Medicinal plants widely used in traditional medicine constitute an important source of new, safer and maybe biologically active compounds against many  disorders  in the herbal medicine in various countries. Furthermore, the scientific interest in the determination of properties associated with medicinal herbs is increasing in the world

(Adisakwattana et al., 2011; Ma et al., 2011). In addition,some authors have studied substances isolated from medicinal herbs, as the Bisabololoxide that is isolated from the Matricaria recutita L. (Ogata et al., 2010).

 

Tabebuia avellanedae is a tree from the Bignoniaceae family and native to Central and South America. It is known as “pau d’arco” , “taheebe”, “lapacho” or “ipe roxo” and its inner bark is used as antimicrobial (Machado et al., 2001), anti-inflammatory (Lira et al., 2008), analgesic, antinociceptive (de Miranda et al., 2001), and anti-tumor drugs (Ueda  et al., 1994). Phytochemical analysis of T. avellanedae have demonstrated the presence of quinones (Sharma et al., 1998), furanonaphthoquinones (Díaz and Medina, 1994), naphthoquinones (Manners and Jurd, 1976), benzoic acid, benzaldehyde derivatives (Wagner et al., 1989), cyclopentene dialdehyde (Koyama et al., 2000), flavonoids and iridoids (Nakano et al., 1993) and phenolic glycosides (Warashina et al., 2004).

 

Lapachol (2-hydroxy-3-(-3-methyl-2-butanyl)-1,4-naphthoquinone) has been isolated from T. avellanedae extracts. There are interest in the studies of this substance due to its anti-tumor (Balassiano et al., 2005), anti-biotic (Santos et al., 2001), anti-leishmanial (Lima et al., 2004), anti-malarial (de Andrade Neto et al., 2004), anti-ulcer (Goel et al., 2004) and anti-inflammatory activities (Lira et al., 2008). Preparation of isolated of lapachol is commercially available and it was used in this study.

 

Radionuclides have been in various clinical evaluations (Saha, 2010) and in experimental models (Bustami et al., 2009; Santos et al., 2013; Frederico et al., 2014,). Technetium-99m (^99mTc) has been widely used in these procedures due to its optimal physical characteristics (6 h physical half-life and gamma emission) that give a negligible environmental impact (Saha, 2010). Several authors have demonstrated the effects of synthetic and natural drugs on the labeling process of blood constituents with ^99mTc (Fonseca et al., 2005; Bustami et al., 2009; Carmo et al., 2011).

 

Blood constituents labeled with ^99mTc have been used as radiobiocomplexes for a number of applications in nuclear medicine. The labeling of blood cells and cell structures is based on the transmembrane transport of a reducing agent (Sn⁺²) and pertecnetate (^99mTcO₄^-) ions into the red blood cells, reduction of ^99mTcO₄^- by Sn⁺², and subsequent binding of the reduced ^99mTc to internal structures. The band-3 anion transport system and calcium channels may be involved in the transportion of ^99mTcO₄^- and Sn⁺², respectively. The fixation of ^99mTc in plasma proteins also depends on the reducing agent action occurring at different proteins sites and albumin is the principal protein involved (Saha, 2010). 

 

The effect of drugs altering the labeling of blood constituents could be due modification of the membrane structure (Braga et al., 2013), decreasing the efficiency of transmembrane transport system of  ^99mTcO₄^-  and  Sn⁺² ions into cells. Redox property and/or metal chelator could be another properties associated with the drugs.

 

In this investigation, the effect of a T. avellanedae extract and of a commercial preparation of lapachol on the labeling of the blood constituents with ^99mTc was evaluated.

 

 

Histological analysis

 

Histological preparations were carried out with blood samples treated with various concentrations of T. avellanedae extract for 60 min at room temperature. Blood smears were prepared, dried, fixed and stained. After that, the morphology of the red blood cells was qualitatively evaluated under optical microscope.

 

Statistical analysis

 

Data are reported as (means ± SD) of %ATI and compared the treated (n=10 for each extract concentration) and control group (n=10) by One way analysis of variance - ANOVA, followed by Tukey 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).

Figure 1 shows the %ATI in blood cells and plasma compartments from whole blood treated with different concentrations of T. avellanedae extract. The analysis of these data indicates that T. avellanedae extract alters significantly (p<0.05) the distribution of radioactivity between the two blood compartments.

 

 

Figure 2 shows the %ATI in insoluble (IF-P) and soluble (SF-P) fractions isolated from plasma separated from whole blood treated with different concentrations of T. avellanedae extract. The analysis of these data indicates that T. avellanedae extract significantly (p<0.05) reduced the radioactivity fixation in IF-P.

 

 

Figure 3 shows the %ATI in insoluble (IF-BC) and soluble (SF-BC) fractions isolated from blood cells separated from blood treated with different concentrations of T. avellanedae extract. The analysis of these data indicates that the incubation with T. avellanedae extract significantly alters the radioactivity fixation on insoluble blood cells fraction at the higher concentrations used (200 mg/ml).

 

 

The qualitative comparison of the shape of the RBC (non-treated and treated with natural extracts) under optical microscopy has revealed strong morphological alterations due to the treatment of blood with T. avellanedae extract in the concentrations of 12.5 and 200 mg/ml. The histological preparation of a sample of blood (control-non-treated) with normal shape of RBC is shown in Figure 4. Figures 5 and 6 show histological preparations of blood treated with T. avellanedae in which are shown qualitative and strong alterations on the shape of the RBC.

 

 

 

 

 

 

 

The evaluation of the influence of drugs on the labeling of blood constituents is highly relevant due to some products, as chocolate, can interfere in the quality of the examinations using red blood cells labeled with ^99mTc (Bustami et al., 2009).

 

The analysis of data presented in Figure 1 show that the aqueous T. avellanedae extract can modify the distribution of ^99mTc between the cellular and plasma compartments almost in all tested concentrations. However, the fixation of ^99mTc in cellular proteins could be altered at high concentrations of this extract (Figure 3).  The fixation of the ^99mTc plasma proteins is also blocked by the presence of the T. avellanedae extract (Figure 2). This finding is interesting and it suggests that the entrance of the stannous and pertechnetate would be blocked on a depended matter (decreasing the radioactivity on the blood cells) (Figure 1). However, only in the highest concentration of the extract, the fixation of the ^99mTc on the blood proteins would be blocked probably due to the anti-oxidant and/or scavenger activities of the substances in the T. avellanedae extract.  These redox properties could be associated with the chemical analysis of T. avellanedae extracts revealed the presence of various compounds as naphthoquinones, flavonoids, quinoid compounds and phenolic glycosides (Warashina et al., 2004). The phenolic compounds presents in different herbal extracts have been described to possess antioxidant and chelating action and be able to inhibit peroxidation reaction in the living systems (Simoes-Pires et al., 2005; Soobrattee et al., 2005). On the other hand, It was described that antimicrobial effects of b-lapachol could be related to the formation of reactive oxygen species (Guiraud et al., 1994). Thus, some compounds present in T. avellanedae  extracts  could  be  capable to impede or facilitate the oxidation of the stannous ions and alter the labeling of cellular proteins with ^99mTc as well interfere with distribution of this radionuclide between plasma and cellular compartments.

 

Other hypothesis that could explain the effects of T. avellanedae extracts on labeling of blood cells with ^99mTc is the interaction of constituents of this extract with ion channels. In fact, it was proposed that the antinociceptive effect of T. avellanedae may be related to an activation of the adenosine receptors (de Miranda et al., 2001).  Other membrane proteins as band-3 and calcium channel may have their function altered by compounds present in T. avellanedae extract decreasing or impeding the transport of Sn⁺² and ^99mTcO₄^- into blood cells and in consequence to modify de distribution of ^99mTc between plasma and cellular compartments.

 

The data obtained in this work show that the labeling of plasma proteins with ^99mTc could be decreased by the aqueous T. avellanedae extract used (Figure 2). Pharmacokinetics data have demonstrated that some compounds (as flavonoids) present in herbal extracts can be transported in blood attached to plasma proteins (Guiraud et al., 1994). Moreover, the already cited oxidant chelating properties of compounds present in T. avellanedae extract also could be related to effect obtained. Taken together, the binding in same proteins sites that the binding sites of ^99mTc and oxidant/chelating properties of T. avellanedae extract compounds could explain the decreasing of labeling of plasma proteins with ^99mTc.

 

In the procedure of labeling RBC with ^99mTc, the stannous and pertechnetate ions pass through the plasma membrane (Gutfilen et al., 1992). Then, as reported to the tobacco extract (Oliveira et al., 2003) and to Maytenus ilicifolia extract (Oliveira et al., 2000), histological alterations of the red blood cells could be responsible for modifications on the labeling of the RBC with ^99mTc. Furthermore, the results obtained with the qualitative comparison of the shape of the RBC (treated and not treated with T. avellanedae extracts) under optical microscopy also justify the modifications in the fixation of ^99mTc by the red blood cells. The achieved results have revealed strong morphological alterations due to the treatment of blood with T. avellanedae extract in two of the concentrations studied (Figures 5 and 6).

 

The analysis of Table 1 suggests that lapachol did not affect the distribution of ^99mTc between cellular and plasma compartments or the binding of this radionuclide in cellular and plasma proteins. The pharmacological actions of lapachol include antitumor, antibiotic, antimalarial, antiinflammatory and antiulceric activities (Subramanian et al., 1998) besides molluscicidal, cercaricidal and trypanocidal activities (Santos et al., 2001; Lima et al., 2004). Oxidative stress and alkylation of cellular nucleophiles have been proposed to explain the lapachol effects on biological system (Bolton et al., 2000). In fact, it was described the generation of reactive oxygen species in the bioactivation of lapachol by P450 reductase (Kumagai et al., 1997) and an electrochemical study (Goulart et al., 2003). However, the absence of effects of lapachol labeling of blood constituents with ^99mTc (Table 1) could be related to the concentrations used in this work, or this substance would be not responsible by our findings. Considering the quantity of lapachol in the T. avellanedae, probably the concentration of lapachol isolated used in the experiments (Table 1) would be small in comparison with the quantity of this molecule extract in the highest concentration. In consequence, the lapachol concentrations would be too low to induce any effect. In addition, the effect of a chemical compound in an extract is associated with an integrative and synergic action among several compounds (Galindo et al., 2010; Carmona and Pereira, 2013). This fact could occur with the lapachol when was used alone. 

 

In conclusion as the labeling of blood constituents with ^99mTc depends on the presence of a reducing, probably the extract of T. avellanedae has substances with redox properties. In addition, probably these properties are not associated with the lapachol or the concentration of lapchol used in this work was not sufficient to promote effect on the labeling process.

The authors declare that they have no conflict of interest.

This study was supported by grants and financial support from CAPES, CNPq and FAPERJ.