I
UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
EFEITO DE PREPARAÇÕES DE PRODUTOS NATURAIS (CRAVO DA ÍNDIA E TOMATE) E DE UM PRODUTO SINTÉTICO NA MARCAÇÃO DE CONSTITUINTES
SANGÜINEOS COM TECNÉCIO-99m E NA MORFOLOGIA DE HEMÁCIAS ISOLADAS DE SANGUE DE RATOS Wistar
SEVERO DE PAOLI
II
EFEITO DE PREPARAÇÕES DE PRODUTOS NATURAIS (CRAVO DA ÍNDIA E TOMATE) E DE UM PRODUTO SINTÉTICO NA MARCAÇÃO DE CONSTITUINTES
SANGÜINEOS COM TECNÉCIO-99m E NA MORFOLOGIA DE HEMÁCIAS ISOLADAS DE SANGUE DE RATOS Wistar
SEVERO DE PAOLI
Tese apresentada à Universidade Federal do Rio Grande do Norte, Centro de Ciências da Saúde, Coordenação do Programa de pós-graduação em Ciências da Saúde para obtenção do título de Doutor em Ciências da Saúde.
Orientador: Mario Bernardo Filho Co-orientador: Aldo da Cunha Medeiros
III
UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
Professor Doutor Aldo da Cunha Medeiros
Coordenador do Programa de Pós-graduação em Ciências da Saúde
IV
SEVERO DE PAOLI
EFEITO DE PREPARAÇÕES DE PRODUTOS NATURAIS (CRAVO DA ÍNDIA E TOMATE) E DE UM PRODUTO SINTÉTICO NA MARCAÇÃO DE CONSTITUINTES
SANGÜINEOS COM TECNÉCIO-99m E NA MORFOLOGIA DE HEMÁCIAS ISOLADAS DE SANGUE DE RATOS Wistar
PRESIDENTE DA BANCA: Prof. Dr. Mário Bernardo Filho – Universidade do Estado do Rio de Janeiro
Prof. Dr. Geraldo Barroso Cavalcanti Junior – Universidade Federal do Rio Grande do Norte
Prof. Dr. Adenilson de Souza da Fonseca – Universidade do Estado do Rio de Janeiro
Prof ª. Drª. Lúcia Maria da Cunha Galvão – Universidade Federal do Rio Grande do Norte
Prof ª. Drª. Ivonete Batista de Araujo – Universidade Federal do Rio Grande do Norte
Natal / RN
V
DEDICATÓRIA
In memoriam, dedico este título aos meus pais Vincenzo de Paoli e Assunta Siciliano de Paoli que ensinaram a ter persistência, paciência e determinação, com o foco humanitário sempre ao alcance dos sonhos.
VI
AGRADECIMENTOS
Ao Professor Mário Bernardo Filho, que como amigo e orientador soube indicar caminhos e sugerir soluções para que esta caminhada pudesse ter êxito, minha amizade, respeito e admiração.
Aos amigos Giuseppe Antonio Presta, Tânia Santos Giani, Adalgisa Ieda Maiworm e Sebastião David dos Santos Filhos meus irmãos na ciência, em todos os momentos difíceis e nos momentos de alegria muito obrigado.
Aos amigos e Professores Laucyr Pires Domingues, Miguel Haroldo Guida, Roberto da Silva Hertal, Edson Gomes de Souza, Eduardo Moiolli e Jayme Leão Guitman, pelos estímulos constantes no magistério e na odontologia.
Aos Amigos e colegas Carlos Guilherme Correa, Neide Lemos de Azevedo, Jorge José de Carvalho, Octávio Marinho Falcão Filho, Tereza Hucks Gallo, Walker André Chagas, Jorge Mamede de Almeida, Luiz Carlos Nogueira, Gilberto Soares Vargas, Raquel Mattos Bernardo e Adenilson de Souza Fonseca meus agradecimentos pela ajuda e compreensão que tiveram para que este sonho fosse realizado.
Ao Professor Doutor Aldo da Cunha Medeiros e ao Professor Doutor José Brandão Neto pelo sempre carinhoso recebimento em Natal e pela atenção constante com nossas necessidades. Minha profunda gratidão
À Doutora Lúcia de Fátima Amorim por toda a ajuda dada aos contatos com a Universidade Federal do Rio Grande do Norte e a hospitalidade carinhosa de seu recebimento em Natal.
VII
LISTA DE ABREVIAÇÕES, SIGLAS E SÍMBOLOS. %ATI Porcentagem de radioatividade
°C Graus Celcius
μg Micrograma
µl Microlitro
99Mo/99mTc Isótopo do elemento molibdênio de número de massa 99/ isótopo metaéstavel do elemento tecnécio de número de massa 99
99mTc Isótopo metaéstavel do elemento tecnécio de número de massa 99 99mTc-RBC Células vermelhas do sangue marcadas com tecnécio-99m
ANOVA Análise de variância
C Células cm Centímetro
CS Células sangüíneas
FI-C Fração insolúvel da célula FS-P Fração solúvel da célula g Grama
kg Quilograma
LDL Lipoproteína de baixa densidade
MBq Mega Bequerel (unidade de atividade de amostra radioativa no Sistema Internacional, sendo que 1 Bq equivale a uma desintegração por segundo)
mg Miligrama
min Minutos
ml Mililitro
Na99mTcO4 Pertecnetato de sodio NaCl Cloreto de sodio
ηm Nanometro
OZE Òxido-de-zinco-eugenol P Plasma
CVS Células vermelhas do sangue
rpm Rotações por minuto
Sn+2 Ion estanoso SnCl2 Cloreto estanoso
VIII SUMÁRIO
Lista de abreviações………...………....viii
Resumo………...Ix 1. Introdução...01
2. Revisão de literatura...03
3. Indexação de Artigos ...06
3.1. Manuscritos aceitos para publicação...06
3.2 Manuscrito submetido para publicação... 29
4. Comentários, críticas e conclusões... 44
5. Anexos...46
6. Referências...47
IX RESUMO
X
1. INTRODUÇÃO
O interesse da comunidade cientifica em estudar efeitos biológicos de produtos naturais e sintéticos vem crescendo ao longo dos tempos. Modelos experimentais empregando radionuclídeos têm sido largamente utilizados nos estudos de fenômenos de natureza biológica, assim como na clínica, na terapia e/ou diagnóstico de doenças (1,2,3)
Em Medicina Nuclear são utilizados radiofármacos, sendo que o radionuclídeo tecnécio-99m (99mTc) tem sido o mais empregado na obtenção de radiobiocomplexos que são usados para auxiliar no diagnóstico de doenças. Constituintes sangüíneos marcados com 99mTc, como hemácias e proteínas plasmáticas, têm sido utilizados como radiofármacos. Tem sido descrito que produtos naturais e sintéticos podem alterar a marcação desses componentes sangüíneos (4,5).
Estudos foram realizados com sangue de ratos Wistar (i) com um extrato de cravo-da-índia (Caryophyllus aromaticus L) que possui eugenol na sua constituição, com (ii) a droga sintética usada como cimento em diversas especialidades odontológicas, o OZE (óxido-de-zinco-eugenol) e (iii) com um extrato de tomate (Solanum lycopersicum) que contém licopeno. (6,7,8)
O objetivo da presente pesquisa foi avaliar com um extrato de cravo da índia e com uma preparação de OZE o efeito desses produtos na marcação de constituintes sangüíneos com 99mTc e na morfologia de hemácias obtidas do sangue de ratos Wistar. Com o extrato de tomate, verificar a ação do mesmo na marcação de constituintes sangüíneos com 99mTc.
2. REVISÃO DA LITERATURA
O tecnécio-99m – (99mTc) é um radionuclídeo, isótopo do tecnécio que é classificado como um metal de transição do grupo VII. Possui uma meia-vida de 6 horas, emissão de radiação gama de 140 keV de energia e apresenta estados de oxidação que variam de –1 a +7. (15.16)
As características físico-químicas do 99mTc, como pertecnetato de sódio, têm permitido a marcação de inúmeras estruturas celulares e moleculares com esse radionuclídeo que são empregados como radiofármacos e também denominados radiobiocomplexos (3,5) Radiobiocomplexos são traçadores radioativos empregados na medicina nuclear para ajudar no diagnóstico e/ou no tratamento das doenças. Células vermelhas do sangue marcadas com 99mTc (99mTc- CVS) são radiobiocomplexos usados freqüentemente na medicina nuclear, para diversas e importantes aplicações clínicas (2,5,9,10).
O desenvolvimento de modelos biológicos para o estudo da influência de drogas medicamentosas na marcação de constituintes sangüíneos com 99mTc é relevante para a pesquisa básica e aplicada. Pode-se estimar a capacidade redutora ou oxidante de substâncias quando o processo de marcação dos componentes sangüíneos com 99mTc é empregado. Da mesma forma, a capacidade de determinadas drogas de alterar a permeabilidade da membrana eritrocitária também tem sido avaliada por esse modelo experimental (9,10,11)
A ligação do 99mTc aos elementos sangüíneos pode ser alterada pela presença de produtos sintéticos e também naturais (13-18). Alguns estudos têm relatado que quando doses clínicas de propranolol, verapamil, clorotiazida são incubadas com sangue, uma redução na eficiência de marcação das hemácias é observada. Drogas anticonvulsivantes como fenitoína e fenobarbital, bem como o ácido acetil salicílico, reduzem a marcação de constituintes sangüíneos (16,17,19).
Existem alguns estudos sobre o efeito de produtos naturais na marcação de hemácias e proteínas plasmáticas e celulares com tecnécio-99m. Extratos de Nicotiana tabacum (15), Shechium edule (18), Maytenus ilicifolia (20), Hiperycum perforatum (21) e Psydium guajava (22) foram capazes de alterar a marcação de constituites sangüíneos com 99mTc.
O OZE é um medicamento largamente utilizado na Medicina e na Odontologia, com propriedades: antiinflamatória, anestésica, germicida, regenerador tecidual e como material cimentante de canais e obturações. (7,28)
O tomate (Solanum lycopersicum) é um fruto rico em ácido ascórbico, beta-caroteno, licopeno (aproximadamente 3,5mg/100g) e sais minerais (cobre, por volta de 3,5mg/100g), sendo consumido cru, cozido, condimentado e em conserva, em larga escala no mundo (8). Sua qualidade precisa ser muito bem avaliada, devido aos pesticidas e às técnicas de agricultura, evitando acréscimos indesejáveis de subtâncias tóxicas ao produto. Diversos estudos demonstram que o licopeno apresenta um efeito preventivo no câncer sendo inversamente proporcional aos fatores de crescimento dos níveis insulínicos no câncer de próstata (24,25).
3. INDEXAÇÃO DE ARTIGOS 3.1 MANUSCRITOS ACEITOS PARA PUBLICAÇÃO
Manuscrito 1:
Effects of clove (
Caryophyllus aromaticus
L
.
) on the labeling
of blood constituents with Technetium-99m and on the
morphology of red blood cells
Severo de Paoli.1,2,4,5*, Tania Santos Giani1,2,4, Giuseppe Antonio Presta 1,2, Marcia Oliveira Pereira2,4, Adenilson de Souza da Fonseca2,5, Sebastião David Santos-Filho 1,2 and Mário Bernardo-Filho2,3.
1Universidade Federal do Rio Grande do Norte, Programa de Pós-Graduação em Ciências da Saúde, Avenida
General Gustavo Cordeiro de Farias, s/n, CEP 59 010 180, Natal, RN, Brasil, severodepaoli@gmail.com
2Universidade do Rio de Janeiro, Instituto de Biologia Roberto Alcantara Gomes, Departamento de Biofísica e
Biometria, Avenida 28 de Setembro, 87 fundos, 4o andar, Vila Isabel, CEP 20 551 030, Rio de Janeiro
3Instituto Nacional do Câncer, Coordenadoria de Pesquisa Básica, Praça Cruz Vermelha, 23, Centro, CEP 20 230
130 Rio de Janeiro, Brasil
4Universidade Estácio de Sá, Rua do Bispo, 83, Rio Comprido, CEP 20 261 063, Rio de Janeiro, Brasil
5Centro Universitário Fundação Educacional Serra dos Órgãos, Faculdades de Odontologia e Fisioterapia,
Avenida Alberto Torres 111, Alto, CEP 25 964 004, Teresópolis, Rio de Janeiro, Brasil
ABSTRACT
Clove (Caryophyllus aromaticus L.) has been used for clinical procedures. Blood constituents labeled with technetium-99m (99mTc) are used in nuclear medicine. The aim of this work was to evaluate the effects of clove extract on the labeling blood constituents with 99mTc and on the morphologic red blood cells. Blood samples were incubated with clove, stannous chloride and 99mTc. Plasma, blood cells, insoluble fractions of plasma and blood cells were separated. The radioactivity was counted and percentage of radioactivity (%ATI) to each blood fraction was calculated. The shape and morphometric parameter (perimeter/area ratio) were evaluated. Clove extract altered significantly (p<0.05) the %ATI of blood constituents and the shape of red blood cells without modifying the perimeter/area ratio. The results indicate that clove extract present chemical compounds that interfere with the radiolabeling of blood constituents and alter the morphology of red blood cells by oxidative/chelating actions or interacting with the cellular membrane structure.
Key words: Technetium-99m, Blood constituents, Red blood cells, Morphology, Caryophyllus aromaticus L.
INTRODUCTION
Cloves (Caryophyllus aromaticus L, or
Syzygium aromaticum) are the aromatic dried flower buds of a tree belonging to the family Myrtaceae. It is native to Indonesia and used as a spice.
The clove tree is an evergreen that grows to a height ranging from 10-20 meters having large oval leaves and crimson flowers in numerous
groups of terminal clusters. The flower buds are at first of a pale color and gradually becoming green, after which they develop into a bright red, when they are ready for collecting. Cloves are harvested when 1.5-2 cm long, and consist of a long calyx, terminating in four spreading sepals, and four unopened petals which form a small ball in the center (Bisset, 2001).
of gastrointestinal, due to increase hydrochloric acid in the stomach and to improve peristalsis (Kumari, 1991), circulatory disturbances (Saeed and Gilani, 1994) and as anti-carcinogen agent (Banerjee et al., 2006).
Clove has been used in humans for dentistry applications for over two thousand years to alleviate the pain of toothache and also widely used to disinfect root canals in temporary fillings and as an oral anesthetic (Duke, 1985).
Eugenol has pronounced antiseptic and anaesthetic properties (Chaieb et al., 2007). Other constituents of the clove are acetyl eugenol, beta-caryophylline, vanillin, crategolic acid, tannins, gallotannic acid, methyl salicylate, flavanoids (eugenin, kaempferol, rhamnetin, and eugenitin), triterpenoids (oleanolic acid, stigmasterol and campesterol) and several sesquiterpenes (Kramer, 1985; Musenga et al., 2006).
Eugenol is known to inhibit the growth of bacteria and is a natural antibiotic (Suresh-Babu and Madhavi, 2001) with broad antimicrobial activities against gram-positive, gram-negative and acid fast bacteria, as well as fungi (Bisset, 1994; Lueng and Foster, 1996).
Red blood cells labeled with technetium-99m (technetium-99mTc-RBC) are widely used in clinical nuclear medicine for several important applications (Wong et al., 2004; Jin et al., 2004; Zaman et al., 2004; Artiko et al., 2004; Harel et al., 2005; Verdu et al., 2005; Olds et al., 2005).
The labeling of RBC with 99mTc has been used as assay to evaluate the properties of different chemical agents (Abreu et al., 2006; Fonseca et al., 2007). The radiolabeling depends of the presence of a reducing agent and stannous chloride is widely utilized. This technique is easily carried out and produces a better and well-controlled product (Harbert et al., 1996; Saha, 2004).
The morphological analysis of the RBC has been of importance in the investigations of diseases (Bielory, 2004) and to evaluate the effects of natural products on membrane structures (Oliveira et al., 2003).
The aim of this work was to evaluate in vitro, the effects of clove on the labeling blood constituents with 99mTc and on the morphology red blood cell.
MATERIALS AND METHODS Animals
All the experimental procedures followed the Ethical Guidelines of the Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro with the protocol number CEA/116/2006.
The animals were kept under environmental conditions (25±2°C, 12h of light/dark cycle), water ad libitum and normal diet. Heparinized whole blood was withdrawn by cardiac puncture from adult male Wistar rats under anesthesia by sodium thiopental, 40mg/kg of weight (n=12, 3 months, 245±35g).
Clove extract preparation
Dry clove flowers (Caryophyllus aromaticus L) (1g) (Fumel Comercial e Industrial Ltda., lot number 12, December 2006, validity December 2007) were triturated and 1g of the triturate was mixed with 10ml of 0.9% NaCl solution (saline). After that, the mixture was boiled at 100ºC and filtered through filter paper (Aldrich Chemical Co, 11cm, Lot number k932). The filtered solution was considered 100mg/ml of aqueous clove extract.
Spectrophotometric measurements
A spectrophotometric analysis (Analyser, 800M, São Paulo, Brazil) of the extract at 10 mg/ml was carried out. The absorbance at 480 nm was considered the marker of the quality control of this extract. All the prepared extracts to be used in the experimental procedures had an optical density of 0.49±0.01 (Figure 1).
Labeling of blood constituents with 99mTc
Samples of whole blood (0.5ml) were incubated with 100µl clove extract at different
concentrations (0, 6.5, 12.5, 25, 50 and 100mg/ml) for 1 hour. After that, 0.5ml of a freshly prepared stannous chloride solution (SnCl2, 1.2μg/ml,
and soluble (SF) and insoluble (IF) fractions were obtained. The radioactivity (% ATI) in P, BC, IF-P, SF-IF-P, IF-BC and SF-BC was determined in a well gamma counter (Clinigamma, gamma counter, Packard Instrument Company, mod C5002, USA). The percentage of incorporated radioactivity (%ATI) was calculated as described previously (Bernardo-Filho et al, 1986).
Morphological evaluation
Blood samples (n=5) for each extract concentration) were incubated with clove extract at different concentrations (6.5, 12.5, 25, 50 and 100mg/ml) for 1 hour. After that, blood smears (n=5, for each extract concentration) were prepared, dried at room temperature and stained by May-Grünwald-Giemsa method. Briefly, the blood smears were fixed with methanol for 5 minutes, then stained with Giemsa (azure eosin methylene blue solution) for 10 minutes and washed in water to remove excess of stain. The slides were dried at room temperature. These stained glass slides were analyzed by optical microscopy, morphometric measurements (perimeter and area) were performed by a software Image pro plus (Cibernetics, USA) to a total of five fields per each slide and the perimeter/area ratios were calculated.
Statistical analysis
Data are reported as means ± standard deviation of %ATI and perimeter/area ratio and they were compared between the treated and control group by One-way analysis of variance - ANOVA, followed by Bonferroni post test with a
p<0.05 as significant level. InStat Graphpad software was used to perform statistical analysis (GraphPad InStat version 3.00 for Windows 95, GraphPad Software, San Diego, California, USA). RESULTS
Figure 1 shows the absorption spectrum of the clove extract used in the experiments. The pattern of the absorption spectra presents the highest measure of the optical density (0.489±0.013) at 480 nm. This parameter has permitted to control the conditions to prepare the extracts and has been used as a marker.
Figure 1: Absorption spectrum of the clove extract.
Table 1 shows the effect of different concentrations of the clove extract on the distribution of radioactivity between cellular and plasma compartments. The clove extract used decreased significantly (p<0.05) the %ATI on the cellular compartment from 96.96±1.06 to 44.45±2.87.
Table 1 – Effect of different concentrations of a clove extract on the distribution of radioactivity in
cellular compartment
Clove extract (mg/ml) %ATI in Cellular compartment
0.0 96.96±1.06
6.5 56.89±3.66
12.5 55.67±4.95 25 54.21±1.86 50 53.94±0.80 100 44.45±2.87*
Blood samples were incubated with clove extract at different concentrations. After that, the labeling of blood constituents with 99mTc was performed. The samples were centrifuged and aliquots of blood cells were separated. The radioactivity in these fractions was determined and the percentage of incorporated radioactivity (%ATI) was determined. (*) p<0.05 when compared to control.
Table 2 shows the effect of different concentrations of the clove extract on the fixation of 99mTc on plasma proteins. The clove extract decreased significantly (p<0.05) the fixation of the 99mTc on the plasma proteins from 64.28±7.55 to 15.11±0.63. 0.00 0.10 0.20 0.30 0.40 0.50 0.60
400 440 480 520 560 600 640 680
Wavelength (nm)
A
b
sor
b
Table 2 – Effect of different concentrations of the clove extract on the labeling of plasma proteins Clove extract (mg/ml) %ATI in Plasma proteins
0.00 73.94±1.04
6.5 33.09±2.82
12.5 33.04±3.97 25 28.15±3.17 50 19.59±5.75 100 15.11±0.63* Blood samples were incubated with clove extract for 1 hour. After
that, the labeling of blood constituents with 99mTc was performed. Aliquots of plasma were precipitated in trichloroacetic acid. The radioactivity in these fractions was determined and the percentage of incorporated radioactivity (%ATI) was determined. (*) p<0.05 when compared to control.
Table 3 shows the effect of different concentrations of clove extract on the fixation of
99mTc on proteins of blood cells. The results show
that the clove extract decreased significantly (p<0.05) the radioactivity fixation on the cellular proteins from 91.39 ±1.17 to 71.16±3.87.
Table 3 - Effect of different concentrations of the clove solution on the labeling of cells proteins
Clove extract (mg/ml) %ATI in proteins of blood cells
0.0 91.39±1.17
6.5 82.03±3.38 12.5 79.65±2.27
25 76.01±3.05
50 73.53±3.47
100 71.16±3.87*
Blood samples were incubated with clove extract for 1 hour. After that, the labeling of blood constituents with 99mTc was performed. Aliquots of blood cells were precipitated in trichloroacetic acid. The radioactivity in these fractions was determined and the percentage of incorporated radioactivity (%ATI) was determined. (*) p<0.05 when compared to control.
Figures 2 and 3 represent the photomicrographies of blood smears from blood samples treated with saline (0.9% NaCl) and clove extract at the higher concentration used (100mg/ml), respectively. The qualitative comparison between these figures indicates that clove extract alters the morphology of RBC.
Figure 2: Photomicrography of blood smear from blood incubated with saline (control). Blood samples were incubated with 0.9% NaCl for 1 hour. Blood smears were prepared and stained by May-Grünwald-Giemsa method. The slides were analyzed by optical microscopy (x1000).
Figure 3: Photomicrography of blood smear from blood incubated with clove extract. Blood samples were incubated with clove extract (100mg/ml) for 1 hour. Blood smears were prepared and stained by May-Grünwald-Giemsa method. The slides were analyzed by optical microscopy (x1000).
0.00 0.20 0.40 0.60 0.80
0.0 6.5 12.5 25.0 50.0 100.0
Clove Extract (mg/ml)
Peri m eter/ area Rati o (1/µµµµ m)
Figure 4 - Effect of clove extract on the perimeter/area ratio of RBC. Morphometric measurements of perimeter/area of RBC from blood smears with a total of five fields per each slide and five slides to each extract were evaluated. The software Image pro plus, media Cibernetics, USA) was used to these evaluations.
DISCUSSION
Some authors have reported that nuclear medicine procedures could be altered by medication treatments that the patient is undergoing. (Hesslewood and Leung, 1994; Owunwanne et al., 1995; Sampson, 1999). Blood constituents labeled with 99mTc have been used in several clinical examinations (Saha, 2004) and also as an experimental assay on an attempt to verify the effect of drugs (Fonseca et al., 2007). This experimental model has permitted to obtain relevant information about properties of various chemical compounds (synthetic and natural) (Abreu et al., 2006; Fonseca et al., 2007).
The analysis of the results shown in tables 1, 2 and 3 indicates that there was an important alteration on the labeling of the blood constituents with 99mTc when the incubation with clove extract
was made in vitro at highest concentration used. These results could be explained, at least in part, by a possible oxidant and citotoxic property of the chemical compounds that are present in the clove solution (Schmalz et al.,
2000). These compounds could oxidize the stannous ions that are necessary to the reduction of the pertechnetate ion, as suggested by other authors to several natural products, as Maytemus icilifolia (Oliveira et al., 2000), Ginkgo biloba
(Moreno et al., 2002), Paullinia cupana (Oliveira
et al., 2002) and Mentha crispa (Santos-Filho et al, 2004).
Other possible explications would be the actions of the clove on the ions transport systems and alteration of the RBC membrane structure. In fact, some flavoids were described as capable of interacting with ions transport channels in membrane (Morales et al., 1994; Re et al., 1999). Bratel et al., (1998) have reported that extractable components of some commonly used root canal sealing materials may interfere with immunocompetent cells in vitro. In addition, the antibacterial effect of compounds presents in clove extract could be related to action on bacterial membrane.
Changes in the optic disc structure and thickness of retinal nerve fiber layer in chronic ocular hypertensive monkeys (Shimazawa et al.,
2006), the relationship between infarct-related artery stenosis and capillary density (Prech et al.,
2005) and the effects of two sex hormones on normal mammal gland of female rats (Pompei et al., 2005) are several important findings obtained by morphometric measurements. Oliveira et al.
(2003) showed possible alterations by morphometric analysis of RBC from blood treated with natural products.
The qualitative morphologic analysis indicated that clove extract induce alterations on shape of RBC (Figure 2). However, this finding was not confirmed by quantitative analysis by mophometric measurements indicating no effect of clove extract at the higher concentration (100mg/ml) on the perimeter/area ratio of RBC (Figure 3).
The morphology of RBC is influenced by function of ion transport systems in membrane (Lew and Bookchin, 2005). Chemical compounds in clove extract may interact with these systems and alter the morphology of RBC observed in this study. This hypothesis could be in agreement with the results of the distribution of radioactivity in cellular compartment (Table 1).
ACKNOWLEDGEMENTS
We are grateful for the biologist Mario Pereira (UERJ) for his technical support and to Mr. Carlos Brown Scavarda (B. A., University of Michigan) for the English language revision. Financial support: CNPq, CAPES and UERJ.
RESUMO
Cravo-da-índia (Caryophyllus aromaticus L.) tem sido usado em tratamentos clínicos. Constituintes sangüíneos marcados com tecnécio-99m (tecnécio-99mTc) são usados em medicina nuclear O objetivo foi avaliar os efeitos de um extrato de cravo-da-índia na marcação de constituintes sangüíneos com 99mTc e na morfologia das hemácias. Amostras de sangue foram incubadas com cravo-da-índia, cloreto estanoso e 99mTc. Plasma, células sangüíneas, frações insolúveis do plasma e das células sangüíneas foram separadas. A porcentagem de radioatividade incorporada (%ATI) nestas frações foi calculada. Forma e relação perímetro/área das hemácias foram avaliadas. O extrato de cravo-da-índia alterou significativamente (p<0,05) a radiomarcação de constituintes sangüíneos e qualitativamente a forma das hemácias. Não foram obtidas alterações na relação perímetro/área hemácias. Os resultados indicam que o extrato de cravo-da-índia apresenta compostos que interferem com a radiomarcação de constituintes sangüíneos e alteram a morfologia de CS através de ações oxidativas/quelantes ou interagindo com a estrutura da membrana celular.
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Manuscrito 2:
Effects of a Tomato (
Solanum lycopersicum
) Extract on the Labeling of
Blood Constituents with Technetium-99m
Severo de Paoli1, 2, 3, 4*, Aline P.M. Dias4, 5, Priscila V.S.Z. Capriles4, 5, Tadeu E.M.M. Costa4,5, Adenilson S. Fonseca4 and Mario Bernardo-Filho4, 5
1Universidade Federal do Rio Grande do Norte, Programa de Pós-Graduação em Ciências da
Saúde, Avenida General Gustavo Cordeiro de Farias, s/n, 59010-180, Natal, RN, Brasil.
2Universidade Estácio de Sá, Rua do Bispo, 83, 20261-063, Rio Comprido, Rio de Janeiro, Brasil, 3Centro Universitário Fundação Educacional Serra dos Órgãos, Faculdades de Odontologia e Fisioterapia,
Avenida Alberto Torres 111, Alto, 25964-004, Teresópolis, Rio de Janeiro, Brasil,
4Universidade do Rio de Janeiro, Instituto de Biologia Roberto Alcantara Gomes,
Departamento de Biofísica e Biometria, Avenida 28 de Setembro, 87 fundos, Vila Isabel, 20551-030, Rio de Janeiro, Brasil,
5Instituto Nacional do Câncer, Coordenadoria de Pesquisa Básica, Praça Cruz Vermelha, 23,
20230130, Rio de Janeiro, Brasil
Abstract
Tomato (Solanum lycopersicum) is the second most produced and consumed vegetable in the world. It has been indicated in the prevention and treatment of cancer, asthma and atherosclerosis. Blood constituents labeled with radionuclides have been used in procedures in nuclear medicine. Data have shown that food and drugs can alter the labeling of blood constituents with technetium-99m (99mTc). This study evaluated the
influence of a tomato extract on this radiolabeling procedure. Heparinized blood (Wistar rats) was incubated in vitro with different concentrations of a tomato extract and 99mTc-labeling was performed. Plasma (P) and blood cells (BC) were separated following soluble (SF-P/SF-BC) and insoluble (IF-P/IF-BC) fractions isolation by precipitation and centrifugation. The radioactivities on blood constituents (P, BC, IF-P, SF-P, IF-BC and SF-BC) were determined and the percentage of radioactivity (%ATI) calculated. The tomato extract used at the highest concentrations (2.00 and 4.00g/ml), reduced significantly (p<0.05) the %ATI in IF-P, although this extract did not modify the radiolabeling on BC, neither the radioactivity fixation on IF-BC. In conclusion, our data suggest that the chemical compounds present in the aqueous tomato extract could have some properties capable of change the fixation of 99mTc on plasma proteins.
Resumo
Efeitos de um extrato de tomate (Solanum lycopersicum) na marcação de constituintes sangüíneos
com tecnécio-99m. Tomate (Solanum lycopersicum) é o segundo vegetal mais produzido e consumido no mundo. Tem sido indicado para prevenção e tratamento de câncer, asma e arteriosclerose. Constituintes sangüíneos marcados com radionuclídeos têm sido usados em procedimentos na medicina nuclear. Dados têm mostrado que alimentos e drogas podem alterar a marcação de constituintes sangüíneos com tecnécio-99m (99mTc). Este estudo avaliou a influência de um extrato de tomate neste procedimento de radiomarcação. Sangue heparinizado (Wistar rats) foi incubado in vitro com diferentes concentrações de um extrato de tomate e a marcação com 99mTc foi realizada. Plasma (P) e células sangüíneas (CS) foram separadas permitindo o
isolamento das frações solúveis (SF-P/SF-CS) e insolúveis (IF-P/IF-CS) por precipitação e centrifugação. A radioatividade nos constituintes sangüíneos (P, CS, IF-P, SF-P, IF-CS e SF-CS) foi determinada e a porcentagem de radioatividade (%ATI), calculada. O extrato de tomate usado, nas maiores concentrações (2.00 and 4.00g/ml), reduziu significativamente (p<0.05) a %ATI na IF-P, embora este extrato não tenha modificado a radiomarcação da CS e fixação da radioatividade na IF-CS. Em conclusão, nossos dados sugerem que os compostos químicos presentes no extrato aquoso do extrato teria algumas propriedades capazes de alterar a fixação do 99mTc nas
proteínas plasmáticas.
Unitermos: Constituintes sangüíneos, Solanum lycopersicum, tecnécio-99m
Introduction
(Rissanen et al., 2002; Frederiksen et al., 2007), reduction of asthma symptoms (Neuman et al., 2000; Wood et al., 2004) and decrease of DNA strand breakages of cells of the immune system (Riso et al., 1999; Porrini et al.; 2005, Riso et al., 2006).
The tomato effects may be related mainly to lycopene which acts on biological mechanisms altering the oxidant status and could be responsible for its positive protective actions (Everson and McQueen 2004; Bose and Agrawal, 2007). Normally, the amount of lycopene in the tomatoes is not always the same and it can vary from 5 mg/kg in the yellow tomatoes to 50 mg/kg in yhe red tomatoes. Reddish foods, such as watermelon, papaya and pink grapefruit may also contain lycopene, but at lower concentrations than in tomatoes (Boyle et al., 2003).
Several theories are being explored to explain the lycopene effects on the prevention of cancer. Lycopene consumption is inversely related to insulin growth factor levels, a factor linked to a greater risk of prostate cancer (Boyle et al., 2003; Jatoi et al., 2007). A second proposed mechanism of lycopene action includes both inhibition of tumor growth by decrease or loss in junctional cell communication (Kucuk et al., 2002; Telef et al., 2006). However, the most widely accepted theory is the antioxidant effects of lycopene acting as a scavenger for singlet oxygen, hydrogen peroxide and nitrogen dioxide that are associated with DNA damage and the development of cancer (Hadley et al., 2002; Bose and Agrawal, 2007). This theory is also used to explain the beneficial effects of lycopene on asthma and atherosclerosis (Neuman et al., 2000, Rissanen et al., 2002; Frederiksen et al., 2007).
Labeled red blood cells (RBC) with 99mTc has come into wide use in clinical nuclear medicine for several important applications, including imaging of cardiovascular system (Niemeyer et al., 1995), peripheral arterial blood flow (Harel et al., 2005), evaluation of gastrointestinal bleeding (Wong et al., 2004, Zaman et al., 2004, Olds et al., 2005), measurement of red cell volume (Hladik III et al., 1987), hepatic hemangiomas (Artiko et al., 2004, Verdu et al., 2005), renal carcinoma (Cortes et al., 2003) and splenic reticuloendothelial system (Jin et al., 2004, Slart et al., 2004).
The use of medicinal plants or natural products for treatment of various diseases has increased in the last decades (Everson and McQueen 2004), justifying the use of accepted experimental models to study some biological properties of various natural products (Reiniger et al., 1999; Fonseca et al., 2005; Freitas et al., 2007).
Natural or synthetic drugs, as well as labeling conditions, can have effect on the labeling of blood constituents (Lima et al., 2002, Frydman et al., 2004, Fonseca et al., 2005, Jesus et al., 2006, Fonseca et al., 2007). The aim of this study is to evaluate the interference of different concentrations of an aqueous tomato extract on the labeling of blood constituents with 99mTc.
Materials and Methods
Animals
Adult male Wistar rats (3-4 months, 250-350g) were maintained in a controlled environment. The animals had free access to water and food and ambient temperature was kept at 25 ± 2ºC. The experimental protocol was approved (CEA/115/2006) by the Ethical
Preparation of tomato extract
Tomato, as fruit, was purchased in a local supermarket. To prepare the extract, 4g of tomatoes (without bark and seeds) were ground in 1ml NaCl 0.9%. The crude extract was filtered and centrifuged (clinical centrifuge, 2000rpm, 10min) to obtain the final extract. This fraction of the extract was considered 4g/ml.
A spectrophotometric analysis (Analyser, 800M, São Paulo, Brazil) of the extract was carried out. The absorbance at 455 nm was considered the marker of the quality control of this extract. All the prepared extracts to be used in the experiments must had the optical density of 0.05±0.004 (Figure 1).
In vitro Radiolabeling of Blood Constituents
Heparinized blood (500µl) was withdrawn by heart puncture from Wistar rats and
incubated with 100µl of different concentrations of a tomato extract (0.05, 0.50, 1.00, 2.00
and 4.00g/ml) or with a saline solution (0.9% NaCl) alone, as control, for 1 hour (room temperature). Afterwards, 500 µl of stannous chloride (1.20 µg/ml) was added and the
incubation continued for further 1 hour. After this period of time, 100 μl of 99mTc (3.7MBq) as sodium pertechnetate (Na99mTcO4), recently milked from a 99Mo/99mTc generator (Instituto
de Pesquisas Energéticas e Nucleares, Comissão Nacional de Energia Nuclear, São Paulo, Brazil) were added and the incubation was continued for 10 minutes. These samples were centrifuged in a clinical centrifuge (1500rpm, 5min) and aliquots of 20 µl of plasma (P) and
blood cells (BC) were isolated. Another aliquots of 20 µl of P and BC were separated and
(Bernardo-Statistical analysis
Data were reported as (means ± standart deviation) of %ATI and compared to the treated (n=10 for each extract concentration) and control group (n=10) by One way analysis of variance - ANOVA, followed by Bonferroni post test, with a p<0.05 as significant level. InStat Graphpad software was used to perform statistical analysis (GraphPad InStat version 3.00 for Windows 95, GraphPad Software, San Diego California, USA).
Results
The figure 1 shows the absorption spectrum of the tomato extract used in the experiments. The pattern of the absorption spectrum presents the highest measure of the optical density (0.055±0.004) at 455 ηm. This parameter has allowed us controlling the experimental
conditions of preparation of the extracts and used as markers. <Figure 1>
The Figure 2 shows the ATI% in blood cells (BC) and plasma (P) compartments from blood treated with different concentrations of tomato extract. The analysis of these data indicates that tomato extract has not altered the distribution of radioactivity in these two compartments (BC and P).
<Figure 2>
The Figure 3 shows the ATI% in insoluble (IF-P) and soluble (SF-P) fractions isolated from plasma separated from whole blood treated with different concentrations of tomato extract. The analysis of this data indicates that tomato extract has significantly (p<0.05) reduced the radioactivity fixation in IF-P in the two highest concentration studied (2.00 and 4.00g/ml).
The Figure 4 shows the ATI% in insoluble (IF-BC) and soluble (SF-BC) fractions isolated from blood cells separated from blood treated with different concentrations of tomato extract. The analysis of this data indicates that tomato extract has not significantly modified the radioactivity fixation in insoluble blood cells fraction.
<Figure 4>
Discussion
It has been described that food and also natural and synthetic drugs can alter the labeling procedure with a radionuclide causing an unexpected behavior of the labeling of the blood constituents with the radiopharmaceutical (Hesslewood & Leung 1994; Sampson, 1999; Gomes et al., 2002).
The changes in the pattern observed when binding the radionuclide 99mTc have been possible through studies carried out with natural or synthetic products interaction (Fonseca et al., 2005). It seems that natural products (terpenoids, isoflavonoids, abajeru) or synthetic drugs (acethylsalicic acid, zinc oxide, eugenol,) as well as food (tomatoes, clove), are capable of modifying the blood constituents labeled with radionuclides (Hesslewood & Leung 1994; Sampson, 1999; Gomes et al., 2002).
The data obtained in this work shown that the tomato extract has reduced the radioactivity fixation on plasma proteins (Figure 3). Yet, the tomato extract has not modified the distribution of radioactivity between plasma and blood cells compartments (Figure 2) neither the fixation of 99mTc on the blood cells proteins (Figure 4). Stannous ion (Sn+2) is used as reducing agent in the 99mTc-labeling of blood constituents and compounds or conditions that interfere with its action can alter the fixation of 99mTc on these constituents (Hladik III et al., 1987; Bernardo-Filho et al., 1994; Moreno et al, 2002). The effect of tomato extract on labeling of plasma proteins could be related to its antioxidant property disturbing the action of Sn+2 on 99mTc and decreasing the radioactivity uptake by plasma proteins. In fact, data have demonstrated that tomato constituents (as lycopene and vitamin C) have antioxidant effects (Hadley et al., 2002; Rissanen et al., 2002; Everson & McQueen 2004; Bose and Agrawal, 2007) and this may explain the alterations of 99mTc-labeling plasma proteins obtained in this work.
On the other hand, in the blood, carotenoids transported by lipoproteins and, more substantially, by low density lipoproteins (LDL), suggest that the increase in LDL resistance to oxidation during consumption of tomato juice may be, at least, partly due to increased content of lycopene (Erdman et al., 1993; Upritchard et al., 2000). In addition, It has been also related a protective effect of beta-carotene and lycopene entrapped in human albumin against the oxidative attack of electronically excited molecular oxygen on 2'-deoxyguanosine (dGuo) (Yamaguchi et al., 1999). So, these interaction between plasma proteins and tomato constituents could decrease the number of the binding sites of 99mTc with plasma proteins and this could be related with the decrease of the radiolabeling of these proteins.
Conclusion
occurs due to chemical substances of the tomato extract that could have action on reducing agent (stannous ion) used in the labeling process and/or the ability to interact with plasma proteins, occupying its binding sites. Although these experiments were performed in rats, the results suggest that caution should be taken with the interpretation of the data obtained from nuclear medical diagnosis and tests when patients consume tomato extracts or its derivatives in food.
Acknowledgements
This research was supported by Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Universidade do Estado do Rio de Janeiro (UERJ).
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FIGURE 1
FIGURE 2
400 420 440 460 480 500 520 540
0,00 0,01 0,02 0,03 0,04 0,05 0,06 0,07 0,08 0,09 0,10
Abs
or
ban
ce
Wavelenght (nm)
0.00 0.05 0.50 1.00 2.00 4.00
0 10 20 30 40 50 60 70 80 90 100
%A
TI
TOMATO EXTRACT CONCENTRATION (g/ml)
P
FIGURE 3
FIGURE 4
0.00 0.05 0.50 1.00 2.00 4.00
0 10 20 30 40 50 60 70 80 90 100
%A
TI
TOMATO EXTRACT CONCENTRATION (g/ml)
0.00 0.05 0.50 1.00 2.00 4.00
0 10 20 30 40 50 60 70 80 90 100
***
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%ATI
3.2. MANUSCRITO SUBMETIDO PARA PUBLICAÇÃO
Zinc-oxide-eugenol alters the labeling of blood constituents with
technetium-99m and the shape of the red blood cells.
S.Paoli MD1, T.S.Giani MD2, G.A.Presta MD3, C.G.Correa4, A.I.Maiworm5, S.D.Santos-Filho MD6 and M.Bernardo-Filho PhD7.
1. Dentistry; Professor, Faculdade de Odontologia (FONF) de Nova Friburgo, Nova Friburgo, RJ, and Centro Universitário Serra dos Órgãos (UNIFESO), Teresópolis, RJ, Brazil; Universidade Federal do Rio Grande do Norte (UFRN), Programa de Pós-Graduação em Ciências da Saúde, Natal, RN, Brazil;
2. Physiotherapist, Professor, Universidade Estácio de Sá, UFRN, Programa de Pós-Graduação em Ciências da Saúde, Natal, RN, Brazil.
3. Medical Doctor; Professor and researcher, UFRN, Programa de Pós-Graduação em Ciências da Saúde, Natal, RN, Brazil.
4. Biologist; Professor and researcher in FONF, Nova Friburgo, RJ, and UNIFESO, Teresópolis, RJ, Brazil. 5. Physiotherapist; Head of the Physiotherapy Department, UFRN, Programa de Pós-Graduação em Ciências da Saúde, Natal, RN, Brazil.
6. Biologist and Physiotherapist Professor in UNIFOA, Volta Redonda, RJ, Brazil; UFRN, Programa de Pós-Graduação em Ciências da Saúde, Natal, RN, Brazil;
7. Biomedical an Physiotherapist, PhD, Instituto Nacional do Câncer, Rio de Janeiro, RJ, Brazil and Universidade do Estado do Rio de Janeiro, Departamento de Biofísica e Biometria. Rio de Janeiro, RJ, Brazil.
Correspondence to author: Severo de Paoli
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, Vila Isabel 20551-030, Rio de Janeiro, Brasil
ABSTRACT. (170 words)
Zinc oxide and eugenol (OZE) mixture produces a cement utilized in almost all the specialities in dentistry all over the world. Blood constituents are labeled with technetium-99m (99mTc) and used in nuclear medicine. Rationale and Objectives: The aim of this work was to study the effect of an OZE solution on the labeling of blood constituents with 99mTc and on the qualitative and quantitative evaluation of the shape of the red blood cells (RBC). Materials and Methods: Blood (Wistar rats) was incubated with OZE, stannous chloride and
99mTc. Samples of blood were isolated and fractions were separated. The percentage of
radioactivity (%ATI) was calculated and the morphology and morphological parameter were evaluated. Results: The %ATI on the blood constituents decreased significantly (p<0.05) due to the treatment with OZE. Qualitative and quantitative alterations were found. Conclusion: Although the experiments had been performed with rats, it is suggested precaution in the interpretation of the examinations of the nuclear medicine in patients that have been treated with OZE in the dentistry procedures.
Keywords: zinc oxide eugenol, dentistry, technetium-99m, blood constituents, red blood cell.
INTRODUCTION
general, this cement is used in procedures of the dentistry in the proportion of 1 g of zinc oxide and up to 0.25 ml of eugenol [5].
OZE has several pharmacological and/or biological properties including: (i) non toxic in vivo and toxic in vitro, (ii) adherent to tissues, (iii) mucostatic or mucocodisplacive (depending on brand used), (iv) good surface detail in thin section, (v) good dimensional stability (little or no dimensional change on setting, 0.1% dimensional change during setting), (vi) can be added to with fresh OZE and (vii) stable on storage and good shelf life. [8]
Radiobiocomplexes, known as radiopharmaceuticals, are radioactive tracers employed in nuclear medicine to help in the diagnosis and/or treatment of diseases. [9, 10]
Red blood cells labeled with 99mTc (99mTc RBC) are radiobiocomplexes widely used in clinical nuclear medicine for several important applications. [11, 12] The labeling of blood constituents with 99mTc depends on the presence of a reducing agent and stannous chloride is widely utilized. The in vitro technique is easily carried out and produces a better and well controlled product. [9]
Some authors have reported that the presence of natural or synthetic drugs might alter the labeling of blood constituents with 99mTc. [13-17] The morphological analysis of RBC has been of importance on the clinical and on the laboratory investigations and has contributed to evaluate possible alterations in the area, shape, volume and perimeter/area ratio of this kind of cellular structure. [18]
MATERIALS AND METHODS Animals
In our laboratory, the experiments have been carried out with rats that are maintained in a controlled environment. The animals had free access to water and food and ambient temperature was kept at 25 ± 2ºC. Heparinized whole blood was withdrawn by cardiac
puncture from adult male Wistar rats (3-4 months of age, 250±15g of weight) and a pool of blood from 6 animals was obtained. The animals are under anesthesia by sodic thiopental 40mg/kg. The protocols of the experiments were performed without sacrificing the animals and was approved (CEA/115/2006) by the Ethical Committee of the Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro.
Zinc oxide eugenol cement preparation
The zinc oxide (500 mg) (Super Dentaria Leão Ltda, Rio de Janeiro, Brazil, lot number 3/058, May 2002, validity May 2008) was mixed with eugenol 100 mg (Biodinâmica Química e Farmacêutica Ltda, Rio de Janeiro, Brazil, lot number 765/00, November/ 2002, validity November/2008) in a glass plaque to obtain a similar cement glass mass [19,20]. This cement was separated in 4 parts of 150 mg. One part was put in 0.9% NaCl (15 ml). After that, it was triturated and mixed in a vortex for 2 minutes. After filtration with a qualitative filter paper (Aldrich Chemical Co, 11cm, Lot number k932), the filtered solution was considered to be 10 mg/ml.
Labeling of blood constituents with 99mTc
sequence, 99mTc (3.7 MBq) was added, as sodium pertechnetate (99Molibdenium/99mTechnetium generator, Instituto de Pesquisas Energéticas e Nucleares, Comissão Nacional de Energia Nuclear, Brazil) and the incubation continued for another 10 min. These samples were centrifuged (clinical centrifuge, 1500 rpm, 5 min and aliquots (20
µl) of plasma (P) and blood cells (BC) were separated. Aliquots (20 μl) of P and BC were also
precipitated in trichloroacetic acid (5%) and soluble (SF) and insoluble fractions (IF) of P and BC were separated. The radioactivity in P, BC, IF-P, SF-P, IF-BC and SF-BC were determined in a well counter (Packard Instrument Company, mod C5002, USA). After that, the percentage of radioactivity (%ATI) was calculated as described previously [21]. All experiments were repeated at least three times.
After the incubation with 99mTc, one drop of each sample was smeared in glass 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 10min and washed in methanol to remove excess of stain. The glass slides were stayed at room temperature to dry. The stained glass slides with MGG were analyzed by optical microscopy and morphometric parameter (perimeter/area ratio) of a total of five fields per each glass slide were evaluated (Software image pro-plus, media Cybernetics, USA).
Statistical analysis
RESULTS
The table 1 shows the effect of different concentrations of the OZE solution on the distribution of the radioactivity between cellular and plasma compartments of the Wistar rats’ blood. The studied OZE solution decreased significantly (p<0.05) the radioactivity on the cellular compartment from 96.00±3.31 to 85.11±1.74.
The table 2 shows the effect of different concentrations of the OZE solution on the fixation of the radioactivity in the soluble and insoluble fraction of the plasma of the Wistar rats’ blood. The studied OZE solution decreased significantly (p<0.05) the fixation of the 99mTc on IF-P from 73.94±1.04 to 62.54±3.34.
The table 3 shows the effect of different concentrations of the OZE solution on the fixation of the radioactivity in the insoluble and soluble fraction of the blood cells obtained from the blood treated with OZE solution. The studied OZE solution decreased significantly (p<0.05) the radioactivity fixation on IF-BC from 91.30±1.17 to 71.16±3.87.
