Influence of Annona muricata ( soursop ) on biodistribution of radiopharmaceuticals in rats 1

PURPOSE: To evaluate the effect of hydroalcoholic extract of A. muricata on biodistribution of two radiopharmaceuticals: sodium phytate and dimercaptosuccinic acid (DMSA), both labeled with 99mtechnetium. METHODS: Twenty four Wistar rats were divided into two treated groups and two controls groups. The controls received water and the treated received 25mg/kg/day of A. muricata by gavage for ten days. One hour after the last dose, the first treated group received 99mTc-DMSA and the second sodium 99mTc-phytate (0.66MBq each group), both via orbital plexus. Controls followed the same protocol. Forty min later, all groups were sacrificed and the blood, kidney and bladder were isolated from the first treated group and the blood, spleen and liver isolated from the second treated group. The percentage of radioactivity per gram of tissue (%ATI/g) was calculated using a gamma counter. RESULTS: The statistical analysis showed that there was a statistically significant decrease (p<0.05) in the uptake of %ATI/g in bladder (0.11±0.01and1.60±0.08), kidney (3.52±0.51and11.84±1.57) and blood (0.15±0.01and 0.54±0.05) between the treated group and control group, respectively. CONCLUSION: The A. muricata hydroalcoholic extract negatively influenced the uptake of 99mTc-DMSA in bladder, kidney and blood of rats.


Introduction
Annona muricata L. (Annonaceae), commonly known as soursop, is found in Central America and South America, including the North, Northeast and Southeast regions of Brazil.
Traditionally, the leaves are used for headaches, insomnia, cystitis, liver problems, diabetes, hypertension and as an anti-inflammatory, antispasmodic and antidysenteric drug.The decoction of the leaves have parasiticide, antirheumatic and antineuralgic effects when used internally, while the cooked leaves, applied topically, treat rheumatism and abscesses 1 .Currently, this plant has been also used as a natural treatment of parasitic infections and cancer 2 .
The A. muricata has essential oils (β-caryophyllene, δ-cadinene and cadinol), chemical components such as alkaloids (reticulin, coreximine, coclarine and anomurine) 3 and antineoplastic substances such as acetogenins, which according Colman-Saizarbitoria et al. 4 , exert a selective cytotoxicity on tumor cells without affecting normal cells.Popularly, soursop leaves are used to treat hypertension, inflammation, liver disease, cystitis, insomnia, headache, hyperglycemia, diabetes and parasitosis.The studies by Luna et al. 5 showed that extract of A. muricata was lethal to the freshwater snail, Biomphalaria glabrata, one of the main transmitters of schistosomiasis (S. mansoni) in Brazil, a parasitic endemic disease in our country.muricata has also been shown to be effective against multi-drug resistant (MDR) cancer cell lines.
Nuclear medicine images have allowed health professionals to measure physiological processes and identify changes related to various diseases.Diseases can alter the biodistribution of a radiopharmaceutical and the analysis of scintigraphic images can help physicians to identify altered biological activity and diagnose clinical disorders.However, other factors such as drug interactions (natural or synthetic compounds) can alter the biodistribution of a radiopharmaceutical 6,7 .If these drug interactions are not anticipated, poor image quality in the nuclear medicine examinations could lead to a misdiagnosis with a possible need to repeat the examination, thereby increasing the radiation exposure to the patient and the staff 8 .
In the applications in nuclear medicine diagnostic, the isotope technetium-99m ( 99m Tc) has preferred choice.Technetium-99m may bind to different substrates or ligands, resulting radiopharmaceuticals with affinity for different organs, systems or receptors in the body [6][7][8] .Technetium-99m in the form of sodium pertechnetate, or labeled with other structures, such as sodium phytate ( 99m Tc-sodium phytate) or dimercaptosuccinic acid ( 99m Tc-DMSA) are the most frequently used radiopharmaceuticals in nuclear medicine 9 .
Dimercaptosuccinic acid labeled with technetium-99m ( 99m Tc-DMSA) has been the radiopharmaceutical of choice for highresolution images of the renal cortex and to estimate the function of the renal parenchyma with better sensitivity and specificity than other techniques 9 .The kidney uptake of 99m Tc-DMSA correlates well with the effective renal plasma flow, glomerular filtration rate and creatinine clearance.Thus, the kidney uptake of 99m Tc-DMSA provides a practical index for assessing renal function.Therefore, 99m Tc-DMSA is currently the standard method for assessing renal lesions in children with infectious diseases of the urinary tract 9 .
The radiocolloid sodium phytate labeled with technetium-99m ( 99m Tc-sodium phytate) is a radiopharmaceutical that has been widely used to study the liver and spleen 10 , especially for diagnosis and progression of disease liver.However, using this radiocolloid has also been used for biodistribution studies, which is useful for determining function parameters 10 .Biodistribution to the liver and bone marrow has shown good correlation with the severity of liver diseases such as cirrhosis and fibrosis of the organs and its prognosis.Thus, quantification of the uptake of 99m Tc-phytate serves as an excellent index of liver function 10 .
Natural products are widely used around the world for a variety of medical and domestic applications.However some of the biological effects and biochemical properties of these products are not yet completely understood.Thus, experimental models can be used to improve our understanding of the cellular and systemic mechanisms of action and biological effects of these natural products.
The present study aimed to evaluate, in experimental animals, the influence of the hydroalcoholic extract of Annona muricata (soursop) on laboratory parameters and biodistribution of two radiopharmaceuticals: dimercaptosuccinic acid labeled with technetium-99m, which is widely used in renal scintigraphy, and sodium phytate labeled with technetium-99m very effective in liver and/or spleen scintigraphy.

Methods
The experimental protocol was submitted to Ethics Committee for Use of Animals in Research (CEUA/UFRN) and approved under the number 035/2012.This protocol followed guidelines for the care and use of laboratory animals.

Extract
The Annona muricata (soursop) extract (prepared from a 10% dye-mother solution and diluted in water, Herbarium Laboratory, Rio de Janeiro/Brazil) was administered orally to the rats in single dose, during 10 days, in the treated groups.The control groups received water by the same way and period.All of the experiments were carried out before the expiration date of this product.

Surgery and sample collection
Twenty four male Wistar rats (100±20 g), were divided into two groups: control (n=12) and treated (n=12) groups.The treated group received daily 0.5 mL of soursop extract (25mg/kg/ day) by gavage for ten days, and the control group received 0. gram of each organ (%ATI/g) was calculated as described elsewhere Bernardo-Filho et al. 6 .

Laboratory analysis
Biochemical parameters were measured using the Konelab 60i spectrophotometer autoanalyzer, in the Onofre Lopes University Hospital.The serum levels of alanine aminotransferase (ALT), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), cholesterol and fractions (LDL and HDL), glucose, creatinine, triglycerides, total protein and fractions (albumin and globulin) were measured using the assay kit from Weiner, São Paulo, Brazil.All data were presented as mean±standard deviation.

Statistical analysis
Data were reported as means±SD.The %ATI/g was compared by Mann-Whitney test and biochemical parameters by the Student's t-test, considering p<0.05 statistically significant in both tests.Stat Graph Pad software was used to perform statistical analysis.

Results
The Table 1 shows the biodistribution of 99m Tc-sodium phytate in organs of control group rats and treated group rats, 40 min after administration of the radiopharmaceutical.The statistical analysis showed that there was no statistically significant difference (p>0.05) in the uptake of radioactivity (% ATI/g) in the spleen, liver and blood, when compared the treated group and control group, despite of the decrease observed in the treated group.The Table 2 shows the biodistribution of 99m Tc-DMSA in organs of control group rats and treated group rats, 40 min after administration of the radiopharmaceutical 99m Tc-DMSA.

Organs
The statistical analysis showed that there was a statistically significant decrease (p<0.05) in the uptake of radioactivity (% ATI/g) in the bladder, kidney and blood comparing the treated group and control group.
The analysis of biochemical parameters (Table 3) shows that there was a statistically significant difference (p<0.05)only in the serum levels of LDL cholesterol (p=0.0141) between the treated group and control group.There was no alteration on hematological blood parameters of the treated group when compared with the control group (data no showed).
related to the presence of acetogenins, characteristic of Annonaceae compounds that exert selective cytotoxicity on tumor cells without affecting normal cells 11 .
There are over 250 known species of Annonaceae, but Annona muricata is one of the most used in Brazil for its local availability, widespread use and alleged healing powers 1 .
However, liver toxicity is a potential complication of these herbal compounds that may lead to liver failure, as demonstrated in studies described by Holanda et al. 7 with the extract of Aloe vera, a medicinal plant widely used by the general population, but despite having facilitated the uptake of sodium pertechnetate (Na 99m TcO 4 ) in rats organs, it was responsible to increasing the levels of hepatic enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT), indicating a possible liver damage.However, in the present study, the A. muricata extract did not significantly alter the levels of AST and ALT, demonstrating no hepatotoxicity.
Adeyemi et al. 12 showed in their study that there was a significant reduction in the concentration of blood glucose in diabetic rats treated with Anonna muricata (soursop), a result not found in our study, possibly due to the low extract concentration used.High levels of unsaturated fatty acids seem to play important role in improving the lipid profile, both in humans and in animals, being appointed more substantial effect on reducing low density lipoprotein (LDL), considered a cardiovascular risk factor 13 .The study done by Adewole and Ojewole 14 showed that diabetic rats treated with streptozotocin showed a decrease in levels of total cholesterol, LDL and triglycerides.Our study showed that treatment of normal rats (non-diabetic) with Anonna muricata also causes a significant decrease in the levels of LDL cholesterol (p<0.05).
Several experimental models have been used to evaluate the properties of synthetic and natural drugs [6][7][8] .The biodistribution of radiopharmaceuticals in the animal organism can be changed with concomitant use of drugs natural and/or synthetic, certain diets, parasitic infections, the use of cigarettes and surgery, as demonstrated in studies described by Araújo-Filho et al. 15 and Rêgo et al. 16 .Changes in biodistribution of sodium pertechnetate (Na 99m TcO 4 ) in organs of rats were observed after treatment with natural products such as Artemisia vulgaris extract 17 and Aloe vera extract 7 .
Nuclear medicine imaging using dimercaptosuccinic acid radiopharmaceutical labeled with technetium-99m ( 99m Tc-DMSA) is currently the standard method for assessing renal lesions especially in children with infectious diseases of the urinary tract 9 .
Study described by Açucena et al. 18 showed that splenectomy in rats altered renal function and uptake of 99m Tc-DMSA.Stokland et al. 9 showed that decreased uptake of 99m

Discussion
Medicinal plants are used to treat a number of diseases around the world and help restore the quality of life of patients.
Over the last few decades, considerable progress has been made towards exploring the biological activities of various plantderived constituents (i.e., phytochemicals).These compounds have been isolated and their pharmacological properties have been evaluated 11 .Annona muricata is used in popular medicine to treat diabetes, cough and hypertension and has analgesic, antiinflammatory and anticancer activities.The antitumor activity is pyelonephritis is caused by dysfunction of renal tubular cells and ischemia.Cortical lesions are characterized by defects in single or multifocal uptake of 99m Tc-DMSA, but can often be observed decrease in uptake diffuse 9 .In our study, it was demonstrated that soursop extract significantly decreased the uptake of 99m Tc-DMSA in kidney, bladder and blood, probably due to biological and/or morphological changes caused by this extract.
Sodium phytate labeled with 99m Tc ( 99m Tc-sodium phytate) is a colloid that has been used as an agent for scintigraphic imaging of the liver and spleen.Parameters such as dimensions and capacity of the liver uptake of the radiopharmaceutical have been used for diagnosis of hepatic disease and its progression and for evaluation of liver function.However, this radiocolloid has also been used for biodistribution studies, which is useful for determining function parameters 19 .
Study in splenectomized rats showed that the biodistribution of 99m Tc-phytate to the liver was higher than in non-splenectomized rats, meaning that the operative process improved the hepatic uptake of radiotracer.According to these authors, this result coincided with the improvement of liver function validated by normal liver enzymes ALT and AST found in splenectomized rats compared with controls.
In our study we demonstrated no statistically significant changes in hepatic and splenic uptake of the radiopharmaceutical sodium phytate labeled with technetium-99m, after the use of A. muricata extract.This fact leads us to believe that this extract, at the studied concentration, does not interfere with normal biodistribution of 99m Tcsodium phytate in organs of Wistar rats.

Conclusions
The results of this study suggest that the A. muricata hydroalcoholic extract negatively influenced the uptake of 99m Tc-DMSA in bladder, kidney and blood of rats.
5 mL of water by the same way and period.Treated animals were subdivided into two subgroups: first group (n=6) was submitted to radiopharmaceutical injection 99m Tc-DMSA and the second (n=6) to 99m Tc-sodium phytate.The controls also suffered the same division and treatment.These radiopharmaceuticals have been labeled with technetium-99m ( 99m Tc-DMSA and 99m Tc-sodium phytate with 0.66 MBq of radioactivity).The technetium-99m was eluted from a 99 Mo/ 99m Tc generator produced by the Institute of Energy and Nuclear Research, São Paulo/Brazil and the kits of DMSA and sodium phytate were kindly donated by the Liga Norteriograndense contra o Câncer, Natal/RN.Each animal for group received 0.1 mL of specific radiopharmaceutical via orbital plexus, on the 10 th day of treatment.After 40 minutes they received the specific dose of the radiopharmaceutical for each group, then, all the animals were sacrificed after anesthesia intramuscular of Ketamin 50mg/ kg (Ketamina ® -Pfizer do Brasil Ltda) associated with Xylazin 10 mg/ml (Ropum ® -Bayer do Brasil Ltda).Blood and organ samples (spleen and liver, from the group treated with sodium phytate, and kidney and bladder, from the group treated with DMSA) were isolated, cleaned with 0.9% saline solution and the radioactivity of each organ was determined by means of an automatic gamma counter (1470 Wizard, Perkin Elmer, Finland) with automatic correction for decay and efficiency of 86%, in the Nucleus of Experimental Surgery-UFRN.The percentage of radioactivity per

TABLE 1 -
Results of effect of A. muricata extract on the uptake of 99m Tcsodium phytate of Wistar rats.

TABLE 2 -
Results of effect of A. muricata extract on the uptake of 99m Tc-DMSA of Wistar rats.
Data are reported as means±SD.*p<0.05,compared with control.
Tc-DMSA in acute

TABLE 3 -
Results of effect of A. muricata extract on biochemical parameters of Wistar rats.