On-line version ISSN 1678-2674
Acta Cir. Bras. vol.27 no.9 São Paulo Sept. 2012
3 - ORIGINAL ARTICLE
EFFECTS OF DRUGS
Anti toxic effect of broccoli extract on stannous dichloride toxicity1
Efeito antitóxico do extrato de brócolis na toxicidade do dicloreto de estanho
Betul CekicI; Fazilet Zumrut Biber MuftulerII; Ayfer Yurt KilcarIII; Necati GunayIV; Serhan SakaryaV; Perihan UnakVI
IFellow Master degree, Department of Nuclear Applications, Institute of Nuclear Sciences, Ege University, Bornova, Izmir-Turkey. Carried out the study and drafted the manuscript
IIAssociate Professor, Department of Nuclear Applications, Institute of Nuclear Sciences, Ege University, Bornova, Izmir-Turkey. Responsible for conception, design, intellectual and scientific content of the study, supervised all phases of the study, manuscript writing and critical revision
IIIFellow PhD degree, Research Assistant, Department of Nuclear Applications, Institute of Nuclear Sciences, Ege University, Bornova, Izmir-Turkey. Involved in technical procedures and extract preparation
IVFellow PhD degree, Research Assistant, ADUBILTEM, Adnan Menderes University, Aydin-Turkey. Involved in technical procedures and bacteria preparation
VFull Professor, ADUBILTEM, Adnan Menderes University Aydin-Turkey. Designed the studies concerning bacteria, analysis and interpretation of data, critical revision
VIFull Professor, Department of Nuclear Applications, Institute of Nuclear Sciences, Ege University, Bornova, Izmir-Turkey. Analysis and interpretation of data, critical revision.
PURPOSE: Since Technetium-99m (99mTc) has favorable physical and chemical characteristics, it is widely used radioisotope in Nuclear Medicine. However, stannous dichloride (SnCl2) has been widely used as a reducing agent in labeling procedure of pharmaceutical with radionuclide, it has been realized that SnCl2 have genotoxic and cytotoxic effects on biological systems. In previous studies, it has been shown that some herbal extract can reduce genotoxic and cytotoxic effects of SnCl2. In the present study, it is aimed to evaluate the effect of the broccoli extract on the survival of E. coli ATCC 25922 strain against to toxic effects of SnCl2.
METHODS: Broccoli was extracted with methanol extraction. HPLC and TLC analysis of broccoli extract were performed. Then antitoxicity and dose response assays were performed on bacterial strain.
RESULTS: The broccoli extract had dose dependent protective effect against SnCl2 toxic effect on E. coli.
CONCLUSIONS: The consumption of broccoli may alter the stannous dichloride toxicity. Broccoli extract may use as a new protective strategies against the toxic effect of SnCl2 on patients who were taken 99mTc radiopharmaceuticals.
Key words: Broccoli. Stannous dichloride. Technetium-99m. Escherichia coli. Toxicity.
OBJETIVO: Em face de suas características físico-químicas, o Tecnécio-99m (99mTc) é um radiofármaco amplamente utilizado na Medicina Nuclear. Todavia, o dicloreto de estanho (SnCl2) tem sido largamente aplicado como um agente redutor no procedimento farmacêutico de marcação com radionuclídeos. Constatou-se que o SnCl2 apresenta efeitos genotóxicos e citotóxicos nos sistemas biológicos. Em estudos prévios, foi demonstrado que alguns extratos de ervas podem reduzir tais efeitos. O estudo atual objetivou avaliar os efeitos do extrato de brócolis na sobrevida da cepa E. coli ATCC 25922, exposta ao efeito tóxico do SnCl2.
MÉTODOS: O extrato de brócolis foi obtido mediante extração com metanol. Analises com HPLC e TLC foram efetuadas. Avaliou-se a antitoxicidade e realizou-se um ensaio dose-resposta para uma cepa de bactérias.
RESULTADOS: O extrato de brócolis mostrou um efeito protetor dose dependente para os efeitos tóxicos do SnCl2 sobre a E. coli.
CONCLUSÕES: O consumo de brócolis pode alterar a toxicidade do dicloreto de estanho. O extrato de brócolis pode ser utilizado como uma nova estratégia para proteção de pacientes contra os efeitos tóxicos do SnCl2, nos quais foi administrado o radiofármaco Tecnécio-99m.
Descritores: Brassica. Compostos Estanho. Tecnécio. Escherichia coli. Toxicidade.
Technetium-99m radiopharmaceuticals have favorable physical and chemical characteristics1,2 and widely used in Nuclear Medicine practice1,3. Since stannous dichloride (SnCl2) is the most common reducing agent in 99mTc radiopharmaceuticals preparation, it has been shown that severe biological effects on central nervous system and oral mucosa in animal studies1,4. Also it has been reported that SnCl2 had lethal effect on Escherichia coli (E. coli) with cytotoxic and genotoxic effects5-8. Genotoxic effect of SnCl2 may be directly on deoxyribonucleic acid (DNA) or indirectly by generating free radicals9.
In environment oxygen and stannous (II) may carry out some reactions and these reactions, leads to mutations in the DNA and it caused death of the cell by the formation of hydrogen peroxide (H2O2), as follows10-12.
Reaction I: Stannous (II) + O2→ Stannous (III) + O2-
Reaction II: Stannous (III) + O2- + 2H+ → Stannous (IV) + H2O2
Reaction III: O2 + Stannous (II) + 2H+ → H2O2 + Stannous (IV)
In recent studies, some authors have also described that genotoxic and cytotoxic effects of SnCl2 can be altered by herbal extract, chemicals and pharmaceutics on E. coli5,6,8-13.
Lima et al.6 demonstrated that cauliflower from Brassicacea family was abolished the lethal effect of SnCl2 on the E. coli strains.
In our study we used broccoli extract which is widely consumed in Turkey14. Broccoli (Brassica oleracea italica) from the Brassicaceae family is a nutrient source of bioactive components including glucosinolates, flavonoids, minerals and antioxidants15-17. In the current study we aimed to evaluate the effect of the broccoli extract on the survival of E. coli ATCC 25922 strains against to toxic effect of SnCl2.
Broccoli was extracted with methanol extraction. HPLC and TLC analysis of broccoli extract were performed. Then antitoxicity and dose response assays were performed on bacterial strain.
Extraction of broccoli
A similar procedure to that for the extraction of broccoli has been applied as described previously19. Broccoli was purchased from local market, dried at room temperature and powdered. The samples (250 mg) were extracted with 5 mL of methanol/water (60:40, v/v) using ultrasonic bath (Ceia P104) for 60 min. at room temperature. The slurry mixture was centrifuged at 2500 rpm for 15 min. The supernatant was collected and 5 mL of methanol/water (60:40, v/v) was added in the remaining parts in the tube. All procedure was repeated 3 times. All the supernatants collected and kept at -20 0C until the experiments.
High performance liquid chromatography (HPLC)
The procedure used for HPLC for broccoli extract was similar to that reported in other studies19. A low pressure gradient HPLC system (LC-10ATvp quaternary pump, SPD-10A/V UV detector, RF-10AXL Fluorescence detector, RAD501 single channel analyzer, a syringe injector equipped with a 1 mL loop and 7-µm VP 250/21 Nucleosil 100-7 C18 column (Macherey - Nagel)) was used for the analytical experiments.
HPLC of Broccoli Extract: Fluorescence was monitored with a model RF-10AXL Shimadzu Fluorescence Detector. The samples were analyzed at a flow rate of 1 mL/min and injection volume was 20 µL. The excitation was monitored at 290 nm and emission at 360 nm. The mobile phase was consisted of two solvents which were water-formic acid (0.33%) (A) and methanol (B). The elution gradient profile used was as 10-17% B in 30 min., 17-40% B in 10 min., 40-100% B in 1 min., 100% B in 5 min. Retention time (Rt) values were given in Figure 1.
Thin layer chromatography (TLC)
Five mL of broccoli extract was applied on ITLC-SG (Merck-5554) plates. As mobile phase, 60% methanol in water was used. Then UV lamp was used to determine location of components in extract. Relative front (Rf) values of the components were calculated.
The E. coli ATCC 25922 strain was provided from Adnan Menderes University Science and Technology Research and Development Center (ADUBILTEM Aydin, Turkey) Epidemiology Laboratory and used in all experiments. The bacteria concentration of ~108 cfu/mL was monitored spectrophotometrically as optical density of 0.1 at A625 nm (Thermolabsystems Multiscan Spectrum).
Bacterial viability assay
For determining the effect of SnCl2 and extract on bacteria viability, bacteria were resuspended in PBS at 108 CFU/ml and incubated with i. SnCl2 ii. Extract + SnCl2 and iii. Extract (as control group) with the 1, 3, 10, 30, 100 µg concentrations for both SnCl2 and extract for 3h at 35ºC. After the incubation bacteria was washed twice in PBS and resuspended. Samples were diluted appropriately and plated on Mueller Hinton Agar (Merck). Following 24h incubation at 35ºC, the total number of bacteria for each dilution was counted and colony-forming units per milliliter (cfu/mL) were determined. The survival fraction (N/N0) was calculated as previously described6.
Dose response of extract
To determine the protective effect of extract; amount of extract was increased 100 µg and 300 µg while SnCl2 concentrations remain stable at 30 µg. Bacterial assay were performed as described above.
All experiments were repeated three times and analysis of variance (ANOVA) was used to compare the mean responses among experimental and control groups. Probability values p<0.05 were considered as significant.
HPLC and TLC studies of broccoli extract were performed. The relevant chromatogram was given in Figure 1. According to HPLC chromatogram, Rt values of broccoli extract were 3.00, 11.50, 14.50 respectively.
Figure 2 represents survival fractions of bacteria in the presence of broccoli extract. In the control group (Extract), there was no difference in survival fraction values for all concentrations. Survival fraction of E. coli was decreased after 3 µg/mL SnCl2 with future dose-dependent decrements at 10 µg/mL as seen in Figure 2.
When we compare SnCl2 group with Extract+SnCL2 group, similarly survival fraction was decreased after 3 µg/mL SnCl2. In Extract + SnCl2 group, E. coli survival fraction is 0.005 at 100 µg. It was seen, at 100 µg/mL, survival fraction of E. coli in Extract + SnCl2 group was more than SnCl2 group. These data suggest that, SnCl2 had toxic effect on E. coli. However, in Extract+ SnCl2 group survival fraction was expected increasing after 100 µg/mL.
Also, survival fraction values of all groups were the same up to 3 µg. Survival fraction value of Extract + SnCl2 group (0.005) was higher than group of SnCl2 (0.00) at 100 µg concentration (p<0.05). These data thought that extract protect E. coli against toxic effect of SnCl2 at high concentrations. So, we decided to do a dose response study for to determine extract protective effect with high concentrations (Figure 3). For this aim, 30 µg SnCl2 + 100 µg extract and 30 µg SnCl2+ 300 µg extract group were prepared addition to 1-1, 3-3, 10-10 and 30-30 µg concentrations were used in bacteria viability assay. As shown in Figure 3, viable bacteria in 30 µg SnCl2 + 300 µg extract group was 10.82 fold (p=0.001) high compared to 30 µg SnCl2 + 30 µg extract group.
Our HPLC results were confirmed with Lin et al.20 LC-MS study results with phenolic components of colloid greens, kale and Chinese broccoli. The results of HPLC and TLC analysis were compatible.
Although its toxic effects were well documented, SnCl2 has been used as 99mTc reducing agent in majority of 99mTc radiopharmaceutical kits. In previous studies it was reported that genotoxic effect mediated by the production of reactive oxygen species on pro- and eukaryotic microbial test systems1,7. So SnCl2 may have harmful effect on E. coli.
In recent studies, it was reported; the genotoxic and cytotoxic effects of SnCl2 can be altered by herbal extract, chemicals and pharmaceutics on E. coli5,8,10-13. One of these studies was performed by using cauliflower which is in the same family with broccoli. In this study, E. coli AB1157 strain was treated with SnCl2 in the presence of cauliflower and it abolished the lethal effect of SnCl2 on the E. coli strains6.
In another study, it was evaluated the influence of the Cymbopogon citratus, Maytenus ilicifolia and Baccharis genistelloides crude extracts on the survival of E. coli AB1157 (wild type) strain. Maytenus ilicifolia extract had high protection effect on E. coli survival compared to other extracts and this protection was highly related with antioxidant and/or oxidant properties of the extracts11. Similarly, the Ganoderma lucidum (reishi) extract had protective effect on E. coli against the oxidative effect of SnCl2, and the chemical compounds in reishi extract had redox/chelating activity5. Although, it has been shown that some other herbal extracts have been eliminated the toxic effects of SnCl25,8,9,11, action of mechanisms are not well known yet.
Broccoli has nutritional antioxidants, such as vitamins C and E, but also a great quantity of non-nutritional antioxidants, such as flavonoids, flavones, and other polyphenolic compounds16,21. In our study broccoli extract had dose dependent protective effect against SnCl2 toxic effect on E. coli. We suggest that consumption of broccoli may alter the SnCl2 toxicity.
The consumption of broccoli may alter the stannous dichloride toxicity. Broccoli extract may use as a new protective strategies against the toxic effect of SnCl2 on patients who were taken 99mTc radiopharmaceuticals.
The authors would like to thank to PhD student Hasan Zora for helping.
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Fazilet Zumrut Biber Muftuler
Department of Nuclear Applications, Institute of Nuclear Sciences
35100 Bornova, Izmir, Turkey
Tel.: +90 232 311 3467
Fax: +90-232-311 3433
Received: April 25, 2012
Review: June 22, 2012
Accepted: July 24, 2012
Conflict of interest: none
Financial source: Ege University Research Fund (contract no 2010 NBE 007)
1 Research performed at Department of Nuclear Applications, Institute of Nuclear Sciences, Ege University, 35100, Bornova, Izmir-Turkey and Adnan Menderes University, Science & Technology Research & Development Central, (ADUBILTEM), 09100, Aydin-Turkey.