Thomas Bartoli

Jean-Marc Joubert
email: joubert@icmpe.cnrs.fr

Karine Provost

Eric Elkaim

Valérie Paul-Boncour

Judith Monnier

Jacques Moscovici

Lotfi Bessais

Site occupancy determination in Th 2 Zn 17 -and TbCu 7 -types Sm 2 Fe 17-x Co x compounds using synchrotron resonant diffraction

Sm 2 Fe 17 compounds are high performance permanent magnets. Cobalt substitution allows to further improve their magnetic properties. Depending on the thermal treatment cobalt substituted compounds can be synthesized either in the TbCu 7 (disordered) or in the Th 2 Zn 17 (ordered) structure type. Rietveld refinement of the number of transition metal dumbbells replacing rare earth atoms from synchrotron powder diffraction data shows that the TbCu 7 disordered structure has the same composition as the ordered one (transition metal to rare earth ratio equal to 8.5). Then cobalt site occupancies have been determined in both structures using synchrotron resonant (anomalous) diffraction. Cobalt is found to be absent from the dumbbell sites. The diffraction results are confirmed by Mössbauer spectroscopy.

Introduction

Today's environment friendly technologies and applications require the development of highperformance permanent magnets with high transition temperatures (T c ) and high specific energy (BH max ).

In this context, there is a growing interest in nanostructured and highly anisotropic intermetallics based on rare earths (R) and transition metals (T). R 2 Fe 17 compounds have proven to be good candidates for such purpose, especially for their relatively low rare-earth content. Their original magnetic properties are related to the presence of Fe-Fe atom pairs (dumbbells) [START_REF] Givord | Magnetic transition and anomalous thermal-expansion in R 2 Fe 17 compounds[END_REF]. However, they present a relatively low Curie temperature (T c < 400K) and exhibit only planar magnetic anisotropy.

Improvement occurs through different mechanisms: electronic and magnetovolumic effects, and modifications of the dumbbells in the material. For Sm 2 Fe 17 based systems, on the one hand, the substitution of Fe by Ga, Si or Mo has a positive impact on the properties [START_REF] Kubis | Intrinsic magnetic properties of Sm 2 Fe 17x M x N y /C y (M=Al, Ga or Si)[END_REF][START_REF] Bessais | Correlation between Sm 2 (Fe,Ga) 17 and its precursor Sm(Fe,Ga) 9[END_REF][START_REF] Bessais | X-ray and intrinsic magnetic properties of nanocrystalline Sm 2 (Fe,M) 17[END_REF]. On the other hand, in the case of Nd 2 Fe 17 , a recent work showed that the magnetic properties can be greatly improved by the substitution of Fe by Co [START_REF] Bouchaala | Influence of Fe-substitution on structural, magnetic and magnetocaloric properties of Nd 2 Fe 17x Co x solid solutions[END_REF].

Furthermore, on the route to Sm 2 Fe 17 synthesis, if the annealing temperature is lower than 800°C, an out-of-equilibrium (metastable) hexagonal phase (TbCu 7 prototype, P6/mmm) is obtained. This hexagonal phase often presents improved magnetic properties compared to the high temperature ordered phase thanks to a slightly improved anisotropy [START_REF] Djéga-Mariadassou | Crystallographic and magnetic study of metastable nanocrystalline Sm(Fe,Si) 9[END_REF][START_REF] Bessais | High coercivity in nanocrystalline carbides Sm(Fe,Ga) 9 C[END_REF][START_REF] Guetari | Structure, magnetic and magnetocaloric properties of new nanocrystalline (Pr,Dy)Fe 9 compounds[END_REF]. The TbCu 7 model (P6/mmm) has been originally proposed by Buschow and van der Goot to account for the non-stoichiometry on the transition metal rich side in CaCu 5 -type compounds [START_REF] Buschow | Composition and crystal structure of hexagonal Cu-rich rare earth-copper compounds[END_REF]. It is accommodated by a random (disordered) replacement of several rare-earth atoms in position 1a by transition metal dumbbells oriented along the c axis (position 2e). Correlatively, a displacement of the transition metal atoms in position 2c to the position 6l around the missing rare-earth atom is also observed. Transition metals in position 3g are unchanged.

Note that the name TbCu 7 is the name of the prototype and that it does not intimate the composition because of the disorder. In this structure, the transition metal to rare earth ratio T/R is not fixed and can change to a large extent (see below).

When the low temperature phase is heated at higher temperature, it evolves towards a rhombohedral phase (Th 2 Zn 17 prototype, R m) which is an ordered variant of the TbCu 7 structure in which the dumbbells are placed in a staircase pattern, replacing every third rare earth atom. The two structures are shown in Figure 1. The rare-earth atoms in position 1a in space group P6/mmm are in position 6c in R m.

For the transition metals, the correspondence is as follows: 2e replaced by 6c, 3g by 9d and 18h (split position) and 6l by 18f. It is very important to determine on which site, and in which amount, the substituting element locates for subsequent theoretical considerations, such as exchange coupling calculations using the Heisenberg model adopting realistic exchange parameters obtained from first principles, and in order to understand the original magnetic properties of these systems.

Site occupancies have already been determined in these structures in several systems. In Sm 2 Fe 17-x M x (M = Ga, Si) systems with Th 2 Zn 17 type, the preferred substitution site of Fe is 18h [START_REF] Bessais | Correlation between Sm 2 (Fe,Ga) 17 and its precursor Sm(Fe,Ga) 9[END_REF]. In other similar cases however (M = Zr, Cr, Mo), the preferred substitution site is 6c [START_REF] Bessais | X-ray and intrinsic magnetic properties of nanocrystalline Sm 2 (Fe,M) 17[END_REF][START_REF] Chen | Effects of substitutions M on the formation, structure and magnetic properties of Sm 2 Fe 15 M 2 C 2[END_REF]. Several structural studies were also done on Nd 2 Fe 17-x Co x giving mixed results between 18h and 9d as site occupancies for Co [START_REF] Bouchaala | Influence of Fe-substitution on structural, magnetic and magnetocaloric properties of Nd 2 Fe 17x Co x solid solutions[END_REF][START_REF] Ryan | Direct determination of cobalt site preferences at infinite dilution in iron-based intermetallic compounds[END_REF][START_REF] Liao | Mössbauer determination of cobalt substitution in iron-based intermetallics[END_REF]. Furthermore, previous works involving X-ray studies on compounds with the TbCu 7 prototype such as metastable SmFe 9-y Ga y and SmFe 9-y Si y concluded in the substitution of Fe on the 3g site (6, 7).

In the case of Co-Fe-Sm compounds, however, precise determination of the substitution sites has never been done before. For this system, conventional X-ray diffraction cannot be used due to the similar atomic numbers of Co and Fe (Z Co =27, Z Fe =26). Neutron diffraction can neither be used because of the prohibitive absorption of Sm. Therefore, resonant diffraction appears to be the technique of choice. By measuring close to the absorption edge of one of the elements, it allows to contrast Fe and Co and determine by Rietveld refinement of powder diffraction data the site occupancies. Mössbauer spectroscopy is a very interesting complementary technique that allows, on the one hand, to study the influence of Co substitution on the different hyperfine parameters and, on the other hand, to confirm the diffraction results. These two techniques have been used in the present work in order to determine the Co site occupancies in various compounds of formula Sm 2 Fe 17-x Co x and in the two structures TbCu 7 and Th 2 Zn 17 .

Experimental section

Synthesis

Sm 2 Fe 17-x Co x compounds (x = 1, 2, 3 and 4) were prepared by using pure elements (99.9 wt%). The prealloys were melted with a slight Sm excess under a high purity argon atmosphere by using an electric arc furnace with a tungsten electrode. To ensure good homogeneity, the obtained ingots were turned over and remelted 5 times.

High energy ball milling for 5 hours under Ar atmosphere in a Fritsch planetary mill was then performed.

The obtained nanopowders were wrapped in tantalum foils and vacuum-sealed in silica tubes. The samples were then annealed for 30 min at two different temperatures in order to obtain the intended hexagonal disordered (750°C, in the following called SmFe 8.5-x/2 Co x/2 ) or the rhombohedral ordered (1200°C, called Sm 2 Fe 17-x Co x ) structures. The thermal treatment was then stopped by quenching the silica tubes in cold water.

Mössbauer

The Mössbauer spectra were obtained using Wissel constant-acceleration spectrometer utilizing a Rh matrix 57 Co source. In order to cancel the parabolic distortion, the spectrometer was operating in the mirror-image mode. The calibration of the spectrometer was performed using a standard of α-Fe. The obtained linewidth was equal to 0.26 mm/s. The Mössbauer spectra were refined according to the model discussed below. The estimated errors are at most ± 0.01 mm/s for isomer shifts () and for quadrupole interaction (2), and ± 0.1 T for hyperfine fields (µ 0 H HF ).

Diffraction

Diffraction measurements were performed on the CRISTAL beamline at SOLEIL synchrotron source at high energy (18470 eV) and selected wavelengths below Fe K-edge (7112 eV). The samples were crushed in agate mortar under inert atmosphere for metastable phases. Due to the relatively high absorption of the samples (= 1017 cm -1 at 7108 eV, below the Fe K-edge, 302 cm -1 at 18470 eV), we mixed the samples powders with 50 wt.% silica and used capillaries of diameter 2R = 0.2 mm. This allows to have the best compromise between a reasonable absorption and a decent signal. We recorded the diagrams in Debye-Scherrer geometry, with rotating capillaries, using a detection system made of 9 linear Mythen modules covering a 50° 2 circle arc. at a distance of 720 mm from the sample. This allowed the obtention of high quality diagrams for all samples at all energies. Silicon powder was measured at all energies in order to refine the wavelength.

The energy was selected using a Si111 monochromator. Prior to diffraction measurements close to the absorption edge, we systematically estimated the average absorption through the capillaries in transmission before edge, and recorded XANES spectra in fluorescence mode (7097-7197 eV energy range, 1 eV resolution), using a SDD Rontec detector, in order to check the Fe K-edge position. A 7 m Fe foil was used as reference. For XANES spectra, normalization and self-absorption correction were performed using MAX-Cherokee and MAX-Absorbix [START_REF] Michalowicz | MAX: Multiplatform Applications for XAFS[END_REF]. The edge was defined as the energy of the first maximum of absorption derivative.

Depending on the sample, the measurement in resonant conditions was done either at 7, 8 or 9 eV below the edge. The dispersion coefficients f' and f'' were obtained from Sasaki's tables [START_REF] Sasaki | Numerical tables of anomalous scattering factors calculated by the Cromer and Liberman method[END_REF] who used Cromer and Liberman method [START_REF] Cromer | [END_REF]16). It was not possible to use calculated f' from the XANES signal and Kramers-Krönig relationship because the energy range that could be accessed was too limited. Towards lower energy, further measurements would have needed a change of the undulator harmonic and at higher energy, we were limited by the presence of Sm L II edge (7312 eV). This is the reason why the measurement was not done too close to the edge, so we could safely use the calculated theoretical values. The f' values obtained are between -6.56 and -6.84 e -for Fe and -2.3 e -for Co warranting a sufficient contrast between the two elements.

The data have been analyzed using the Rietveld method using Fullprof [START_REF] Rodríguez-Carvajal | Fullprof: a program for Rietveld refinement and pattern matching analysis[END_REF][START_REF] Rodríguez-Carvajal | Recent developments of the program FULLPROF[END_REF]. The secondary phases detected in each sample have been considered. The line shape was pseudo-Voigt. The internal coordinates and an overall B displacement parameter were refined. The site occupancies were refined as follows. For the Th 2 Zn 17 phase, the pattern at low energy is sufficient to solve the multi-site occupancy problem. Each transition metal site was considered as fully occupied by a mixing of Fe and Co. The global composition was constrained to the nominal composition, so only the distribution of the substituting element was refined (3 occupancy parameters). The way to constrain the occupancy parameter of the 4 th site to match the nominal composition is detailed in Ref. [START_REF] Joubert | Site occupancies in LaNi 5 three-substituted compound determined by means of multiwavelength X-ray powder diffraction[END_REF].

For the TbCu 7 structure, there is an additional complexity in having to refine the number of dumbbells.

We can take advantage of the high energy pattern because the resonant effect is not necessary to refine the dumbbell ratio. But, it is impossible to set the constraints in order to make a joint refinement because the presence of dumbbells does not correspond to a simple substitution scheme as in Ref. [START_REF] Joubert | Site occupancies in LaNi 5 three-substituted compound determined by means of multiwavelength X-ray powder diffraction[END_REF].

Therefore, we used the high energy pattern to determine the number of dumbbells, and then fixed this number for the refinement of the low energy pattern which was therefore refined separately using the same composition constraint as mentioned above. This is not a real problem because we do not expect correlation between the two refinements i.e. the refinement of dumbbells is independent on the nature of the atom on the dumbbell because at high energy Co and Fe have similar form factors.

It is extremely difficult to estimate the effective absorption of the samples in the capillary especially because it is almost impossible to determine the quantity of material inside the capillary. Measurements of the transmission direct beam through the capillary gave only approximate results because of geometrical aspects. To solve the problem, and in order to obtain reliable values of the displacement parameters, we calculated the theoretical absorption ratio between high and low energies. Finally, we made the refinements of the high and low energy patterns with the following constraints: keeping the ratio for the effective absorption equal to the theoretical ratio of absorption coefficients and having equal displacement parameters in the two refinements.

Results

XANES results

Figure 2 presents the XANES of all samples. Since it proved almost impossible to get a precise estimation of the capillaries absorbance, it is difficult to assess whether the differences observed between the diagrams are significant or just due to uncertainties in the auto-absorption correction. However, no change in the edge position is observed compared with Fe foil as illustrated by the XANES derivatives.

Changes as a function of the structure or as a function of composition are neither observed. The first maximum of the XANES derivative was used to determine the edge position and set at 7112 eV. The exact energies used for the diagrams measured in resonant conditions were determined based on the distance to the edge: -7 eV, -8 eV or -9 eV, depending on the sample. We observed a 1 eV difference in the energies as determined from the edge or from the refinement of silicon diagrams, which is the order of magnitude of the resolution of the XANES spectra. For the refinement of the diagrams recorded in resonant conditions, we therefore used the wavelength determined by the silicon refinement, and f' and f'' values based on the edge position. The R/T ratio in the Th 2 Zn 17 phase is fixed by the crystal structure to 2/17. Non-stoichiometry has not been reported in this ordered phase. This ratio corresponds also to the nominal composition of our samples. On the other hand, the cobalt to iron ratio is fixed by the nominal composition.

The situation is less obvious for the disordered phase because, due to the disorder, the R/T ratio cannot be guessed from the structure. However, it can be obtained from the structural refinement. This refinement has been done on the high energy pattern. The refined composition, according to Buschow's model [START_REF] Buschow | Composition and crystal structure of hexagonal Cu-rich rare earth-copper compounds[END_REF][START_REF] Joubert | A structural study of the homogeneity domain of LaNi 5[END_REF] is as follows:

(1) This model derives from the CaCu 5 model in which sites 1a, 2c and 3g are completely occupied. Rare earth atoms in position 1a are replaced by dumbbells of transition metals in position 2e. Correlatively a corresponding amount of the transition metals in position 2c is displaced towards the missing rare earth atom (position 6l). It has been shown by total scattering experiments that this displacement is indeed locally correlated [START_REF] Cerny | Local atomic order in the vicinity of Cu 2 dumbbells in TbCu 7type YCu 6.576 studied by Bragg and total scattering techniques[END_REF]. The parameter s can change from 0 to 1/3, in this model, able to describe compositions from RT 5 , corresponding to the CaCu 5 structure, up to RT 8.5 with no atom in position 2c.

Note that higher T/R compositions can be obtained with different models [START_REF] Bessais | Structure and magnetic properties of nanocrystalline Sm(Fe 1x Co x ) 11 Ti (x<~2)[END_REF].

Within this scheme only one parameter, s, was refined. In each case, the refinement gives a value of s extremely close to 1/3. The convergence of this parameter towards this value, without any constraint, is remarkable. This ratio corresponds to the maximum number of dumbbells that can be obtained in the above model. Site 2c (in green in Fig. 1) which is completely vacant has therefore not been considered in the subsequent refinements. This result is also important because it indicates that the composition is R 0.666 T 5.666 , i.e. corresponding exactly to a ratio R/T=2/17. This indicates that the two phases with the structures Th 2 Zn 17 and TbCu 7 have indeed the very same compositions and transform one into each other as a function of the temperature treatment. As indicated in the experimental section, the number of dumbbells has been fixed in the subsequent refinement of the site occupancies.

To summarize, the lattice parameters, B, atomic positions and number of dumbbells for the SmFe 8.5- x/2 Co x/2 series, have been refined from the high energy pattern, while the cobalt to iron substitution has been obtained from the low energy pattern using the resonant effect. The complete results can be found in Tables 1 and2. Figures 3 and4 show examples of the refinements for the two structures and at the two different energies. The lattice parameters of the Sm 2 Fe 17-x Co x samples phase depend regularly on the Co composition with an increase of the c/a ratio as a function of x (Figure 5) which is not the case for the SmFe 8.5-x/2 Co x/2 samples (Figure 6). It reveals that the Co substitution expands the lattice along the c axis only in the case of ordered dumbbels, whereas the effect is negligible in the disordered structure. The B parameter dependence as a function of composition is shown in Figure 7 for the two phases. The observed increase can be related to the higher static displacement caused by the substitution. On the other hand, only slight variation of atomic coordinates is observed with composition (not shown). The occupancy parameters are plotted for Sm 2 Fe 17-x Co x (Figure 8) and SmFe 8.5-x/2 Co x/2 series (Figure 9)

phases. Higher estimated standard deviations are observed for the ordered phase which are related to its higher structural complexity. We can nevertheless observe clear trends in the occupancy parameters with a significantly higher cobalt occupancy in site 18h and an almost null Co occupancy in site 6c. For the disordered phase, the partition of cobalt is also very different among the different sites with a clear preference for site 3g and no occupancy in the dumbbell position in site 2e.

Mössbauer results

Mössbauer spectrometry is the recoil-free emission and resonant absorption of -rays in solids, i.e.

 -ray emission and absorption without the loss of energy to the lattice. The Mössbauer spectra of the Sm 2 Fe 17-x Co x and SmFe 8.5-x/2 Co x/2 series are complex. The solution chosen for their analysis must correspond to a model justified by experimental approaches resulting from other techniques or based on relevant theoretical considerations. The reliability of the proposed model for the interpretation of the experimental spectra must be demonstrated by the monotonic evolution of the hyperfine parameters, isomer shift (δ) and hyperfine field (µ 0 H HF ). The simulation is based on several criteria outlined below:

-The environments of Fe atoms have been established based on a multinomial distribution law as a function of the distribution of atoms, obtained after the crystallographic study.

-The assignment of the hyperfine parameters to the different inequivalent crystallographic sites is determined by the correlation between the isomer shift and the volume of the Wigner-Seitz cells (WSCV). We have used the crystallographic characteristics (unit cell parameters and atomic positions) for the calculation of the WSCV.

-The assumption of relative intensities of the sextuplet lines in the 3:2:1 ratio of a randomly oriented powder system was considered due to the absence of crystallographic texture.

-Lamb-Mössbauer absorption factors were considered equal for all sites.

In Figure 10, the experimental spectra for Sm 2 Fe 15 Co 2 and SmFe 7.5 Co 1 at room temperature are presented. One can notice a shoulder in the region corresponding to the velocities +5.5 mm/s for Sm 2 Fe 15 Co 2 and -5.5 mm/s for SmFe 7.5 Co 1 , which is attributable to the Fe-Fe dumbbells at sites 6c and 2e, respectively. Further refinement shows that the abundance of these sites corresponds to a total occupation by iron of site 6c for Sm 2 Fe 15 Co 2 and of site 2e for SmFe 7.5 Co 1 . This result agrees with our 20 finding that cobalt neither occupies sites 6c nor 2e. We will explain the results obtained from the structural refinement, that cobalt is preferentially distributed among sites 18h and 3g.

- A single line width of 0.28 mm/s has been imposed to limit the number of sites needed to fit Mössbauer spectra. We neglected theoretical abundances lower than 1.5% for Sm 2 Fe 17-x Co x and 2% for SmFe 8.5-

x/2 Co x/2 .
During the refinement of the hyperfine parameters, the abundances of the different sites were considered as fixed, while the hyperfine parameters δ, H and 2ε were released.

In the first step of the refinement, the assignment of the hyperfine field to all the subspectra associated with a particular crystallographic site was based on the physical fact that the highest hyperfine field 22 belonged to site 6c (2e) and the lowest to site 9d (3g) according to their respective theoretical abundances. At this stage, no distinction was made between sites 18f and 18h. In the second step, a mean value of δ was assigned to each hyperfield taking into account the correlation between δ and WSCV volume. Then, the refinement was performed with all free parameters except abundance and finally, in the last step, we freed all hyperfine parameters. The sets of hyperfine parameters resulting from such a refinement are given in Table 3 and 4 for x=2 as an example.

Figures 11 and12 show the evolution of the isomer shift of each of the sites as a function of x.

Discussion

Due to the planar orientation of the magnetization in both phases for sites 9d, 18h and 18f in space group R m, and 3g and 6l in space group P6/mmm, the angle between the hyperfine field and the electric field gradient is not zero. Each of these sites will be decomposed into two non-equivalent magnetic sites with relative abundances equal to 2:1, as a consequence, during the refinement of the Mössbauer spectra we have used several sextets relating to the different magnetic sub-sites.

The isomer shift δ of an individual site, can be predicted qualitatively from the perturbation brought by Co 3d electrons at the s electron density to the iron nuclei of the site considered. Indeed, according to the expression of the isomer shift δ is a function of the electron density s at the nucleus multiplied by a nuclear factor , negative in the case of iron:

(

Where is the non-relativistic Schrödinger wave function (at r = 0), and is the change in nuclear radius. The larger is, the smaller δ is and vice versa. The result is that the Co d electrons that fill the Fe d band will shield the 4s electrons of Fe atoms. As a result, the density at the iron core will be reduced, so the isomer shift will increase as a function of the Co content. Furthermore, for a given value of Co content, the correlation between δ and WSCV volume shown above must be considered, the greater the WSCV volume, the greater δ [START_REF] Forker | Mossbauer study of the hyperfine interaction of Fe-57 in Y 1-s Co 5+2s and related compounds[END_REF][START_REF] Bessais | Crystallographic and hyperfine parameters of PrTi(Fe,Co) 11 and their carbides[END_REF].

Whatever the Co content, the observed sequence is δ9d < δ18f < δ18h < δ6c for Sm 2 Fe 17-x Co x and δ6l < δ3g < δ2e for SmFe 8.5-x/2 Co x/2 , which corresponds to the evolution of WSCV.

The increase of the isomer shift for 6c, 18f and 9d (2e and 6l), with Co content, is the consequence of the shielding of Fe 4s electrons by the additional Co 3d electrons. As explained above, the evolution of the isomer shift for the different crystallographic sites with cobalt content is the consequence of the shielding of Fe 4s electrons by the additional cobalt 3d electrons. δ6c, δ18f and δ9d (δ2e and δ6l), show an increase with cobalt substitution, whereas δ18h (δ3g) remains quasi-constant. This result can be understood in terms of preferential cobalt atom occupation. For the hexagonal TbCu 7 structure, the 2e and 6l Fe atoms have six adjacent 3g neighbors while the 3g atom site has only four. The 3g atoms with the fewest 3g neighbors are the least affected by cobalt substitution and their s electron charge density is then quasi modified. Concerning the rhombohedral Th 2 Zn 17 structure, the 6c, 9d, and 18f sites have three, four and four adjacent 18h neighbors whereas the 18h iron site has only two. The 18h atoms with the smallest 18h neighbors are the least affected by cobalt substitution and consequently δ18h is less modified.

The structure model obtained from diffraction was mandatory to refine the Mössbauer results because of the high complexity of the spectra. However, the good quality of the Mössbauer refinements and the monotonic variation of the hyperfine field (not shown) and isomeric shift (Figures 11 and12) testifies, in turn, for the relevance and accuracy of the structure model.

The refinement of the high energy diffraction patterns of SmFe 8.5-x/2 Co x/2 samples, gives a s value always converging exactly to 0.333 which is the maximum number of dumbbells in a disordered model giving an explanation why the stable phase actually orders when activated by temperature without any composition change. The refinement of this parameter with such an accuracy to this particular value has not been reported previously, as far as we are aware.

The two crystal structures are related, the stable Th 2 Zn 17 derives from the disordered TbCu 7 structure by ordering of the dumbbells. Each site in the Th 2 Zn 17 therefore derives from a position in the TbCu 7 structure i.e. sites 18h and 9d derive from site 3g, 18 f from 6l and 6c from 2e. Therefore, similar cobalt site occupancies are expected. From a comparison of Figures 8 and9, it is shown that it is the case (maximum occupancy for sites 18h and 3g, null occupancy for the dumbbell position 6c and 2e). It is remarkable that the site occupancies (expressed in percentage not in atom per site) are found identical in the two sites 9d and 18h originating from the symmetry splitting of site 3g in space group P6/mmm. . Concerning the Th 2 Zn 17 type, the results found for Sm 2 Fe 17-x Co x compounds are in good agreement with previous works on the same system [START_REF] Bessais | A Fe-57 Mossbauer study of nanostructured Sm 2 Fe 17-x Co x C 3[END_REF] and with literature on other systems (3-5, 10-12, 26). Regarding the disordered SmFe 8.5-x/2 Co x/2 series, the resulting preference of cobalt for site 3g is also shared by gallium and silicon atoms with similar structures [START_REF] Djéga-Mariadassou | Crystallographic and magnetic study of metastable nanocrystalline Sm(Fe,Si) 9[END_REF][START_REF] Bessais | High coercivity in nanocrystalline carbides Sm(Fe,Ga) 9 C[END_REF] even though gallium and silicon atoms have slightly different radii compared to that of cobalt. The main reason explaining the preferred site occupancies of site 3g by the larger atoms (here Co) in the TbCu 7 structure and of sites 9d and 18h in the Th 2 Zn 17 structure seems to be the larger size of this site.

The precise determination of the cobalt site in the Sm 2 Fe 17-x Co x and SmFe 8.5-x/2 Co x/2 series is necessary for the understanding of the intrinsic magnetic properties (e.g. exchange interaction, Curie temperature) as well as for the calculation of the electronic structure by first-principles approach. This step is essential before optimizing the microstructure in order to obtain the optimal energy product (BH)max for hard magnets. The correlation between Co position and magnetic properties will be done in an upcoming paper.

Conclusion

We have refined from resonant diffraction data obtained at the Fe K-edge the distribution of substituting Co in ordered and disordered Sm 2 Fe 17 compounds. We found, from the structural refinement, that the two phases have the same composition for each Co amount. The Co site occupancies between the two phases follow the site equivalence between the two structures. The results are also in perfect agreement with both our own Mössbauer spectroscopy measurements on this system and other results previously obtained on other systems. This new and relevant structural analysis, of the two phases, will allow us to make DFT calculations under optimal conditions. This will give us the magnetic moment for each inequivalent crystallographic site.

Figure 1 .

 1 Figure 1. Th 2 Zn 17 and TbCu 7 crystal structures. Note that the position 2c in green is not occupied in our

Figure 2 .

 2 Figure 2. Normalized absorption and first derivative of Sm 2 Fe 17-x Co x and SmFe 8.5-x/2 Co x/2 series.

Figure 3 .

 3 Figure 3. Rietveld plot of Sm 2 Fe 13 Co 4 (x=4) at 7104 eV (8 eV below the Fe edge). Experimental (points),

Figure 4 .

 4 Figure 4. Rietveld plot of SmFe 7 Co 1.5 (x=3) at 18470 eV. Experimental (points), calculated (line) and

Figure 5 .

 5 Figure 5. lattice parameters of the Sm 2 Fe 17-x Co x series as a function of x (error bars are smaller than the

Figure 6 .

 6 Figure 6. lattice parameters of the SmFe 8.5-x/2 Co x/2 series as a function of x (error bars are smaller than

Figure 7 .

 7 Figure 7. displacement parameters as a function of x for Sm 2 Fe 17-x Co x and SmFe 8.5-x/2 Co x/2

Figure 8 .

 8 Figure 8. Cobalt site occupancies (in atom per site) for the Sm 2 Fe 17-x Co x series.

Figure 9 .

 9 Figure 9. Cobalt site occupancies (in atom per site) for the SmFe 8.5-x/2 Co x/2 series.

Figure 11 .Figure 12 .

 1112 Figure 11. isomer shift for the Sm 2 Fe 17-x Co x series.

Table 1 .

 1 TbCu 7 phase, Th 2 Zn 17 phase, α-Fe, SmO, SmO 2 , Sm 2 O 3 , SmFe 3 . Results for the Sm 2 Fe 17-x Co

	Composition	Sm 2 Fe 16 Co 1	Sm 2 Fe 15 Co 2	Sm 2 Fe 14 Co 3	Sm 2 Fe 13 Co 4
	x	1	2	3	4
	Secondary	SmO (4%)	SmO (3%)	SmO (2%)	SmO (2%)
	phases (wt.%)	Sm 2 O 3 (10%)	monoclinic-		SmFe 3 (9%)
		α-Fe (0.3%)	Sm 2 O 3 (4%)
			SmFe 3 (4%)
	a (Å)	8.5518(1)	8.5445(1)	8.5408(1)	8.5320(1)
	c (Å)	12.4574(2)	12.4696(2)	12.4716(2)	12.4765(2)
	x 18h	0.5012(1)	0.5014(1)	0.5011(1)	0.5010(2)
	z 18h	0.1570(2)	0.1569(2)	0.1575(2)	0.1572(2)
	x 18f	0.2911(2)	0.2922(2)	0.2922(2)	0.2923(3)
	z 6c (Fe,Co)	0.0964(2)	0.0962(2)	0.0959(2)	0.0956(3)
	z 6c (Sm)	0.3425(1)	0.3425(1)	0.3420(1)	0.3424(1)
	Co in 18h (atom) 2.0(4)	2.7(7)	3.7(2)	5.8(3)
	Co in 18f (atom)	0.2(4)	1.3(7)	3.0(2)	2.3(3)
	Co in 9d (atom)	1.2(3)	1.6(6)	2.2(2)	3.0(3)
	Co in 6c (atom)	-0.4(4)	0.6(4)	0.0(2)	0.9(3)
	B (Å 2 )	0.415(6)	0.473(7)	0.513(8)	0.506(9)
	R B (%) HE	2.6	3.6	6.5	4.2
	R B (%) LE	5.4	7.0	8.2	7.3

x samples, space group R-3m. Estimated standard deviations calculated by Fullprof (in brackets referring to the last digit) have been multiplied by the Bérar parameter (between 3 and 7). Bragg R factors are given for the high (HE) and low (LE) energy pattern refinements.

Table 2 .

 2 Results for the SmFe 8.5-x/2 Co x/2 samples, space group P6/mmm. Estimated standard deviations calculated by Fullprof (in brackets referring to the last digit) have been multiplied by the Bérar parameter

(between 3 and 10). Bragg R factors are given for the high (HE) and low (LE) energy pattern refinements.

Table 3 .

 3 Room temperature Mössbauer spectra for Sm 2 Fe 15 Co 1 and SmFe 7.5 Co 1 compounds. The thick lines and the symbols correspond to the calculated and experimental spectra, respectively. The different sextets used in the refinement are presented by colored thin lines. Mössbauer hyperfine parameters for Sm 2 Fe 15 Co 2 sample. µ 0 H HF is the hyperfine field, isthe isomer shift, 2 is the quadrupole interaction and WSCV is the Wigner-Seitz cell volume.

	Megacounts
		SmFe 7.5 Co 1
	Sm 2 Fe 15 Co 2
	8	-6	-4	-2	0	2	4	6	8
			Source velocity (mm/s)

Megacounts

Figure 10.

Table 4 .

 4 Mössbauer hyperfine parameters for SmFe 7.5 Co 1 sample. µ 0 H HF is the hyperfine field, isthe isomer shift, 2 is the quadrupole interaction and WSCV is the Wigner-Seitz cell volume.

Synopsis

The cobalt site occupancies in substituted Sm 2 Fe 17 (Th 2 Zn 17 -type) and SmFe 8.5 (TbCu 7 -type) have been determined by Rietveld refinement of resonant diffraction data measured at the iron K-edge.

Results are compared with Mössbauer spectroscopy measurements.

Table of content graphics


Abdelkhaleq Legssyer

Hassane Mekhfi

Mohamed Bnouham

Vincent Roumy

Christine Herrenknecht

Christophe Fourneau

Alain Laurens

Jacqueline Hoerter

Abderrahim Ziyyat

Rodolphe Fischmeister

Tannins and catechin gallate mediate the vasorelaxant effect of Arbutus unedo on the rat isolated aorta

Keywords: Arbutus unedo, Ericaceae, Tannins, Vasorelaxation, Rat thoracic aorta

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

contracted with 0.1 µM noradrenaline. At 10 -2 g/L, AE produced an endothelium dependent relaxation of 665%, (n=8). Leaves of Arbutus were then extracted successively with different solvents (hexane, dichloromethane, ethyl acetate, methanol and water) and the extracts obtained were tested on pre-contracted aorta. The methanolic and the ethyl acetate extracts were the most active. When tannins (primarily condensed tannins) were precipitated from the methanolic extract, they showed a strong vasorelaxant activity (874%, n=5) whereas the elimination of tannins in the methanolic extract reduced significantly its vasorelaxant activity (428%, n=8, p<0.005).

Methanolic extract was further separated semi-preparatively by reversed-phase HPLC and fractions were collected and tested for their vasorelaxant activities. Four fractions (Fr2,Fr3,Fr4 and Fr6) were most active and produced 88±2% (n=5), 75±6% (n=5), 76±3% (n=7) and 76±3% (n=9) relaxation, respectively. These four fractions mainly correspond to polyphenols compounds. The analysis of Fr6 chromatogram indicates that this fraction contains catechin gallate. Dose-response curves for the vasorelaxant effects of Fr6 and commercial catechin gallate were superimposable confirming the likely contribution of catechin gallate to the effect of Fr6. In conclusion, the vasorelaxant activity of Arbutus is present both in leaves and roots. This activity is likely due to

INTRODUCTION

Arbutus unedo L. (Ericaceae) is a species widely spread in Mediterranean basin and used in traditional medicine as astringent, diuretic and urinary antiseptic (Grieve, 1967).

In Morocco, the roots of Arbutus are employed in decoction form for several uses [START_REF] Bellakhdar | La pharmacopée marocaine traditionnelle: médecine ancienne et savoirs populaires[END_REF] specially in the therapy of hypertension and diabetes [START_REF] Ziyyat | Phytotherapy of hypertension and diabetes in oriental Morocco[END_REF]. In the spontaneously hypertensive rat (SHR), oral administration of the root aqueous extract of Arbutus produces vascular, diuretic and natriuretic effects [START_REF] Ziyyat | Cardiovascular effects of Arbutus unedo L. in spontaneously hypertensive rats[END_REF]. Recently, we have shown that root-aqueous extract also induces an endothelium-dependent vasorelaxation on rat isolated aorta [START_REF] Ziyyat | Arbutus unedo induces endothelium dependent relaxation of the isolated rat aorta[END_REF], giving a rationale for its use in folk medicine against hypertension. This effect could be attributed to polyphenolic compounds likely to be present in the extract. Indeed, phytochemical studies on Arbutus have led to the isolation of several polyphenolic compounds, mainly tannins in the roots [START_REF] Garnier | Ressources Médicinales de la Flore Française[END_REF] and flavonoids such as afzelin, quercitrin and hyperoside in the aerial part [START_REF] Dauguet | Les flavonoïdes de Arbutus unedo L. (Ericacées)[END_REF]. The present study was thus undertaken to isolate some of the fractions present in Arbutus extract which might be responsible for its endothelium-dependent vasorelaxant activity and to examine the implication of some polyphenolic compounds, such as tannins, in this effect.

MATERIALS AND METHODS

Vegetal material

Leaves of

Plant extract preparation

Aqueous extract (AE): 20 g of dried and powdered leaves of Arbutus unedo were extracted by infusion with 1 L of water for 30 min. The aqueous extract was obtained after filtration and evaporation to dryness in vacuo.

Soxhlet extraction: 79 g of dried and powered leaves of Arbutus unedo were extracted successively with different solvents (hexane, dichloromethane, ethyl acetate, methanol and water). The extracts were evaporated to dryness in vacuo to afford respectively 5.0 g, 1.15 g, 3.75 g, 19.0 g and 2.53 g of dry extract. (Rendement : 6.4%, 1.5%, 5.2%, 24.1%, 3.2%) Semi-preparative HPLC separation of the methanolic extract: a final purification of the methanolic fraction was performed using repeated reversed-phase semi-preparative HPLC on Whatman (Maidstone; UK) Partisil 10 ODS-3 column (25×0.94 cm i.d., 10 µm), flow rate 5 mL/min, UV detection 280 nm, 30 mg injection and step gradient elution as follows: 0-16 min (methanol/water/formic acid 25/75/0.02 v/v), 16-96 min (methanol/water/formic acid 35/65/0.02 v/v).

Equipment: semi-preparative separations were carried out with a Waters HPLC preparative system (St Quentin en Yvelines, France) equipped with a 590 pump coupled to a solvent select valve, a 484 detector and a ABB SE120 recorder.

Tannins separation

Separation by caffeine: 1 g of methanolic extract was dissolved in 2 mL of water.

Tannins were precipitated by 4 mL of a 2% caffeine aqueous solution. After filtration the caffeine tannate precipitate was washed by water and then dissolved in 50 mL of methanol. 50 mL of water was then added to the solution witch was extracted four times by 50 mL of chloroform and then evaporated under reduce pressure to give 288 mg of dry extract. In the same manner, to remove caffeine, the filtrate was extracted by chloroform and evaporated to dryness under reduce pressure to give 686 mg of dry extract.

Precipitation of tannins by skin powder: 100 mg of methanolic extract was dissolved in 20 mL of water. 200 mg of skin powder were added under stirring conditions during 1 hour. Tannins were removed by adsorption on skin powder after filtration. The filtrate led to 40 mg of dry extract.

Methanolic extract analysis

Analytical HPLC procedure: the methanolic extract and the different fractions isolated from methanolic extract were analyzed by using reversed-phase HPLC with a Waters µBondapak C18 (30×0.39 i.d. cm, 10 µm), 2 mL/min, UV detection 280 nm and a gradient elution with water (A) and methanol (B) both added with 0.05% (v/v) formic acid as mobile phases with linear gradient from 5 to 60% B in 30 min, 60% B during 5 min.

Equipment: chromatographic analysis were performed using an HPLC system equipped with a Merck L-6200A pumpFontenay sous Bois, France), a Waters 717 autosampler, a Merck L-4000 detector and a Shimadzu C-R3A integrator(Croissy Beabourg, France).

Preparation of aorta and experimental device

The animal investigation conforms with the European Community guiding principles in the care and use of animals (86/609/CEE, CE Off J n°L358, 18 December 1986) and the French decree n°87/748 of October 19, 1987(J Off République Française, 20 October 1987, pp. 12245-12248). Authorisations to perform animal experiments according to this decree were obtained from the French Ministère de l'Agriculture et de la Forêt (n°04226, April 12, 1991).

Male Wistar rats weighing 300-350 g were anaesthetised with sodium pentobarbital (50 mg/kg of body weight, i.p.), and the thoracic aorta was removed carefully in physiological salt solution (PSS). An aortic ring of about 3 mm in length was suspended between two stainless steel hooks in 10-mL water-jacketed bath containing PSS of the following composition (mM): NaCl 119, KCl 4.7, CaCl2 1.6, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25 and glucose 11. The tissue bath solution was maintained at 37°C and gassed with 95% O2 + 5% CO2 (pH 7.4). The isometric contraction was recorded via a force-displacement transducer connected to an amplifier and a paper recorder. Data were also recorded on computer. A tension of 1 g was initially applied to the ring which was equilibrated in the medium for 30 min. Before each experiment, vasoconstriction was initiated by 0.1 µM noradrenaline (NAdr) in normal PSS and when contraction reached steady-state, 10 µM carbachol (CCh, a cholinesterase-resistant analogue of acetylcholine) was added to induce endotheliumdependent relaxation. This step was necessary to verify the endothelium integrity. For relaxation studies, precontraction of the aortic rings was induced by 0.1 µM NAdr, the extract of Arbutus unedo was then added to the organ bath, and relaxation was evaluated as a percentage of the initial tension induced by NAdr alone. In denuded aorta, the endothelium was removed mechanically by rubbing the lumen of the artery with plastic tubing. In this case, relaxation was induced by 1 µM of sodium nitroprusside.

Chemicals. (-)-Norepinephrine hydrochloride (Noradrenaline, NAdr), carbamylcholine chloride (carbachol, CCh), catechin, gallic acid and catechin gallate were purchased from Sigma Chemicals Co. (St Quentin-Fallavier, France); Sodium nitroprusside (SNP) from Farco Chemicals (ville, pays); Arbutin, epicatechin, quercetin and hyperoside from Extrasynthèse(Genay, France); Formic acid from Prolabo (Fontenay sous Bois).

Methanol was HPLC grade purchased from Carlo Erba (Val de Reuil, France). Water was purified by Milli-Q water purification system (Millipore,(St Quentin en Yvelines, France).

Statistics.

Results are expressed as the means ± SEM for n separate experiments. Data were analysed by Student's t-test and a difference was considered as statistically significant when p value was less than 0.05.

RESULTS

Figure 1A shows a typical experiment in which an intact thoracic aortic ring was exposed to 0.1 µM NAdr after equilibration in control solution. NAdr produced a dramatic increase in tension. As expected, application of CCh (10 µM) in the continuing presence of NAdr strongly (~80%) reduced the effect of NAdr. The tension obtained in the presence of 0.1 µM NAdr and the relaxation produced by 10 µM CCh were used as references in subsequent experiments evaluating the relaxant effects of plant extracts.

On average, intact aortic rings developed a tension of 1.30.1 g in response to 0.1 µM NAdr (n=25), and 10 µM CCh induced a 832% relaxation (n=20). As shown in Fig. 1B, aqueous extract of Arbutus leaves (AE, 10 -2 g/L) also induced a strong relaxation of pre-contracted aortic rings. The relaxation was preceded by a small rise in tension. On average, AE (10 -2 g/L) produced a 665% (n=8) relaxation. As the relaxant effect of CCh requires the presence of an intact endothelium, we tested whether this was also the case for the relaxant effect of AE. Figure 1C shows an experiment performed in a denuded aortic ring. NAdr still produced a strong contraction of the aorta but, as expected, CCh had no effect. Interestingly, addition of AE (10 -2 g/L) to the cocktail had no effect either, while application of the NO-donor SNP strongly relaxed the muscle. This result, which was obtained in all of 3 similar experiments, indicates that, similarly to root extracts [START_REF] Ziyyat | Arbutus unedo induces endothelium dependent relaxation of the isolated rat aorta[END_REF], leave extracts from Arbutus also require an intact endothelium to produce a relaxant effect.

(Figure 1 near here)

The subsequent experiments were aimed at identifying the active compounds present in AE which might be responsible for the relaxant effect. To do this, leaves of Arbutus were extracted successively with solvents of increasing polarity. Lipids were first removed with hexane, and four successive extracts were obtained with dichloromethane, ethyl acetate, methanol and water. These extracts were then tested on NAdr pre-contracted aortic rings at concentrations ranging from 10 -3 to 10 -1 g/L. As shown in Fig. 2, all four extracts caused a vasorelaxant effect. However, ethyl acetate and methanol extracts appeared most active, since at 10 -2 g/L concentration they fully relaxed NAdr pre-contracted rings, while dichloromethane and water extracts required a 10-fold larger concentration to produce a similar effect (Fig. 2).

(Figure 2 near here)

In the following, we focused our attention to the methanolic extract, which caused a 794% relaxation (n=9) of NAdr pre-contracted aortic rings at 10 -2 g/L and was selected for the purification of active compounds. Preliminary chemical research on this extract showed the abundant presence of condensed tannins (green coloration with FeCl3 and precipitation with Stiasny reagent). Therefore, it was logical to question the implication of tannins in the vasorelaxant effects observed with this extract. We addressed this question by treating the methanolic extract with caffeine, a procedure used to precipitate most of the tannins [START_REF] Okuda | Effects of the interaction of tannins with coexisting substances. III. Formation and solubilization of precipitates with alkaloids[END_REF]. Methanolic extract was also treated with skin powder, which is commonly used to eliminate the tannins present in solution(European Pharmacopoeia 4 th edition, Council of Europe, Strasbourg, France).

The fraction precipitated by caffeine and the filtrate of the fraction treated with skin powder were tested on aortic rings pre-contracted by NAdr. Vasorelaxation was still robust (874%, n=5) when the fraction precipitated by caffeine was tested at 10 -2 g/L.

On the contrary, elimination of tannins by skin powder strongly reduced the vasorelaxant effect (428%, n=8, p<0.005). These experiments clearly indicate that tannins participate in the vasorelaxant effect of Arbutus leaves. However, the presence of a residual vasorelaxant activity in the solution treated by skin powder also demonstrates that other components besides tannins may also be involved.

(Figure 3 near here) Methanolic extract was further separated semi-preparatively by reversed-phase HPLC. A complete chromatogram of the total methanolic extract is shown in Fig. 3A. This chromatogram was separated in thirteen fractions, named Fr1 to Fr13 (Fig. 3A).

Each fraction was collected and tested at 10 -2 g/L on pre-contracted aorta. Figure 3B shows that Fr2, Fr3, Fr4 and Fr6 were the most active, as they produced a relaxing effect of 88±2% (n=5), 75±6% (n=5), 76±3% (n=7) and 776±3% (n=10), respectively.

The nine other fractions produced a 4 to 10 fold lower relaxation at the same concentration, and none of their effects was statistically significant. The four active fractions were further examined for their concentration-dependent effects. As shown in Fig. 4A, Fr2, Fr3, Fr4 and Fr6 produced a concentration-dependent relaxation. Fr2, Fr4 and Fr6 had a threshold value near 10 -3 g/L and their effect was half maximal around 3x10 -3 g/L. Fr3 was about 3-fold less active.

(Figure 4 near here) Despite the complexity of the methanolic extract chromatogram (Fig. 3A), several components were identified by HPLC coupled with mass spectrometry and confirmed by retention time of standard. These include arbutoside, quercitin, epicatechin, catechin, catechin gallate, hyperoside and gallic acid (see Fig. 5 for chemical structures). Thus, we tested all these compounds at 10 -2 g/L for their effects on NAdr pre-contracted aortic rings. As shown in Table 1, only catechin gallate induced a strong vasorelaxant activity. Arbutoside, quercitin and epicatechin had no significant effect whereas catechin and hyperoside induced a moderate vasorelaxant effect (Table 1).

(Figure 5 near here) Analysis of Fr6 chromatogram indicated that catechin gallate was actually present in this fraction. Therefore, we compared the vasorelaxant effect of Fr6 and catechin gallate. As shown in Fig. 4B, Fr6 and commercially available catechin gallate produced a similar concentration-dependent vasorelaxant effect on NAdr pre-contracted aortic rings. In order to determine structure-activity relationships, this activity was compared that of catechin and gallic acid. As shown above, the vasorelaxant effect of catechin was weaker than that of catechin gallate (Table 1). Moreover, gallic acid exerted a strong vasoconstriction (5113% at 10 -2 g/L, n=5, p<0.05).

(Table 1 near here)

DISCUSSION

Arbutus unedo is one of the most popular medicinal plants used in oriental Morocco [START_REF] Ziyyat | Phytotherapy of hypertension and diabetes in oriental Morocco[END_REF]. Traditionally, roots of the plant are used in decoction to treat hypertension. In this study, we focused our interest on Arbutus leaves, because they can be collected more easily and do not require the destruction of the shrub, which is of ecological relevance. We found that the aqueous extract of Arbutus leaves produces a strong relaxation of pre-contracted aorta. This effect was endothelium-dependent and comparable, in potency and efficacy, to that of the root extract [START_REF] Ziyyat | Arbutus unedo induces endothelium dependent relaxation of the isolated rat aorta[END_REF].

Thus, Arbutus leaves may be used instead of roots in folk medicine, provided that, like the roots, they do not present any toxic effect.

As we found that the vasorelaxant effect of Arbutus roots extract involves activation of endothelial NO-synthase and cGMP synthesis [START_REF] Ziyyat | Arbutus unedo induces endothelium dependent relaxation of the isolated rat aorta[END_REF], it is likely that Arbutus leaves also contain one or several active compounds which cause the endothelial cells to produce NO. Extraction of dried and powdered leaves by different successive solvents of increasing polarity led us to the observation that these compounds were likely present in the ethyl acetate and methanolic extracts. Indeed, these two extracts accounted for most of the vasorelaxant activity of Arbutus. Bioguided fractionation of these extracts, using Stiasny reagent and the green coloration with FeCl3, led us to unmask the presence of polyphenolic compounds such as tannins.

Tannins and related polyphenols include more than 4000 identified compounds, and represent one of the largest groups of active phytochemicals [START_REF] Waltner-Law | Epigallocatechin gallate, a constituent of green tea, represses hepatic glucose production[END_REF]. The participation of condensed tannins in the vasorelaxant effect of Arbutus leaves extract was suggested by the observation i) that tannins precipitated by caffeine from the methanolic extract showed a strong vasorelaxant activity, whereas ii) the adsorption of tannins by skin powder in the methanolic extract reduced significantly its effect. Thus, the endothelium-dependent vasodilator effect of Arbutus leaves extract was mainly due to oligomeric condensed tannins. However, the presence of a residual vasorelaxant activity in the solution treated by skin powder also demonstrates that other components besides tannins may also be involved. These are likely to include flavonic heterosides (flavonoids), which are present in Arbutus leaves [START_REF] Dauguet | Les flavonoïdes de Arbutus unedo L. (Ericacées)[END_REF]. Both flavonoids and tannins have been shown earlier to produce endotheliumdependent relaxation of rat aortic rings [START_REF] Fitzpatrick | Endothelium-dependent vasorelaxing activity of wine and other grape products[END_REF][START_REF] Chen | Vasorelaxing activity of resveratrol and quercetin in isolated rat aorta[END_REF][START_REF] Chan | Relaxation of flavones and flavonols in rat isolated thoracic aorta: mechanism of action and structure-activity relationships[END_REF], an effect which resembles the effect of Arbutus leaves extract. Moreover, tannins and related polyphenols were also shown to produce an endothelium-dependent, NO-derived vasorelaxation through an extracellular Ca 2+ -dependent mechanism [START_REF] Andriantsitohaina | Regulation of vascular tone by plant polyphenols: role of nitric oxide[END_REF]Andrianbeloson et al., 1998;[START_REF] Andriantsitohaina | Regulation of vascular tone by plant polyphenols: role of nitric oxide[END_REF]. Oral administration of wine polyphenol compounds produces a decrease in blood pressure in normotensive rats associated with an endothelium-dependent relaxation and an induction of gene expression of inducible NO-synthase and cyclooxygenase 2 [START_REF] Diebolt | Wine polyphenols decrease blood pressure, improve NO vasodilatation, and induce gene expression[END_REF].

Recently, provinol, a phenolic compound, was found to accelerate the regression of blood pressure and improve structural and functional cardiovascular changes produced by chronic inhibition of NO synthesis in L-NAME rats [START_REF] Bernatova | Wine polyphenols improve cardiovascular remodeling and vascular function in NO-deficient hypertension[END_REF]. This resembles the effect of an oral administration of the root aqueous extract of Arbutus in the SHR rat [START_REF] Ziyyat | Cardiovascular effects of Arbutus unedo L. in spontaneously hypertensive rats[END_REF]. Thus, there is ample evidence to suggest that tannins or related polyphenols also mediate the vasorelaxant effect of Arbutus leaves extract.

Semipreparative separation of the methanolic extract by reversed-phase HPLC led to the identification of thirteen fractions. Out of these, four fractions (Fr2, Fr3, Fr4 and Fr6) were particularly active. These four fractions correspond to polyphenol compounds, primarily oligomeric condensed tannins. Interestingly, we found that Fr6 fraction contains catechin gallate, an oligomeric tannin. Test of its activity on aortic rings identified this compound as a potent vasorelaxant compound, similar to the complete Fr6 fraction. While this is the first report on the isolation of catechin gallate from Arbutus, we conclude that this compound represents one of the main compounds responsible for the vasorelaxant effect of the plant.

Catechins are generally known to produce vasorelaxant effects [START_REF] Huang | Involvement of endothelium/nitric oxide in vasorelaxation induced by purified green tea (-) epicathechin[END_REF]). Yet, catechin derivatives were found to potentiate the contractile response to phenylephrine at low concentrations and to produce vasorelaxation only when used at high concentration [START_REF] Sanae | Effects of catechins on vascular tone in rat thoracic aorta with endothelium[END_REF]. Although structurally related to catechin gallate, we found here that catechin exerted a much weaker relaxation than catechin gallate and gallic acid was even vasoconstrictive (see also [START_REF] Sanae | Endothelium-dependent contraction of rat thoracic aorta induced by gallic acid[END_REF].

In addition to fraction Fr6, a strong vasorelaxant activity was also found in fractions Fr2, Fr3 and Fr4 of the methanolic extract. These fractions correspond to oligomeric condensed tannins. Oligomerization of tannins may be required for tannins to produce a vasorelaxant activity. Indeed, (+)-catechin, which is with (-)-epicatechin one of the basic units of oligomeric condensed tannins, produces no vasorelaxation while oligomeric tannins present in red wine [START_REF] Andriambeloson | Natural dietary polyphenolic compounds cause endothelium-dependent vasorelaxation in rat thoracic aorta[END_REF] or Arbutus (this study) relax pre-contracted aorta. Fraction Fr11, which contains heterosides of quercetin (hyperoside and quercitrin), had a much weaker activity. Genin alone (quercitin) showed a very weak activity, while the corresponding heteroside (hyperoside) was more active. Finally, arbutoside (present in Fr1), which is responsible for the antiseptic activity of Arbutus leaves [START_REF] Frohne | Urinary disinfectant activity of bearberry leaf extracts[END_REF], had no vasorelaxant activity.

In conclusion, like the root extract, Arbutus leaves extract possesses a strong endothelium-dependent, vasorelaxant activity which provides a rationale for the use of this plant in folk medicine to treat hypertension. A chromatographic fractionation of Arbutus leaves methanolic extract allowed to assign this vasorelaxant activity to polyphenolic compounds such as oligomeric condensed tannins and catechin gallate.

  Arbutus unedo were collected from Jbel Tazekka in oriental Morocco in October 2000. Taxonomic identification was performed by Pr B. Haloui from the Biology Department of Oujda Sciences Faculty (Morocco) where voucher specimen has been deposited (collection N° ZL 14).

Figure 1 .

 1 Figure 1. Vasorelaxant effect of Arbutus leaves aqueous extract Original tracings showing the effect of carbachol (CCh, 10 µM, A, C), aqueous extract of Arbutus leaves (AE, 10 -2 g/L, B, C) or SNP (1 µM, C) in intact (A, B) or denuded (C) rat aortic rings pre-contracted by 0.1 µM NAdr. The rings were first exposed to control solution and the solid lines indicate the period of drug perfusion.

Figure 2 .

 2 Figure 2. Vasorelaxant effect of Arbutus leaves extracts Original tracings showing the vascular effect of four extracts of Arbutus leaves obtained by soxhlet extraction with solvents of increasing polarity. Dichloromethane (A), water (B), ethyl acetate (C) and methanol extracts (D) of Arbutus leaves were tested at 10 -3 -10 -1 g/L on noradrenaline (NAdr, 1 µM) pre-contracted intact rat aortic rings. The rings were first exposed to control solution and the solid lines indicate the period of drug perfusion.

Figure 3 .

 3 Figure 3. Separation and test of different fractions from methanol extract of Arbutus leaves A) complete chromatogram of the total methanolic extract of Arbutus leaves obtained by semi-preparative reversed-phase HPLC. This chromatogram was separated in thirteen fractions, named Fr1 to Fr13. B) Each fraction was collected and tested at 10 -2 g/L on 0.1 µM NAdr pre-contracted rat aortic rings. The effect is expressed as percent relaxation. Each bar indicates the mean  SEM of the number of experiments shown in

Figure 4 .

 4 Figure 4. Concentration-response curves for the effects of different fractions from methanol extract of Arbutus leaves A) Concentration-response curves for the effects of fractions Fr2 (), Fr3 (), Fr4 (triangles) and Fr6 () separated as shown in Fig. 3A. Aortic rings pre-contracted by 0.1 µM NAdr were exposed to 10 -3 , 3x10 -3 or 10 -2 g/L of each fraction. B) Concentration-response curves for the effects of Fr6 (, same data as in A) and catechin gallate (CG, ). Each symbol indicates the mean  SEM of the number of experiments

Figure 5 .

 5 Figure 5. Chemical structures of compounds identified in methanolic extract.

Table 1 .

 1 Mean values of the vasorelaxant effect (expressed as percent of relaxation on intact NAdr pre-contracted aorta) of the compounds identified in methanolic extract

	(n=4).
					1 0 -2 g/L
		Arbutoside Quercitin Epicatechin Hyperoside Catechin	Catechin gallate
	% relaxation	11	51	31	236*	354*	816*
	Values are means  SEM.

* p<0.05 vs. Controle (NAdr. Alone).

Acknowledgements

We gratefully acknowledge the help of P. Lechêne (INSERM U-446, Université Paris Sud, France) for computer programming and Pr. B. Haloui (Faculté des Sciences, Université Mohamed Ier, Morocco) for botanical determination. This work was supported by grants from: the Centre National de Recherche (CNR, Morocco), projet PARS Médecine 081; the Institut National de la Santé et de la Recherche Médicale (INSERM, France), projet Franco-Marocain INSERM-CNR 2000.

Relaxation (%)

(3)