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Keywords:

  • MRI;
  • oral contrast agent;
  • manganese;
  • I. paraguayensis;
  • gastrointestinal

Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. REFERENCES

In this article we demonstrate the potential of herbal extracts from yerba mate (Ilex paraguayensis) as an oral contrast agent for MRI. At typical drinking concentrations, yerba mate acts as a “biphasic” contrast agent with T1 weighting at short echo times and T2 weighting at echo times greater than about 40 ms. Based on data obtained from X-ray fluorescence elemental analysis, NMR relaxometry, and ESR we identify the relaxation agent in the extract as a low-molecular-weight manganese complex. Yerba mate exhibits an unusually high manganese content that is readily available for hot water extraction. Despite the high elemental manganese levels in I. paraguayensis extract, no manganese-related toxicity of yerba mate has been observed even among heavy yerba mate drinkers, indicating that the manganese in the extract has only a very low bioavailability. Imaging results on staff and patient volunteers demonstrate good contrasting of the GI tract. The relaxation studies of the contrast agent show a sensitivity to pH that is consistent with imaging results from stomach and small bowel. Magn Reson Med, 2006. © 2006 Wiley-Liss, Inc.

The use of contrast agents in abdominal MRI applications is still a developing field, both in research and in clinical routine. A recent overview of the field is given by Giovagnoni et al. (1). For most MRI applications, contrast agents are administered i.v. In abdominal MRI, however, orally administered contrast agents are also used, often in combination with i.v. contrast agents. Gd-DTPA, which dominates the market in i.v. contrast agent applications, is not suitable for oral application due to instability of the complex under acidic pH conditions in the stomach.

The role of oral contrast agent preparations for abdominal imaging is twofold. In addition to providing the desired contrast properties for imaging, the agents also produce some dilatation of the lumen of the gastrointestinal organs. According to Giovagnoni et al. (1), the action of contrast agents can be classified as positive, negative, and “biphasic” (i.e., positive contrast for moderate T1 weighting and negative for stronger T2 weighting). Contrast agents based on synthetic complexes of gadolinium, manganese, or iron typically provide positive contrast at low concentration and biphasic contrast action at higher concentrations. Suspensions of superparamagnetic nanoparticles or mineral particles such as barium sulfate or clay typically lead to negative contrast.

Water or aqueous solutions of methyl cellulose act as a biphasic contrast agent with isointense signal in T1 weighting and hyperintense signal in T2 weighting.

Furthermore, several different foodstuffs have been proposed for use as gastrointestinal contrast agents (2), including blueberry juice (3), pineapple juice (4), and green tea (5), as well as materials with nonaqueous phases such as infant formula, dietary supplement formula, and vegetable oil. Major advantages of food-based contrast agents are their low toxicity, environmental degradability, low price, and good availability. However, there are also potential problems, including possible absorption in the gastrointestinal tract, especially of lipid components, and natural or preparation-related variations in the concentration and relaxivity of paramagnetic ions in the foodstuffs. The latter problem can be avoided when working with a food material available in a mass-produced instant, lyophilized form.

Based on some experiments with a nonclinical application we identified yerba mate (Ilex paraguayensis) as a foodstuff with very promising properties for possible contrast agent applications. It exhibits a high relaxivity, which is mainly due to manganese bound to natural complexing agents (about 16.5 mg/liter at normal drinking concentrations). Both the herbal tea and the instant formulations offer a high yield and long shelf life. Both are available at low price and the manufacturers are working toward more uniform product quality, which is associated with the mineral inventory of the product (6).

Yerba mate is consumed in high quantities as a tea especially in the southern parts of South America. In ethnopharmacology, several positive effects are ascribed to mate (7, 8). Negative effects of regular mate consumption known from some epidemiologic studies are mostly explained as thermal lesions due to special drinking customs (9). The high mineral content of yerba mate hot water extract has been known for a long time (6); results on the bioavailability of the minerals suggest that they are bound quite strongly to tannins (10). Yerba mate is registered in several countries' official lists of foods and nutrients and pharmacopoeia.

To our knowledge, the NMR properties of yerba mate extracts have not been investigated previously. Here, we present results from in vitro relaxation studies and first imaging experiments that demonstrate the potential of this complex for use as an oral contrast agent.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. REFERENCES

Yerba Mate Material

Mate extract for both NMR relaxometry and for chemical analysis was prepared by hot water extraction from ground yerba mate obtained from several suppliers. Furthermore, an instant preparation (“Pajarito yerba mate soluble instantaneo,” Lauro Raatz SA, Asunción, Paraguay) was used. For the in vivo studies, only the instant preparation was used.

In Vitro Studies

Longitudinal relaxation times of instant mate solutions and hot water extracts of ground yerba mate were studied by means of a saturation-recovery sequence at 22 MHz in a simple MRS 6 relaxometer (JSI, Lubljana, Slovenia).

In order to assess the accessibility of the manganese in yerba mate to hot water extraction and the kinetics of the exctraction, a time series of hot water extraction samples was performed for a sample of ground yerba mate, in which the extract was sampled after various extraction times up to 24 min. For these samples, both the elemental manganese content of the extract (by TXRF) and the longitudinal NMR relaxation time at 22 MHz were determined. Before the measurements, the extract samples were stored at room temperature for about 2 days. Furthermore, the variation of the relaxation time with pH was studied using NaOH and HCl to adjust the pH. For the samples from the extraction series and for several instant mate solutions, the concentration of dissolved heavy elements was determined by total X-ray fluorescence (TXRF) analysis using an Extra IIA unit (Atomika Instruments, Oberschleissheim, Germany).

For the instant solution, further NMR frequency-dependent relaxometry data (dispersion spectra, NMRD) were obtained using a homebuilt electronic field-cycling NMR relaxometer (11) and a commercial field-cycling relaxometer (Spinmaster FFC 2000, Stelar, Mede [PV]). Both in the constant-field experiments at 22 MHz and in the NMRD measurements, the relaxation of the solutions was monoexponential. The evaluation of the relaxometry data was performed by simple exponential regression in a spreadsheet program. Furthermore, ESR spectra of dry and dissolved Pajarito instant powder and of redried solution of the instant powder were recorded on a modified Bruker cw-ESR spectrometer with a rectangular cavity resonator.

In Vivo Studies and Image Processing

In vivo studies were performed on staff (N = 4) and patient (N = 2) volunteers, all of whom provided written informed consent to the study. In the volunteers, nothing was done to suppress peristalsis. Patients received 20 mg of bisacodyl (Laxbene, Merkle, Germany) for purging and 100 mg of pirencepine (Gastrozepin 50, Boehringer Ingelheim, Germany) as an acid inhibitor on the day before the exam. About 1 h before MRI, patients and volunteers consumed 1 liter of the 10 g/liter yerba mate instant solution. Immediately before the exam, the patients received an i.v. injection of 40 mg of N-butylscopolamine bromide (2 mL Buscopan, Boehringer Ingelheim) in order to reduce peristalsis artifacts. None of the patients reported negative side effects from the yerba mate.

MRI was performed on a Siemens Magnetom Vision clinical MRI scanner operating at 63 MHz proton resonance frequency. Abdominal imaging was performed using a Siemens CP body array coil. Table 1 gives an overview of the imaging protocols used in the studies together with references to other studies where the same protocols were used (12, 13).

Table 1. Imaging Protocols Used in the in MRI Studies Presented in This Work
Protocol nameSequence typeTR/msTE/msExcitation angleReference
FL1FLASH100.84.130°13
FL2FLASH72.54.170°12
TS1TSE2730138 12

Postprocessing and evaluation of the images was performed by a combination of pv-wave (Visual Numerics, Inc., Boulder, CO) procedures (conversion of the DICOM data to 3D analyze) and MRIcro (a freeware package available from the University of Nottingham (14)). ROI intensities were evaluated using MRIcro. The ROIs were manually drawn into the images. Changes in ROI position between images recorded at different times were made only when structural changes of the stomach geometry made it necessary.

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. REFERENCES

NMR Relaxivity, EPR Spectra, and Extraction Kinetics

Longitudinal relaxation of water was studied for both hot water extracts from ground yerba mate and solutions of instant yerba mate formulations. For the Pajarito instant formulation, additional experiments were performed on samples in which the pH was manipulated by adding HCl or NaOH. The relaxation rate constant R1 shows a maximum at pH 7 with a steep decrease to higher and a moderate decrease to lower pH values. For details see Fig. 1.

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Figure 1. pH dependence of the proton longitudinal relaxation rate in a solution of 10 g/liter Pajarito instant yerba mate at 22 MHz. The error bars indicate the possible range of kinetic phenomena which became obvious during the experiments but were not fully studied.

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In the TXRF analysis of the metal elements in the solutions, calcium and potassium were found in high quantities of about 100–200 mg/liter (for 10 g/liter instant powder or 20 g/liter hot water extract from ground yerba mate). Beside these two elements, manganese was the predominant metal in the solutions at concentrations of about 15 mg/liter. By contrast, the iron concentration was determined to be less than 0.5 mg/liter in all samples studied by TXRF, and other paramagnetic species were present only in minor quantities. The only other element that could be detected in all samples was zinc at concentrations of 0.5–2.0 mg/liter.

In Fig. 2, the results from the time-resolved extraction studies are given. The extraction kinetics proved to be roughly monoexponential with a rate constant of 0.2 l/min. As can be seen from the plot, the relaxation rate increases linearly with the extracted manganese concentration. The relaxivity was determined as 155 ± 7 s−l liter/g.

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Figure 2. (a) Manganese extraction kinetics for yerba mate in hot water. The line corresponds to an exponential extraction model fitted to the data and suggests a time constant of 4.9 min for the extraction. (b) Relaxivity (at 22 MHz) of manganese extracted from yerba mate and in a Mn(NO3)2 solution. The numbers close to the yerba mate data points indicate the duration of the extraction in minutes (see graph a). The straight line corresponds to a relaxivity of 155 ± 7 s−lliter/g.

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During NMR relaxometry experiments performed on hot water extract samples, we found that the relaxation times of the extract samples decreased significantly during the first day after extraction and that this decrease was faster for samples stored under good air access than for those in closed vials.

Figure 3 shows results from field-cycling relaxometry on two samples of Pajarito and from a 2 mM MnCl2 solution in the form of manganese millimolar relaxivities as a function of the proton NMR frequency. As can be seen from the graph, the general shape of the dispersion and the relaxivities is quite similar.

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Figure 3. Dispersion of relaxivities for the mate complex and the MnCl2 in aqueous solution.

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Figure 4 presents the ESR spectra obtained from a powder sample and from a solution of the instant mate powder. A further spectrum recorded on a spot of the instant solution dried on paper was found to be almost identical to that of the solution.

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Figure 4. X-band ESR spectra of Pajarito instant mate in powder form and in an aqueous solution of 10 g/liter (corresponding to 16.5 mg/liter Mn content). Note the complete absence of the Mn2+ hyperfine pattern and the free radical signal from the solution spectra.

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In Vivo Imaging

Figure 5 shows T1-weighted images (protocol FL1) obtained in two sessions on the same volunteer after drinking 1 liter of water and 1 liter of 10 g/liter instant yerba mate solution. Both the significant increase in signal intensity in the gastric lumen and a much better delineation of intestinal structures are obvious. In both cases, the subject consumed the water or yerba mate solution while in the magnet, and the signal intensities in the gastric lumen were monitored as a function of time after ingestion. The recorded signal intensities along with the signal intensity of reference ROIs positioned in the liver are given for both cases in Fig. 6.

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Figure 5. Coronal slices from T1-weighted images (protocol FL1, slice thickness 6 mm, in-place resolution [1.17 mm]2). Data sets were obtained from the same volunteer 5 min after drinking of 1 liter of water (a) and 1 liter of instant yerba mate at a concentration of 10 g/liter (b). The signal enhancement in the stomach and small bowel is obvious.

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Figure 6. Time dependence of signal intensities for ROIs in the stomach and liver in T1-weighted images (protocol FL1) in a volunteer after drinking water or instant yerba mate solution. The rather strong scatter in the water data from the stomach originates from problems in finding ROIs without disturbance from floating solid food particles in the stomach.

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Figure 7 shows imaging results obtained in a patient using protocols FL2 and TS1. These images clearly show the biphasic nature of the yerba mate contrast agent. In both patients the mesenteric fat was clearly visualized and infiltrating inflammation of the bowel wall could be excluded. In addition, possible complications from inflammation such as fistulas and abscesses could be safely excluded.

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Figure 7. MRI data obtained in a patient about 1 h after drinking of 1 liter of 10 g/liter instant yerba mate extract. Image a was recorded using protocol FL2 and image a using protocol TS1.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. REFERENCES

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.

CONCLUSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. REFERENCES

I. paraguayensis contains high amounts of water-extractable manganese. Since I. paraguaensis is consumed in large quantities as a herbal tea in certain areas of the world and no symptoms of manganese toxicity are reported for regular mate drinkers, the bioavailability of manganese contained in the extract seems to be very low. This fact, along with a molar relaxivity of the complex close to free Mn2+ ions or better at typical MRI field strengths, makes the complex an interesting candidate for use as an MRI contrast agent. Gastrointestinal applications of mate extract as a contrast agent look promising. Further research is needed for isolating the manganese complex from mate extract in order to further develop the complex as a clinical contrast agent. The clinical application of the untreated extract is problematic due to the rather high content in several xanthines (which can, for example, pose a risk for patients undertaking MAO inhibitors).

Acknowledgements

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. REFERENCES

The authors are grateful to O. Lips (Darmstadt), G. Ferrante (Stelar), and P. Lewitz (Palaiseau) for the relaxation-dispersion spectra; to C. Sternkopf and T. Baumann (Munich) for the TXRF data; and to G. Denninger and M. Schulte (Stuttgart) for the ESR spectra.

REFERENCES

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. Acknowledgements
  8. REFERENCES
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