Nascimento, todo o meu amor.
DEUS reconhece a dignidade de teu servo.
v
AGRADECIMENTOS
Obrigado Senhor por ter me concedido a oportunidade de colaborar com mais um
conhecimento para a humanidade.
Ao Professor Mario Bernardo Filho, que como orientador e amigo, soube indicar
caminhos e sugerir soluções para que esta caminhada pudesse ter êxito, minha
amizade, respeito e admiração.
Ao Professor Aldo da Cunha Medeiros por todo incentivo e acompanhamento dos
trabalhos realizados.
Ao Professor José Brandão Neto pela acolhida no programa de Pós-Graduação em
Ciências da Saúde e por todo incentivo a que o doutorado fosse concluído.
Ao Programa de Pós-graduação em Ciências da Saúde. Ao auxílio dado pelo CNPq.
Aos amigos Adalgisa Ieda Maiworm, Giuseppe Antonio Presta, Severo de Paoli e
Tânia Santos Giani, meus irmãos na ciência, em todos os momentos difíceis e nos
momentos de alegria, muito obrigado.
Ao serviço de Medicina Nuclear, ao Laboratório de Endocrinologia e ao Laboratório
Central do Hospital Universitário Pedro Ernesto pelo suporte aos resultados obtidos.
A todos que direta ou indiretamente possibilitaram a realização deste trabalho.
vi
LISTA DE ABREVIAÇÕES, SIGLAS E SÍMBOLOS.
DNA ácido desoxirribonucléico
LB Luria Broth
O-2 radical superóxido
OH radical hidroxila
H2O2 peróxido de hidrogênio
O2 oxigênio
ANOVA análise de variância
ATP adenosina trifosfato
Bq Bequerel (unidade de atividade de amostra radioativa no Sistema
Internacional, sendo que 1 Bq equivale a uma desintegração por segundo)
BC Blood Cell (célula sanguínea)
Ca++ íon cálcio
Cl- íon cloreto
FI-C fração insolúvel da célula
FS-P fração solúvel da célula
FI-P fração insolúvel do plasma
FS-P fração solúvel do plasma
radiação gama
Hb hemoglobina
HCO3- íon bicarbonato
MBq megabequerel
g micrograma
l microlitro
Mo molibdênio
NaCl cloreto de sódio
P plasma
PP proteína plasmática
% ATI porcentagem de radioatividade
rpm rotações por minuto
Sn+2 íon estanoso
vii
SnCl2 cloreto estanoso
99mTc tecnécio-99m
TCA ácido tricloroacético
TcO4- íon pertecnetato
Na99mTcO4 pertecnetato de sódio
UI Unidade internaciomal
IPEN Instituto de Pesquisas Energéticas e Nucleares de São Paulo
CNEN Comissão Nacional de Energia Nuclear
RBC Célula Vermelha do Sangue
viii
SUMÁRIO
Lista de abreviações……………………………………….....……………………vii
Sumário...…………………………………………………………………………....ix
Resumo……………………………………………………………………………....x
1 Introdução.....................................................................................................01
2 Revisão de literatura.....................................................................................03
3 Artigos anexados..........................................................................................08
3.1 Artigo publicado.........................................................................................10
3.2 Manuscritos no prelo.................................................................................11
3.3 Manuscritos submetidos............................................................................47
4 Comentários, críticas e conclusões..............................................................64
5 Anexos.........................................................................................................68
6 Referências..................................................................................................71
7 Abstract........................................................................................................76
ix
RESUMO
Avaliações clínicas têm sido possíveis com radiobiocomplexos marcados com
tecnécio-99mTc (99mTc). Drogas naturais ou sintéticas são capazes de interferir na
marcação de estruturas sanguíneas com 99mTc, assim como na biodistribuição de
radiobiocomplexos. Também tem sido descrita a toxicidade de vários produtos
naturais. O objetivo deste estudo foi comparar o efeito dos extratos de Mentha
crispa (hortelã) e de Hypericum perfloratum (hipérico) em diferentes modelos
experimentais. Na marcação de estruturas sangüíneas com 99mTc verificou-se que
ambos os extratos foram capazes de diminuir a radioatividade no compartimento
celular, nas proteínas plasmáticas e celulares. Na morfometria das hemácias,
apenas a hortelã foi capaz de alterar a forma e a relação perímetro/áreas das
hemácias. Na biodistribuição do radiobiocomplexo pertecnetato de sódio
(Na99mTcO4) a hortelã aumentou a captação do Na99mTcO4 no rim, no baço, no
fígado e na tireóide, enquanto que o hipérico diminuiu a captação do Na99mTcO4 no
osso, no estômago, no pulmão e na tireóide, e aumentou no pâncreas. Na
sobrevivência de culturas bacterianas o hipérico foi capaz de proteger a bactéria do
efeito danoso do cloreto estanoso (SnCl2). O hipérico não alterou a topologia nem
protegeu o DNA plasmidial da ação do SnCl2. Provavelmente os efeitos
apresentados por ambos os extratos poderiam ser explicados por substâncias
presentes nos extratos que poderiam alterar a morfologia das hemácias, o
transporte de íons pela membrana e/ou formar fitocomplexos. O estudo teve caráter
multidisciplinar com a participação das seguintes áreas do conhecimento:
Radiobiologia, Botânica, Endocrinologia, Fitoterapia e Hematologia.
Palavras-chave: hemácias; plasma; proteínas, tecnécio-99m; radiobiocomplexos;
ratos; Hypericum perforatum; Mentha crispa.
x
1
1 INTRODUÇÃO
O uso de produtos naturais é uma prática generalizada na
medicina popular. A pesquisa acadêmica trouxe novos conhecimentos sobre as
plantas e suas propriedades terapêuticas. O crescente aumento do interesse
da comunidade acadêmica em estudar os medicamentos naturais tem
contribuído para o uso cada vez mais freqüente de produtos naturais. Existem
alguns estudos sobre o efeito de plantas medicinais na marcação de hemácias
com tecnécio-99m (99mTc) (1, 2).
Nos procedimentos em medicina nuclear, o cloreto estanoso
(SnCl2) é freqüentemente usado como um agente redutor na marcação de
moléculas e células com 99mTc para obtenção de imagens cintilográficas.
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 (3). Foi
relatado que o efeito do SnCl2 também dependeria da presença de mecanismo
de restauração (4, 5) e que o SnCl2 também é capaz de promover a quebra de
DNA plasmidial (3, 6, 7).
O mecanismo de ação do Hypericum perforatum (erva de
São João) e da Mentha crispa (hortelã) ainda não estão bem esclarecidos, e
até hoje não há descrição na literatura de sua influência na biodistribuição dos
radiofármacos empregados na Medicina Nuclear, nem os seus efeitos na
radiomarcação de elementos sangüíneos e em nível morfológico nos diversos
tecidos do organismo animal.
2
Este estudo teve como objetivo teórico e prático comparar
efeitos dos extratos de Hypericum perforatum (Hipérico) e de Mentha crispa
(Hortelã) em diferentes modelos experimentais.
Os modelos experimentais utilizados na presente pesquisa
avaliaram os efeitos biológicos da hortelã e do hipérico na marcação de
elementos sangüíneos com o tecnécio-99m, na biodistribuição do radiofármaco
(radiobiocomplexo) pertecnetato de sódio (Na99mTcO4) e na morfologia de
hemácias e em culturas bacterianas tratadas com SnCl2.
A pesquisa foi realizada no Laboratório de Radiofarmácia
Experimental do Departamento de Biofísica e Biometria, e no Departamento de
Histologia do Instituto de Biologia Roberto Alcantara Gomes da Universidade
do Estado do Rio de Janeiro. Os experimentos foram possíveis através de
convênio firmado entre a Universidade do Estado do Rio de Janeiro e a
Universidade Federal do Rio Grande do Norte, sob a orientação do Professor
Doutor Mario Bernardo Filho, e na vigência dos auxílios concedidos pela
CAPES, FAPERJ e CNPq.
3
2 REVISÃO DE LITERATURA
A descoberta das radiações ionizantes e de elementos
radioativos despertou de imediato o interesse de suas aplicações na Biologia e
nas Ciências Médicas. Como a origem do fenômeno radioativo é nuclear, os
nuclídeos que emitem radiação são chamados mais apropriadamente de
radionuclídeos (8). Alguns nuclídeos existentes na natureza já são radioativos
(naturais) enquanto outros podem ser produzidos pelo homem (artificiais) (8,9).
Em medicina nuclear os radionuclídeos podem ser utilizados como fonte de
radiação ou como traçador. No primeiro caso, o material biológico recebe
apenas as radiações emitidas pelo radionuclídeo usado. No segundo, o próprio
radioisótopo é incorporado no meio biológico que se deseja estudar (10, 11,
12).
Os radionuclídeos quando utilizados na área biomédica têm
possibilitado a elucidação de inúmeros fenômenos que ocorrem nos seres
vivos, inclusive no homem, onde os mesmos são administrados como
radiofármacos (radiobiocomplexos), que são células ou moléculas marcadas
com o radionuclídeo, amplamente utilizadas em medicina nuclear, para
diagnóstico e para terapia (10, 13, 14). A utilização em medicina nuclear é
interessante pelo fato das doses administradas em diagnóstico acarretarem
uma baixa exposição do paciente e produzirem imagens de excelentes
qualidades (15).
O processo de marcação de células e moléculas com o
radionuclídeo tecnécio-99m (99mTc) normalmente necessita da utilização de
um agente redutor. A redução do radionuclídeo pode ser obtida através de
4
diferentes agentes químicos, sendo que o cloreto estanoso (SnCl2) é o agente
redutor mais freqüentemente utilizado para esta finalidade. O SnCl2 possui uma
eficiência de marcação do traçador radioativo superior a de outros agentes
redutores, justificando sua preferência, não só na medicina nuclear, mas
também na marcação de diversas estruturas de interesse biomédico (14, 16).
Estudos clínicos de diagnóstico em procedimentos estáticos
e avaliações dinâmicas têm sido possíveis com os radiofármacos marcados
com o 99mTc (8, 17, 18, 19) que é obtido como pertecnetato de sódio
(Na99mTcO4) por meio de um gerador 99Mo/99mTc.
A marcação de hemácias com 99mTc representa uma das
técnicas de relevância na medicina nuclear, e é aperfeiçoada em resposta ao
grande número de exames clínicos em que é utilizada (20,21). Hemácias
marcadas com 99mTc são utilizadas em medicina nuclear para a obtenção de
imagens do pool sangüíneo, avaliação do sistema cardiovascular, volemia e
hemorragias gastrintestinais (14,22). Além disso, novas aplicações têm sido
realizadas com este radiofármaco incluindo a determinação do fluxo sangüíneo
no miocárdio e no cérebro, de imagens do osso e para o diagnóstico da função
excretora do fígado e dos rins (21).
Estudos realizados demonstraram que na marcação de
hemácias com 99mTc, o ânion pertecnetato atravessa o espaço intracelular por
troca com os íons cloreto e/ou bicarbonato (23,24). Estes fatos indicam que o
processo de marcação ocorre em nível intracelular. 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 (5,25). Na
marcação de hemácias com 99mTc utilizando-se SnCl2, o 99mTc também pode
5
se ligar com as proteínas plasmáticas dependendo da concentração do agente
redutor, do radiobiocomplexo utilizado e do tempo de incubação considerado,
como pode ser observado pela técnica de precipitação com ácido
tricloroacético (2). Ao administrar-se um radiofármaco a um paciente sempre
uma fração do mesmo é encontrada ligada às proteínas plasmáticas (14, 26).
Quando um radiofármaco é administrado a um paciente,
ocorre o processo de biodistribuição. Este processo consiste de: (i) absorção,
(ii) distribuição, (iii) metabolismo e (iv) excreção da substância (27). Os
radiobiocomplexos são designados a ter uma biodistribuição específica e/ou
um padrão de eliminação quando administrados a indivíduos normais. Na
presença de alterações fisiopatológicas e/ou bioquímicas este padrão normal
de biodistribuição e eliminação são alterados. A biodistribuição ou
farmacocinética dos radiobiocomplexos pode ser alterada por uma variedade
de drogas administradas aos pacientes (28) e por procedimentos cirúrgicos (12,
29).
Com administração intravenosa, o Na99mTcO4 é distribuído
no compartimento vascular, 70 a 80% dos íons pertecnetato se ligam
inicialmente às proteínas plasmáticas. A eliminação plasmática é muito rápida e
o equilíbrio entre o compartimento vascular e o fluido intersticial é completado
em um curto tempo, entre 2 a 3 minutos. A meia-vida de eliminação do plasma
é de aproximadamente 30 minutos (29). Íons pertecnetato livres difundem
através das membranas dos capilares para o líquido intersticial, de onde são
captados por diferentes órgãos como o estômago (30), intestino (31), glândulas
salivares (32), tireóide (33), plexo coróide e rins (29). A captação pela tireóide é
por transporte ativo, representando cerca de 2 a 4% da dose injetada. Nos rins
6
os íons pertecnetato são filtrados nos glomérulos, mas 86% são reabsorvidos
nos tubos proximais. Apenas 30% da dose injetada são excretados na urina em
24h. O íon pertecnetato é facilmente absorvido pelo sistema digestório após
administração oral ou após injeção intramuscular por processo de difusão
simples (8, 9).
O uso de drogas sintéticas e naturais é fundamental na
obtenção de um resultado terapêutico desejado no tratamento de doenças.
Neste caso existe a possibilidade de que uma droga possa alterar a
intensidade dos efeitos farmacológicos de outra droga administrada
concomitantemente. O resultado final pode ser o aumento ou diminuição dos
efeitos de uma droga ou ainda aparecimento de um novo efeito não observável
durante a administração dos fármacos isoladamente (27). O mecanismo
sinergístico, ou não, entre drogas pode alterar todo o comportamento
farmacocinético (absorção, metabolismo e excreção) e farmacodinâmicos
(afinidade pelo mesmo sítio de ligação) da droga, por meio de diversas
possibilidades que acabam por modificar a resposta farmacológica esperada
(12,27). Os mecanismos de ação envolvendo os produtos sintéticos têm sido
de modo geral, bem estabelecidos (27). Entretanto não é o que acontece com
muitos dos produtos naturais usados na clínica médica (34).
A interação medicamentosa com radiobiocomplexos pode
ser devido à possibilidade do medicamento: (i) modificar a natureza química do
radiobiocomplexo, (ii) alterar o meio bioquímico do alvo no qual o
radiobiocomplexo se concentra, (iii) alterar o meio bioquímico do não alvo para
o radiobiocomplexo, (iv) favorecer ou dificultar a ligação do radiobiocomplexo
às proteínas plasmáticas e aos elementos sanguíneos e (v) em excesso pode,
7
ainda, exercer seu efeito terapêutico mimetizando sintomas de doenças (35).
Alterando a distribuição normal dos radiotraçadores, podem-se obter
diagnósticos imprecisos podendo haver a necessidade da repetição de
exames, expondo o paciente a doses de radiação desnecessárias (12).
A hortelã (Mentha crispa) é uma planta medicinal muito
utilizada por possuir propriedades carminática, eupéptica, estimulante,
colagoga, estomáquica, anti-séptica, antiemética, antiespasmódica e
analgésica (36). Como fitoterápico, é indicado para fadiga geral, para atonia
digestiva, cólicas, flatulência, vômitos durante a gravidez, intoxicações de
origem gastrintestinal, afecções hepáticas, palpitações, enxaquecas, tremores,
asma, bronquite crônica (favorece a expectoração), sinusite, dores dentárias
(bochechos) e nevralgias faciais provocadas pelo frio (36).
A erva de São João ou hipérico (Hypericum perforatum) tem
suas ações descritas como sedativa, diurética, útil para afecções nervosas com
depressão, hemorragias, diarréia e problemas urinários crônicos. De particular
interesse para cientistas é o uso potencial para diminuir a depressão com
poucos efeitos colaterais melhorando a terapia de drogas antidepressivas (37).
Extraída da erva de São João a hipericina é o principal composto responsável
pelo efeito antidepressivo (38).
Estudos com culturas bacterianas com diferentes
capacidades de 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 (3,9). Foi relatado que o efeito do SnCl2 também
dependeria da presença de mecanismo de restauração (4,7). O SnCl2 também
é capaz de promover a quebra da molécula de DNA plasmidial (40).
8
3 INDEXAÇÃO DE ARTIGOS
3.1 ARTIGO PUBLICADO
a- Efeito de um extrato de Hipérico (Hypericum perforatum) na marcação
in vitro de elementos sanguíneos com tecnécio-99m e na
biodisponibilidade do radiofármaco pertecnetato de sódio em ratos
Wistar, publicado em 2005, no periódico “Acta Cirúrgica Brasileira”, Qualis
Internacional C.
3.2 MANUSCRITOS NO PRELO
a- Aqueous extract of the medicinal plant Mentha crispa alters the
biodistribution of the radiopharmaceutical sodium pertechnetate in Wistar
rats. Aceito para publicação em 2007, no periódico “Medicinal Chemistry
Research”, Qualis Internacional C.
b- Influence of an aqueous extract of Hypericum perforatum (Hypericin)
on the survival of Escherichia coli AB1157 and on the electrophoretic
mobility of pBSK plasmid DNA. Aceito para publicação em 2007, no periódico
“Revista Brasileira de Farmacognosia”, Qualis Nacional A.
c- The male reproductive system and the effect of an extract of a
medicinal plant (Hypericum perforatum) on the labeling process of blood
constituents with technetium-99m. Aceito para publicação em 2007, no
periódico “Brazilian Archives of Biology and Technology”, Qualis Internacional
B.
9
3.3 MANUSCRITOS SUBMETIDOS
a- Morphologic and morphometric in vitro evaluation of red blood cell
labeled with technetium-99m: effects of the Hypericum perforatum and
Mentha crispa extracts. Submetido ao periódico “Biological Research”, Qualis
Internacional B.
10
3.1 ARTIGO PUBLICADO
Separata
Acta Cirúrgica Brasileira
v. 20, suplemento 1, pp. 76-80, 2005.
Efeito de um extrato de Hipérico (Hypericum perforatum) na marcação in
vitro de elementos sanguíneos com tecnécio-99m e na biodisponibilidade
do radiofármaco pertecnetato de sódio em ratos Wistar,
Sebastião David Santos-Filho, Mario Bernardo-Filho
76 - Acta Cirúrgica Brasileira - Vol 20 - Supl no 1 2005
Efeito de um extrato de Hipérico (Hypericum perforatum) na marcaçãoin vitro de elementos sangüíneos com tecnécio-99m e na biodisponibilidade
do radiofármaco pertecnetato de sódio em ratos Wistar1
Sebastião David Santos-Filho2, Mario Bernardo-Filho3
Santos-Filho SD, Bernardo-Filho M. Efeito de um extrato de Hipérico (Hypericum perforatum) na marcação in vitro de elementossangüíneos com tecnécio-99m e na biodisponibilidade do radiofármaco pertecnetato de sódio em ratos Wistar. Acta Cir Bras [serialon line] Available from: URL: htt://www.scielo.br/acb.
RESUMO - Objetivo: Avaliar o efeito de um extrato de hipérico (Hypericum perforatum) na marcação de elementos sanguíneos comtecnécio-99m (99mTc) e na biodisponibilidade do radiofármaco pertecnetato de sódio em ratos Wistar. Métodos:Sangue (heparinizado)de ratos Wistar é incubado com um extrato de hipérico, com cloreto estanoso e a seguir com Tc-99m, como pertecnetato de sódio(99mTcO
4Na). Plasma (P) e células (CS) são isolados por centrifugação. Amostras de P e CS também são precipitadas com ácido
tricloroacético 5%, e separadas as frações solúveis (FS-P e FS-CS) e insolúveis (FI-P e FI-CS). Para a análise da biodistribuição, 0,3mL do radiofármaco 99mTcO
4Na foi administrada em ratos Wistar que receberam por gavagem extrato ou salina (NaCl 0,9%) por 15
dias. Após 10 minutos os animais foram sacrificados e os órgãos isolados para contagem da atividade radioativa. Resultados: Oextrato de hipérico reduziu de forma significativa (P<0,05) a %ATI ligada às células, à fração insolúvel celular e à fração insolúvel doplasma. A biodistribuição foi diminuída significativamente (P<0,01) no osso, no músculo e na tireóide. No pâncreas o percentual deradioatividade aumentou significativamente (P<0,05). Conclusão: No extrato vegetal estudado podem existir substâncias queoxidariam o íon estanoso, reduzindo a fixação do 99mTc às hemácias e proteínas plasmáticas e celulares. Da mesma forma poderiamproduzir alterações metabólicas com conseqüente influência na captação do radiofármaco pertecnetato de sódio no osso, músculo,pâncreas e tireóide.
DESCRITORES: Hipérico. Tecnécio-99m. Biodisponibilidade. Cirurgia.
Introdução
1. Laboratório de Radiofarmácia Experimental, Departamento de Biofísica e Biometria, Instituto de Biologia Roberto Alcantara Gomes, Univer-sidade do Estado do Rio de Janeiro.
2. Doutorando do Programa de Pós-Graduação em Ciências da Saúde da Universidade Federal do Rio Grande do Norte.3. Professor Titular, Laboratório de Radiofarmácia Experimental, Departamento de Biofísica e Biometria, Instituto de Biologia Roberto Alcantara
Gomes, Universidade do Estado do Rio de Janeiro.
Hemácias podem ser marcadas com tecnécio-99m (99mTc) eempregadas em diagnóstico por imagem de sangramentos in-ternos, localização de hemangiomas e de obstruções no sistemacirculatório que necessitariam de intervenção cirúrgica
1,2,3. Es-
sas hemácias marcadas também têm sido de importância emvários estudos clínicos e também experimentais de possívelinteresse para a pesquisa na medicina nuclear
4-11. Essa marca-
ção é baseada na capacidade de redução do cloreto estanoso(SnCl
2) que atua sobre o 99mTc, na forma de pertecnetato de
sódio. Tem sido descrito que vários fatores interferem na mar-cação de hemácias e proteínas plasmáticas com 99mTc, e dentreeles estão os produtos naturais
8,10,12,13.
O radiofármaco pertecnetato de sódio é distribuído atravésdos líquidos vasculares e intersticiais e apresenta normal-mente uma captação preferencial na tiróide, o estômago, tratointestinal, e glândulas salivares
14. Vários fatores, como a tera-
pia com drogas, a terapia com radiação, processos cirúrgicos,condições de dieta, além de doenças podem afetar abiodistribuição dos diferentes radiofármacos
9,16-19. Esses fa-
tores levam a uma repetição do exame, resultando numairradiação desnecessária ao paciente.
Plantas medicinais têm sido utilizadas largamente no mundono tratamento de doenças, ou mesmo visando potencializaras ações terapêuticas, diminuindo doses e efeitos adversos.Muitos dos efeitos adversos e interações potenciais destasplantas são pouco estudados, assim como grande parte dasaplicações médicas e populares é difícil de ser interpretadacientificamente
19.
A erva de São João (hipérico; Hypericum perforatum) temsuas ações descritas como sedativa, adstringente e diurética,sendo também útil para afecções nervosas com depressão,hemorragias, diarréia e problemas urinários crônicos
20. Tam-
bém tem sido sugerida para tratar danos espinhais e é usadapara contusões, feridas, tumores e ulcerações. Hypericumperforatum parece ter uma significante atividadeantidepressiva tendo sido proposto ser o melhor indicadorde estresse clínico do que estresse agudo, e pode indicaratividade adaptogênica
21.
O objetivo deste trabalho é avaliar o efeito de extrato dehipérico (Hypericum perforatum) na marcação de elementossanguíneos com tecnécio-99m e na biodistribuição doradiofármaco pertecnetato de sódio em ratos Wistar.
Acta Cirúrgica Brasileira - Vol 20 - Supl no 1 2005 - 77
Métodos
Sangue foi colhido de ratos Wistar (17 animais) machos, compeso de 336±11g, após injeção intraperitoneal compentobarbital PA na dose de 50 mg/kg de peso. Amostras de3 mL de sangue foram retiradas por punção cardíaca, em se-ringas com anticoagulante (0,2 mL de heparina). Após a coletade sangue os ratos foram separados para serem novamentemanipulados passados 15 a 20 dias. Isto é importante paraprevenir infecções e reduzir o estresse e sofrimento dos ani-mais.
Para preparar o extrato, hipérico em pó (Herbarium, Brasil,lote 954661, validade: 08/2006, data de fabricação: 23/04/2002)foi misturado com salina (NaCl 0,9%) na concentração finalde 50 mg/mL e homogeneizado em agitador tipo vortex por 30segundos. Após filtração, essa solução do extrato foi deno-minada 100%, sendo diluído para as concentrações de: 6,25;12,5; 25, 50 e 100% (estudos de marcação de constituintessanguíneos) que foram usadas nos estudos in vitro e 100%no estudo in vivo (biodisponibilidade).
Os experimentos (total de 3) de marcação dos constituintessanguíneos foram realizados em triplicata, havendo a neces-sidade de um total de 9mL de sangue que foram obtidos deum pool de sangue coletado de 3 animais. Amostras de 0,5 mlde sangue foram incubadas, a temperatura ambiente, com oextrato vegetal (100 ìL) em diferentes concentrações (6,25;12,5; 25; 50 e 100%), por uma hora. Após, foi adicionado 0,5mL de solução de cloreto estanoso (SnCl
2; 1,2 mg/mL; Reagen
S.A., Brasil) e realizou-se a incubação por mais uma hora.Após, foram adicionadas 0,1 mL de 99mTc (3,7 MBq) na for-ma de pertecnetato de sódio (99mTcO
4Na), em todos os tubos,
continuando a incubação por mais 10 minutos. As amostrasforam centrifugadas (1500 rpm, 5 minutos) em centrífuga clí-nica Alpha ICA (Instrumentos Científicos Alpha Ltda.).Alíquotas de plasma (P) e células (C) foram então separadase a porcentagem de atividades (% ATI) calculada. Para avaliaro efeito na ligação do Tc-99m às proteínas plasmáticas e celu-lares, alíquotas de P e C (20 mL) foram também precipitadas
em 1ml de ácido tricloroacético (TCA 5%), sendo então,centrifugadas e isoladas a fração solúvel (FS) e insolúvel (FI)de P e C. No controle foi adicionada solução salina no lugardo extrato vegetal. A porcentagem de radioatividade incorpo-rada aos elementos sanguíneos foi calculada.
Nos experimentos de biodisponibilidade (in vivo), um extratode hipérico 100% (50mg/mL) foi administrado por gavagem emratos Wistar (5 ratos), com intervalos de 24 horas, entre cadadose por 15 dias. Nos animais controle (3 ratos), foi administra-da salina. Uma hora após a última dose (15º. dia), 0,3ml doradiofármaco pertecnetato de sódio (7,4 MBq) é injetado atra-vés do plexo venoso orbital. Após 10 minutos os animais foramsacrificados e os órgãos isolados para contagem da atividaderadioativa em cintilador sólido de NaI(Tl) (Automatic GammaCounter, modelo C 5002, Packard, Canadá). A seguir foram cal-culadas: (i) as percentagens de radioatividade total (%ATI)nos órgãos e (ii) as percentagens de radioatividade por gramade tecido (%ATI/g) em cada órgão. As %ATI/g foram determi-nadas dividindo-se as percentagens de radioatividade porgrama de tecido pela massa de cada órgão.
Os valores experimentais obtidos foram comparados com osresultados do grupo controle usando o teste ANOVA segui-do pelo teste de comparações múltiplas de Dunnett.
Resultados
Na Figura 1 é mostrada a distribuição de radioatividade noscompartimentos celular (C) e plasmático (P) e nas fraçõesinsolúvel e solúvel celular e plasmática de amostras de san-gue tratadas com diferentes concentrações de extrato aquosode hipérico (6,25, 12,5, 25, 50 e 100%). Os resultados mostramque o extrato de hipérico reduziu de forma significativa(P<0,05) a %ATI ligada às células de 95,03 ± 0,51 (controle)para 4,16 ± 0,92 (100% do extrato), à fração insolúvel celularde 87,46 ± 5,07 (controle) para 14,82 ± 0,61 (100% do extrato)e à fração insolúvel do plasma de 76,24 ± 6,15 (controle) para26,67 ± 1,70 (100% do extrato).
FIGURA 1 – Distribuição de radioatividade nas frações sangüíneas de amostras de sangue tratadascom diversas concentrações de um extrato de hipérico
78 - Acta Cirúrgica Brasileira - Vol 20 - Supl no 1 2005
Amostras de sangue foram obtidas de ratos Wistar atravésde punção cardíaca e incubadas in vitro durante uma horacom diferentes concentrações do extrato de hortelã (macerado- folhas e caule) (100; 50; 25; 12,5; 6,25 %). No controle foiutilizada solução salina 0,9%. A seguir foi adicionado cloretoestanoso e Tc-99m. Após centrifugação, amostras de célula(C) e Plasma (P) foram isoladas e também precipitadas comTCA 5 %. As amostras foram contadas em cintilador sólido eas %ATI calculadas.
FIGURA 2 – Distribuição da radioatividade do radiofármaco pertecnetato de sódio
Na Figura 2 são mostrados os percentuais de radioatividadepor grama dos órgãos retirados de animais tratados comhipérico ou com solução salina (controle). Como pode serobservado alguns órgãos tiveram a biodistribuição diminuí-da significativamente (P<0,01): (a) no osso de 0,45 ± 0,01 para0,16 ± 0,06; (b) no músculo de 0,18 ± 0,004 para 0,10 ± 0,02; (c)na tireóide de 6,09 ± 1,66 para 1,54 ± 1,07. No pâncreas opercentual de radioatividade aumentou significativamente(P<0,05) de 0,16 ± 0,04 para 0,24 ± 0,03.
Extrato de hipérico 100% (50mg/mL) foi administrado porgavagem em ratos Wistar (n=5), com intervalos de 24 horas,entre cada dose por 15 dias. Uma hora após a última dose(15º. dia), 0,3ml do radiofármaco (7,4 MBq) foi injetado atra-vés do plexo venoso orbital. Após 30 minutos os animaisforam sacrificados, a contagem da atividade radioativa emcada órgão isolado obtida e foram calculadas as percenta-gens de radioatividade (%ATI) nos órgãos.
Discussão
A marcação de constituintes sanguíneos com 99mTc temmuitas aplicações. Tem sido descrito que extratos obtidos deplantas medicinais podem alterar a marcação de elementossanguíneos com 99mTc e também a morfologia doseritrócitos
6,7,10,13.
A distribuição, a fixação e a eliminação de radiofármacos emum organismo dependem de vários fatores, tais como o fluxosanguíneo, o metabolismo tecidual e a ligação aos elementos
sanguíneos4,15
. Vários autores têm demonstrado que abiodisponibilidade de radiofármacos pode ser modificado porprodutos naturais e sintéticos
6-13,22.
Nos resultados apresentados na figura 1, o extrato de hipéricofoi capaz de diminuir a marcação de constituintes celularescom 99mTc e nesse processo de marcação tanto os íonsestanoso e pertecnetato parecem atravessar a membranaeritrocitária
23. Seria altamente sugestivo que os produtos exis-
tentes no extrato de hipérico poderiam comprometer o trânsitodesses íons através da membrana através de proposta apre-sentada por Krishtal em outro tipo celular
24. Esses autores
descreveram que a hiperforina e outros constituintes dohipérico têm a capacidade de modular canais iônicos da mem-brana celular. Em relação aos canais de cálcio esse efeito seriadevido a interação com a calmodulina ou através das vias deativação da calmodulina. à ação da hiperforina.s e plasmáticosdo sangue.
O extrato de hipérico usado (ver figura 2) diminuiu a captaçãodo 99mTcO
4Na no osso, no músculo e na tireóide, assim como,
aumentou a fixação desse radiofármaco no pâncreas. Esses
Acta Cirúrgica Brasileira - Vol 20 - Supl no 1 2005 - 79
resultados poderiam, em parte, serem atribuídos à capacida-de de interação dos produtos presentes no hipérico àmembranas lipídicas como descrito por Neagoe
25. Esses au-
tores têm demonstrado que os componentes de um extratoetanólico de hipérico seriam capaz de interagir com membra-nas lipídicas acarretando alterações na absorção ebiodisponibilidade, assim como efeitos farmacodinâmicos naexcitabilidade neuronal.
Na técnica de marcação dos constituintes sanguíneos com99mTc é necessário um agente redutor. Uma vez que ocor-reu redução da marcação dos constituintes sanguíneoscom 99mTc, poderia ser especulado que os produtos exis-tentes no extrato de hipérico estariam oxidando o íonestanoso impedindo sua ação. Os compostos químicospresentes nesse extrato também poderiam atuar com agen-tes quelantes levando à formação de complexos com o99mTcO
4Na e SnCl
2, sendo que esse fato também poderia
justificar a diminuição na fixação de radioatividade noselementos sanguíneos. Possíveis alterações morfológicaspromovidas pelo extrato do hipérico, à semelhança às ob-servadas nos estudos com a hortelã
13 poderiam também
justificar a não captação pelas células vermelhas do san-gue.
A evidência que um extrato de hipérico pode alterar a marca-ção de células vermelhas do sangue, assim como abiodisponibilidade de radiofármaco pode ser de grande rele-vância no momento da interpretação de imagenscintilográficas. Por outro lado, deve-se considerar que osachados experimentais obtidos nesse trabalho foram em con-dições experimentais controladas e realizadas com ratos.
Conclusão
De acordo com os resultados obtidos nesse estudo pode-seespecular que o extrato de hipérico seria capaz de alterar invitro a marcação de elementos sanguíneos com 99mTc, assimcomo também a biodistribuição do radiofármaco 99mTcO
4Na.
A ação biológica desse extrato poderia estar relacionada a (i)propriedades redox do extrato, (ii) efeitos quelantes, (iii) alte-rações na morfologia das hemácias, (iii) modificação notransporte de íons através da membrana eritrocitária, (iv)metabolização no organismo e (v) atuação em órgãos especí-ficos. Embora os resultados tenham sido obtidos com animais,sugere-se precaução com os exames em medicina nuclear empacientes que utilizem Hypericum perforatum como medica-mento.
Referências
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9- Diré G, Lima E, Mattos D, Oliveira MB, Pereira MJ, Moreno S,Freitas R, Gomes ML, Bernardo-Filho M. Effect of chayotte(Sechium edule) extract on the biodistribution of technetium-99mand on the morphometry of red blood cells. J Labelled CompRadiopharm 2001; 44:648-50.
10- Lima EA, Dire G, Mattos DM, Freitas RS, Gomes ML, Olivei-ra MB, Faria MV, Jales RL, Bernardo-Filho M. Effect of an extractof cauliflower (leaf) on the labeling of blood elements withtechnetium-99m and on the survival of Escherichia coli AB1157submitted to the treatment with stannous chloride. Food ChemToxicol 2002; 40: 9149-53.
11- Moreno SRF, Diré G, Freitas RS, Farah MB, Lima-Filho GL,Rocha EK, Jales RLC Bernardo-Filho M. Effect of Ginkgo bilobaon the labeling of blood elements with technetium-99m: in vitrostudy. Rev Bras Farmacogn 2002; 12:62-3.
12- Moreno S, Rocha EK, Pereira M, Mandarim-Lacerda C, FreitasRS, Nascimento ALR, Carvalho JJ, Lima-Filho GL, Diré G, LimaEAC, Bernardo-Filho M. Ginkgo Biloba: Experimental model toevaluate it’s action on the labeling of blood elements withtechnetium-99m and on the morphometry of red blood cells. PakistanJ Nut 2004; 3:68-71.
13- Santos-Filho SD, Diré GL, Lima E, Oliveira MN, Bernardo-Filho M. Effect of Mentha crispa (mint) extract on the labeling ofblood elements with technetium-99m: A possible evaluation of freeradicals. J Biol Sci 2004; 4: 266-70.
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Santos-Filho SD, Bernardo-Filho M. Effect of Hypericum perforatum extract on in vitro labelling of blood elements with technetium-99mand on biodisponibility of sodium pertecnetate in Wistar rats. Acta Cir Bras [serial on line] Available from: URL: htt://www.scielo.br/acb.
ABSTRACT – Purpose: To evaluate the effect of a hiperico extract (Hypericum perforatum) on the labeling of blood elements withtechnetium-99m (99mTc) and in the bioavailability of the radiopharmaceutical sodium pertechnetate in Wistar rats. Methods: Blood(heparinized) withdrawn from Wistar rats is incubated with a hiperico extract, with a stannous cloride and with 99mTc, as sodiumpertechnetate (99mTcO
4Na). Plasma (P) and cells (C) are isolated by centrifugation. Samples of P and C are also precipitated with
trichloroacetic acid (TCA 5%) and soluble (FS-P; FS-C) and isoluble (FI-P; FI-C) fractions are separated. In the bioavailabilityanalysis, the extract or NaCl 0.9% solution is administrated into Wistar rats (gavage) during 15 days. Sodium pertechnetate wasadministered and after 10 min, the animals are sacrificed, the organs were isolated, the radioactivity determined in a well counter, andthe percentages of radioactivity per gram (%ATI/g) in the organs are calculated. Results: The hiperico extract decreased significantly(P<0.05) the %ATI in the cells, cellular insoluble fraction and plasma insoluble fraction. The biodistribution was significantly (P<0.01)decreased in bone, muscle and thyroid and significantly (P<0.05) increased in pancreas. Conclusion: The analysis of the resultsindicates that in studied extract should have substances that should oxidize the stannous ion, reducing the fixation of the 99mTc onthe erythrocytes and plasma and cellular proteins. Moreover, it could produce metabolic alterations with influence in the uptake ofthe radiopharmaceutical 99mTcO4Na in bone, muscle, pancreas and thyroid.
KEYWORDS:Hiperico. Technetium-99m. Bioavailability. Surgery.
Correspondência:Sebastião David Santos-FilhoUniversidade do Estado do Rio de JaneiroInstituto de Biologia Roberto Alcantara GomesDepartamento de Biofísica e BiometriaLaboratório de Radiofarmácia ExperimentalAv. 28 de Setembro, 8720551-030. Rio de Janeiro – RJTel.: [email protected] de interesse: nenhumFonte de financiamento: CNPq, FAPERJ, UFRN, UERJ
11
3.2 MANUSCRITO NO PRELO
a- Medicinal Chemistry Research, Qualis Internacional C.
Aqueous extract of the medicinal plant Mentha crispa alters the biodistribution of
the radiopharmaceutical sodium pertechnetate in Wistar rats.
Sebastião David Santos-Filho1, 2, 3, Adalgisa Ieda Maiworm1, 3, Giuseppe Antonio
Presta1,3, Severo de Paoli1,3, Tânia Santos Giani1,3, Mario Bernardo-Filho1,4.
1Departamento de Biofísica e Biometria, Instituto de Biologia Roberto Alcantara Gomes,
Universidade do Estado do Rio de Janeiro, 20551030, Rio de Janeiro, RJ, Brasil
2Ciências Biomédicas, Centro Universitário de Volta Redonda, 27251970, Volta
Redonda, RJ, Brasil
3Programa de Pós-Graduação em Ciências da Saúde, Universidade Federal do Rio
Grande do Norte, 59010180, Natal, RN, Brasil
4Coordenadoria de Pesquisa, Instituto Nacional do Câncer, 20230130, 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, 20551030
Vila Isabel, Rio de Janeiro, RJ, Brazil.
Phone/Fax: 55-021-21-2587-6432
12
Running Title: Mint and radiopharmaceutical biodistribution
Financial support: CAPES, CNPq and UERJ
Abstract
Mentha crispa extract or NaCl 0.9% were daily (15 days) administered by gavage into
Wistar rats. After that, animals have received by ocular plexus via, the Na99mTcO4 and
they were sacrificed. Organs were isolated, the radioactivity determined, the
percentages of radioactivity in each organs calculated and statistical analysis
performed. Significant (p=0.0061) increase of the radioactivity was observed in kidney,
spleen, and thyroid, that could be justified by effects of the extract and/or by generation
of active metabolites.
Key-words: Mentha crispa, biodistribution, sodium pertechnetate, radiobiocomplex.
Introduction:
The relevance of the techniques used in the nuclear medicine is mainly related
with their ability to provide suitable information on physiology rather than on anatomy1.
Several radiopharmaceutical or radiobiocomplex2 have been used to obtain single
photon emission computed tomography (SPECT) and positron emission tomography
(PET) images3,4.
Sodium pertechnetate (Na99mTcO4) is a radiobiocomplex that has a long and
successful history of use in several clinical applications involving SPECT, such as, (i)
labeling of various 99mTc-radiobiocomplexes, (ii) imaging of the thyroid, (iii) imaging of
the gastric mucosa to diagnose Meckel´s diverticulum, (iv) labeling (in vivo) of red blood
cells for blood pool imaging and first-pass cardiac angiography, (v) evaluating of the
nasolacrimal drainage, (vi) studying of the parathyroid adenoma in conjunction with
201Tl+, (vii) evaluating of the testicular torsion and infection, (viii) imaging of the salivary
13
gland and (ix) imaging of the brain to investigate possible damage to the blood-brain
barrier4-7.
Some physiological and/or pharmacological properties related with the
radiobiocomplex Na99mTcO4 have been reported. After intravenous injection, it is weakly
bound to serum proteins (70-80%). Unbound pertechnetate ion (99mTcO4-) diffuses
slowly through the capillary membranes to the interstitial fluids, from where it is cleared
by various organs such as stomach wall, intestines, salivary glands, thyroid, choroid
plexus, sweat glands, kidneys and mucous membrane. The thyroid uptake of 99mTcO4-
is by active transport, as a sodium-iodide symporter, and its uptake is 2-4%. In the
kidneys, 99mTcO4- is filtered in the glomeruli, but 86% are reabsorved in the proximal
tubes. Only 30% of the injected dose is excreted in urine in 24 h. Lactating women
secrete 10% of 99mTcO4- in milk. 99mTcO4
- is easily absorbed by the digestive system
after oral administration or after intramuscular injection by simple diffusion1,6,8.
Many factors, as drug therapy, radiation therapy, dietary conditions, besides
pathological process could affect the biodistribution of the different
radiobiocomplexes2,9-14 or the labeling of blood constituents15-18. These factors may lead
to (i) a poor visualization of the organs and to induce inadequate interpretation of the
examination and misdiagnosis or (ii) the repetition of the examination procedure
resulting in unnecessary irradiation to the patient and the staff1,2,19.
It is well established that medicinal plants contain mixtures of various
pharmacologically active compounds, and thus they may have the potentiality to
interfere with the labeling of radiobiocomplexes10,20-28 and/or with their
biodistribution2,10,11,24,29.
14
Mentha crispa (mint; M.crispa) is utilized in herbal medicine due to various
pharmacological actions. Mint is part of a genus of the Labiatae family, which
comprises a wide number of species, varieties and hybrids. Mint flowers and leaves are
used for tea infusions, which have digestive, calming, antiseptic and anti-asthmatic
properties30. The extract and leaves of the Mentha species are described as biological
additives31. Rosmarinic acid, a natural phenolic compound contained in many
Lamiaceae herbs such as mint, inhibits complement-dependent inflammatory processes
and may have therapeutic potential32. Mint was also capable of iron(III) reduction, 1,1-
diphenyl-2-picrylhydrazyl radical scavenging, and to inhibit iron(III)-ascorbate-catalyzed
hydroxyl radical-mediated brain phospholipid peroxidation33. Santos-Filho28 have
described that an extract of mint was capable to interfere on the labeling of blood cells
with the Tc-99m, as well as, the fixation of this radionuclide on the plasma and blood
cells proteins. The main pharmacodynamic effect of mint oil is a dose-related
antispasmodic effect on the smooth musculature due to the interference of menthol with
the movement of calcium across the cell membrane, relevant to the gastrointestinal
tract34.
As the extract of mint is commonly used in the world, the aim of this work
was to evaluate the effects of a mint extract on the biodistribution of the
radiobiocomplex sodium pertechnetate in Wistar rats.
Materials and methods:
All 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/113/2006.B
15
The experiments were performed without sacrificing the animals. The animals
were maintained under controlled conditions (22±5°C, 12h of light/dark cycle). Food and
water were available ad libitum.
Wistar rats (male, 3 months, 11 animals, 293±13g) were separated in 2 groups:
mint group (n=5) and control group (n=6).
The mint group was treated with an extract of mint prepared with 4g of crushed
leaves of Mentha crispa (Mãe Terra Produtos Naturais – http://www.maeterra.com.br -,
Brazil, lot 05FEV2002) in 100mL of 0.9% NaCl solution (saline). The preparation was
centrifuged (clinical centrifuge, 3000 rpm, 10 min), filtered through paper (quality filter
paper) and the filtered solution was considered to be 40mg/mL. The control group
received only saline.
The extracts and saline were daily administered by intragastric via during 15 days
with 24 hours intervals. After that, the animals received by ocular plexus via 0.3mL of
the Na99mTcO4 (3.7MBq) and were rapidly sacrificed after 10 min following the criteria of
the Ethical Committee of the Institution.
The organs (pancreas, testis, stomach, kidney, spleen, duodenum, heart, lung,
liver, thyroid, bone, muscle and brain) were isolated and the mass of each organ was
determined in an analytical balance. The radioactivity in each organ was determined in
a well counter (mod C5002, Packard, USA).
The activity in each organ was divided by the total activity administered to
determine the percentage of radioactivity (%ATI) in each organ. The %ATI was divided
by the mass of each tissue to determine the percentage of radioactivity per gram
(%ATI/g).
16
Statistical analysis was performed by Wilcoxon test. P< 0.05 was considered
significant. All data are expressed as mean standard deviation (SD).
Results:
The results of the biodistribution of the radiobiocomplex sodium pertechnetate in
different organs isolated from male rats are shown in the Table 1. The comparison of
the %ATI/g of these tissues isolated from the control group and from the mint group has
revealed that the mint extract increased the fixation of this radiobiocomplex in the
pancreas, kidney, spleen, liver and thyroid (P = 0.0061). The results also show that the
mint extract did not modify the fixation of the radiobiocomplex sodium pertechnetate in
other organs.
Discussion:
In nuclear medicine the clinical procedures to obtain image, normally, are
associated with a low radiation dose level to the patient and to the staff when compared
with other similar procedures using X rays. Moreover, the environmental impact is
negligible due to the half-life and the energy of the radiation emitted in the decay of the
main radionuclides used in the nuclear medicine procedures. In addition, the high
quality of the scintigraphic images in the SPECT and in the positron emission
tomography (PET) has helped in a rapid and efficient elucidation of the diagnosis of
various diseases35-38.
The radiobiocomplexes can be utilized to study the blood flow, the metabolism,
the morphology of an organ3,36, and to evaluate the drug formulation and drug delivery
systems37.
Unexpected patterns of the biodistribution of a radiobiocomplex have normally
been associated with a disease and this fact aid in the clinical diagnosis1. However,
17
some authors have reported that many factors, besides the disease, can interfere in the
bioavailibility of a radiobiocomplex. When these factors are unknown, it is undesirable
and the consequences are (i) the possibility of misdiagnosis (misleading information that
can either mask or mimic certain disease symptoms) and/or (ii) the repetition of the
examination with an increase of radiation dose to the patient and staff1,39.
The drug therapy, radiation therapy besides and surgical interventions could also
affect the biodistribution of the radiobiocomplexes1,12,40. The drug interaction with
radiobiocomplexes has been studied and it can be know or unknown6. Passos41 and
Passos42 have also demonstrated that the dietary conditions can also interfere with the
biodistribution of radiobiocomplex. Several authors have also reported that some
chemical compounds due to their pharmacological properties can alter the
biodistribution of various radiobiocomplexes9,11,12,29,34,40,46,47,48,49.
Some authors have described that synthetic or natural products in the blood, as
well as, the labeling conditions can have an effect on the labeling of red blood cells with
technetium-99m (99mTc)11,25-28,43-45.
The mint extract (Table 1) increased the fixation of the studied radiobiocomplex
in the pancreas, kidney, spleen, liver and thyroid, and did not modify the fixation in the
other organs. Szentmihalyi and collaborators50 have reported that some components of
the mint extracts could interfere with the movement of calcium ions across the cell
membrane51. It is possible to speculate that, as the radiobiocomplex studied is an ion,
the effect of the components of the mint extract would act also in the transport6,8 of the
pertechnetate ion through the cellular membrane of determined organs. Moreover, the
extract of mint seems to produce damage on the red blood cell membrane with
morphological changes28 that could be related with physiological/pharmacological
18
modifications in determined organs and could interfere in the uptake of ions, as the
pertechnetate.
In conclusion, these results can be justified by the presence of determined
chemical compounds in the extract and/or by the generation of active metabolites
capable to interfere with the biodistribution of the studied radiobiocomplex. As the use of
extracts is increasing in the world, the knowledge about the drugs and the other factors
capable to interfere on the biodistribution of the radiobiocomplexes is highly worthwhile
to permit secure diagnosis of diseases. In addition, the development of biological
models to study this phenomenon is relevant and desired. Although results described in
this work have been obtained with rats, it is suggested to consider the possibility of an
unexpected effect of the mint in the uptake of the radiobiocomplex sodium
pertechnetate in several organs in the examinations in nuclear medicine.
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26
TABLE
Table 1: The effect of a Mentha crispa extract on the biodistribution of sodium
pertechnetate in Wistar rats.
Percentage of radioactivity per gram of tissue
(%ATI/g) (MEAN±SD) Organs
Control Treated
Pancreas (*) 0.15±0.03 0.28±0.07
Testis 0.14±0.02 0.23±0.10
Stomach 3.51±0.03 3.17±0.23
Kidney (*) 0.55±0.01 0.90±0.08
Spleen (*) 0.42±0.05 0.69±0.09
Duodenum 0.88±0.18 1.06±0.21
Heart 0.31±0.02 0.33±0.09
Lung 0.84±0.05 0.92±0.17
Liver (*) 0.61±0.03 0.72±0.02
Thyroid (*) 6.09±0.09 12.83±0.82
Bone 0.45±0.01 0.41±0.11
Muscle 0.18±0.01 0.19±0.08
Brain 0.09±0.04 0.06±0.01
(*) P=0.0061. An extract of mint (40g/L) was administered (ig) in rats for 15
days. Na99mTcO4 (0.3mL, 3.7Mbq) was administered for ocular plexus way.
After 10 min the animals were sacrificed and the organs were isolated. The
radioactivity in each organ (%ATI/g) was determined in a well counter and the
total percent of radioactivity was calculated.
27
b- Revista Brasileira de Farmacognosia, Qualis Nacional A.
Influence of an aqueous extract of Hypericum perforatum (Hypericin) on the
survival of Escherichia coli AB1157 and on the electrophoretic mobility of pBSK
plasmid DNA
Sebastião D. Santos-Filho1, 2, 3 *, Raquel M. Bernardo3, Kelly C.M. Santos3,
Adenilson S. Fonseca1, 3, Mario Bernardo-Filho3
1- Centro de Ciências da Saúde, Centro Universitário de Volta Redonda, Fundação
Oswaldo Aranha, Av Paulo Erlei Alves Abrantes, 1325, Três Poços, 27240-560,
Volta Redonda, RJ, Brazil,
2- Programa de Pós-graduação em Ciências da Saúde, Universidade Federal do Rio
Grande do Norte, Av. Gen. Gustavo Cordeiro de Farias, s/n, 59010-180, Natal, RN,
Brazil
3- Departamento de Biofísica e Biometria, Instituto de Biologia Roberto Alcantara
Gomes, Universidade do Estado do Rio de Janeiro, Av. 28 de setembro, 87, Vila Isabel,
20551-030,
Rio de Janeiro, RJ, Brazil
Abstract
Hiperico (Hypericum perforatum or St John’s worth) has been widely used as an herbal
medicine to treat depression. Hypericin is the main chemical compound of the hiperico.
Stannous chloride (SnCl2) is the most used reducing agent in nuclear medicine. The aim
of this work was to verify the effect of a hiperico extract on the survival of Escherichia
coli AB1157 and on the plasmid DNA topology. Exponentially E. coli AB1157 cultures
were incubated with SnCl2 in presence or absence of hypericin. Aliquots were spread
28
onto Petri dishes containing solidified rich medium; the colonies units were counted
after overnight and the survival fraction was calculated. Plasmid DNA samples were
incubated with SnCl2 in presence or absence of hypericin extract during 40 minutes;
0.8% agarose gel electrophoresis was performed, the gel was stained with ethidium
bromide and the plasmid topological forms (bands) were visualized. The results reveal
that hiperico extract is neither capable of altering the survival of E. coli cells nor the
plasmid DNA topology but it may have protected these cells against the SnCl2 action.
The data suggest absence of cytotoxic and genotoxic effects of the aqueous hiperico
extract and a protective effect on E. coli cells against the action of SnCl2.
Keywords: Escherichia coli, DNA, Electrophoresis, Hypericum perforatum, Stannous
chloride
Introduction
The use of natural products, as medicinal plants, is very frequent worldwide.
Hiperico (Hypericum perforatum or St John’s wort) has been widely used as an herbal
medicine. Hypericin is the main chemical compound of the hiperico (McGarry et al,
2007). Authors have reported several pharmacologic actions of this chemical compound
(hypericin)(Wurglics and Schubert-Zsilavecz, 2006). Several studies have demonstrated
the efficacy of St John's wort as an antidepressant agent in human beings, besides of
its testing in animal behavioral models of depression (Hunt et al, 2001; Werneke et al,
2004; Linde et al, 2005; McGarry et al, 2007) and dysthymia (Rotblatt and Ziment,
2002). This natural product has been also used in folk medicine to treat hemorrhoids
and bruises, as well as to induce vasodilatation (Hunt et al, 2001; Siepmann et al, 2004;
Jaric et al, 2007). St John's wort reduces the effects of several drugs including
immunosuppressants, oral contraceptives, oral anticoagulants and HIV protease
29
inhibitors (Mannel, 2004; Busti et al, 2004; Bobrov et al, 2007). Pharmacodynamic
effects on neuronal excitability also were reported (Neagoe et al, 2004). Recently, a
anti-inflammatory action was demonstrated in an experimental model (Sosa et al.,
2007).
Free radicals (an unpaired electron in their outer valence shell) are highly
reactive species of molecules that could be produced by some oxidative cellular
mechanisms (Infanger et al, 2006). If these molecules are produced in excess, they can
induce tissue damage. There are internal mechanisms in the living organisms
constituted by enzymes and other molecules to protect the cells of these free radicals
(Hsieh et al, 2005; Kinoshita et al, 2005; Bao and Lou, 2006; Marcus et al, 2006). In
addition to these defense mechanisms, certain medicinal compounds, including
vitamins and other nutrition products could inhibit the production of free radicals
(Infanger et al, 2006).
Stannous chloride (SnCl2) is the most used reducing agent in nuclear medicine to
label cellular and molecular structures with biological interest with technetium-99m to be
used as radiobiocomplexes. Some authors have performed studies about the
citotoxic/genotoxic potentials of SnCl2 (Pungartnik et al, 2005; Almeida et al, 2007).
Using bacterial cultures and plasmid DNA has been suggested that stannous chloride
appears to induce damages in the deoxyribonucleic acid (DNA) by oxidative
mechanisms related to free radicals generation (Dantas et al., 2002; El-Demerdash et
al, 2005). Data from assays with Escherichia coli (E. coli) deficient in DNA repair
mechanisms suggested that this chemical agent could induce different lesions in DNA
(El-Demerdash et al, 2005; Almeida et al, 2007). Thus, the aim of this work was to verify
30
the effect of the hiperico aqueous extract on the survival of E. coli AB1157 and on the
electrophoretic mobility of pBSK plasmid DNA.
Materials and methods
Preparation of the extract The extract of hiperico was prepared with 5g of a purified dust of Hypericum
perforatum (Herbarium Laboratório Botânico LTDA, Brazil, lot 954661) in 100ml of 0.9%
NaCl (saline). The quality controls of this hiperico preparation performed by the
Herbarium Laboratório Botânico LTDA revealed that the major chemical compound is
hypericin (95%). The preparation was homogenized in a vortex mixer and filtered
through paper (quality filter paper) and 1ml was considered to contain 50mg of the
hypericin extract and regarded as 100% of the filtered solution. The quality control of
this extract was evaluated with the analysis of the absorption spectrum of a hypericin
recorded in the range from 400 up to 700nm with intervals of 20nm using a
spectrophotometer (Analyser 800M, Analyser Comércio e Indústria LTDA, São Paulo,
Brazil), using a cuvette of 1cm pathlength. All spectrophotometric measurements were
performed on the same spectrophotometer and the value of the absorbance
(0.414±0.016) at 580 nm was used as marker and the quality control of each prepared
hypericin in the all experiments.
Bacteria inactivation The E. coli AB1157, a wild-type strain, proficient to repair damage in the DNA,
was used in this work. From stock (in glycerol 50% v/v) a sample (50µl) of the culture
was grown on liquid LB medium (5ml, 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 (200µl) was taken from this culture and further
31
incubated (20ml; liquid LB medium) under the same conditions to exponential growth
(108 cells/ml). The cells were collected by centrifugation, washed twice in 10ml of saline
and suspended again in the same solution until they reached 108 cells/ml. Samples (1.0
ml) of these washed cultures (108 cells/ml) were incubated on the shaking water bath
with (i) 0.5ml of SnCl2 (75 µg/ml) and 0.1ml of saline, or (ii) 0.1ml of hiperico extract (50
mg/ml) and 0.5ml of saline, or (iii) 0.1ml of hiperico extract (50 mg/ml) and 0.5ml of
SnCl2 (75 µg/ml), or (iv) 0.6ml of saline as a control, on initial time and after 60min , at
37°C. During the assay, at 0 and 60 min, aliquots (100µl) were diluted with saline and
spread onto Petri dishes containing solidified LB medium (1.5% agar). Colonies units
formed after overnight incubation at 37°C were determined. 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 (N0).
Analysis of DNA mobility alterations Preparation of plasmids was performed using alkaline method, described by
Sambroock et al (1989). Experiments were done with plasmid DNA (200ng) exposed to
hiperico extract (50 mg/ml). Aliquots were also incubated with 200µg/ml of SnCl2 in
order to evaluate the influence of hiperico extract in the DNA lesions induced by
stannous ion. The experiments were carried out at room temperature for 40min.
Aliquots of each sample (10µl) were mixed to 2µl of 6x concentrated loading buffer
(0.25% xylene cyanol FF, 0.25% bromophenol blue, 30% glycerol in water), applied in a
horizontal gel electrophoresis chamber in Tris-acetate-EDTA buffer at pH 8.0 and the
electrophoresis were performed. Following, the gel was stained with ethidium bromide
(0.5µg/ml), the DNA bands were visualized by fluorescence in an ultraviolet
transilluminator system and the gel was recorded using a Polaroid system.
32
Results
Figure 1 shows the survival fractions of E. coli AB1157 cultures treated with
SnCl2 in presence or absence of hiperico extract during 60 minutes. The data show no
alteration on the survival fraction of E. coli incubated with hiperico extract at the used
concentration (50mg/ml). The results reveal inactivation of E. coli cells induced by
SnCl2. The data also show a protective effect induced by the extract of hiperico against
the inactivation produced by the treatment with the SnCl2.
<Figure 1>
The electrophoretic profile of pBSK plasmid DNA in different experimental
conditions is shown in figure 2. In lane 1, the plasmid DNA alone is found mostly as a
supercoil form (form I). In lane 2 is shown the efficient cleavage of the plasmid DNA by
SnCl2 evidenced by formation of open circular (form II). Lanes 3 to 5 show the
electrophoretic profile of plasmid DNA incubated with hiperico at different
concentrations (50, 5, 0.5mg/ml) suggesting no modifications in plasmid topology when
compared with control (lane 1). In lanes 6 to 8 is shown that hiperico extract was not
capable of protect the plasmid DNA against the stannous chloride action.
<Figure 2>
Discussion
Free radicals (FR) have been related to the primary destructive intermediates
molecules in a wide range of environmental conditions, as well as these species are
involved in various biological phenomena, as mutagenesis, apoptosis and aging
(Ozben, 2007).
Although cytotoxic and genotoxic effects of SnCl2 have been demonstrated in
different experimental models and these appeared to be mediated by free radicals (El-
33
Demerdash et al, 2005; Almeida et al, 2007). Moreno et al. (2004) related that an
extract of Ginkgo biloba protected the plasmid DNA from the lesions induced by SnCl2.
The results herein obtained revealed absence of cytotoxic effect of hiperico
extract on E. coli wild type in the concentration tested (figure 1). The data also show
that the aqueous hiperico extract protected these bacterial cells against the lethal action
of SnCl2. This result may well be explained by: (i) its antioxidant properties; (ii) its free
radicals scavenger action; or (iii) its metal ions chelating action related to this hiperico
extract.
Some authors have reported an antioxidant action to the hiperico extract (Breyer
et al, 2007; Gioti et al, 2007). Hunt et al (2001) have found that hiperico could be a
potent inhibitor of the superoxide radical in a cell-free, as well as in the human vascular
system. In addition, hiperico extract, standardized to both hypericin and hyperforin,
appears to have significant free radical scavenging properties in cell-free and human
vascular systems (Hunt et al, 2001; Wahlman et al, 2003). Our results indicated no
protective action of the aqueous hiperico extract in plasmid DNA treated with SnCl2
(figure 2). Moreover, hiperico was not also capable to induce modifications in the DNA
mobility in agarose gel.
In conclusion, our experimental data suggest absence of cytotoxic and genotoxic effects
of the aqueous hiperico extract and a protective effect on E. coli cells against the lethal
action of SnCl2. However, additional studies should be performed to try to elucidate the
action mechanisms involved in the effects of hiperico extract obtained in this work.
Acknowledgments: CNPq, CAPES and UERJ supported this work.
34
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Figure 1
0,00001
0,0001
0,001
0,01
0,1
1
10
0 10 20 30 40 50 60
Tempo (min)
Fraç
ão d
e so
brev
ivên
cia
(N/N
0)
ControlSnCl2SnCl2 + ExtratExtrat
Figure 2
1 2 3 4 5 6 7 8
Form II
Form I
39
Figures Legends
Figure 1: Effect of the hiperico extract on the inactivation induced by stannous chloride
on E. coli AB1157. Exponential growth of E.coli suspended in saline and treated with
stannous chloride for different incubation times (min) related with the presence or
absence of the extract and with the extract alone.
Figure 2: Agarose gel electrophoresis of pBSK Plasmid DNA (100ng) treated with
different concentrations of hiperico, alone or associated with stannous chloride
(100µg/ml). Lane 1: control; Lane 2: stannous chloride (100µg/ml); Lane 3: hiperico
(50mg/ml); Lane 4: hiperico (5mg/ml); Lane 5: hiperico (0.5mg/ml); Lane 6: hiperico
(50mg/ml) and stannous chloride (100µg/ml); Lane 7: hiperico (5mg/ml) and stannous
chloride (100µg/ml); Lane 8: hiperico (0.5mg/ml) and stannous chloride (100µg/ml);
Form I: DNA supercoil (white band); Form II: open circular and/or linear.
40
c- Brazilian Archives of Biology and Technology, Qualis Internacional B.
The male reproductive system and the effect of an extract of a medicinal plant (Hypericum perforatum) on the labeling process of blood constituents with technetium-99m Sebastião David Santos-Filho1,2*, Adenilson de Souza da Fonseca1 and Mario Bernardo-Filho1,3
1- Laboratório de Radiofarmácia Experimental, Departamento de Biofísica e Biometria, Instituto de BiologiaRoberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Av. 28 de setembro, 87, 20551-030, Rio deJaneiro, RJ, Brasil, [email protected] Programa de Pós-Graduação em Ciências da Saúde, Centro de Ciências da Saúde, Universidade Federal do RioGrande do Norte, A. Gal. Gustavo Cordeiro de Farias, s/n, 59010-180, Natal, RN, Brasil3- Coordenadoria de Pesquisa, Instituto Nacional do Câncer, Praça Cruz Vermelha, 23, 20230-130Rio de Janeiro,RJ, Brasil
ABSTRACT
Hypericum perforatum (hiperico) is a plant that has been used to treat diseases and also inhibits rat andhuman vas deferens contractility. In nuclear medicine, stannous chloride (SnCl2) is used as a reducing agent to obtain radiopharmaceuticals labeling with technetium-99m. As the SnCl2 seems to have adverseeffects related with the reproductive performance of male rabbits as well as the human consumption ofhiperico might affect sexual function. In the present work, consistent results show significant changes on the blood constituents labeled by technetium-99m obtained from young rats under the effect of anhiperico extract as opposed to blood samples equally treated taken from elderly rat.. Supposedly, this extract could protect the male reproductive system against action of SnCl2 at least in young rats. Thefindings described in this work allow introducing a simple assay to evaluate the action of products that could interfere with the male reproductive system. Key words: stannous chloride, young animals, elderly animals, sexual function, Technetium-99m,Hypericum perforatum.
INTRODUCTION
Sexual dysfunctions are common amongthe general population and can affect the malereproductive system, as the sperm production andthe ejaculation. Several authors (Wu et al 1996;Naha and Chowdhury 2006; Kumar et al, 2006)have reported that metal ions such as iron, lead,chromium, manganese, tin and cadmium adversely affect the male reproductive. Agarwal et al (2006)have published a review about the role of free radicals and oxidative stress in the pathophysiology of human reproduction and theysuggest that the oxidative stress and sperm DNAdamage have been implicated in male infertility.Elevated levels of oxygen reactive species arecorrelated to the poor fertility outcomes seen in the assisted reproductive technology setting and it is indicated that an accurate evaluation of seminaloxidative stress by standardized assays may help in the diagnosis and management of male
infertility. There is evidence in the literature on thebeneficial effects of oral antioxidantsupplementation of vitamins in male infertility(Agarwal et al, 2006; Menezo et al, 2007).
Tin is a heavy metal, which has long beenregarded as a contaminant of the environment(Wood, 1974). One of its inorganic salts, stannous chloride (SnCl2), has been widely used in dailyhuman life, to conserve preserve soft drinks, in food manufacturing, as a result of processing and packaging and in biocidal preparations (de Mattos et al, 2000). In nuclear medicine, SnCl2 is used asa reducing agent of technetium-99m (99mTc), as sodium pertechnetate, the most used radionuclide to obtain several radiopharmaceuticals which are also used in the single photon emission computedtomography (SPECT). In addition, it has been alsoused as a radiotracer to label different cells andmolecules of interest to scientific research (Saha,2004). It was demonstrated that tin, as SnCl2, can
41
facilitate the neuromuscular transmission by accelerating the transmitter release from the nerveterminals in the mouse (Hattori et al, 1989). Whenthis salt is injected into laboratory animals, it canproduce stimulation or depression of the centralnervous system. Because calcium (Ca2+) influx into the cytoplasm is indispensable to release thetransmitter, it would be possible that SnCl2increases the Ca2+ influx at the nerve terminalsbut not by blocking the K+ channels (Hattori et al,1994). SnCl2 is known to (i) inhibit the immuneresponse in rodents (Hayashi et al, 1984), (ii) alter the gene expression (Helvig et al, 1998) and (iii)induce tumor generation in thyroid gland (Babar et al, 1991). There is no general agreement regardingits genotoxicity and it was discussed that the effects of this salt might depend on thephysicochemical conditions and the route of its administration. This salt is directly administered to human beings, intravenously, when it is used as areducing agent to prepare 99mTc-radiopharmaceuticals (Saha, 2004). Stannous chloride is capable of inducing the generation of free radicals that seem to be implicated with theoxidative stress (Caldeira-de-Araujo et al, 1996;Dantas et al, 1996; Dantas et al, 1999; Yousef etal, 2007).
Yousef (2005) has studied the protective role of ascorbic acid on reproductive performanceof male rabbits given sublethal dose of tin, asstannous chloride. The results showed that treatment with SnCl2 caused a statistical decreasein the libido, ejaculate volume, spermconcentration, total sperm output, percentage of the sperm motility, total motile sperm per ejaculate, packed sperm volume, total functional sperm fraction, normal and live sperm and semeninitial fructose. Dead sperm and initial hydrogenion concentration were increased. While, relative weights of testes and epididymis were decreased.The results obtained suggest that assessment of reproductive toxicity of SnCl2 needs to be addressed, and may presently be underestimated.Also, the beneficial influence of ascorbic acid in counteracting the toxic effects of SnCl2 and improved the reproductive performance of malerabbit was demonstrated.
Due to the high relevance of the stannoussalts, several authors have reported different experimental models in various levels of organization to try to study its redox properties, as well as to try to identify products (natural and synthetic) that could reduce or abolish the effect of
the stannous salts. Melo et al (2001) have reportedstudies with Escherichia coli cultures evaluating the influence of crude extracts on the survival of the strain submitted to SnCl2. Fonseca et al (2005)have described evaluation of the effect ofAcetylsalicylic acid on the labeling of blood constituents with 99mTc. Yousef (2005) has reported studies with the protective effect of theascorbic acid to enhance reproductive performanceof male rabbits treated with stannous chloride.
As natural products are also increasinglyavailable in most industrialized countries of theworld, the use of the techniques allows to evaluate their biological and chemical properties becomehighly relevant. The labeling of blood constituents with 99mTc is a simple technique that has beenused to investigate redoxi properties of different natural products, as Paullinia cupana (Oliveira etal, 2002), propolis (Jesus et al, 2006), Fucusvesiculosus (Oliveira et al, 2003). In this technique is used a reducing agent, the stannous chloride. The compounds present in the natural extract withoxidant property will oxidize the stannous ion and the labeling of blood constituents will decrease.
Hypericum perforatum (St John's wort orhiperico) is an herbal medicine that has been widelyused to treat several diseases. Several studies have beenperformed to try to identify properties of thisphytotherapeutic agent. Meta-analytical studies show that St John's wort extracts are more effective than placebo in patients with mild and moderate depression (Newall et al, 1996; Werneke et al, 2004) anddysthymia (Rotblatt and Ziment, 2002). This plant alsohas been used as antiviral and diuretic, and to treatdiarrhea, dyspepsia, parasites, neuralgia, sciatica and rheumatism (Rotblatt and Ziment, 2002). Wang et al(2001) have reported that some components of St John'swort interfere with CYP3A4, one of the maincytochrome P450 isoenzymes. CYP3A4 is involved in the metabolism of many commonly used drugs. StJohn’s wort can interact with medicines that can resultin serious toxicological effects (Cordeiro et al, 2005).Mannel (2004) and Wada et al (2002) have describedthat the St John's wort reduces the efficacy of severalpharmacological compounds including,immunosuppressants (risk of graft rejection), oralcontraceptives (risk of pregnancy), oral anticoagulants(risk of thrombosis), and HIV protease inhibitors. It haspharmacodynamic effects on neuronal excitability(Neagoe et al, 2004). Sosa et al (2007) suggested thatdifferent constituents of St John’s wort have beeninvolved in anti-inflammatory activity. Capasso et al(2005) have reported that the St John’s wort directlyinhibits rat and human vas deferens contractility.Moreover, it is supposed that, if confirmed in vivo,these results might affect sexual function in humans
42
and, moreover, these results might explain the delayedejaculation described in patients receiving St John’swort. Also, the SnCl2 seems to generate free radicalsthat may be related to undesirable effects in thereproductive performance of male rabbits as well as theHypericum perforatum might affect sexual function inhumans.
The aim of this work is to present results aboutthe effect of an extract of this medicinal plant(Hypericum perforatum) on the labeling process of blood constituents with 99mTc, in which is usedstannous chloride as reducing agent to further correlatewith other physiological changes.
MATERIAL AND METHODS
The protocols of the experiments were performed without sacrificing the animals and was approved (CEA/113/2006) by the Ethical Committee of the Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Riode Janeiro.
Heparinized whole blood was withdrawnby cardiac puncture from male Wistar rats, 9animals, 3 months of age (young animals) and 293±13g of weight, and from male Wistar rats, 9animals, 6 months of age (elderly animals),359±22g of weight.
The extract of St John’s wort was preparedwith 5g of dust of Hypericum perforatum(Herbarium Laboratório Botânico LTDA, Brazil, lot 954661) in 100ml of 0.9% NaCl. The preparation was homogenized in a vortex mixerand filtered through paper (quality filter paper) and1ml of the filtered solution was considered to have50mg of the extract and it was denoted as 100%.The quality control of this extract was evaluatedwith the analysis of the absorption spectra of aHypericum perforatum extract recorded in the range from 400 up to 700nm with intervals of 20nm using a spectrophotometer (Analyser 800M,Analyser Comércio e Indústria LTDA, São Paulo,Brazil), using a cuvette of 1cm pathlength. Allspectrophotometric measurements were performedon the same spectrophotometer and the value of the absorbance at 580 nm (0.414±0.016) was usedas a marker and the quality control of eachprepared hiperico extract in the all experiments.
The tubes used in these experiments werepreviously closed with a rubber cap and a syringewas used to reduce the air atmosphere (vacuum)inside the vials.
Blood samples (0.5ml) either from youngor elderly animals were incubated with 100 l of St John’s wort extract (50mg/ml) for 1 hour at roomtemperature. Samples of heparinized whole blood were incubated with saline solution (0.9% NaCl) as control. Then, 0.5ml of a freshly prepared stannous chloride solution (1.2 g/ml), as SnCl2(Sigma, USA) was added and the incubationcontinued for another 1 hour. After this period of time, 99mTc (0.1ml), as sodium pertechnetate, recently milked from a 99Mo/99mTc generator(Instituto de Pesquisas Energéticas e Nucleares,Comissão Nacional de Energia Nuclear, Brazil),was added and the incubation continued foranother 10 min. These samples were centrifuged and plasma (P) and BC cells were separated.Samples (20 l) of P and BC were also precipitatedwith 1 ml of trichloroacetic acid (TCA) 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 wellcounter (Automatic Gamma Counter, Packard Instrument Co, Illinois, USA). After that, the percentage of radioactivity (%ATI) was calculatedas described elsewhere (Bernardo-Filho et al,1990; Bernardo-Filho et al, 1994; Lima-Filho etal, 2003).
Statistical analysis involved one-wayANOVA, followed by the Turkey-KramerMultiple 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).
RESULTS
Figure 1 shows the distribution of theradioactivity in the cellular and plasmacompartments isolated from whole blood fromyoung or elderly animals treated with the St John’swort extract and saline solution. The resultsindicates that there is a significant decrease(P<0.05) in radioactivity distribution of the 99mTcin the cellular compartment in presence of St John’s wort extract from 95.02±0.51 to 4.15±0.91when blood from young rats was used. However, when the blood from elderly rats was used, the St John’s wort extract was not capable to interfere inthe distribution of radioactivity in the cellular and plasma compartments.
43
0
10
20
30
40
50
60
70
80
90
100
young eldery young eldery
Control Treated
%A
TI CellPlasma
Figure 1 – Assessment of the effects of Hypericumperforatum extract on the distribution of theradioactivity in the cellular (cel) and plasmatic(plasma) compartments on blood of young andelderly animals.
The results in Figure 2 indicate that thereis a significant (P<0.05) decrease in theradionuclide fixation by the plasma proteins (IF-P) isolated from whole blood (young animals) treated with St John’s wort extract from 76.24±6.15 to26.67±1.70. However, when the blood from
Figure 2 – Assessment of the effects ofHypericum perforatum extract in the labeling with 99mTc of soluble (SF) and insoluble (IF) fractions of the plasma (P) on young and elderly animals.
elderly rats was used, the St John’s wort extract was not capable to interfere in the fixation of radioactivity in the plasma proteins (IF-P).
The results shown in Figure 3 indicate that there is a significant (P<0.05) decrease in theradioactivity fixation in the cellular proteins (IF-BC) isolated from whole blood (young animals)treated with St John’s wort extract from95.73±0.33 to 14.82±0.61. The fixation of radioactivity in the cellular proteins (IF-BC) obtained form elderly animals has also diminished,
and although this decrease would be significant(P<0.05), it was only a small alteration (from90.75±1.25 to 82.12±5.05).
Figure 3 – Assessment of the effects of Hypericum perforatum extract in the labeling with 99mTc of soluble (SF) and insoluble (IF) fractions of the BC of young and elderlyanimals.
DISCUSSION
The physiology of the ejaculation includesemission of sperm with the accessory gland fluid into the urethra, simultaneous closure of the urethral sphincters, and forceful ejaculation ofsemen through the urethra. The quality of thissperm can be affected in several conditions. Due tothe industrialization processes, the human beings have an important direct or indirect exposition to some metals that can adversely affect the malereproductive system by directly acting on the sperm plasma membrane or by catalyzing thegeneration of free radicals (Wu et al 1996; Naha and Chowdhury 2006; Kumar et al, 2006). Metal chelators control lipid peroxidation of the spermplasma membrane and protect the integrity of thespermatozoon, and also prevent DNA damage(Guthrie and Welch, 2007). Albumin,ceruloplasmin, and metallothionein are proteins that interact with iron and cooper and decrease the free radicals formation (Yousef; 2005; Agarwal etal, 2006).
The development of experimental modelsto try to verify the capability of a product to avoid the oxidation of the metals or to be a metalchelator is worthwhile. A model using a reducingagent could be useful due to it would be possible to study the oxidant property of some chemical
44
products, as the medicinal plants. The oxidation ofa reducing agent, as the stannous chloride, it mightbe convenient to prevent about their biologicaleffects as the generation of free radicals. Freeradicals generated due to the presence of stannous chloride could cause deterioration in the semen quality and could be undesirable to the malerabbits reproductive performance (Yousef, 2005).
The extract of hiperico was capable to interfere on the fixation of the Tc-99m on the blood constituents when blood withdrawn from young rats was used (Figure 1, 2 and 3). This extract could have an action similar to the vitamin C, as reported by Agarwal et al (2006) that hassuggested that vitamin C would neutralize hydroxyl, superoxide, and hydrogen peroxide radicals. In addition, it would also help recyclevitamin E, which would neutralize H2O2 and wouldprotect the plasma membrane from lipid peroxidation. It is also possible to argue thathiperico extract could act directly or indirectly(generation of free radicals), leading to the oxidation of the stannous salt. In this case, it would be possible to speculate that the stannouschloride could have a similar action to the vitamin C in the experimental model used by Yousef (2005). Moreover, Capasso et al (2005) havereported that the St John’s wort directly inhibits rat and human vas deferens contractility.Moreover, it is supposed that, if confirmed in vivo,these results might affect sexual function in humans and, these results might explain thedelayed ejaculation described in patients receivingSt John’s wort.
Some authors have reported that the free radical hypothesis states that aging results fromrandom deleterious events, and that self-inflicted oxidative damage is the primary contributor tosuch a stochastic degeneration of organisms. The hypothesis has been supported by experimentaldata that demonstrate that steady-state levels ofoxidation-damaged macromolecules increase with age (Orr and Sohal, 1994; Parkes et al, 1998; Sunet al, 2002). Some attempts have been made tocorrelate oxidation with a reduced activity of theoxidative defense systems. However, theseattempts have generated conflicting results and catalases have been demonstrated either increaseor decrease with the age, depending on the tissuesor organisms analyzed. Other studies have demonstrated that the abundance of someantioxidant defense proteins may actually increasewith age in some tissues. Similarly, in a
reproductively arrested population of E. coli cells,the levels of oxidative defense proteins increase and the population becomes increasingly resistant to external oxidative stresses (Matin, 1991; Hengge-Aronis, 2000). These findings could aid tounderstand that the extract of hiperico was not capable to interfere on the fixation of the Tc-99mon the blood constituents when blood withdrawn from elderly rats was used (Figures 1 and 2).
In conclusion, the findings described hereallow to set the bounds for a new assay to evaluate the action of products that could interfere with the male reproductive system. Moreover, the hipericoseems to alter the labeling of blood constituentswith 99mTc isolated from whole blood of younganimals. Then, it is supposed that this hiperico extract could protect the male reproductive systemagainst action of metals, as the stannous chloride, at least in young rats.
ACKNOWLEDGMENTS:We are gratefulness to Mr. Carlos Brown Scavarda (University of Michigan) for the English language revision. Financial support: CNPq,CAPES and UERJ.
RESUMO
Hypericum perforatum (hiperico) tem sidoutilizado para tratar diferentes distúrbios e tambéminibir a contractilidade do ducto deferente em ratose em humanos. Na medicina nuclear, o cloreto estanoso (SnCl2) é usado como um agente redutorpara obter radiofármacos marcados com tecnécio-99m. Como o SnCl2 parece acarretar efeitosindesejáveis relacionados com o desempenhoreprodutivo de coelhos machos e o hiperico pode afetar a função sexual em humanos, o objetivodesse trabalho é apresentar resultados sobre oefeito de um extrato de hiperico na marcação de constituintes sangüíneos com o tecnécio-99m retirados de ratos jovens e idosos. O hipericoparece alterar a marcação de constituintessangüíneos com tecnécio-99m isolados de sangue de animais jovens. Embora, esse resultado não seja observado em ratos idosos. Provavelmente, oextrato poderia apresentar uma ação protetora para o sistema reprodutivo contra a ação do SnCl2, pelo menos em ratos jovens. Os resultados descritosnesse trabalho permitem introduzir um ensaiosimples para avaliar a ação de produtos que
45
poderiam interferir com o sistema reprodutormasculino.
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47
3.3 MANUSCRITO SUBMETIDO
a- Biological Research, Qualis Internacional B
Morphologic and morphometric in vitro evaluation of red blood cell labeled with
technetium-99m: effects of the Hypericum perforatum and Mentha crispa extracts
Sebastião David Santos-Filho1,2, Adalgisa Ieda Maiworm1,2, Severo de Paoli1,2, Tânia
Giani1,2, Giuseppe Antonio Presta1,2 and Mario Bernardo-Filho1,3
1Departamento de Biofísica e Biometria, Instituto de Biologia Roberto Alcantara Gomes,
Universidade do Estado do Rio de Janeiro, 20551030, Rio de Janeiro, RJ, Brasil
2Programa de Pós-Graduação em Ciências da Saúde, Universidade Federal do Rio
Grande do Norte, 59010180, Natal, RN, Brasil
3Coordenadoria de Pesquisa, Instituto Nacional do Câncer, 20230130, Rio de Janeiro,
RJ, Brasil.
Running Title: Morphologic and morphometric in vitro evaluation of erythrocyte
Abstract
Morphologic and the morphometric analysis of red blood cells (RBC) have permitted to
evaluate alterations of area, shape and volume of the RBC. Technetium-99m(99mTc)-
labeled RBC are widely used in nuclear medicine. Hypericum perforatum(hiperico) and
Mentha crispa(mint) are utilized in herbal medicine. The aim of this work is to evaluate
possible morphologic and morphometric alterations on 99mTc-labeled RBC due to the
treatments with the hiperico or mint extract. Blood withdrawn from Wistar rats was
incubated with different concentrations of hiperico or mint extract. Stannous chloride
48
and sodium pertechnetate were added. In control, NaCl 0.9%(saline) was used. After
the incubation, one drop of each sample was smeared, stained and analyzed by optical
microscopy(morphologic and the morphometric evaluations). Statistical analysis was
performed. The qualitative comparison of the shape of the RBC(no treated and treated)
revealed only significant(p<0.05) alterations with the treatment with 6.5 and 25%
Mentha crispa extract. Perimeter/area of the RBC treated with mint extract showed
significant(p<0.05) alterations on 12.5%(0.77±0.03), 25%(0.73±0.04) and
50%(0.76±0.04) of mint extract when compared with control cells (0.67±0.05). Hiperico
extract has not altered the perimeter/area ratio and the shape of the RBC. The effects
may be due to the specific products of the mint extract that would be action on the RBC
membrane.
Key words: Red blood cell, Hypericum perforatum, Mentha crispa, Morphology,
Morphometric, Technetium-99m.
Corresponding author:
Sebastião David Santos-Filho
Universidade do Estado do Rio de Janeiro
Instituto de Biologia Roberto Alcântara Gomes
Departamento de Biofísica e Biometria
Laboratório de Radiofarmácia Experimental
Av 28 de setembro, 87,
20551-030 – Rio de Janeiro – RJ – Brasil
E-mail: [email protected]
Introduction:
49
The morphologic and the morphometric analysis of red blood cell (RBC) is an
important field of the clinical and laboratory investigations and have permitted to
evaluate possible alterations in the area, shape, volume and perimeter/area ratio of the
RBC. These findings are relevant in study of different pathological disorders (1-4).
Moreover, some authors have used these analyses to study the effect of plant extracts
on the morphology and the morphometry of the RBC (5-7).
Red blood cell labeled with technetium-99m (99mTc) scan is a nuclear study best
suited for identifying slow-bleeding sources as gastrointestinal bleeding (8, 9). Other
applications of this radiopharmaceutical (radiobiocomplex) (10) are (i) in the
determination of the left ventricular function by measuring the ejection fractions (11) and
(ii) in the evaluation of wall motion abnormalities (12, 13).
The RBC has been labeled with 99mTc for in vitro, in vivo or in vivo/ in vitro
techniques. The labeled process with 99mTc depends on a reducing agent and
stannous ion (Sn+2) is usually used for this purpose (13, 14). When whole blood is
employed on the labeling of RBC with 99mTc, radioactivity is mainly found on RBC (13),
however it is also bound on plasma proteins (15).
The labeling of blood constituents with 99mTc has been successfully used as an
experimental model to assess some important biological properties of natural products
extracts (5, 17-22).
Hypericum perforatum (hiperico) and Mentha crispa (mint; M.crispa) are utilized
in herbal medicine due to various pharmacological actions (23-26). Furthermore, the
extracts of these natural products have some chemical compounds that are common to
both, as phenylpropanoids (27) and flavonoids (28). Authors have reported that the
crude extracts of hiperico and mint are capable to interfere on the fixation of a
radionuclide on blood constituents (21, 22).
50
Mint flowers and leaves are used for tea infusions, which have digestive, calming,
antiseptic and anti-asthmatic properties (29). The extract and leaves of the Mentha
species are indicated as biological additives (30). Mint inhibits complement-dependent
inflammatory processes and may have therapeutic potential (25). Santos-Filho et al (21)
have described that an extract of mint was capable to interfere on the labeling of blood
cells with the Tc-99m, as well as, the fixation of this radionuclide on the plasma and
blood cells proteins. The main pharmacodynamic effect of mint oil is a dose-related
antispasmodic effect on the smooth musculature due to the interference of menthol with
the movement of calcium across the cell membrane, relevant to the gastrointestinal tract
(26).
Published meta-analyses show that hiperico extracts are more effective than
placebo in patients with mild and moderate depression (31). Trials show that hiperico is
effective as tricyclic and serotonin reuptake inhibitor antidepressants (24, 32). Hiperico
reduces the efficacy of several pharmacological groups including: immunosuppressant,
oral contraceptives, oral anticoagulants and HIV protease inhibitors (23). It has
pharmacodynamic effects on neuronal excitability (33). Santos-Filho et al (22) have
reported that an extract of hiperico leads to a significant (p<0.05) alteration on the
labeling of blood cells with 99mTc and in the fixation of the radioactivity on the plasma
and cells proteins.
As the morphologic alterations of the RBC could be related with some
undesirable effects, the aim of this work is to evaluate if hiperico or mint extract could
induce to morphologic and morphometric alterations on RBC labeled with 99mTc.
Material and methods:
The hiperico extract was prepared with 5g of dust of Hypericum perforatum
(Herbarium – http://www.herbarium.com.br -, Brazil, lot 954661, validity 06/08/06) in
51
100mL of 0.9% NaCl. The preparation was homogenized in vortex mixer, filtered
through paper (quality filter paper) and the filtered solution was considered to be
50mg/mL and 100%.
The mint extract was prepared with 4g of crushed leaves of Mentha crispa (Mãe
Terra Produtos Naturais – http://www.maeterra.com.br -, Brazil, lot 05FEV002, validity
06/05/2006) in 100mL of 0.9% NaCl. The preparation was centrifuged (clinical
centrifuge, 3000 rpm, 10 min), filtered through paper (quality filter paper) and the filtered
solution was considered to be 40mg/mL and 100%. The experiments with these lots of
these products were finished in the end of March 2006.
The protocols of the experiments were performed without sacrificing the animals
and was approved by the Ethical Committee of the Instituto de Biologia Roberto
Alcantara Gomes, Universidade do Estado do Rio de Janeiro (CEA/113/06).
Heparinized whole blood was withdrawn by cardiac puncture from adult male Wistar
rats, 3 animals, 3 months of age, 293±13g of weight following the Ethical guidelines of
the Institution.
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) hiperico or mint 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, 99mTc (0.1 ml), as sodium pertechnetate,
recently milked from a 99Mo/99mTc generator (Instituto de Pesquisas Energéticas e
52
Nucleares, Comissão Nacional de Energia Nuclear, Brasil), was added and the
incubation continued for another 10 min.
After the incubation with 99mTc, one drop of each sample was smeared in slides
(5 slides for each sample) and the May-Grünwald-Giemsa (MGG) method was
performed. The smear blood was fixed with methanol (Vetec, Brazil) for 5 min, then
stained with Giemsa (azure eosin methylene blue solution, Isofar, Brazil) for 10 min and
washed in methanol to remove excess of stain. The slides were stayed at room
temperature to dry. The stained slides with MGG were analyzed by optical microscopy
and for morphometric measurements a total five fields per each slide were evaluated. A
spherical shape and normal size distribution were assumed to RBC on control samples.
The following morphometric parameters were calculated: area ( m2); diameter ( m);
perimeter ( m) and sphericity (adimensional-[/]). A perimeter/area ratio was also
calculated (“Software” image pro plus, media Cibernetics, USA).
Statistical analyses 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, USA).
Results:
The comparison of the shape of the RBC (no treated and treated with natural
extracts) under optical microscopy has revealed only morphological significant (p<0.05)
alterations due to the treatment of blood with 6.5 and 25% Mentha crispa extract
(Figures 1, 2 and 3).
Morphometric values of perimeter/area of the RBC treated with mint extract
showed significant (p<0.05) alterations on 12.5% (0.77 ± 0.03), 25% (0.73 ± 0.04) and
53
50% (0.76 ± 0.04) concentrations of mint extract when compared with control cells (0.67
± 0.05) (Figure 4).
The hiperico extract has not significantly altered the morphology (Figure 5) and
the perimeter/area ratio (Fig 6) of the RBC.
Discussion:
In the clinical and in the laboratory investigations, the morphologic and the
morphometric analysis of the RBC are worthwhile and can aid to understand deviations
of the physiological conditions. The shape of a normal RBC is well known: under resting
conditions it is that of a biconcave discocyte. However, RBC can easily undergo
transformation to other shapes with stomatocytes and echinocytes as extremes. Various
anticancer agents, generally reactive and labile substances as the oxazaphosphorines
and the fluoropyrimidines, can induce severe deformation of shape. Shape changes in
RBC can induce rheological disturbances, which occasionally have pathophysiological
consequences. (4, 34).
The morphometric parameters, as area, shape and volume have used for several
authors. Engstrom and Lofvenberg (1) have described that after hydroxyurea treatment,
the RBC membrane area increased 24% and the cell volume increased 39% (P <.005)
with a 12% increase in the minimum cylindrical diameter of the RBC. Berezina et al (2)
showed RBC shape alterations appear within the first hours after trauma and persist for
at least 7 to 10 days. These changes are more severe in patients with secondary septic
complications. Panis and Souza (3) compared methods of analyzing changes in RBC
volume when exposed to osmotic challenge. The results reveal that hematocrit ratio,
mean corpuscular volume, optical density and mean cellular hemoglobin yield
significant volume changes and that hematocrit ratio is the most sensitive assay. Vaya
et al (4) showed a statistically significant correlation between RBC elongation indices
54
(EI) at 12, 30, and 60 Pa and the hematimetric indices, suggesting that the alteration in
shape which characterizes the iron deficiency anaemia, accounts in part for the
decreased EI. Concerning to other important approach related with the analysis of
morphologic and morphometric parameters, Oliveira et al (5) demonstrated that roast
coffee beans (Coffea arabica) extract altered the shape of RBC. Lima-Filho et al (6)
established that the phytic acid and the stannous fluoride solution, in different
concentrations, were capable of to alter the morphological properties of RBC. Oliveira et
al (7) evaluated that an aqueous extract of Pfaffia sp. did not alter the morphology of the
RBC.
The labeling of blood constituents with 99mTc has been successfully used as an
experimental model to assess some important biological properties of natural products
extracts. Extracts of Maytenus ilicifolia (16), Ginkgo biloba (17), Paullinia cupana (19),
Coffea arabica (5), Mentha crispa (21) and Hypericum perforatum (22) can alter the
labeling of RBC with 99mTc.However, extracts of Brassica olerace (18) and Sechium
edule (20) have not promoted alterations on the labeling of RBC with 99mTc.
The analysis of data presented in Figures 1, 2 and 3 shows that in the
concentrations of 6.25% (2.5mg/ml) and 12.5% (5mg/ml) the mint extract produced
significant (p<0.05) alterations on the shape of RBC. The hiperico extract did not induce
modifications on this morphological parameter (Figure 5).
The membrane bilayer and the network of membrane-associated proteins
together regulate the characteristic shape and elastic properties of RBC (35). The
primary cellular defect is loss of membrane surface area relative to intracellular volume,
leading to spheroidal shape and decreased deformability (36). Membrane loss is due to
defects in one of several membrane proteins, including ankyrin, band 3, spectrin and
protein 4.2 (37). The analysis of Figures 4 and 6 suggest that RBC have significant
55
(p<0.05) morphometric alterations in the perimeter/area ratio on the presence of the
mint extract in the concentrations of 12.5% (5mg/ml), 25% (10mg/ml) and 50%
(20mg/ml). The mint extract could have components that act in the RBC membrane
structure, probably with an interaction with the membrane proteins, inducing
deformation in RBC shape. In addition, RBC inflates at a rate proportional to their
surface area, in agreement with a constant flux model, and lyse after attaining a
spherical morphology. Spherical RBC display increased alkaline hemolysis fragility
(shorter lifetimes), providing an explanation for the increased osmotic fragility of the
RBC from patients who have spherocytosis (38). It should justify the perimeter/area
ratio of the 100% of mint extract be almost similar to control data.
The hiperico extract did not alter the perimeter/area of the RBC (Figure 6).
Although Hypericum perforatum and Mentha crispa have common chemical
compounds, as phenylpropanoids (27) and flavonoids (28) and these crude extracts
have some similar effects on the labeling of the blood constituents with Tc-99m, the
action on the morphology and on the morphometry of the RBC seems to be different.
The effects observed with the mint may be due to the specific products presents
in this extract that may (i) alter the morphology of RBC possibly with an action in the
erythrocyte membrane structure (membrane proteins) and/or (ii) change the
permeability of the RBC membrane showed with the perimeter/area ratio alteration.
Moreover, probably the method used to obtain the extracts could isolate different
chemical components of each tested natural products, independently on the chemical
agents present in each extract.
In conclusion, although these experiments were carried out with blood withdrawn
from rats and in vitro assays have been used, we suggest precaution on the
56
morphologic and morphometric analysis of blood obtained from patients that are using
Mentha crispa extracts.
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17- 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 2001; 44: 639-641.
18- Lima E.A.C., Diré G., Mattos D.M.M., Freiras R.S., Gomes M.L., Oliveira M.B.N.,
Faria M.V.C., Jales R.L., 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
2002; 40:919-23.
19- Oliveira, J. F.; Avila, A. S.; Braga, A. C. S.; Oliveira, M. B. N.; Boasquevisque, E. M.;
Jales, R. L.; Cardoso, V. N. and 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 2002; 73: 305-312.
20- 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. African J Biotechnol 2004; 3: 484-488.
21- Santos-Filho SD, Diré GL, Lima E, Oliveira MN, Bernardo-Filho M. (2004) 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.
22- Santos-Filho, S. D., Bernardo-Filho, M. Efeito de um extrato de Hipérico (Hypericum
perforatum) na marcação in vitro de elementos sanguíneos com tecnécio-99m e na
biodisponibilidade do radiofármaco pertecnetato de sódio em ratos Wistar. Acta Cir
Bras 20: 121-125, 2005.
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24- Mannel M. Drug interactions with St John's wort : mechanisms and clinical
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25- Renzulli C, Galvano F, Pierdomenico L, Speroni E, Guerra MC. Effects of
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26- Grigoleit HG, Grigoleit P. Pharmacology and preclinical pharmacokinetics of
peppermint oil. Phytomedicine. 2005; 12: 612-616.
27- Tognolini M, Barocelli E, Ballabeni V, Bruni R, Bianchi A, Chiavarini M, Impicciatore
M. Comparative screening of plant essential oils: phenylpropanoid moiety as basic core
for antiplatelet activity. Life Sci 2006; 78: 1419-1432.
28- Fiamegos YC, Nanos CG, Vervoort J, Stalikas CD. Analytical procedure for the in-
vial derivatization extraction of phenolic acids and flavonoids in methanolic and aqueous
plant extracts followed by gas chromatography with mass-selective detection. J
Chromatogr A. 2004; 1041: 11-18.
29- Park KJ, Vohnikova Z, Brod FPR. Evaluation of drying parameters and desorption
Isotherms of garden mint leaves (Mentha crispa L.). J Food Eng. 2002; 51: 193-199.
30- Davis EM, Ringer KL, McConkey ME, Croteau R. Monoterpene metabolism.
Cloning, expression, and characterization of menthone reductases from peppermint.
Plant Physiol. 2005; 137: 873-881.
31- Newall CA, Anderson LA, Phillipson JD. Herbal medicines: a guide for health-care
professionals. London: Pharmaceutical Press, 1996.
32- Beckmann SE, Roger WS, Switzer J. Consumer use of St. John’s wort: a survey of
effectiveness, safety and tolerability. Pharmacotherapy 2000; 20: 568-574.
33- Neagoe I, Macri BM, Flonta ML. Hyperici herba extract interaction with artificial lipid
bilayers. J Pharm Pharmacol. 2004; 56: 1283-1289.
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34- Dumez H, Guetens G, De Boeck G, Highley MS, Maes RA, van Oosterom AT, de
Bruijn EA. The relevance of therapeutic drug monitoring in plasma and erythrocytes in
anti-cancer drug treatment. Clin Chem Lab Med. 2004; 42: 1219-1227.
35- Khanna R, Chang SH, Andrabi S, Azam M, Kim A, Rivera A, Brugnara C, Low PS,
Liu SC, Chishti AH. Headpiece domain of dematin is required for the stability of the
erythrocyte membrane. PNAS 2002; 99: 6637-6642.
36- Eber S, Lux SE. Hereditary spherocytosis – defects in proteins that connect the
membrane skeleton to the lipid bilayer. Semin Hematol. 2004; 41:118-141.
37- Gallagher PG. Update on the clinical spectrum and genetics of red blood cell
membrane disorders. Curr Hematol Resp. 2004; 3:85-91.
38- Ionescu-Zanetti C, Wang LP, Di Carlo D, Hung P, Di Blas A, Hughey R, Lee LP.
Alkaline hemolysis fragility is dependent on cell shape: results from a morphology
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FIGURES
Figure 1: Samples of whole blood were incubated with NaCl 0.9% solution for 60 min.
After that, stannous chloride solution was added and the incubation continued for 60
min. Then, 99mTc, as sodium pertechnetate was added. Blood smears were prepared,
dried, fixed and staining with the May-Grünwald-Giemsa method. After that, the
morphology of the red blood cells was evaluated under optical microscope (x 1000).
61
Figure 2: Samples of whole blood were incubated with mint extract (6.25%) for 60 min.
After that, stannous chloride solution was added and the incubation continued for 60
min. Then, 99mTc, as sodium pertechnetate was added. Blood smears were prepared,
dried, fixed and staining with the May-Grünwald-Giemsa method. After that, the
morphology of the red blood cells was evaluated under optical microscope (x 1000).
Arrows indicate some erythrocytes with morphological alterations.
Figure 3: Samples of whole blood were incubated with mint extract (25%) for 60 min.
After that, stannous chloride solution was added and the incubation continued for 60
min. Then, 99mTc, as sodium pertechnetate was added. Blood smears were prepared,
dried, fixed and staining with the May-Grünwald-Giemsa method. After that, the
morphology of the red blood cells was evaluated under optical microscope (x 1000).
Arrows indicate some erythrocytes with morphological alterations.
62
Figure 4: Samples of whole blood were incubated with different concentrations of mint
extract for 60 min. After that, stannous chloride solution was added and the incubation
continued for 60 min. Then, 99mTc, as sodium pertechnetate was added. Blood smears
were prepared, dried, fixed and staining with the May-Grünwald-Giemsa method. The
MGG glass blades were analyzed by optical microscopy and for morphometric
measurements a total five fields/blade were evaluated. A spherical shape and normal
size distribution were assumed to erythrocyte on control blades. The following
morphometric parameters were calculated: area ( m2); diameter ( m); perimeter ( m)
and sphericity (adimensional-[/]). A perimeter/area ratio was calculated (“Software”
image pro plus, média Cibernetics, USA).
Figure 5: Samples of whole blood were incubated with hiperico extract (100%) for 60
min. After that, stannous chloride solution was added and the incubation continued for
63
60 min. Then, 99mTc, as sodium pertechnetate was added. Blood smears were
prepared, dried, fixed and staining with the May-Grünwald-Giemsa method. After that,
the morphology of the red blood cells was evaluated under optical microscope (x 1000).
Figure 6: Samples of whole blood were incubated with different concentrations of
hiperico extract for 60 min. After that, stannous chloride solution was added and the
incubation continued for 60 min. Then, 99mTc, as sodium pertechnetate was added.
Blood smears were prepared, dried, fixed and staining with the May-Grünwald-Giemsa
method. The MGG glass blades were analyzed by optical microscopy and for
morphometric measurements a total five fields/blade were evaluated. A spherical shape
and normal size distribution were assumed to RBC on control blades. The following
morphometric parameters were calculated: area ( m2); diameter ( m); perimeter ( m)
and sphericity (adimensional-[/]). A perimeter/area ratio was calculated (“Software”
image pro plus, média Cibernetics, USA).
64
4 COMENTÁRIOS, CRÍTICAS E CONCLUSÕES.
A experiência como Professor de Biofísica no ensino superior no
Centro Universitário de Volta Redonda, na cidade de Volta Redonda, Rio de Janeiro,
despertou o interesse do autor desse estudo na busca por uma maior qualificação
profissional, primeiramente no curso de mestrado, em seguida no curso de doutorado.
Na tentativa de aprofundar os conhecimentos referentes aos efeitos
produzidos por produtos naturais, apresentamos junto ao Programa de Pós-Graduação
em Ciências da Saúde o projeto de tese intitulado “Comparação de efeitos dos extratos
de Hypericum perforatum (Hipérico) e Mentha crispa (Hortelã) em diferentes modelos
experimentais”.
O desenvolvimento desse trabalho teve como base estudos de
marcação com tecnécio-99m e de biodistribuição do pertecnetato de sódio como bons
indicadores dos efeitos produzidos pela hortelã e pelo hipérico. O aprendizado das
técnicas não foi difícil pois são simples e de fácil execução, e as condições laboratoriais
favoráveis. Obtive resultados preliminares, que apresentei no projeto de doutorado
encaminhado ao programa de pós-graduação em Ciências da Saúde. Posteriormente
conclui cada um dos experimentos satisfatoriamente de acordo com cronograma pré-
estabelecido e constante do projeto.
Modelos experimentais em níveis molecular e celular foram
utilizados para avaliar os efeitos biológicos produzidos pelos extratos estudados.
Com parte dos resultados obtidos com os estudos com o hipérico
elaboramos um artigo, intitulado “Efeito de um extrato de hipérico (Hypericum
perforatum) na marcação in vitro de elementos sangüíneos com tecnécio-99m e na
65
biodisponibilidade do radiofármaco pertecnetado de sódio em ratos Wistar”, que foi
publicado no periódico “Acta Cirúrgica Brasileira”, indexado no Medline, Qualis
Internacional C.
Como parte dos resultados obtidos nos estudos sobre a hortelã
elaboramos um outro manuscrito, intitulado “Aqueous extract of the medicinal plant
Mentha crispa alter the biodistribution of the radiopharmaceutical sodium pertechnetate
in Wistar rats”, que foi aceito para publicação no periódico “Medicinal Chemistry
Research”, indexado no Medline, Qualis Internacional C.
Os estudos feitos com o hipérico sobre sobrevivência bacteriana e
mobilidade eletroforética de DNA plasmidial originou um outro manuscrito intitulado
“Influence of an aqueous extract of Hypericum perforatum (Hypericin) on the survival of
Escherichia coli AB1157 and on the electrophoretic mobility of pBSK plasmid DNA” que
foi aceito para publicação no periódico “Revista Brasileira de Farmacognosia”, indexado
no Scielo, Qualis Nacional A.
Os estudos feitos com o hipérico sobre diferenças no processo de
marcação de constituintes sanguíneos com tecnécio-99m, entre animais com 3 e 6
meses de idade, originou o manuscrito intitulado “The male reproductive system and
the effect of an extract of a medicinal plant (Hypericum perforatum) on the labeling
process of blood constituents with technetium-99m”, que foi aceito para publicação no
periódico “Brazilian Archives of Biology and Technology”, indexado no Scielo, Qualis
Internacional B.
Com os resultados encontrados na elaboração dos manuscritos
anteriores ficamos motivados a realizar novos estudos e conhecer melhor o efeito dos
extratos na marcação com tecnécio-99m, na morfologia e morfometria das hemácias.
Estudos feitos com esfregaços sanguíneos seguidos de captura de imagens em
66
microscopia de luz e análise dos resultados através da determinação da relação
perímetro/área originaram mais um manuscrito intitulado “Morphologic and
morphometric in vitro evaluation of red blood cell labeled with technetium-99m: effects
of the Hypericum perforatum and Mentha crispa extracts” submetido ao periódico
“Biological Research”, indexado no Medline, Qualis Internacional B.
Ao concluirmos nosso estudo, verificamos que compostos químicos
existentes nos extratos estudados são os responsáveis pelos efeitos produzidos e que
caracterizam o mecanismo de ação da planta medicinal nos diferentes níveis de
organização estrutural, da molécula a célula e ao tecido biológico.
A alteração da distribuição normal dos radiotraçadores devido a
interação medicamentosa pode comprometer o diagnóstico e/ou levar a necessidade
de repetição de exames, expondo o paciente a doses de radiação desnecessárias.
Esta interação medicamentosa é de importância para profissionais
de saúde que utilizam os exames da medicina nuclear para tratamento e diagnóstico de
diversas doenças como os médicos e os fisioterapeutas. O fisioterapeuta é profissional
de nível superior que prescreve, ministra e supervisiona terapia física com o objetivo de
presenciar, manter, desenvolver ou restaurar a integridade de um órgão, sistema ou
função do corpo humano, através de terapias físicas e terapias cinéticas. Para um bom
êxito do tratamento utilizado o fisioterapeuta 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 fisioterapeuta, tenham novos parâmetros
sobre os efeitos das plantas medicinais estudadas sobre o organismo humano.
67
Deseja-se também que as informações levantadas por esta pesquisa
sirvam 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 nesses anos do
Curso de Doutorado 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 Doutorado e a
aquisição de novos conhecimentos poderão ser relevantes para ações em projetos
futuros junto a grupos de pesquisa da Universidade do Estado do Rio de Janeiro e/ou
do Centro Universitário de Volta Redonda, e na orientação de novos alunos nos
programas de pós-graduação.
68
5 ANEXOS
ANEXO 1
Carta de aceite do manuscrito I
----- Original Message -----From: "Medicinal Chemistry Research" <[email protected]>To: <[email protected]>; <[email protected]>Sent: Thursday, July 12, 2007 4:01 PMSubject: Decision on your manuscript #MCRE-51R1
Dear Santos-Filho:
I am pleased to inform you that your manuscript, "Aqueous extract of the medicinal plant Mentha crispa alters the
biodistribution of the radiopharmaceutical sodium pertechnetate in Wistar rats." has been accepted for publication in
Medicinal Chemistry Research.
If you haven't already sent the signed Copyright Transfer Form, we will need to receive it. You can locate the form
on the journal's Welcome Page at:
http://mcre.edmgr.com/
Please print the form, sign it, and return it to us by fax at 781-878-0449. Thank you. Your manuscript cannot be
published until we receive the signed form.
Sincerely yours,
Stephen Cutler
Editor-in-Chief, Medicinal Chemistry Research
69
ANEXO 2
Carta de aceite do manuscrito 2
De: “José Maria Barbosa Filho” <[email protected]>
Para: <[email protected]>
Cc: <[email protected]>
Enviada em: terça-feira, 17 de julho de 2007 18:04
Assunto: Revista Brasileira de Farmacognosia - CARTA DE ACEITAÇAO
Prezado Prof. Sebastião Santos-Filho,
Informo que o manuscrito intitulado “Influence ofan aqueous extract ofHypericum perforatum (Hypericin)
on the survival of Eicherichia coli ABI 157 and on the electro phoretic mobility ofpBSK plasmid DNA”, de
autoria de Sebastião D. Santos-Filho, Raquel M. Bernardo, kelly C.M. Santos, Adenilson 5. Fonseca e
Mário Bernardo-Filho, foi aceito para publicação na REVISTA BRASILEIRA DE FARMACOGNOSIA
Atenciosamente,
José Maria Barbosa Filho
Editor Chefe
70
ANEXO 3
Carta de aceite do manuscrito 3
Brazilian Archives of Biology and Technology
DECLARAÇÃO
Declaramos para os devidos fins que o artigo: “The male reproductive system and the effect of an extract of a medicinal plant (Hypericumperforatum) on the labeling process of blood constituents with technetium-99m”, de autoria de Sebastião David Santos-Filho, Adenilson
de Souza da Fonseca and Mario Bernardo-Filho, foi aceito e será
publicado no Brazilian Archives of Biology and Technology.
Curitiba, 19 de julho de 2007
Prof. Dr. Carlos Ricardo Soccol Editor
71
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7 ABSTRACT
Several clinic evaluations have been possible with radiobiocomplexes labeled with
technetium-99m (99mTc). Some natural and synthetic drugs are capable of to interfere
on the labeling of blood constituents with 99mTc, as well as on the biodistribution of
radiobiocomplexes. Authors have also reported about the toxicity of several natural
products. The aim of this study was to compare the effects of the Mentha crispa
(hortelã) and of the Hypericum perforatum (hipérico) in different experimental models.
On the labeling of red blood cells (RBC) and plasma and cellular proteins with 99mTc,
both extracts were capable of to decrease the radioactivity percentage on the cellular
compartment and on the fixation on plasma and cellular proteins. On the morphometry
of the RBC, only the hortelã was capable to alter the shape and the perimeter/area ratio
of the RBC. On the biodistribution of the radiobiocomplex sodium pertechnetate
(Na99mTcO4), the hortelã increased the Na99mTcO4 distribution in the kidney, spleen,
liver and thyroid, meanwhile the hipérico decreased the Na99mTcO4 distribution in the
bone, stomach, lungs and thyroid, and increased the Na99mTcO4 distribution in the
pancreas. On the bacterial cultures survival, the hipérico was capable of to protect the
bacteria against the stannous chloride (SnCl2) effect. The hipérico did not alter the
topology of plasmidial DNA and did not protect the plasmidial DNA against the SnCl2
action. Probably, the effects presented by both extracts could be due to chemical
compounds of the extracts that could alter the morphology of the RBC and the plasma
membrane ions transport, and/or by phytocomplexes that could be formed with different
effects dependent on the biological system considered.
Key-words: red blood cells; plasma proteins, technetium-99m; radiobiocomplexes; rats;
Hypericum perforatum; Mentha crispa.