Guava extract (Psidium guajava) alters the labelling of blood constituents with technetium-99m

Guava extract ( Psidium guajava ) alters the labelling of blood constituents with technetium-99m 1 first-author 1 1 1 1 2 3 †E-mail:adenilso@uerj.br †E-mail:adenilso@uerj.br adenilso@uerj.br 1 Department of Biophysics and Biometry, Institute of Biology Roberto Alcantara Gomes, University of Estate of Rio de Janeiro, Rio de Janeiro 20551030, Brazil Department of Biophysics and Biometry, Institute of Biology Roberto Alcantara Gomes, University of Estate of Rio de Janeiro, Rio de Janeiro 20551030, Brazil Department of Physiology, Biological Sciences and Health Center, Federal University of Sergipe, São Cristóvão, Sergipe 49100000, Brazil Department of Physiology, Biological Sciences and Health Center, Federal University of Sergipe, São Cristóvão, Sergipe 49100000, Brazil Department of Pharmacology and Psychobiology, Institute of Biology Roberto Alcantara Gomes, University of Estate of Rio de Janeiro, Rio de Janeiro 20551030, Brazil Department of Pharmacology and Psychobiology, Institute of Biology Roberto Alcantara Gomes, University of Estate of Rio de Janeiro, Rio de Janeiro 20551030, Brazil Psidium guajava (guava) leaf is a phytotherapic used in folk medicine to treat gastrointestinal and respiratory disturbances and is used as anti-inflammatory medicine. In nuclear medicine, blood constituents (BC) are labelled with technetium-99m ( 99m Tc) and used to image procedures. However, data have demonstrated that synthetic or natural drugs could modify the labelling of BC with 99m Tc. The aim of this work was to evaluate the effects of aqueous extract of guava leaves on the labelling of BC with 99m Tc. Blood samples of Wistar rats were incubated with different concentrations of guava extract and labelled with 99m Tc after the percentage of incorporated radioactivity (%ATI) in BC was determined. The results suggest that aqueous guava extract could present antioxidant action and/or alters the membrane structures involved in ion transport into cells, thus decreasing the radiolabelling of BC with 99m Tc. The data showed significant ( P < 0.05) alteration of ATI in BC from blood incubated with guava extract. Antioxidant Blood constituents Psidium guajava Technetium-99m INTRODUCTION 1 Psidium guajava (guava), belonging to the family of Myrtaceae , is a native of tropical America and has long been naturalized in Southeast Asia. The positive effects of guava extracts on human ailments have been described (Lozoya, 1999 1999 ). The pharmacological actions and the medicinal uses of aqueous extracts of guava leaves in folk medicine include the treatment of various types gastrointestinal disturbances such as vomiting, diarrhea, inhibition of the peristaltic reflex, gastroenteritis, spasmolytic activity, dysentery, abdominal distention, flatulence and gastric pain (Lutterodt, 1992 1992 ; Aguilar et al., 1994 1994 ; Lozoya et al., 1994 1994 ). These extracts have also been indicated to cause disturbances of the central nervous system: insomnia, convulsions and epilepsy (Lutterodt and Maleque, 1988 1988 ; Meckes et al., 1996 1996 ). Bronchitis, asthma attacks, cough, pulmonary diseases could be also treated with guava teas (Batick, 1984 1984 ; Khan and Ahmad, 1985 1985 ) and could also be useful as anti-inflammatory and hemostatic agent (Liu, 1988 1988 ). Moreover, aqueous extracts of guava leaves were described to be effective against a number of microbial strains: Aeromonas hydrophila , Shigella spp. and Vibrio spp. (Chulasiri et al., 1986 1986 ), Staphylococus aureus and β - streptococcus group A (Jaiarj et al., 1999 1999 ), Escherichia coli , Pseudomonas aeruginosa , Bacillus subtilis (Abdelrahim et al., 2002 2002 ). In addition, anti-rotavirus activity has also been reported to exist in these extracts (Gonçalves et al., 2005 2005 ). Phytochemical studies have identified more than 20 compounds in guava extracts (Osman et al., 1974 1974 ; Begum et al., 2002 2002 ). The major constituents of its leaves were identified to be tannins, β -sitosterol, maslinic acid, essential oils, triterpenoids and flavonoids (Osman et al., 1974 1974 ; Arima and Danno, 2002 2002 ; Begum et al., 2002 2002 ; 2004 2004 ). Technetium-99m ( 99m Tc) has been the most used radionuclide in nuclear medicine diagnosis procedures (single photon emission computed tomography, SPECT) and in labelling of molecular and cellular structures that can be used as radiopharmaceuticals (radiobiocomplexes) (Saha, 2003 2003 ). Red blood cells labelled with 99m Tc ( 99m Tc-RBC) are radiobiocomplexes widely used in clinical nuclear medicine for several important applications (Wong et al., 2004 2004 ; Zaman et al., 2004 2004 ; Olds et al., 2005 2005 ; Harel et al., 2005 2005 ; Artiko et al., 2004 2004 ; Verdu et al., 2005 2005 ; Cortes et al., 2003 2003 ; Jin et al., 2004 2004 ; Slart et al., 2004 2004 ). Sequential steps of the intracellular labelling process of blood constituents include: (1) transmembrane transport of reducing agent (Sn 2+ ) and 99m TcO 4 − ions into the internal compartment of the RBC, (2) reduction of 99m TcO 4 − by Sn 2+ , and (3) subsequent binding of the reduced 99m Tc to hemoglobin (Dewanjee et al., 1982 1982 ). The band-3 anion transport system and calcium channels may be involved in transport of these ions (Callahan and Rabito, 1990 1990 ; Gutfilen et al., 1992 1992 ; Sampson, 1996 1996 ). Several authors have reported the effect of artificial and natural drug on the radiolabelling process (de Oliveira et al., 2003a 2003a ; 2003b 2003b ; Frydman et al., 2004 2004 ; Moreno et al., 2004 2004 ; Fonseca et al., 2005 2005 ). The drugs could alter the labelling of blood constituents acting as antioxidant agent, modify the membrane structure or decrease the efficiency of transmembrane transport system of stannous and pertecnetate ions into cells. In this context, compounds in aqueous extracts of guava leaves have been reported to present antioxidant action and some of their pharmacological effects could be related to interaction in calcium channels (Morales et al., 1994 1994 ; Qian and Nihorimbere, 2004 2004 ). However, to the authors’ knowledge, there is no report on the possible effects of guava extracts on radiolabelling of blood constituents as RBC or plasma proteins. This work was aimed at evaluating the effects of aqueous extract of guava leaves on the labelling of blood constituents with technetium-99m. MATERIALS AND METHODS 1 Animals 2 Adult male Wistar rats (3~4 months of age, body weight 250~350 g) were maintained in a controlled environment and allowed free access to water and food and ambient temperature was kept at (25±2) °C. Experiments were conducted in accordance with the rules of the Institutional Committee of Animal Care. Preparation of guava extract 2 The guava extracts were prepared with leaves of Psidium guajava L. (Conde Garcia et al., 2003 2003 ) obtained from the Campus of the Federal University of Sergipe (Aracaju, Brazil), and collected between June and July of 2000 (plants were free of toxic compounds). A botanic specimen was compared with the exsicata at Federal University of Sergipe Herbarium, Brazil (code ASE 03304, collectors T.S. dos Santos and V.S. Paes, collectors No. 4122, registering book No. 1, page 138, exsicata No. 04531). To prepare the extracts, 20 mg of dry leaves were ground in 2 ml of 0.9% NaCl (100 °C, 5 min). The crude extract was filtered, centrifuged (1500 r/min, 5 min) to obtain the final extract. The supernatant was considered as the concentration at 10 mg/ml of guava leaves. Radiolabelling in vitro of blood constituents 2 Heparinized blood (500 μl), was withdrawn from the Wistar rats and incubated with 100 μl of guava extract at different concentrations (6.25, 12.5, 25.0, 50.0 and 100.0 mg/ml) or with a saline solution alone, as control, for 1 h (room temperature). After this the radiolabelling of blood constituents with 99m Tc (Na 99m TcO 4 , Institute of Energetic and Nuclear Research, National Commission of Nuclear Energy, São Paulo, Brazil) was performed as described in (Bernardo-Filho et al., 1983 1983 ). The samples were centrifuged (1500 r/min, 5 min) and aliquot of plasma (P) and blood cells (BC) were isolated. Another aliquots of P and BC were separated and precipitated in trichloroacetic acid (5%) and centrifuged (1500 r/min, 5 min) to isolate soluble (SF) and insoluble fractions (IF). The radioactivity in P, BC, SF-P, IF-P, SF-BC and IF-BC were determined in a well counter (Packard, model C5002, Illinois, USA) and the percentage of incorporated radioactivity (%ATI) was calculated as described in (Bernardo-Filho et al., 1983 1983 ; 1986 1986 ). Statistical analysis 2 Data obtained are reported as means±standard deviation of %ATI. To compare the treated (5 blood samples for each extract concentration) and control groups (10 blood samples), the one way analysis of variance (ANOVA) test was performed to verify possible statistical difference between the experimental groups. After this, a rigorous statistical post test (Tukey) was chosen to identify the P value ( P < 0.05 as lesser significant level) and compare each treated group with control. InStat GraphPad software was used to perform statistical analysis (GraphPad InStat version 3.00 for Windows 95, GraphPad Software, San Diego California, USA). RESULTS 1 Fig. 1a 1a shows the %ATI in insoluble (IF-P) and soluble (SF-P) fractions isolated from plasma separated from whole blood treated with different concentrations of guava extract. Analysis of these data indicates that guava extract significantly ( P < 0.05) reduced the radioactivity uptake in IF-P at the highest concentration studied (100.0 mg/ml) but no alteration was obtained with lesser concentration used. # P ≤0.05; ## P ≤0.01; ### P ≤0.001, when compared to control group of plasma; * P ≤0.05; ** P ≤0.01; *** P ≤0.001, when compared to control group of blood cells Fig. 1b 1b shows the %ATI in insoluble (IF-BC) and soluble (SF-BC) fractions isolated from blood cells separated from blood treated with different concentrations of guava extract. Analysis of these data indicates that incubation with guava extract at low concentrations (6.0 and 12.5 mg/ml) did not reduce the %ATI of IF-BC. However, when the highest concentrations (25, 50 and 100 mg/ml) were used significant alterations of radioactivity uptake in insoluble fractions of blood cells resulted. Fig. 1c 1c shows the %ATI in blood cells (BC) and plasma (P) from blood treated with different concentrations of guava extract. Analysis of these data indicated that guava extract alters the distribution of radioactivity in these two compartments (BC and P) at all guava extract concentrations studied. The data in this figure present an inverse dose-dependent relation with greater decreasing of cells %ATI at highest concentrations of guava extract. DISCUSSION 1 Analysis of data presented in this study showed that aqueous guava extract could modify the fixation of 99m Tc in plasma proteins only at high concentrations (Fig. 1a 1a ). In cellular proteins, guava extract could affect the uptake of 99m Tc at high but also at lesser concentrations (Fig. 1b 1b ). However, the effects of this extract were more evident on the distribution of 99m Tc between the blood compartments. The data analysis results suggest that aqueous extract of guava leaves used in this work could decrease the fixation of radioactivity in blood cell in a typical dose-dependent relation (Fig. 1c 1c ). Some authors have described that natural and synthetic products can alter the labelling of blood constituents with 99m Tc (Frydman et al., 2004 2004 ; Valenca et al., 2005 2005 ; Fonseca et al., 2005 2005 ). The labelling of blood constituents could decrease due to the action of drugs in (1) binding at the same sites on the blood constituents, (2) direct inhibition (chelating action) of the stannous and pertechnetate ions, (3) direct oxidation or generation of free radicals that could oxidize the stannous ion, (4) antioxidant action impeding or decreasing the stannous ion oxidization, and (5) alteration of the plasma membrane structure or modifying the transport systems of stannous and pertechnetate ions into cells. Several compounds (as flavonoids) present in guava extracts are transported in blood attached to plasma proteins (Podhajcer et al., 1980 1980 ). The fixation of 99m Tc in plasma proteins occurs at different proteins sites with albumin being the principal protein involved (Early and Soddee, 1995 1995 ). The binding sites in these proteins could be the same binding sites of 99m Tc and thus in the presence of guava extract compounds the plasma protein labelling with 99m Tc could be reduced. However, the data shown in this work suggest that at only highest concentration of aqueous guava extract these compounds (flavonoids and others phenolic compounds) could compete with the binding of 99m Tc with plasma proteins. The antioxidant activity of some compounds could be used to prevent various chronic diseases such as heart-disease, diabetes, cancer, arterial thrombosis, cataract and may provide health-promoting effects (Kimura et al., 1985 1985 ; Qian and Nihorimbere, 2004 2004 ). Chemical analysis of guava extracts has revealed the presence of essential oils, tannins, saponins, carotenoids, flavonoids and triterpenes (Cuellar et al., 1984 1984 ; Mercadante et al., 1999 1999 ; Arima and Danno, 2002 2002 ; Begum et al., 2002 2002 ). The different phenolic phytochemicals in guava leaves have been described to possess antioxidant action and able to inhibit peroxidation reaction in living systems (Kimura et al., 1985 1985 ; Vanacker et al., 1995 1995 ; Qian and Nihorimbere, 2004 2004 ). Thus, some compounds present in guava extracts can inhibit the oxidation of the stannous ions and to alter the labelling of plasma and cellular proteins with 99m Tc as well as interfere with the distribution of this radionuclide between plasma and cellular compartments. Moreover, data have demonstrated that aqueous and hidroalcoholic extracts of guava leaves present anti-microbial activity (Arima and Danno, 2002 2002 ; Qa’dan et al., 2005 2005 ). Flavonoid glycosides and flavonoids as guaijavarin and quercetin have been isolated and present activity against Salmonella enteritidis and Bacillus cereus (Arima and Danno, 2002 2002 ). This anti-microbial activity may be related to action on plasma membrane modifying its structure and decreasing or impeding the transport of stannous and pertechnetate ions into blood cells that can alter the labelling efficiency of blood constituents with 99m Tc. On the other hand, the above cited compound quercetin has been described to decrease the spasmolytic activity in experimental models (Morales et al., 1994 1994 ). This effect was related to a possible narcotic (opioid) component in the guava leaves (Lutterodt and Maleque, 1988 1988 ; Lutterodt, 1992 1992 ) while other data have indicated inhibition of acetylcholine release in neuromuscular junctions by calcium-antagonistic action of quercetin decreasing the inward calcium membrane current leading to a decrease of smooth muscle contractile force (Lozoya et al., 1994 1994 ; Morales et al., 1994 1994 ; Re et al., 1999 1999 ) and depression of myocardial inotropism (Conde Garcia et al., 2003 2003 ). The radiolabelling process of red blood cells with 99m Tc depends on the entry of stannous and pertechnetate ions into these cells through ionic channels (Callahan and Rabito, 1990 1990 ; Gutfilen et al., 1992 1992 ). The action of quercetin and/or other compounds present in aqueous guava extract blocking and/or binding on calcium and anions (as band 3) channels could make difficult the transport of stannous ions through plasma membrane and to decrease the labelling of blood cells with 99m Tc explaining in part the data obtained in this work. In conclusion, our data suggest that aqueous extracts of guava leaves could present antioxidant action and/or effects on the membrane structures involved in ions transport altering the radiolabelling of blood constituents with 99m Tc and that precaution should be taken in examinations of nuclear medicine based on this procedure in patients using guava extracts. References SI Abdelrahim , AZ Almadboul , MEA Omer , A Elegami . 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