Abhishake Mondal

Sonja Durdevic

Lise-Marie Chamoreau

Yves Journaux

Miguel Julve

Laurent Lisnard

Rodrigue Lescouëzec

Sonja Durdevik

A cyanide and hydroxo-bridged nanocage: a new generation of coordination clusters

published or not. The documents may come

solvent molecules). 16 For example, S. Holmes et al. reported in 2008 a cubic {Fe4Co4} cage exhibiting interesting thermallyand photo-induced electron transfer properties. 16a Actually, such discrete cyanide-bridged {Fe-Co} systems are currently attracting a strong interest because of their photo-switchable magnetic properties. It is also worth noticing that these complexes may exhibit SMM-like behaviour in the photo-induced paramagnetic state, as illustrated by the very recent works from O. Sato et al. and H. Oshio et al. 17 The reaction of the [Co2(OH2)(piv)4(Hpiv)4] and [Fe(Tp)(CN)3] -building blocks in acetonitrile yielded prismatic red crystals of complex 1 by slow evaporation. The crystal structure of 1 is made of an anionic {Fe4Co12} 2-cubic nanocage cryptating a [Co II (CH3CN)6] 2+ complex, and crystallisation molecules (Figure 1). The corners of the cube are alternatively occupied by fac-[Fe II/III (Tp)(CN)3] -/2-complexes and [Co II 3(OH)(piv)4(L)] + (where L= MeCN or Hpiv) trimetallic clusters whereas the edges are defined by the Fe-CN-Co bonds, all cyanide acting as bridging ligands. Each cobalt cluster has a µ3-OH capping group that lies below the metallic plane and µ:η1:η1 bridging pivalate groups are found along the edges of the triangle defined by the cobalt atoms (See SI). Structural differences between the tricobalt units arise from the µ:η1:η1 or µ3:η2:η1 coordination modes observed for the fourth bridging pivalate group in each cluster as well as the presence of pivalic acid or acetonitrile as terminal ligand (see SI for more details). As a result, six-, fiveand four-coordinate cobalt(II) ions are found in the polymetallic cage. The observed Fe-C and Fe-N bond distances indicate the presence of two reduced iron(II) ions which is consistent with BVS calculations and charge balancing that support the presence of thirteen cobalt(II) ions, four hydroxide anions and three non-deprotonated pivalic acid (See SI). The acetonitrile molecules in the encapsulated [Co II (CH3CN)6] 2+ complex points toward the six faces of the cube. Within the cube the {Fe-C-N-Co} edges are slightly bent with average angles close to linearity for {Fe-C-N} (175. 8° [173.6-176.9]) but smaller for {C- N-Co} (163.2° [153.8-167.9]). The values of the Fe-Co distances along the edges range from 4.98 to 5.09 Å with a mean value of 5.03 Å and the shortest intermolecular metal-metal distance found is of 10.16 Å, which actually compares to the longest intracube metal-metal distances (from 11.08 to 11.99 Å). In comparison to the largest {Fe(Tp)(CN)3}-based heteropolycyanometalates, 1 represents a fine example of nanocage with sixteen metal centres and it sets the highest nuclearity observed so far. 18 The magnetic properties of 1 have been investigated measuring the thermal dependence of the χMT product (χM being the magnetic molar susceptibility per {Fe4Co13} unit) in the 2-300 K temperature range at 2.5 kOe (Figure 2). The χMT value at 300 K, 39.6 cm3 mol -1 K, lies within the expected range for the following set of non-interacting ions: (i) two low-spin Fe(II) (S = 0), (ii) two low-spin Fe(III) ions (S = ½; χMT ~ 0.7 cm 3 mol -1 K per ion, with g = 2.6) 19 and (iii) thirteen high-spin Co(II) ions (S = 3/2; 2.7 < χMT < 3.4 cm 3 mol -1 K per ion), 20 with paramagnetic ions exhibiting a significant orbital contribution to their magnetic moment. On lowering the temperature, the χMT value first smoothly decreases, reaching a plateau between 20 and 10 K, (χMT = 26.7 cm 3 mol -1 K) and finally decreases more abruptly down to 2.0 K (χMT = 17.7 cm 3 mol -1 K). The decrease of χMT from room temperature to 50 K can be ascribed to the effect of spin-orbit coupling (Co(II) and Fe(III) ions exhibiting 4 T1g and 2 T2g ground terms, respectively). Indeed, a good fit is obtained with a model taking into account 13 Co(II) and 2 Fe(III) ions, spin-orbit interaction and no exchange interaction between ions. 20, § The plateau around 20 K could be the signature of the weak ferromagnetic interaction between the low-spin iron(III) and cobalt(II) ions. 14 Finally, the sharp decrease at low temperature may be assigned to anisotropy effects. Indeed the isofield lines are not superposable in the reduced magnetisation versus H/T plot (insert, Figure 2), which usually, comes from a significant magnetic anisotropy (see SI). Nonetheless, no out-of-phase AC magnetic susceptibility signal was observed down to 2 K, discarding so the occurrence of slow magnetic relaxation. Beside, 1 does not show any photomagnetic effect under laser light irradiation in the visible range. Overall, the thorough analysis and the simulation of the magnetic data are precluded because of the size of the system. &

Conclusions

We have started a promising synthetic approach, which gives access to a new generation of cluster-based molecular magnets where are married, for the first time, two popular ligands traditionally used separately in molecular magnetism: the oxo/hydroxo and cyanide bridges. In the present example, the use of a versatile oxo-based cluster toward a partially blocked cyanide building block has led to an unprecedented hydroxo-cyanide based supramolecular cubic nanocage. Beyond its use in molecular magnetism, the novel synthetic approach that we have developed is also relevant for the molecular chemists interested in the design of discrete polymetallic systems. § best fit led to λCo= -120 cm -1 , DCo = 468 cm -1 , αCo = 0.81, λFe = -372 cm -1 , DFe = -324 cm -1 , αFe = 0.77 and TIP = 0.025 cm 3 mol -1 where λM, DM, αM and TIP are the spin-orbit constant, the axial distortion, the orbital reduction factor and the temperature independent paramagnetism, respectively & Neglecting the spin-orbit interaction, the size of the Hamiltonian matrices to be considered are already equal to 67108864 x 67108864 By taking into accounts the spin-orbit coupling using the T-P isomorphism, 27 the size becomes intractable with size larger than 320 10 12 .

Notes and references

Scheme 1 .

 1 Scheme 1. Different synthetic strategies based on the self-assembly of cyanide building blocks leading to Fe-Co MMMs: (a) three dimensional (3D) structure of photomagnetic PBAs; (b) one-dimensional (1D) structure of SCMs; (c) Discrete cubic model (0D) complex of photomagnetic PBAs; (d) new approach for designing hydroxo/oxo-cyanide mixed systems.

Figure 1 .

 1 Figure 1. Structure of 1 and its hydroxo-and cyanide-bridged core. The free solvent molecules were omitted for clarity.

Figure 2 .

 2 Figure 2. χMT vs. T plot for 1 (solid line is the best fit curve, see text). Insert: M vs. H/T curve for 1 in the 2-7 T magnetic field range and between 1.8 and 8 K (solid lines are eye guides).

a

  Institut Parisien de Chimie Moléculaire, UMR 7201, UPMC, Univ Paris 06, Paris, 75005 Paris, France. Fax: 00 33 (0)1 44 27 38 41; Tel: 00 33 (0)1 44 27 55 62; E-mail: rodrigue.lescouezec@upmc.fr b Instituto de Ciencia Molecular (ICMol), Facultat de Química, Universitat de València, C/ Catedrático José Beltrán 2, 46980 Paterna, València, Spain. ‡ This work was supported by the Ministère de l'Enseignement Supérieur et de la Recherche (MESR, France), the Erasmus Mundus program (lot 13), the Agence Nationale de la Recherche (Project : ANR-08-BLAN-0186-01) and the Ministerio Español de Ciencia e Innovación (Project CTQ 2010-15364) and generalitat Valenciana (PROMETEO/2009/108 and ISIC2012/002).

Figure

  Figure S1a: representation of the {Co3m3-OH} clusters with atom labels and BVS calculations for the oxygen atoms assuming cobalt(II). Top: Co(1) to Co(3). Bottom: Co(4) to Co(6).

Figure

  Figure S1b: representation of the {Co3m3-OH} clusters with atom labels and BVS calculations for the oxygen atoms assuming cobalt(II). Top: Co(7) to Co(9). Bottom: Co(10) to Co(12).

Table 2 :

 2 BVS calculations 1

	Atom Label	BVS calculated for Co(II)	BVS calculated for Co(III)
	Co(1)	1,9971	2,1326
	Co(2)	1,8311	1,9892
	Co(3)	2,0189	2,1489
	Co(4)	1,8964	2,0516
	Co(5)	1,9375	2,0636
	Co(6)	1,8984	2,0476
	Co(7)	1,8724	2,0160
	Co(8)	1,9340	2,1344
	Co(9)	1,9113	2,0620
	Co(10)	1,8940	2,0491
	Co(11)	1,9492	2,0796
	Co(12)	1,9029	2,0411
	Co(13)	1,8578	2,4344

1 a) I. D.

Brown, D. Altermatt, Acta Crystallogr. 1985, B41, 244, b)

N. E. Brese, M. O'Keeffe, Acta

Crystallogr. 1991, B47, 192, c)

M. O'Keeffe, N. E.

Brese, Acta Crystallogr. 1992, B48, 152.

Table 3 :

 3 Fe-C and Fe-N bond length distances in Å.

	Fe(III)
	Fe(1)	C3	1.9256(40)	Fe(3)	C8	1.9147(37)
		C2	1.9270(41)		C9	1.9236(39)
		C1	1.9369(44)		C7	1.9299(37)
		N105	1.9644(35)		N304	1.9712(38)
		N101	1.9661(41)		N302	1.9752(35)
		N103	1.9775(40)		N300	1.9832(34)
	Fe(II)
	Fe(2)	C4	1.8918(37)	Fe(4)	C10	1.8850(36)
		C6	1.8923(37)		C11	1.8861(38)
		C5	1.8942(38)		C12	1.8945(38)
		N203	2.0023(35)		N403	2.0102(36)
		N205	2.0100(34)		N401	2.0119(36)
		N201	2.0175(34)		N405	2.0152(35)

Supplementary information

Cyanide-and hydroxo-bridged nanocage: a new generation of magnetic coordination clusters.

Abhishake Mondal, † Sonja Durdevik, † Lise-Marie Chamoreau, † Yves Journaux, † Miguel Julve, § Laurent Lisnard,* , † , Rodrigue Lescouëzec* , † † Institut Parisien de Chimie Moléculaire (IPCM), UMR7201, UPMC Paris 6, 4 place Jussieu, 75252 Paris cedex5 (France); § Instituto de Ciencia Molecular (ICMol), Facultat de Química, Universitat de València, Catedrático José Beltrán 2, 46980 Paterna, València (Spain).

Synthesis of 1.

Crystal structure: data collection and refinement.

Table 1: Structural description of the Co3 clusters.

Table 2: BVS calculations.

Table 3: Fe-C and Fe-N bond length distances in Å.

Figure S1a: representation of the {Co3m3-OH} clusters with atom labels and BVS calculations for the oxygen atoms assuming cobalt(II). Top: Co(1) to Co (3). Bottom: Co(4) to Co (6).

Figure S1b: representation of the {Co3m3-OH} clusters with atom labels and BVS calculations for the oxygen atoms assuming cobalt(II). Top: Co(7) to Co (9). Bottom: Co (10) to Co(12).

Fit of the magnetic data: details and comments.

Figure S2: M vs. H/T plot for 1.

Synthesis of 1.

[PPh4][Fe III (Tp)(CN)3].H2O [18] (70.3 mg, 0.1 mmol) and [Co2(H2O)(piv)4(Hpiv)4] [13] (285 mg 0. Crystal structure: data collection and refinement.

Crystal data for 1 (C180H279B4Co13Fe4N52O45. 7). There is a quite large void (400 Å 3 ) in the crystal structure, as underlined by Checkcif.

There is residual density in this region and there must be missing solvent molecules in the proposed model. However, the low intensity of the residual peaks (<1 e.Å -3 ) prevented any sensible refinement. Moreover, the most important residues are located around the pivalates of the Co1-Co2-Co3 triangle, showing that the disorder of this part is not fully modelled. As mentioned above, no sensible model could be obtained. Some tertio-butyl moieties of pivalates are disordered over two positions and refined isotropically. CCDC 890505 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or deposit@ccdc.cam.ac.uk).

Table 1: Structural description of the Co3 clusters. 4 bridging pivalates, µ:η 1 : η 1 .

2 terminal pivalic acids.

2 octahedral cobalt ions.

1 tetrahedral cobalt ion.

[Co II 3(OH)(piv)4(Hpiv)] +

. {Co(4), Co( 5), Co( 6)}.

4 bridging pivalates, three µ:η1: η1 mode and one µ3:η 2 : η 1 .

1 terminal pivalic acid.

1 octahedral cobalt ion.

2 trigonal bipyramidal cobalt ions.

[Co II 3(OH)(piv)4(Hpiv)] +

. {Co (10), Co (11), Co(12)}.

4 bridging pivalates, three µ:η1: η1 mode and one µ3:η 2 : η 1 .

1 terminal pivalic acid.

1 octahedral cobalt ion.

2 trigonal bipyramidal cobalt ions.

[Co II 3(OH)(piv)4(MeCN)] 1+

. {Co(7), Co( 8), Co( 9)}.

4 bridging pivalates, three µ:η 1 : η 1 mode and one µ3:η 2 : η 1 .

1 terminal acetonitrile.

1 octahedral cobalt ion.

2 trigonal bipyramidal cobalt ions.

Fit of the magnetic data: details and comments.

The system being composed of weakly coupled high-spin cobalt(II) ions and low-spin iron(III) ions, the magnetic data was modelled in the 300-50 K temperature range using the following Hamiltonian where the interaction between the magnetic ions are neglected [eq (1-3)]:

with

The first term in the Hamiltonian ( 2) corresponds to the spin-orbit coupling effects, where lM with M=Co, Fe is the spin-orbit coupling constant and aM being the orbital reduction factor due to the existence of covalent character of bonds involving metal and ligands. The second term in this Hamiltonian accounts for the axial distortion of the six-coordinated Co II or Fe III ions. The last term represents the Zeeman interaction. The numerical coefficient n depends on electronic configuration and comes from the isomorphic transformation which convert the hamiltonian for a 2S+1 T2 or 2S+1 T1 state into an hamiltonian for a 2S+1 P term (L=1). For high-spin cobalt(II) and low-spin iron(III) these coefficients are equal to n = -3/2 and n = -1 respectively.

[1]

Adding a TIP term to take into account the effect of unpopulated exicted states, the least-squares fit of the experimental data of 1 using full diagonalisation of the Hamiltonian matrix performed in the 300-50 K temperature range led to lCo= -120 cm -1 , DCo = 468 cm -1 , aCo = 0.81, lFe = -372 cm -1 , DFe = -324 cm -1 , aFe = 0.77 and TIP = 0.025 cm 3 mol -1 . These values are in the expected range for high-spin cobalt(II) and low-spin iron(III) ions.

[1]R. M. Golding, Applied wave mechanics Van Nostrand Co, London, 1969 p 243.

Reduced magnetisation plot M(H/T)

The saturation magnetization value of 23.2 BM for 1 (figure S2) is close to the calculated one (25 BM) with the best parameters obtained by the fit of c.T vs. T plot in the high temperature range. These reduce magnetisation M versus H/T curves are recorded in the 1.8K-8K temperature range where it is impossible to neglect the interaction between the magnetic ions as shown by the c.T versus T plot. Given the size of the cluster (see ref 28), it is impossible to model these curves. However, the absence of a unique curve for the reduce variable H/T can have two origins. Firstly, the anisotropy of the iron and cobalt ions results in nonsuperposable isofield curve. Secondly, the exchange interactions through the cyanide and through the oxo bridges are quite weak (as observed in numerous previously reported Fe-CN-Co based systems, and, in agreement with the treatment of the c.T vs. T plot). Thus numerous close-lying excited states lie close to the ground state, so that even at low temperature, the magnetization value results from a thermal distribution of various states. This distribution of close-lying levels also leads to nonsuperposable isofield curves.


Enrique Mejías

Carlos Gomez

Pierre Gareil

Nathalie Delaunay

Gloria Montenegro

Enrique Mejías1

Carlos J Gómez3

Pierre Gareil4

Nathalie Delaunay4

Gloria Montenegro6

Characterization of phenolic profile alterations in metal-polluted bee pollen via capillary electrophoresis

Keywords: Antioxidant, antiradical activity, capillary electrophoresis, metals, phenol

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

Introduction

Bee pollen is a conglomerate of plant pollen that is combined in the corbicula, a special cavity on the hind legs in honeybees (Apis mellifera) [START_REF] Montenegro | Biological evaluation of bee pollen from native Chilean plants[END_REF]. The quantity of water in bee pollen varies between 4 and 10%, and this water content ensures good preservation over time and the nondegradation of organic compounds, which consequently maintain original inherent properties [START_REF] Gergen | Determination of water content in ee's pollen samples by Karl Fischer titration[END_REF][START_REF] Nogueira | Commercial bee pollen with different geographical origins: A comprehensive approach[END_REF]. The chemical composition of bee pollen varies according to the plant origin, geographical location, and environmental conditions [START_REF] Komosinska-Vassev | Bee pollen: Chemical composition and therapeutic application[END_REF][START_REF] Nogueira | Commercial bee pollen with different geographical origins: A comprehensive approach[END_REF]. Despite the variations, bee pollen is normally acidic, with a pH between 3.4 and 5.1, and composed of 20% proteins (including essential amino acids), 55% total carbohydrates with 25% reducing sugars (primarily fructose and glucose), 5% lipids, 1.6% phenolic compounds, 0.7% vitamins, and 1.6% bioelements (macro and micronutrients) [START_REF] Campos | Pollen composition and standardization of analytical methods[END_REF][START_REF] Komosinska-Vassev | Bee pollen: Chemical composition and therapeutic application[END_REF][START_REF] Nogueira | Commercial bee pollen with different geographical origins: A comprehensive approach[END_REF]. Of the metals, the most predominant are iron, magnesium, manganese, copper, zinc, calcium, sodium, and potassium [START_REF] Vit | Composición química de polen apícola fresco recolectado en el páramo de Misintá de los andes venezolanos[END_REF]. Considering this diverse composition, bee pollen is a nutritional supplement with a variety of properties that can be determined from the chemical nature of the compounds within bee pollen [START_REF] Taha | Chemical composition and amounts of mineral elements in honeybeecollected pollen in relation to botanical origin[END_REF][START_REF] Yang | Characterization of chemical composition of bee pollen in China[END_REF]. One important group of compounds is polyphenols, which are directly related to the antioxidant capacity of several bee pollens [START_REF] Denisow | Biological and therapeutic properties of bee pollen: a review[END_REF].

The three most important groups of compounds with antioxidant properties in bee pollen are flavonoids, phenolic acids, and tannins [START_REF] Mǎrghitaş | In vitro antioxidant capacity of honeybee-collected pollen of selected floral origin harvested from Romania[END_REF][START_REF] Pascoal | Biological activities of commercial bee pollens: Antimicrobial, antimutagenic, antioxidant and antiinflammatory[END_REF]. The mechanisms of antiradical activity are different according to the chemical structure of each compound [START_REF] Denisow | Biological and therapeutic properties of bee pollen: a review[END_REF]. In bee pollen, these compounds act together as antioxidants, with each contributing toward the total activity [START_REF] Almaraz-Abarca | Antioxidant activity of polyphenolic extract of monofloral honeybee-collected pollen from mesquite (Prosopis juliflora, Leguminosae)[END_REF][START_REF] Leblanc | Antioxidant activity of Sonoran Desert bee pollen[END_REF][START_REF] Leja | Antioxidative properties of bee pollen in selected plant species[END_REF][START_REF] Rzepecka-Stojko | Polyphenols from Bee Pollen: Structure, absorption, metabolism and biological activity[END_REF]. Usually, the total polyphenol content, antioxidant activity, and antiradical activity are related to the physiological and bioactive roles of substances in terms of human nutrition [START_REF] Kroyer | Evaluation of bioactive properties of pollen extracts as functional dietary food supplement[END_REF].

The chemical profiles of natural extracts obtained from a complex organic matrix, such as pollen or honey, have traditionally been determined through chromatographic techniques (e.g., HPLC, FTIR, HPLC-UV, and HPLC-MS) [START_REF] Dominguez | Capillary electrophoresis method for the simultaneous determination of carbohydrates and proline in honey samples[END_REF][START_REF] Liang | Determination of carbohydrates in honey and milk by capillary electrophoresis in combination with graphene-cobalt microsphere hybrid paste electrodes[END_REF].

Nevertheless, capillary electrophoresis (CE) and related techniques, such as electrokinetic chromatography, are interesting alternatives because they possess several advantages, including decreased analysis time, high separation efficiency, ultra-small sample volume, and low reagent consumption [START_REF] Arráez-Román | Identification of phenolic compounds in rosemary honey using solid-phase extraction by capillary electrophoresis-electrospray ionization-mass spectrometry[END_REF][START_REF] Moreno-González | Determination of aminoglycosides in honey by capillary electrophoresis tandem mass spectrometry and extraction with molecularly imprinted polymers[END_REF][START_REF] Nicolaou | Analysis of polyphenols using capillary zone electrophoresis -Determination of the most effective wine sample pretreatment method[END_REF][START_REF] Rizelio | Fast determination of cations in honey by capillary electrophoresis: A possible method for geographic origin discrimination[END_REF].

Several apiculture products, such as honey, pollen, and propolis, are suitable materials for monitoring environmental contamination [START_REF] Formicki | Metal content in honey, propolis, wax, and bee pollen and implication for metal pollution monitoring[END_REF][START_REF] González-Martín | Determination of the mineral composition and toxic element contents of propolis by near infrared spectroscopy[END_REF]. This attribute is due to the direct contact that honeybees have with environmental contaminants during flight and through food sources (e.g., plant pollen, nectar, and water) (Lambert et al., 2012;r l o s i and ilc s a, 2001). Because the botanical origin is the primary conditioner for the composition of apiculture products, any contaminating compounds can likely be traced to the originating plant species [START_REF] Almeida-Muradian | Chemical composition and botanical evaluation of dried bee pollen pellets[END_REF][START_REF] Campos | Pollen composition and standardization of analytical methods[END_REF]. Several studies have assessed contaminants in honeys, including antibiotics [START_REF] Al-Waili | Antibiotic, pesticide, and microbial contaminants of honey: human health hazards[END_REF], heavy metals [START_REF] Fredes | Heavy metal and other trace elements contents in honey bee in Chile[END_REF][START_REF] Tuzen | Trace element levels in honeys from different regions of Turkey[END_REF], pesticides [START_REF] Panseri | Occurrence of pesticide residues in Italian honey from different areas in relation to its potential contamination sources[END_REF][START_REF] Rodríguez López | Evaluation of pesticide residues in honey from different geographic regions of Colombia[END_REF], and others [START_REF] Cortopassi-Laurino | Global meliponiculture: challenges and opportunities[END_REF]Lambert et al., 2012). Volcanic activity can also affect the mineral composition of honeys and pollens produced in nearby areas [START_REF] Mejías | The Antioxidant Activity of Chilean Honey and Bee Pollen roduced in the Llaima Volcano's Zones[END_REF].

Despite these links, pollen is an understudied apiculture product in terms of its chemical composition, biological activity, and possible contaminants.

The aim of this study was to assess the relationship between bioaccumulation of metals and the effect on the antioxidant activity of native pollen samples. The first step determined the botanical origins of bee pollen collected in an area affected by volcanic activity and consequently metal pollution. Immediately after this determination, the most predominant Chilean native species detected in the assessed bee pollen was isolated and subsequently evaluated via CE and mass spectrophotometry to determine the phenolic contents. The selected native samples were also fortified by aliquots of certified reference materials for lead (Pb), cadmium (Cd), and chromium (Cr). Electropherograms from the fortified and control groups were then compared to established behavioral differences in phenolic compounds as a result of increased metal presence.

Materials and methods

Pollen samples

Pollen samples were collected near the town of Vilcún in the Araucanía Region of southern Chile (38°41'59.80" S, 71°45'22.70" ). Samples ere obtained near or distant from (>30 km) the Llaima Volcano, with these samples constituting the Llaima and control groups, respectively. Importantly, both collection areas contained the same botanical species.

Complete bee pollen samples were collected from five beehives located in each sampling zone.

Mellisopalynological analysis for determining the botanical origin of bee pollen

The botanical composition of bee pollen samples was quantitatively counted following methods described by [START_REF] Louveaux | Methods of melissopalynology[END_REF]. Briefly, bee pollen (20 g) was placed on acetolyzed slides [START_REF] Montenegro | Aplicación de la Norma Chilena Oficial de denominación de origen botánico de la miel para la caracterización de la producción apícola[END_REF]. Then, a sample aliquot was diluted with warm distilled water (20 mL at 40 °C), and the solution was transferred to an appropriate tube and centrifuged at 3,500 rpm for 10 min. The supernatant was discarded, and the pollen residue was deposited at the bottom of the tube for re-suspension in distilled water (100 mL). An aliquot (20 mL) was then added to a slide ith Cal erla's solution (10 mL), hich was either basic fuchsine or diamond. The slide was gently dried. Finally, melted glycerinated gelatin (15 mL) was added to the mixture. For each sample, pollen grain residues were identified using an optical microscope at 400X and 1000X magnifications. Both the Llaima and control samples were analyzed using the above criteria.

Preparation of E. rubra pollen solutions

Mellisopalynological analysis revealed Escallonia rubra to be a primary component (45%,

Table 1) of the collected bee pollen samples. This species belongs to the native endemic flora of Chile and in this case, was the primary native component of the selected bee pollen samples Therefore, E. rubra pollen was isolated from every collected pollen sample and solutions prepared for subsequent colorimetric, ferric reducing/antioxidant power (FRAP),

1,1-diphenyl-2-picrylhydrazyl radical (DPPH), metal content, and CE assays. Specifically, E.

rubra pollen (2 g) was suspended in distilled water (4.0 mL, pH 4) and then vortexed. The pH range for all suspensions was between pH 4.8 and 5.5.

Colorimetric assays for determining Total Phenolic Compounds

The procedures described by [START_REF] Singleton | Colorunetry of total phenolics with phosphomolybdicphosphotungstic acid reagents[END_REF] and [START_REF] Buratti | Evaluation of the antioxidant power of honey, propolis and royal jelly by amperometric flow injection analysis[END_REF] were used with minor modifications. Briefly, E. rubra pollen solution (200 μL) as mixed ith the Folin-Ciocalteu reagent (50 μL) and 20% Na2CO3 (150 μL). Finall , distilled ater (600 μL) was added. Absorbance at 765 nm was determined after 30 min in a Shimadzu (Brazil) UV-1700 UV-visible spectrophotometer. Gallic acid was used as the standard to derive the calibration curve (0-150 mg mL-1). The results revealed the phenolic contents of E. rubra pollen (expressed as the mg equivalent of gallic acid kg-1 of sample). All samples were analyzed in triplicate.

FRAP assays for determining antioxidant activity

FRAP assays were performed according to [START_REF] Bertoncelj | Evaluation of the phenolic content, antioxidant activity and colour of Slovenian honey[END_REF]. Briefly, the FRAP reagent was prepared by mixing 2.5 mL of 2,4,6-tripyridyls-triazine (TPTZ; 10 mM TPTZ 40 mM-1

HCl) with 2.5 mL of 20 mM FeCl3. Then, 0.3 M acetate buffer (25.0 mL, pH 3.6) was added to the mixture. The FRAP reagent was freshly prepared for each assay run. To measure antioxidant capacity, E. rubra pollen (0.200 mL) was mixed with the FRAP reagent (1.8 mL).

Absorbance was read at 593 nm after 10 min. FeSO4•7H2O as used as the standard to derive the calibration curve (50-1000 mM). The values are expressed as mM Fe+2 equivalents g-1 of sample. Samples were analyzed in triplicate.

DPPH assays for determining antiradical activity

The procedures described by [START_REF] Meda | Determination of the total phenolic, flavonoid and proline contents in Burkina Fasan honey, as well as their radical scavenging activity[END_REF], as modified by [START_REF] Mejías | The Antioxidant Activity of Chilean Honey and Bee Pollen roduced in the Llaima Volcano's Zones[END_REF], were used to determine antiradical activity. The DPPH assay establishes the antiradical properties of the chemical compounds in pollen by inhibiting or decreasing the oxidant activity of DPPH. Briefly, E. rubra extracts (750 μL) ere mixed with the DPPH radical (1.5 mL) in methanol (0.02 mg DPPH mL-1 MeOH). Absorbance was read at 517 nm after 15 min. A blank sample was prepared with methanol. Ascorbic acid (Calbiochem, Germany) was used as the standard to derive the calibration curve (1-10 mg mL-1). The values for antiradical activity are expressed as mg of ascorbic acid equivalents/g of sample.

Samples were analyzed in triplicate.

Determining metal contents

Heavy metal contents in E. rubra pollen samples were determined following protocols defined in the Chilean standard (INN, 2008). Briefly, E. rubra pollen (5 g) was placed in a porcelain crucible that was subsequently placed in a muffle furnace for 3 h at 105 °C.

Subsequently, the temperature of the muffle oven was gradually increased to 130 °C, with samples kept at this temperature for 12 h. Then, the samples were digested by 65% HNO3 and in the crucible, placed on a heating plate at 50 °C for 30 min. Subsequently, aliquots of acid (2 mL) were added to prevent boiling. The aliquots were added until achieving total dissolution of the sample or the absolute disappearance of turbidity inside the crucible. The excess acid was evaporated until the sample was completely dried. Finally, the residue in the crucible was resuspended in 2% HNO3 (10 mL), and the resulting solution was fully transferred to a volumetric flask (10 mL) for further readings via inductively coupled plasma optical emission spectrometry.

Fortification assays

Escallonia rubra pollen samples were fortified using the certified reference material CMRCMQ-FCHILE-007 lot No. 012 bottle No. 006. The manufacturer reported concentrations of this material as follo : 49 μg L-1, Cr 36 μg L-1 and Cd 32 μg L-1.

Escallonia rubra pollen samples collected near the Llaima Volcano were fortified in increasing order of concentration with certified reference material aliquots of Pb, Cr, and Cd.

For this fortification, the pollen samples were separated into five subsample groups (termed A1, B1, C1, D1, and E1), each containing 500 mg of pollen. The A1-D1 subsample groups received certified reference material aliquots, with the weights recorded to establish the final concentration for each metal in μg g-1. The E1 group was not fortified and was used as the control sample.

Capillary electrophoresis with diode-array detection for determining phenolic contents

Capillary electrophoresis with diode-array detection (CE-DAD) experiments were performed at the Laboratoire de Physicochimie des Electrolytes, Colloides et Sciences Analytiques (PECSA)-Chimie ParisTech (École Nationale Supérieure de Chimie de Paris). Specifically, an Agilent G1600A Capillary Electrophoresis System equipped with an on-column diode-array detection system was used. Instrument conditions were set according to [START_REF] Nicolaou | Analysis of polyphenols using capillary zone electrophoresis -Determination of the most effective wine sample pretreatment method[END_REF]. Briefly, the fused silica capillaries had internal diameters of 50 μm, a total length of 64 cm, and an effective length of 55 cm. New capillaries were rinsed with water for 10 min, followed by 1 M NaOH for 1 h, water again for 20 min, and finally a background electrolyte (i.e., 0.1 M sodium tetraborate decahydrate) for 15 min. Samples were injected by applying a pressure of 50 mbar for 5 s. The separation voltage was 25 kV.

Statistical analyses

Exploratory data analyses were performed to evaluate patterns, detect anomalous values, and verify assumptions of statistical tests, including normality, independence, and homoscedasticity. Differences in phenol contents, antioxidant activity, and FRAP values between Llaima and control samples were established via one-way ANOVA with a post hoc Tu e 's test.

Results

Botanical origin of bee pollen

Mellisopalynological analysis was used to separate and identify the distinct plant pollens in complete bee pollen collected near the Llaima Volcano (Table 1). Based on the results, the most abundant native pollen was that of E. rubra, which was chosen for subsequent determinations of antioxidant properties and phenolic compound profiles via CE.

Composition of Total Phenolic Compounds of E. rubra pollen

Colorimetric analyses using the Folin-Ciocalteu reagent revealed no statistically significant differences in total hydrosoluble phenolic compounds between Llaima and control samples (Table 2). However, the control samples had 32% and 25% higher antioxidant and antiradical capacities, respectively, than did the samples collected near the Llaima Volcano (Table 2).

Metal contents of E. rubra pollen

Metal content analyses revealed no significant differences between Llaima and control samples in terms of their Zn and Cr concentrations. However, the concentrations of Cu, Fe, Pb, and Cd were higher in Llaima samples, but the Mn concentrations were significantly lower in the Llaima samples than they were in their control counterparts (Table 3).

Effects of fortification on the composition of Total Phenolic Compounds of E. rubra pollen

Four subsample groups (i.e., A1-D1) of E. rubra pollen collected near the Llaima Volcano were fortified with increasing amounts of certified reference material for Pb, Cd, and Cr. An additional subsample (i.e., E1) was left unfortified and used as the control. Compared with the E1 control, the final metal concentrations in the least (i.e., A1) and most (i.e., D1) fortified subsamples were as follow: A1 subsample: 177 times more Pb, 805 times more Cr, and 99 times more Cd; and D1 subsample, 705 times more Pb, 3220 times more Cr, and 392 times more Cd. In other words, the D1 subsample presented four times higher metal contents than the A1 subsample. The fortification results for all subsample groups are presented in Table 4.

No relationship was found between total phenolic compounds and fortification in the distinct subsample groups. In evaluating the linear relationship, the R2 value was 0.1657 (Fig. 1).

The fortified samples of E. rubra pollen were also evaluated to determine their antioxidant and antiradical capacities. Relationship assessments between final metal concentrations and antioxidant capacity (Fig. 2) revealed an exponential tendency, with an adjustment of 0.9343.

Therefore, as metal concentrations increased, the antioxidant capacity of pollen subsamples decreased. Similarly, increased metal concentrations were correlated with decreased antiradical capacity (Fig. 3), with an exponential tendency presenting an adjustment of 0.9689.

Effects of fortification on phenolic profiles in E. rubra pollen

Using CE-DAD analysis, changes in the phenolic profiles of fortified E. rubra subsamples were detected (Table 5). The migration times of the distinct evaluated standards were displaced, specifically for naringenin, rutine, p-coumaric acid, ferulic acid, caffeic acid, and gallic acid.

Discussion

Species from the Escallonia family include small trees and bushes primarily distributed in mountainous and humid regions, with a range from Costa Rica to Tierra del Fuego in Argentina and Chile. Nevertheless, most Escallonia species are located within Andean zones in Argentina, Chile, and Peru [START_REF] Morello | Análisis preliminar de la variabilidad genética de escallonia alpina y E. rubra (Escalloniaceae)[END_REF]. This family is widely reported between the 30° and 53° latitudes, which would account for the high prevalence of E. rubra pollen in the collected bee pollen samples (Table 1). Escallonia rubra pollen has also been reported in other nectar and honey samples collected from Andean zones in South America [START_REF] Chalcoff | Nectar concentration and composition of 26 species from the temperate forest of South America[END_REF][START_REF] Mejías | The Antioxidant Activity of Chilean Honey and Bee Pollen roduced in the Llaima Volcano's Zones[END_REF][START_REF] Montenegro | Multivariate analysis of pollen frequency of the native species Escallonia pulverulenta (Saxifragaceae) in Chilean honeys[END_REF].

Heavy metals are elements with a relative density higher than the density of water (Tchounwou et al., 2012). Some of these elements are considered micronutrients, but others, such as lead, cadmium, chromium, and mercury, can be toxic to plants at high levels of concentration, which are unable to metabolize these elements [START_REF] Michalak | Phenolic Compounds and Their Antioxidant Activity in Plants Growing under Heavy Metal Stress[END_REF]. The primary toxic effects of heavy metals result from the production of reactive oxygen species and reactive nitrogen species [START_REF] Bhaduri | Antioxidant enzyme responses of plants to heavy metal stress[END_REF][START_REF] Sytar | Heavy metal-induced oxidative damage, defense reactions, and detoxification mechanisms in plants[END_REF]. At a cellular level, heavy metals inhibit antioxidant enzymes, damage DNA, oxidize proteins and lipids, and cause a redox imbalance, among other effects [START_REF] Nagajyoti | Heavy metals, occurrence and toxicity for plants: A review[END_REF]Tchounwou et al., 2012). Two types of cellular mechanisms are used to confront these negative effects. First, mechanisms using enzymes, such as superoxide dismutase, catalases, peroxidases, and reductases, are activated. Second, non-enzymatic mechanisms are used, which include some vitamins (e.g., A, E, and D), amino acids (e.g., proline), and carotenoids and flavonoids [START_REF] Gill | Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants[END_REF].

Chemical analysis of E. rubra pollen revealed that although the concentrations of total phenolic compounds were similar between Llaima and control samples, antioxidant and antiradical activities were lower in pollen samples collected near the Llaima Volcano (Table 2). Furthermore, Llaima samples also showed higher concentrations of heavy metals, including copper, iron, lead, and cadmium (Table 3). Antioxidant potential can also be predicted by the availability of phenolic and flavonoid compounds, which donate hydrogen radicals that neutralize free radicals [START_REF] Buratti | Evaluation of the antioxidant power of honey, propolis and royal jelly by amperometric flow injection analysis[END_REF]. Heavy metal contamination originates from various sources, including industrial, agricultural (pesticides), domestic (wastewater), atmospheric, and natural (volcanoes) sources [START_REF] Nagajyoti | Heavy metals, occurrence and toxicity for plants: A review[END_REF].

Nonetheless, the metals in bee products are related to the botanical origin, which depends on the geographical origin and the composition of soils in the areas surrounding beehives [START_REF] Bilandzic | Essential and toxic element concentrations in monofloral honeys from southern Croatia[END_REF].

To determine whether the decreased antioxidant activity in the pollen samples was effectively due to the high heavy metal concentrations, fortification assays were performed with increasing amounts of certified reference materials in E. rubra pollen subsamples (Table 4).

The previously observed pattern was effectively maintained: total phenol concentrations were the same between subsamples, but antioxidant and antiradical activities decreased exponentially as heavy metal concentrations increased (Figs. 2 and3). These results were further corroborated via CE-DAD, which revealed changes in the phenolic profile as a result of variation in migration times for the distinct applied standards (Table 5). A plausible reason for the shift in migration times of pollen polyphenols in the metal-fortified samples might be complex formation between polyphenols and metals. Polyphenols are well known for their ability to chelate heavy metals such as Fe, Pb, and Cu. By chelating heavy metals, polyphenols prevent free radical formation (antioxidant effect) by a possible Fenton-like reaction. Interaction of heavy metals with polyphenols/flavonoids involves the same hydroxyl groups that are involved in the FRAP or DPPH antioxidant assays. Complexation of these groups by metal ions renders them unavailable for hydrogen transfer from the phenolic OH group (followed by an electron transfer) in these assays, which is then observed as a decrease in antioxidant capability [START_REF] Bautista | An overview of the application of Fenton oxidation to industrial wastewaters treatment[END_REF][START_REF] Mahal | Radical Scavenging and Catalytic Activity of Metal-henolic Complexes[END_REF]. [START_REF] Crane | Bees, honey and pollen as indicators of metals in the environment[END_REF] was the first to propose using apiculture products as biomarkers for environmental contamination. Honeybees can be used to identify chemical disruptions in the environment via two signals: hive death and detection of residues of contaminated substances in apiculture products [START_REF] Barga S A | Honey bees and their products: Bioindicators of environmental contamination[END_REF]. A highly notable advantage in using apiculture products as biomarkers is the early and spatial detection of patterns for distinct environmental contaminants [START_REF] Van Der Steen | Assessment of the potential of honeybees (Apis mellifera L .) in biomonitoring of air pollution by cadmium , lead and vanadium[END_REF]. This strategy is founded on the fact that botanical origin is the primary conditioner for the chemical composition of apiculture products. Therefore, the plant species of origin dictates the presence of antioxidant compounds and subsequent bioactive traits, in addition to contaminants such as pesticides and heavy metals [START_REF] Almeida-Muradian | Chemical composition and botanical evaluation of dried bee pollen pellets[END_REF][START_REF] Campos | Pollen composition and standardization of analytical methods[END_REF]. Various studies exemplify the use of apiculture biomarkers in the detection of heavy metals (i.e., lead, cadmium, chromium, and nickel), pesticides, and other anthropogenic environmental contaminants, including aromatic hydrocarbons, perfluorinated compounds, and phthalates [START_REF] Formicki | Metal content in honey, propolis, wax, and bee pollen and implication for metal pollution monitoring[END_REF][START_REF] Gómez-Ramos | Screening of environmental contaminants in honey bee wax comb using gas chromatography-high-resolution time-of-flight mass spectrometry[END_REF][START_REF] Porrini | Honey Bees and Bee Products As Monitors of the Environmental Contamination[END_REF][START_REF] Tuzen | Trace element levels in honeys from different regions of Turkey[END_REF][START_REF] Üren | Distribution of elements in honeys and effect of a thermoelectric power plant on the element contents[END_REF].

The effects of contaminants on human health are a growing concern. Although some elements are required in low doses to maintain biological functions, chronic exposure, even at low doses, can cause functional damage to distinct organs. This damage is in addition to the associations of exposure ith diseases such as cancer, ar inson's, and Alzheimer's [START_REF] Jaishankar | Toxicity, mechanism and health effects of some heavy metals[END_REF]. Plants absorb heavy metals and other contaminants from a contaminated environment; however, because plants are unable to metabolize metabolize these compounds, bioaccumulation occurs [START_REF] Liu | Human health risk assessment of heavy metals in soil-vegetable system: A multi-medium analysis[END_REF][START_REF] Michalak | Phenolic Compounds and Their Antioxidant Activity in Plants Growing under Heavy Metal Stress[END_REF]. Given that plants are one of the primary food sources for humans, heavy metal exposure through plants accounts for 90% of total metal intake, with only 10% occurring via dermal contact or [START_REF] Khan | The uptake and bioaccumulation of heavy metals by food plants, their effects on plants nutrients, and associated health risk: a review[END_REF].

The results of this study establish a foundation for future studies on bee pollen and other apiculture products. Continued research in this field is critical, because these products can be applied as a useful tool for studying the environmental effects of anthropogenic contamination, with the data collected used to aid in developing efficient monitoring and control protocols.

Disclosure statement

No potential conflict of interest was reported by the authors.

Acknowledgements

This work was supported by the Office of the Vice-Rector for Academic Affairs, Universidad Tecnológica Metropolitana under Grant VRACUTEM L216-11, awarded to Dr. Enrique Mejías.