NMR Relaxivity, EPR Spectra, and Extraction Kinetics
The pH dependence of the relaxivity in yerba mate solutions is in good agreement with the observation in some of the volunteer studies where good filling of the bowel was achieved and the positive contrast in the bowel was higher than that in the stomach. The dependence of the relaxation rate on the pH value may offer a diagnostic potential for pH mapping during the passage of the contrast agent from the stomach into the bowel. However, it should be noted that both pH and concentration changes may affect the local signal intensities, which might in turn complicate the interpretation of the data. Systematic studies of pH sensitivity in vivo have not yet been performed.
The concentration ratio between manganese and iron found in the elemental analysis of the yerba mate extract is quite similar to that reported in blueberry juice and pineapple juice (3, 4). The overall manganese content reported for these materials was slightly higher (about 20 mg/liter for blueberry juice and about 26 mg/liter for pineapple juice). However, these juices typically are turbid formulations, and no information is available regarding the percentage of manganese available in dissolved form and the percentage of manganese bound to colloidal particles. Comparing the relaxation time and elemental content of manganese in pineapple juice reported in (4) with our data in the yerba mate, the relaxivities of both agents as a function of the elemental manganese content seem to be quite similar.
As indicated in other studies on the manganese content of food and medicinal plants (15, 16), both the absolute elemental content of manganese and its availability for hot water extraction vary significantly among different species or cultivars and even within single cultivars. Studies on the mineral content of yerba mate have reported total Mn contents between 328 mg/kg and 1.24 g/kg for different samples of yerba mate (6).
Both the linear increase of the relaxation rate and the roughly monoexponential extraction behavior with time of the manganese indicate a homogeneous manganese reservoir in yerba mate. The oxygen sensitivity of freshly prepared yerba mate extracts suggests that the manganese in the mate extract may be associated with the molecules responsible for the antioxidant action of mate (8) and that the oxidation state of these molecules dramatically influences the relaxivity of the manganese ions. In instant preparations no comparable time dependence of the relaxivity could be observed. This again is consistent with the oxidation effect as the complexes in the instant preparation have a long history of air exposure.
In comparing the analytical information on the total manganese content in yerba mate as found in the literature and the measured manganese content of the instant solutions, hot water extracts, and macerations prepared in our studies, we find that most of the manganese content in yerba mate is actually available for extraction and that the extractable manganese is conserved during the production of instant formulations. The high availability of manganese for aqueous extraction is different from the findings for Helichrysum arenarium (16).
In order to gain further insights into the nature of the manganese contained in mate, 1H relaxation dispersion studies and EPR spectroscopy were performed. In order to keep the experiments simple, they were limited to the Pajarito instant mate.
By carefully comparing the NMRD results of instant yerba mate and MnCl2 solution, we see lower values for the molecular relaxivity at low resonance frequencies below 5 MHz while the relaxivity at high resonance frequency is slightly higher than for the MnCl2 solution. In the MnCl2 solution, the Mn2+ ions are complexed by water molecules into a hexaquo complex (17). While this complex readily exchanges water between the coordination sphere of the Mn2+ ion and the free water phase, the complex formed between Mn2+ ions and ligands from the yerba mate extract can be expected to be stable. However, this complex is still surrounded by an additional water coordination sphere. The low-frequency part of the dispersion for the hexaquo complex is due to contact relaxation of water protons in the inner coordination sphere of the Mn2+ ion (17). Only a minor dispersion can be observed for the yerba mate extract in this field region. This suggests that contact relaxation is strongly reduced in the case of the manganese-containing yerba mate extract compared to the hexaquo complex. This is in agreement with the NMRD behavior observed in other manganese complexes such as Mn–EDTA, where contact relaxation was even negligible in many cases. The dispersion between 1 and 10 MHz is present both for the MnCl2-solution and for the yerba mate extract solution. This indicates that the dipolar relaxation between electron spin and water protons is also active in the yerba mate solution, as it is in the hexaquo complex. The higher relaxivity at higher frequencies can be attributed to slightly longer rotational correlation times of the complex compared to the hexaquo complex and/or the electronic relaxation time dispersion of Mn2+ in the complex. Depending on the strength of these effects, even more dramatic increases in the relaxivity of manganese complexes can be observed for manganese complexes with other substances such as concanavalin A (17).
On the basis of the rather similar relaxation time dispersion curves for the Pajarito solution, one might also expect that the ESR spectra for Mn2+ ions and the instant mate powder or the solution should be quite similar. The main features in the ESR spectrum of the powder sample (Fig. 4) are quite similar to other manganese-rich dry plant materials such as tobacco and tea (18, 19). At g factors close to 2 we find a narrow line corresponding to free radicals, some indications of the typical Mn2+ hyperfine sextet, and a very broad line with a width of about 40 mT in which most of the signal intensity of the system is contained. An additional, less intense broad line with a g factor of about 2.35 and a width of about 20 mT can also be observed. No similar line was reported in the studies on tea and tobacco.
The two broad lines can be also seen in the spectra of the solution and the redried solution on paper. However, in none of these latter measurements can any indication for the Mn hyperfine sextet be seen. This is very different from the observations on solutions obtained from green or black tea or extracts of green tea (5, 19), where the Mn hextet was reported to be the main feature of the ESR spectrum. In Ref. (5) an intensity loss of the Mn ESR signal in the presence of isolated polysaccharide ligands from green tea is reported. Because the spectra in that publication appear to have been recorded only over a small magnetic field range, it might be that a similar broad line containing most of the manganese signal intensity was overlooked. Even if this were the case, however, the tea spectra are different from the mate spectra reported here as there was at least some hyperfine structure observable in those spectra while any such structure is absent in the solution spectra of the dissolved mate samples. The absence of a resolved manganese hyperfine splitting in the solution spectrum of the mate powder suggests that the inner ligand sphere of the Mn–ion in the complex leads to considerable broadening of the manganese resonance lines. The absence of free radical signal from the solution spectra is again an indication for the presence of effective antioxidants in the solution that eliminate the free radicals that have probably been formed during drying and storage of the lyophilized instant formulation.
The broad spectrum without indications for hyperfine structure in the manganese complex from the yerba mate can be attributed to the existence of a static zero field splitting and enhanced electron relaxation caused by the ligands compared to the hexaquo complex, where only a transient zero field splitting exists due to the high symmetry of the complex.
In Vivo Imaging
The signals from the liver reference ROIs in both cases show only minor variation (Fig. 6), and the values for both the water session and the yerba mate session are nearly the same. The fact that no signal change in the liver was observed in the experiment with the yerba mate solution gives further evidence for the low bioavailability of the manganese from the yerba mate solution.
While the signal intensity of the water in the stomach shows no clear tendency with time (the evaluation was complicated by the presence of residual solid food particles in the stomach, which may have resulted in additional scatter in the data points), there is a significant decrease in intragastric signal intensity with time in the case of the yerba mate solution. However, even after more than 60 min there is still a strong hyperintensity of the gastric lumen. The observed decrease in intragastric signal intensity can be understood as the result of the measured pH sensitivity of the relaxation rate in yerba mate and increasing acidification of the gastric environment within the observation period.
As can be seen in Fig. 5, the hyperintense signal due to the yerba mate solution extends to the small bowel. Because the volunteer studied in these images was not requested to fast for a sufficiently long period, the spread of the yerba mate solution in the bowel is not spatially homogeneous as it is impeded by solid fecal objects. Nevertheless, sufficiently large intestinal regions for comparisons of the signal intensity by placing ROIs could be found in both the water and the yerba mate images.
The use of extracts from dexanthinated mate may be possible even without full isolation of the complex from the extract. A promising approach is dexanthination by means of supercritical CO2 (20). Furthermore, it was recently discovered that Ilex brevicuspis, a species closely related to I. paraguayensis and used as a component in many yerba mate formulations, contains no xanthines, while its other constituents are quite similar to I. paraguayensis (21). As those findings indicate, extracts from this Ilex species could be used as contrast agents without possible problems of CNS-active components. An advantage of yerba mate extract over fruit juices is the fact that it is free of macronutrients. Furthermore, the observed pH sensitivity of the complex may be useful in gaining further diagnostically relevant insights on gastrointestinal pH.
In addition to medical concerns, there is also an environmental consideration that creates interest in gadolinium-free contrast agents. Gadolinium-based contrast agents persist for a long time in ground water and have led in some places to an increase in the elemental gadolinium concentration in the aqueous environment by several orders of magnitude (22). For a complex of herbal origin, we can expect rapid natural degradation. Furthermore, natural manganese concentrations are sufficiently high so that the medical use of a manganese-based contrast agent will not have a noticeable effect on the natural manganese balance.