Nassim Bouchoul

Elodie Fourré

Jean-Michel Tatibouët

Catherine Batiot-Dupeyrat
email: catherine.batiot.dupeyrat@univ-poitiers.fr

Plasma-catalytic dry reforming of CH4 over calcium oxide: catalyst structural and textural modifications

Keywords: nonthermal plasma, methane, carbon dioxide, calcium oxide

The coupling of catalyst and nonthermal plasma for the dry reforming of methane was investigated with a special attention to the textural and structural catalyst modifications under plasma discharge. The reaction was performed using calcium oxide as material located into the DBD plasma reactor, while the deposited power was fixed at 8W and the total gas flow at 40 mL/min (75% helium as diluent). The results obtained showed that CaO grain size affects the reactant transformation in the range: 250-1000µm. CH4 and CO2 conversion increases from 18.1 to 21.1% and 8.7 to 11.2% respectively from the biggest to the smallest catalyst grain.

Ethane formation is favored when the biggest particles are used, corresponding to the largest gas space between grains, suggesting the preferential recombination of CH3 radicals to form C2H6 in gaseous phase and not at the surface of the solid. The reaction was performed from room temperature to 300°C, little effect were observed for methane conversion while high CO2 conversion was observed during the 20 minutes at 300°C. The characterization of the catalyst after reaction under plasma shows structural catalyst modification and the carbonation of CaO at the highest temperatures. The amount of carbonate species was quantified and the results show that 59 monolayers of CaCO3 are obtained after one hour of plasma at P=8W using a mixture with a CH4/CO2 ratio of 2.

Introduction

Dry reforming of methane reaction has been the subject of many studies since in this process two significant greenhouse contributors are valorized, producing syngas with low H2/CO ratio suitable for the production of methanol and liquid hydrocarbons using Fischer-Tropsch processes [1,2].The reaction is usually carried out at temperatures higher than 700°C due to thermodynamic limitations and by using catalysts based on noble metals, such as (Rh, Ru, Pt, and Pd), known for their significant resistance to carbon coking [3]. Research efforts were performed on lower cost transition metals such as (Ni, Co, Fe) that can overcome carbon deposition limitation [4]. However, despite the considerable progress made in this field, dry reforming of CH4 using conventional catalytic processes still faces two major drawbacks that limit commercial application: the high operating temperatures and the catalyst deactivation due to coke deposition at the surface of active catalytic species [5]. Nonthermal plasma is considered as an attractive alternative method for methane and carbon dioxide transformation [6]. Many reactions are initiated at low temperature under the plasma discharge leading to valuable chemicals such as hydrocarbons [7] or oxygenates [8]. However the selectivity towards targeted products is often difficult to achieve due to the occurrence of a large number of reactions in gaseous phase. The coupling of catalyst and plasma was proposed to overcome this drawback [9]. In many studies, the selected catalysts were similar to those used in conventional thermal catalytic processes, and the influence of Ni/Al2O3 located into the plasma zone was investigated using different plasma reactor geometry. The presence of the reduced Ni/Al2O3 in the discharge lead to a decrease in conversions of CH4 and CO2 [10]. It was proposed that the presence of conductive Ni active sites decreased the electric field strength and consequently the electron density reducing thus reactant conversion [11,12]. From these works it is possible to conclude that the combination of plasma and catalyst requires the development of specific materials able to transform reactants present under active species form, such as excited species, radicals and ions [13].

The combination of nonthermal plasma with different metal oxides was investigated, while alumina possesses no catalytic activity in the thermal dry reforming of methane reaction, a significant improvement in CH4 and CO2 conversion over Al2O3 was observed when coupled with plasma [14]. Gadzhieva [15] showed that CH4 adsorption sites were formed on the γ-Al2O3

surface by molecular and dissociative mechanisms under the action of an electric-discharge plasma at room temperature.

Spinel nickel ferrite nanoparticles NiFe2O4, embedded in silica was successfully used by Zheng et al. [16]. The authors exhibited excellent catalytic performance and high resistance to carbon formation during dry reforming under ambient conditions. Jiang et al. [17] showed that CH4 and CO2 conversions are reduced with the use of a zeolite A, but the formation of carbon black was inhibited. A higher CO2 conversion and a lower CH4 conversion was reported by Zhang et al. using La2O3/Al2O3 under pulse corona plasma [18].

There are needs to develop more deeply the coupling of plasma and catalysis and the main goal of the present study was to investigate particularly the structural and textural modifications of materials under plasma discharge.

In this respect, we decided to study the influence of the presence of calcium oxide located in a packed bed DBD reactor. This material was chosen due to its relatively low dielectric constant , since as shown by Wang et al. [19] a too high dielectric constant of the packing material limits the discharge to the contact point of the beads and consequently is not favorable to develop reactions at the surface of a catalyst. Moreover the basic properties of CaO, was expected to favor CO2 adsorption and activation, since in thermal catalytic processes (T>700°C) it is known that carbonate species are involved in the reaction of dry reforming of methane.

Experimental part

The reaction was performed at room temperature, 100, 200, 300°C and atmospheric pressure in a coaxial dielectric barrier discharges (DBD) reactor (figure 1). The nonthermal plasma reactor (dielectric barrier discharge) was composed of an alumina tube (ID: 4mm; OD: 6mm), a stainless steel electrode inside the reactor (1.0mm) and a copper electrode wrapped around the alumina tube (100 mm long).

For plasma reactions performed at room temperature, an optic fiber was used to measure the temperature rise closed to the plasma zone. For that purpose, a fiber optic temperature sensor Optocon (TS2: 200µm of diameter) was introduced right below the plasma zone. It was checked that the temperature did not exceed 373K under the experimental conditions used.

Helium, methane and carbon dioxide were flowed through the plasma reactor at a total flow rate of 40mL.min -1 using different ratio CH4/ CO2 (from 0.5 to 2) with a constant concentration in He: 75%, corresponding to a residence time of 1.8s without catalyst and 1.6s in the presence of a catalyst.

A sinusoidal supply of power was applied across the electrode (TG1010A Aim-TTi, Thurlby Thandar Instruments Brand). The discharge power, calculated through the figure of Lissajous (Fig. 2), was fixed at 8 watts, keeping the frequency constant at 800Hz and varying the voltage from 12 to 14kV. At room temperature the voltage was equal to 14kV and was decreased at 12kV when the reaction was performed at 300°C .The electrical signals were monitored with high voltage probes (PMK, model PHV4-2757) connected to an oscilloscope (waveRunner 62 Xi, Lecroy). The applied voltage and current intensity of the DBD at an operating frequency of 800Hz are shown in Fig. 3.

The oxide used in this study are commercial products supplied by ACROS. The powder occupies all the plasma region, the solid was packed between two glass wool plug, the gas flow coming from the top of the reactor. The solids were characterized before and after plasma treatments.

Surface areas were measured according to the BET procedure. The nitrogen adsorptiondesorption isotherms were determined with a Micromeritics Flowsorb II 2300 appartus at -196°C.

Thermogravimetric analysis were performed with a SDTQ600TA analyser under a 100mL/min flow of air up to 1173K.

The catalysts were characterized by X-Ray Diffraction (XRD) using a Siemens D-5005 diffractometer with CuK=1.5417Å, operated at 40kV and 30mA. The diffraction patterns were recorded in the 2 range of 10-90° with a step interval of 0.02° and period of 1s.

Gas phase components were analyzed on-line by gas chromatography equipped with FID (oxygenated products) and TCD detectors (hydrogen, carbon monoxide, carbon dioxide, methane). The reaction was performed during one hour.

All the experiments were performed three times after cleaning the inner electrode and changing the catalyst (when used), a margin error of ±3% was calculated.

The conversion, selectivity, yields and energy efficiency were defined as:

Preliminary results

First experiments were performed by filling the plasma zone with quartz wool at room temperature, 100, 200 and 300°C, with different CH4/CO2 ratio (0.5, 1, 2). It was decided to fill the plasma reactor with quartz wool instead to keeping the plasma zone empty in order to obtain more accurate data in terms of residence time when compared with the presence of catalysts.

Moreover the nature of plasma discharge is also modified in the presence of materials.

Compared with an empty reactor, the presence of quartz wool (dielectric constant close to 4) causes an increase of the average electron energy but a decrease of electron density, improving the electric field strength but decreasing the amount of microdischarges [20,21]. A higher conversion is expected with dielectric materials in the discharge, but it could be compensated by the lower contact time of reactant induced by the filling of the plasma zone [22]. Moreover all the experiments were performed using helium as a dilution gas (75% vol.). Using a constant flow of helium as diluent allow to obtain a more homogeneous plasma discharge. Indeed, Ozkan et al. [23], showed that a glow discharge was obtained with helium while a filamentary discharge is observed in the presence of argon. Moreover, the authors showed that the glow regime (with He) favors CH4 dissociation since the number of electrons participating to C-H bond breaking is much higher in a glow discharge than in a filamentary one. The role of helium to perform activation under plasma discharge is also explained by a transfer of charges and energy from He species such as He + or He 2+ to CH4 and CO2, leading to the formation of vibrationally excited species such as CH4 + and CO2 + increasing, then, their reactivity [24]. CH4, CO2 conversions and selectivity to products are gathered in Figures 4 and Table 1 respectively. It appears that methane conversion is always higher than CO2 conversion independently of the reaction temperature and the ratio CH4/CO2. The excited oxygen atomic species generated from CO2 promotes conversion of CH4 leading to methyl and hydroxyl radicals [25]. Moreover conversions are not significantly modified within the temperature range used in this study, while the selectivity to products are strongly influenced by the temperature and CH4/CO2 ratio. As expected and reported elsewhere [26,27] the selectivity to CO is maximum using CO2 in excess and the selectivity to hydrocarbons (mainly ethane) is the highest in an excess of methane in the feed. Note that carbon balance is maximum for the ratio CH4/CO2 = 0.5, closed to 99% at room temperature, while for CH4/CO2 = 2 the carbon balance is lower than 90% and a deposition was visible at the surface of the inner electrode. An increase of the temperature until 300°C leads to a significant decrease in CO selectivity for both experiments in excess of CO2 and CH4 while with the stoichiometric mixture, the influence of temperature is not clear. The selectivity to oxygenate compounds decreases when the temperature increases and the carbon deposition occurs more readily at higher temperature.

Methane and carbon dioxide transformation over calcium oxide under plasma discharge 2.2.1 Influence of the grain size and role of CaO material

The influence of the grain size was studied using CaO oxide after calcination at 800°C in order to eliminate carbonate and hydrogeno-carbonate species. The CaO powder was sieved and separated into four different samples with diameters d1: 250-355µm, d2: 355-650µm, d3:650-800 and d4: 800-1000µm. Note that if we consider a compact filling of the reactor with spherical particles, the weight of materials and the total volume of gas between the grains are not modified. The parameters that are affected by the grain size are the accessible surface, the average size of space between particles and the number of contact point between the particles.

The results reported in Figure 5, obtained in the presence of an excess of CO2 (CH4/CO2 = 0.5), show that conversion of CO2 and CH4 increased with decreasing particle size. Methane conversion is closed to 30% in the presence of the smallest CaO grains, while with quartz wool methane conversion was lower than 20%, CO2 conversion was 16% and 12% with and without CaO respectively. Kasinathan et al. [28] obtained a similar effect of particle size for CH4 conversion into C2, C3 and C4 hydrocarbons over MgO/Al2O3. The authors, using an ICCD camera, showed that the smaller the catalyst particle used, the lighter the plasma observed on the catalyst surface. An improved interaction between the surface of the oxide and the plasma was proposed for the smallest catalyst grains. The exact role of the surface of the oxide is difficult to determine since some reactions involved can be performed in gas phase as at the surface of the oxide, by example CO can be obtained directly by CO2 splitting and also after CO2 adsorption at the surface of the basic CaO according to:

CO2 + * →CO2ads (1)
CO2ads → CO + Oads (under plasma)

(CHx) + Oads → COads + x/2H2 (2)
The selectivity to products depends on the particle size (Table 2), ethane formation is favored when the biggest particles are used, so when the space between grains is the largest, suggesting that the recombination of CH3 radicals to form C2H6 is favored in gaseous phase and not at the surface of the solid. Note that the carbon balance is high, always higher than 90%, the carbon deposition is limited under the experimental conditions used (excess of CO2). As expected, CO is the main product formed while the amount of oxygenated products is low whatever the grain size.

According to the results obtained here, it is believed that the reactivity is governed by the characteristics of the discharge at the external surface of the packing material possessing a low dielectric constant (≈11 [29]). CaO calcined at 800°C is characterized by the presence of meso and macropores with porous volume of 0.01 cm 3 /g and an average pore diameter of 20 nm (determined by adsorption/desorption of nitrogen). The specific surface area of CaO is low after calcination at 800°C: 1.8 m 2 /g (Table 4). In DBD plasma conditions, the formation of microdischarges in pores is possible when their diameter is larger than the Debye length. This value depends on electron density and temperature in the plasma streamer but is typically in the order of 100 nm to 1µm. According to calculations performed by the group of A. Bogaerts, plasma can penetrate in pores of about 50nm but only in some extent and at very short time [30,31].

Consequently, using CaO the penetration of micro-discharges within pores can be excluded.

Influence of the ratio CH4/CO2

The reaction was carried out at room temperature by filling the plasma zone with calcium oxide powder calcined at 800°C, sieved in the range: 355 µm< d < 650 µm, the deposited power was fixed at 8W and the helium concentration was 75%. A similar trend, as reported for the experiments using quartz wool, is observable: the carbon balance and the selectivity to CO is maximum with an excess of CO2 (Table 3). Besides CO and hydrocarbons, oxygenated products were formed from CH4 and CO2 mixture. The main oxygenates quantified were formaldehyde, methanol, acetaldehyde and ethanol, however the selectivities are very low under the experimental conditions used in this study. An opposite trend is visible concerning formaldehyde and methanol formation: CH3OH is slightly favored when an excess of CO2 is used, while the formation of HCOH is favored in an excess of methane. These results are in agreement with the reported research of De Bie et al. using a one-dimensional fluid model [32].

The dominant reaction pathways for the production of methanol was indicated by the authors, methanol being produced through the following three-body reaction between CH3 and OH radicals: CH3 + OH + M → CH3OH + M. It is also supported by the work of Wang et al. based on density functional theory (DFT) calculations [33] and Istadi who used a hybrid artificial neural networkgenetic algorithm technique [34]. The production of formaldehyde comes from the reaction between CO2 and CH2 radicals and acetaldehyde is obtained by O atom (from CO2 dissociation) reaction with ethyl radical [32].

Influence of temperature

A previous study performed in our team [14] shows that an increase of temperature at 300°C favors the conversion of CH4 and CO2 at the surface of balls composed of La2O3/Al2O3. It was thus interesting to investigate the influence of temperature on reactant conversion and product selectivities in the presence of calcium oxide calcined at 800°C. The results obtained for grain size: 355 µm < d < 650 µm, P= 8W, from 100 to 300°C and a CH4/CO2 ratio of 2 are gathered in figure 6. It is observed that methane conversion is relatively stable during time on stream and is closed to 20% whatever the reaction temperature, while with quartz wool methane conversion decreases regularly at 100 and 200°C, until 13.6 % after 50 minutes at 100°C. The carbon balance is significantly higher in the presence of CaO where it is maintained closed to 80% at 100 and 200°C, while it falls at 68.2% without catalyst after 30 minutes. Consequently, the results show that the presence of CaO limits carbon deposition, which is clearly observable at the surface of the electrode when the reactor is filled with quartz wool. At 300°C, the behavior is completely different: CO2 conversion is strongly increased at the beginning of the experiment performed at 300°C, reaching 40%, then it decreases regularly with time and stabilizing at around 15% after 30 minutes. The carbon balance follows an opposite trend suggesting a reaction of CO2 with the basic CaO material. The selectivity to the main products: CO and C2H6

is not modified by the increase of the reaction temperature. Note that methane conversion is not significantly increased in the presence of CaO compared to quartz wool, so the adsorption of CO2 during the first minutes of reaction at 300°C is not favorable for the activation of methane at the surface of the catalyst. It suggests that the carbonation of CaO (evidenced by TDA-TGA analysis, see below) leads to the formation of stable species that do not favor CO2 dissociation, nor the further reaction between adsorbed oxygen species and activated methane, contrary to what was proposed when using La2O3/Al2O3 [14].

Moreover, little modifications are observable on oxygenates, an increase in the reaction temperature lead to a decrease of the selectivity to methanol and formaldehyde while the selectivity to acetaldehyde is not strongly modified.

2.3 Characterization of the oxide after reaction at different temperatures

XRD analysis and surface area

The catalyst was characterized by XRD before and after experiments under plasma in an excess of methane at 100, 200 and 300°C. The results are gathered in Figure 7. Before reaction, as expected, the phase identified was CaO (ICSD 98-009-0486). After exposure to a mixture of CH4 and CO2 under plasma at 100°C, no significant differences are observed, the main phase identified being CaO. At 200 and 300°C, peaks assigned to Ca(OH)2 (ICSD 98-007-3467) and CaCO3 (ICSD 98-004-0543) are clearly visible. The results show undoubtedly that hydration and carbonation of calcium oxide proceeds under plasma discharge at 200 and 300°C while the catalyst seems not significantly modified at room temperature and 100°C after one hour under plasma. The specific surface area of the materials used was very low: 1.8 m 2 /g (Table 4). It seems that after reaction under plasma, the surface area slightly increased reaching 2.7 m 2 /g when the reaction was performed at 200°C, the calcium hydroxide particles formed during reaction occupying a larger molar volume. Note that the pore size was very low for the starting material (20 nm) and no significant differences were observed in porosity after reaction under plasma whatever the reaction temperature.

TGA-TDA analysis and carbonation

The catalyst recovered after reaction at room temperature, 100, 200 and 300°C did not exhibit significant differences in colors (Figure 8), only the catalyst after reaction at 300°C appears a little bit greyer, it attests that the carbon deposition is very limited when using CaO and plasma.

TGA-TDA analysis were performed after one hour of reaction under plasma discharge using calcium oxide calcined at 800°C, with grain size: 355 µm < d < 650 µm, P= 8W, from room to 300°C and a CH4/CO2 ratio of 2 (Figure 9). No significant differences are observed before reaction and after reaction at room temperature as at 100°C. The weight loss between 500-700°C is attributed to the presence of stable carbonate species, the carbonation of CaO observed for the solid before reaction proceeds during the transfer step from the calcined oven to the plasma reactor. When the reaction is performed at 200°C, TGA profile exhibits an important weight loss between 300-500°C (6.1%) corresponding to an endothermic peak. It is attributed to the decomposition of Ca(OH)2 [35]. The formation of calcium hydroxide can result from H2O dissociation to H + , OH -in gas phase since water is widely formed under our experimental conditions through the reverse water gas shift reaction.

After reaction at 300°C, the presence of calcium carbonate is preponderant. The absorption capacity of CaO, corresponding to the ratio of CO2 captured/CaO (weight %), under plasma, determined after one hour on stream is equal to 7.5% at 300°C.

From the data collected by TGA analysis it was possible to indicate the number of theoretical monolayers of CaO covered by Ca(OH)2 and CaCO3. The approximation made is that the coverage is homogeneous at the surface of the solid, while an irregular coverage is more likely.

However, such calculations allow to show how phase modifications occurs under plasma depending on the experimental conditions used. The calculation was performed knowing that CaO crystallizes with a face-centered cubic structure (NaCl type) with a lattice parameter a= 4.81 Å. The results reported in Table 4 show that the amount of absorbed CO2 corresponds to 59 monolayers, showing that CO2 or activated CO2 species are able to diffuse within CaO under plasma discharge at 300°C. The diffusion of reactive plasma species deep into porous catalysts, with pore size of 10 nm was proposed by Holzer et al. [36].

It is known that the carbonation reaction of CaO is exothermic (CaO(s) + CO2(g) = CaCO3 (s)

Hr,298K = -178 kJ/mol) and is possible within a large range of temperatures. Kinetically the carbonation of CaO proceeds in two successive steps, a fast one followed by a slow one. The reaction is controlled by the reaction between CaO and CO2 leading to the formation of a layer of CaCO3 covering the remaining CaO, that constitutes a diffusional resistance for CO2 reaction, switching carbonation reaction from a fast step to a slow one controlled by gas diffusion [37]. Practically, CO2 sorption is performed in the so called carbonator [38] at temperatures above 500°C in order to limit the effect of diffusion limitation. Moreover, the steam effect at low temperature was reported by Wang et al. [39], the authors showed that carbonation of CaO is possible between 250 and 400°C, while no CaO carbonation would occur without the presence of water at these temperatures. A two-step route is proposed when H2O vapor is present in the feed gas: CaO →Ca(OH)2 → CaCO3. However at low temperature (300°C), the kinetic of CaO carbonation is slow since 200 minutes are required to reach a conversion rate of 8%, in the presence of water (8vol%) [39], while under nonthermal plasma carbonation of CaO proceeds rapidly during the first minutes of experiments.

Conclusion

In this study calcium oxide was used in association with plasma discharge to perform the transformation of methane and carbon dioxide. We showed that the CaO grain size influences strongly CH4 and CO2 conversion, the lowest particle size favoring reactant transformation, while the selectivity to oxygenates remains low (< 2%) whatever the size of CaO grains for a CH4/CO2 ratio = 0.5. Nevertheless, the selectivity to ethane is maximum as soon as the space between grains is maximum (bigger particles) which is attributed to the recombination of radical species in gas phase. The use of methane in excess (CH4/CO2 = 2) favors the formation of oxygenates compounds even if the selectivity remains low: 2.4%, the main oxygenated product being acetaldehyde. When performing the reaction at 300°C, catalyst structural modification are observed and the carbonation of CaO proceeds. The amount of carbonate species was quantified and the results show that 59 monolayers of CaCO3 are obtained after one hour of plasma at P=8W using a mixture with a CH4/CO2 ratio of 2. It proves that the CO2 molecules are able to diffuse rapidly into the small CaO pores at 300°C under the plasma discharge.

List of Tables:

Table1:
Influence of the CH4/CO2 ratio and temperature on product selectivity and hydrogen yield (average value, reaction time: 30 minutes), P=8W, total flow: 40mL/min, He: 75%.

  Conversion (%) of CH4 and CO2 = 100  mole of CH4 (or CO2) converted/ mole of CH4 (or CO2) in the feed The selectivity was calculated based on carbon atoms: Selectivity to CnHy (%) = 100  n  mole of CnHy / (mole of CH4 converted + mole of CO2 converted) Selectivity to CO (%) = 100  mole of CO / (mole of CH4 + mole of CO2) converted Yield in H2 (%) = 100 mole of H2 /2*(mole of CH4) introduced Carbon Balance: CB (%) = 100 (mole of CO +∑ n  mole of CnHy)/ (mole of CH4 converted + mole of CO2 converted) 2 Results and discussion

Figure 1 Figure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 7 :Figure 8 :Figure 9 :-

 123456789 Figure 1: Reactor scheme

Table 2 :

 2 Influence of the grain size on product selectivity and hydrogen yield (average value,

	reaction time: 30 minutes), CaO calcined at 800°C before reaction, d1: 250-355µm, d2: 355-
	650µm, d3:650-800, d4: 800-1000µm, P=8W, total flow: 40mL/min, He: 75%, ratio CH4/CO2
	=0.5
	CH4/CO2	Conversion (%) CO2 CH4	H2 yield (%)	CO	Selectivity (%) C2H6 C3H8 HCOH AcOH MeOH EtOH	CB (%)
	0.5	12.0 24.4 10.6 80.9 10.8	1.2	1.5	0.3	1.0	0.3	96.0
	1	12.4 21.4	7.6	63.6 17.8	2.1	1.8	0.7	0.8	0.3	88.5
	2	10.3 17.5	6.1	45.5 27.1	3.6	1.9	1.3	0.7	0.4	83.1

Table 3 :

 3 Influence

	of the CH4/CO2 ratio on product selectivity and hydrogen yield (average
	value, reaction time: 30 minutes), CaO calcined at 800°C before reaction, grain size: 355-
	650µm, P=8W, total flow: 40mL/min, He: 75%.

Table 4 :

 4 Characterization of the CaO catalyst after plasma treatment, number of layers formed according to TGA analysis, reaction conditions: CaO calcined at 800°C, grain size: 355-650µm, P=8W, total flow: 40mL/min, He: 75%, ratio CH4/CO2 =2

Acknowledgements

The authors grateful acknowledge the ANR for the financial support of the PRC program VALCO2PLAS


Nassim Bouchoul

Elodie Fourré

Alysson Duarte

Nathalie Tanchoux

Christophe Louste

Catherine Batiot-Dupeyrat
email: catherine.batiot.dupeyrat@univ-poitiers.fr

Plasma-metal oxides coupling for CH4-CO2 transformation into syngas and/or hydrocarbons, oxygenates

Dry reforming of methane was investigated by non-thermal plasma coupled with different metal oxides: BaO, La2O3, ZnO, CaO, -Al2O3, MgO, γ-Al2O3, TiO2 and CeO2. The deposited power was fixed at 8W and the total gas flow at 40 mL.min -1 (75% helium as diluent). Electrical characterization showed that the CO2 and CH4 conversions were enhanced (from 5.6 to 30.6% for CH4 and from 1.9 to 16.1 % for CO2) when the permittivity was reduced from 2903 to 4.1, respectively. Methanol selectivities were favored for the oxides presenting low permittivities, indicating that reaction is favored under a low electric field, thus low density of reactive species.

The effect of reaction temperature was evaluated on MgO catalyst. The increase of the temperature favored CH4 conversion, while reducing methanol selectivity. The oxide characterization by TGA revealed the re-hydroxylation of MgO at low temperature, which was correlated to the improved oxygenated compounds selectivities.

Introduction

The simultaneous transformation of methane and carbon dioxide into synthesis gas is a wellknown reaction, largely studied for decades. The reaction is very endothermic and requires temperatures higher than 700°C and the use of a catalyst to favor the reactant activation. Several catalytic processes have been yet proposed composed of a metal (Pt, Pd, Ru, Ni…) deposited at the surface of a support (Al2O3, La2O3…) [1]. Despite the progress realized, the process still faced two main drawbacks: the high energy cost and the catalyst deactivation due to carbon deposition [2]. Consequently, new concepts and breakthrough processes are still needed to perform the reaction.

Non-thermal plasma is considered as an attractive alternative method for methane and carbon dioxide transformation since it allows this thermodynamically limited reaction to proceed at ambient temperature. The main advantages of non-thermal plasma are the almost absence of starting delay and the direct energy transfer to gas molecules without the need to heat the gas, lowering thus the energetic cost.

The transformation of CH4 and CO2 was studied using a variety of electrical discharges or electron beams leading to plasmas such as corona discharges, gliding arc discharges, dielectric barrier discharges (DBD), plasma needle, plasma jets and micro hollow cathode discharges [3].

Within the variety of non-thermal plasma, DBD plasma presents the advantage of avoiding the formation of an electric arc, due to the presence of a dielectric material between the two electrodes. The application of a high electrical potential difference between two electrodes (few kV) leads to the formation of an intense electric field, where electrons are accelerated and collides with molecules of the gas phase creating plasma filaments. It results in the formation of excited species, such as electrons, radicals, ions or excited molecules. The most of the primary energy is transferred to the electrons (instead of heating the entire gas stream), leading to the formation of a non-equilibrium state where electrons are highly energetic, while the ions, excited and neutral molecules remain at temperature close to ambient. This results in a highly reactive environment favorable to various chemical reactions. Starting from CH4 and CO2, valuable chemicals such as hydrocarbons or oxygenates are commonly obtained [4][5][6]. However the selectivity towards targeted products is often difficult to achieve due to the occurrence of a large number of reactions in gaseous phase. The coupling of catalyst and plasma was proposed to overcome this drawback [7][8][9][10][11]. Nevertheless, coupling plasma and catalysis in a same reactor is not trivial since the plasma discharge propagation is modified by the presence of a solid possessing dielectric properties. In a classical thermal catalytic process, reactions proceed at the surface of the material into the micro-porosity of the solid, while under plasma discharge, it has been shown that plasma can be generated only in pore sizes larger than the Debye length (~50nm) [12]. The influence of the grain size was also evidenced by authors [13], an improved interaction between the plasma and the surface of the material was shown for the smallest catalyst grains leading to higher reactants conversion.

In many studies, the selected catalysts were similar to those used in conventional thermal catalytic processes, such as Ni/Al2O3, but it was shown that the presence of conductive Ni active sites decreased the electric field strength and consequently the electron density, reducing thus reactants conversion [14]. The combination of non-thermal plasma with different metal oxides was investigated. While alumina possesses no catalytic activity in the reaction of thermal dry reforming of methane, a significant improvement in CH4 and CO2 conversion over Al2O3 was observed when coupled with plasma [15].

Combining catalysis to plasma requires the development of specific materials able to interact with the reactants active species (such as metastable species, radicals and ions). In this respect, the influence of different metal oxides, located in a packed bed DBD reactor, were investigated. These materials were selected due to their physical and chemical properties, in terms of BET surface, permittivity and basicity. Materials possessing a relatively low dielectric constant were chosen, since a too high dielectric constant of the packing material limits the discharge to the contact point of the catalyst beads [16].

Experimental part

The reaction was performed at room temperature and atmospheric pressure in a coaxial dielectric barrier discharges (DBD) reactor (Fig. 1). The non-thermal plasma reactor consisted in an alumina tube (ID: 4 mm; ED: 6 mm), with a stainless steel electrode centered inside (1.0mm diameter) and a copper electrode wrapped around the alumina tube (100 mm long).

Helium, methane and carbon dioxide were flown through the plasma reactor via fine-regulation valves, at a total flow rate of 40 mL.min -1 using a ratio CO2/CH4 = 2, with a constant concentration in He: 75 %, corresponding to a contact time of 1.6s.

A sinusoidal supply of voltage was applied across the electrodes (TG1010A Aim-TTi, Thurlby Thandar Instruments Brand). The discharge power, calculated from the Lissajous figures, was fixed at 8 watts (frequency at 800Hz and voltage at 13.5kV).

Dielectric constants of oxides were determined using an Ametek ®, Solartron Analytical apparatus: a sinusoidal voltage of 50 V was applied with a scan in frequency from 0,1 Hz to 1 MHz. Prior to measurements, the oxides were pressed at 5 tons, producing wafers of 2 mm thick, to neglect the contribution of air permittivity. Wafers were placed in an oven at 120°C before analysis to avoid water adsorption. Thermogravimetric analysis were performed with a SDTQ600TA analyzer under a 100 mL.min -1 flow of air up to 1173K. Surface areas were measured according to the BET procedure. The nitrogen adsorptiondesorption isotherms were determined with a Micromeritics Flowsorb II 2300 apparatus at -196°C.

The product gases were analyzed using two chromatographs, one equipped with a TCD (3 way µGC Varian Quad CP-4900) and the other one with a FID (Agilent 7220A). The three-way gas chromatograph is equipped with the columns: PoraPLOT Q, CP-Sil 5CB, and a-molecular sieve 5A columns. It allows to analyze on-line within 3 min the gases: H2, CO, CO2, CH4, C2H6, and C2H4. The Agilent chromatograph is equipped with U-bond Restek column for the analysis of oxygenate compounds.

The reaction was performed during one hour, methane and carbon dioxide conversion being stable during this time on stream. The experiments were performed three times by changing the catalyst (when used), a margin error of ±3% was calculated.

The conversion, selectivity, yields and energy efficiency were defined as:

Results and discussion

Influence of the oxide chemical nature

The list of oxides and their chemical and physical properties are gathered in Table 1. The oxides possess different surface areas from below 1 m 2 .g -1 (for BaO and La2O3) to 79 m 2 .g -1 for CeO2.

The dielectric constant (εr) of the oxides measured at a frequency of 800 Hz and 1.10 6 Hz are compared with values reported in literature in Table 1. Lin et al. [17] used a similar method to measure the dielectric constant of fine grain size CaCu3Ti4O12 materials. The authors showed that εr tends to increase as raising sintering temperature of the material. Moreover, the presence of dopants or impurities strongly affects the value of dielectric constant, which increases from 8300 to 12000 when CuO and Cu2O traces are detected.

Considering TiO2, different values for the dielectric constant are reported in literature, from 58 to >1000. Results show that the dielectric constant depends on the method of synthesis. By example, Rathore et al. [18] obtained different dielectric constants of TiO2 at 1 kHz: 584.3, 176.9 and 218.9 for three samples prepared by a sol-gel method and calcined at 400, 500 and 600°C respectively. Note that as expected the amount of rutile phase was higher in the sample calcined at 600°C. In another study, Wypych et al. [19] concluded that dielectric parameters strictly depended on the rate of anatase-rutile transformation, morphology of investigated powders, and relative density of measured pellets. The highest value of dielectric permittivity (>1000 at 1 kHz) were obtained for rutile phase (TiO2 calcined at 850°C). In our study a high value was obtained for the commercial rutile TiO2 used (εr = 2903).

Note that  values obtained at 800Hz differ strongly from those obtained at 1 MHz for TiO2 and -Al2O3. This discrepancy is generally associated with strong absorption at low frequencies due to dipole relaxation [20]. Moreover, -Al2O3 possesses a higher dielectric constant than γ-Al2O3 at 800Hz, while similar values are generally given for  and  alumina: 9-10 [16,21]. Literature data gives εr of about 5 for ZnO [22] and 52 for CeO2 [23], which differ significantly from our measurements.

Bhargava et al. [24] obtained a value of approximately 30 for MgO using a method similar to ours, while a value of 9 was proposed by Robertson [25], who calculated the permittivity of a series of metal oxides using an equation as a sum of electronic and lattice contributions. The differences observed between our values and those of Robertson could be due to the interfacial polarizations originating from sub-grain boundary and grain boundary barriers [17], which is not taken into account in the calculation of Robertson. However, a common result is that TiO2 exhibits the highest dielectric constant among the metal oxides studied (εr = 58 for Robertson).

Depending of the published work, significant differences are observed for dielectric constants of various metal oxides. It was thus important to perform our own measurements, in similar experimental conditions (same grain size, room temperature) in order to obtain reliable data and classify materials according to their εr values.

The electrical aspect of the DBD reactor filled with -Al2O3 and γ-Al2O3 was considered. As shown in Fig. 2, low current peaks are observed in the presence of -Al2O3 when compared to γ-Al2O3. Similar results were obtained by Jo et al. [26] using spheres of alumina (16-20 mesh).

The Lissajous Q-V figures exhibited significant differences for the two alumina oxides, an indication of different effective capacitances. These values were calculated from the slope of the lines AB and CD (as shown in Fig. 3 for -Al2O3) and reported in Table 2. The total capacitance of the reactor without plasma (Ccell) was also calculated from the slope of lines DA and CB. The higher effective capacitance for -Al2O3 (than for γ-Al2O3) proves that the electrical properties of the two oxides are different, while similar grain size are used (355-650µm). Thus, the effective capacitance can be related to the dielectric constant obtained at 800Hz.

The selected oxides were introduced in the plasma reactor after sieving all the materials in the range of 355-650 µm. Previous studies showed that the grain size affected significantly the reactants conversion due to their impact on the accessible surface, average size of space and number of contact points between the particles. An improved interaction between the surface of the oxide and the plasma was proposed for the smallest catalyst grains [27,28]. Methane and CO2 conversions for the different oxides are reported in Figure 4. The values when the plasma zone is filled with quartz wool are also indicated, as a reference value. According to the volume occupied by the quartz wool or by the catalyst grains, the residence times are considered similar, at approximately 1.2 s. The results show that ZnO and TiO2 have a negative effect on the conversion of both reactants. CO2 conversion is slightly improved in presence of CeO2 and BaO when compared to quartz wool, while no increase in CH4 conversion is observed. The presence of the metallic oxides La2O3, -Al2O3, CaO, MgO and γ-Al2O3 favors reactant transformation, the best results being obtained with γ-Al2O3 with CH4 and CO2 conversions of 30.6 % and 16.1 %, respectively. As expected, CO2 conversion is always lower than methane conversion [29][30][31].

Comparing the reactivity of -Al2O3 and γ-Al2O3, it is important to note that the surface of the two oxides differ strongly as -Al2O3 possesses less OH surface groups than γ-Al2O3. The role of OH sites in the presence of a discharge was discussed by Liu et al. [32]. The authors suggested that materials containing numerous OH groups become the most easily charged; dehydration occurs, leaving oxygen vacancies, which may react to form an active site for methyl radical production.

The results gathered in Fig. 6 show that the best performances in terms of CH4 and CO2 conversion are obtained for oxides with low dielectric constant values. The best results are reached with -Al2O3, MgO and CaO, followed by La2O3, BaO, -Al2O3 and CeO2, while a very low CH4 transformation is observed over TiO2. A negative linear correlation for CO2 conversion with the increasing dielectric constant was shown by Debek et al. [33] using Ni supported catalyst in the glow discharge plasma-catalyst assisted CO2 hydrogenation. The authors attributed this trend to the different packed bed void volumes obtained in presence of catalysts.

In our case, the grain size was calibrated so the results cannot be explained according to this parameter. It is accepted that the main plasma parameter affected by the presence of a solid is the electric field, which is enhanced [5,34]. The field enhancement results from the high local curvature of the grain surface. A clear increase in the high-energy tail of the electron energy distribution was demonstrated in the presence of TiO2 pellets into the plasma reactor by Tu et al. [14]. As a consequence, we believe that, even if the reactive species density is increased with TiO2, the volume of efficient discharge is reduced, and the global transformation of reactants is limited [4,35].

Among the products formed under plasma discharge, CO and C2H6 are the major ones (Table 3), a maximum CO selectivity of 81% being obtained with CaO, which is associated with a high carbon balance: 97%. The lowest selectivity to CO is of 62% with ZnO and the carbon balance only reaches 75%, suggesting carbon deposition. It seems that the selectivity to CO depends slightly on the material permittivity (Fig. 7), CO being favored over materials possessing the lowest ε value: CaO, BaO. Note that it is also correlated with carbon balance. The results support the hypothesis that, in the localized strong electric field, methane conversion is limited and is transformed into heavy products and/or coke.

In addition to hydrocarbons, a series of oxygenates was detected and quantified: methanol, formaldehyde, acetaldehyde and acetone. Other products (than those listed in table 3) were detected using a mass spectrometer connected at the reactor exit. They were mainly heavier hydrocarbons (C3, C4) and oxygenates, such as acetic acid, but no quantification was possible due to their low concentrations. The ratio H2/CO is far from unity, limited between 0.35 and 0.50, due to the excess of CO2 used in this study (CO2/CH4= 2). Under non-thermal plasma electrons generated possess an energy in the range 1-10 eV, high enough to perform the direct dissociation of CH4 and CO2 according to [36]:

CO2 + e  CO+ O + e CH4 + e  CH3 + H + e 2 CH3  C2H6 …
The dehydrogenation of methyl radicals can continue leading to the formation of coke, from which CO is produced:

C + O  CO
The active oxygen species generated by CO2 dissociation can recombinate to O2 or react with methane:

CH4 + O  CH3 + OH
In order to estimate the contribution of methane to CO formation, the ratio (R) between the amount of CO produced and CO2 transformed was calculated (a value of unity would indicate that CO is produced by CO2 dissociation).

Under the experimental conditions used in this study, R is always higher than unity confirming that CO is not only obtained through direct CO2 decomposition but also from methane (Fig. 5). R value reaches a maximum with quartz wool suggesting that the presence of metal oxides favors the recombination of oxygen species and consequently inhibits, to a small extend, methane activation through oxygen species.

The selectivity to CO depends strongly on the nature of catalyst, the highest value being obtained with CaO, La2O3 and MgO. Note that the lowest selectivity to CO is accompanied by a low carbon balance for TiO2, CeO2 and ZnO suggesting carbon deposition through methane cracking (CH4 CH3  CH2  CH C) and/or CO and CO2 dissociation (CO → C + O, CO2  C + O2) [37].

Consequently, it confirms that packing materials with high permittivity (TiO2, CeO2) not only is unfavorable for methane conversion but decreases the selectivity to products. The results support the hypothesis that, in the localized strong electric field, methane conversion is limited and is transformed into heavy products and/or coke.

Influence of temperature

The influence of the reaction temperature was investigated using MgO with a grain size: 355 µm < d < 650 µm, a power of 8W, a temperature range from 100 to 300°C and a CO2/CH4 ratio of 2. This oxide was chosen based on its CO2 and CH4 conversion, relatively low permittivity (18.2) and selectivities to oxygenated compounds. A small increase of CO2 conversion is observed while CH4 conversion is significantly enhanced as a function of the reactor temperature (Table 4). In addition to CO and C2H6, oxygenated products quantification showed that their concentrations depend on the reaction temperature. At low temperature, methanol and ethanol formation is favored while at 300 °C, low selectivities are obtained. Methanol synthesis is favored at lower temperature under plasma discharge as shown by Eliasson et al. [38]. It is believed that the production of methanol mainly depends on the formation of CH3 and OH radicals [39]. At higher temperature, the formation of -OH groups is limited at the surface of the catalyst (see TGA analysis) and the coupling of CH3 is favored, decreasing the selectivity into methanol.

MgO oxide characterization after reaction at different temperatures

TGA-TDA analysis of MgO before and after reaction under plasma are displayed in Fig. 8. The weight loss between 250-350 °C is associated with an endothermal effect, suggesting the elimination of hydroxyl groups from the surface according to [40]:

Mg(OH)2 → MgO + H2O
Note that a small weight loss is also present in the temperature range: 500-600 °C, which can be attributed to decarbonation following [41]:

MgCO3 → MgO + CO2
The coupling of TGA with mass analysis confirmed that the first weight loss corresponds to water elimination (m/z=18) and the second one to CO2 elimination (peak corresponding to m/z = 44). Before reaction, TGA analysis of MgO exhibited a weight loss between 500-600 °C indicating that the carbonation of MgO proceeds via atmospheric CO2 adsorption after calcination under air at 800 °C (Fig. 8).

After one hour of reaction at room temperature under plasma, significant differences are observed in the TGA profiles indicating that more hydroxyl groups are present than before reaction. This increase at low temperature could be explained by the involvement of the reverse water gas shift reaction (CO2+ H2→ CO+ H2O) [42] and the subsequent dissociation of water at the surface of MgO.

When the reaction is performed at higher temperature, water desorption from the surface of the oxide is favored and the number of -OH groups is significantly reduced (Table 5). It appears that re-hydroxylation of MgO is favored at room temperature and to a lesser extend at 100°C, which corresponds to the highest oxygenates selectivities.

Reaction mechanism under plasma in the presence of MgO

Under the experimental conditions used in this study, the transformation of CH4 and CO2 can proceed by electron collision in the gas phase and also at the surface of the material introduced in the plasma zone. It is not possible to indicate precisely the involvement of homogeneous reactions and heterogeneous ones. But, taking into consideration the change in products selectivity and characterization of the material after experiments, surface reactions can be proposed.

The formation of the main product, CO, would occur through direct CO2 dissociation as the most important channel due to the excess of CO2 used in this study [43].

Ethane is obtained by methyl coupling, while methanol can be formed by reaction between radical species CH3 and OH [44].

By comparing the results on MgO oxide with those obtained on La2O3/Al2O3 catalyst [8], it is proposed that the dissociation of CH4 at the surface of MgO oxide is favored through molecular and dissociative mechanisms under electric discharge plasma.

-At high temperature, the plasma assisted catalytic dissociation of CH4 would be favored [45,46]. The selectivity to ethane is also maximum at high temperature, showing that the coupling of methyl radicals at the surface MgO is enhanced according to:

MgO-CH4 ads →MgO-CH3 and 2CH3 → C2H6 -At low temperature, we suggest that surface -OH groups are involved in the formation of oxygenates during plasma treatment, with the best selectivities at low or room temperature, corresponding to the highest concentration of surface hydroxyl groups (see TGA analysis). The dissociation of water (produced through the reverse water gas shift reaction) proceeds according to [47,48]:

H2O + e  H + OH + e H2O + e  OH + H - It is believed that the dissociation of water is favoured at the surface of MgO, as proposed by Nozaki et al. [49] leading to the formation of chemisorbed oxygen-related intermediates such as O and OH. It is also in accordance with the results we obtained since hydroxylation of MgO under plasma is supported by TGA analysis. The presence of such -OH groups would favour the formation of methanol, but to a low concentration since methanol molecule is not stable under plasma discharge, and is easily oxidized into CO and/or CO2 [50].

It is proposed that methanol can be formed through water activation in 2 ways [24]:

In the gas phase:

CH4 + He* → CH3  + H + He H2O + e -→ OH  + H  CH3  + OH  →CH3OH
At the surface of hydroxylated MgO:

CH3  + Mg-(OH)ads → CH3OH

Conclusion

A selection of different oxides: BaO, La2O3, ZnO, CaO, -Al2O3, MgO, γ-Al2O3, TiO2 and CeO2 were characterized and tested in the CO2 and CH4 reaction for the production of syngas, hydrocarbons and oxygenated compounds. The best results were obtained in the presence of γ-Al2O3 with methane and carbon dioxide conversions of 30.6% and 16.1% respectively, while using TiO2, CH4 and CO2 conversions reach only 5.6 and 1.9% respectively. The selectivity to CO and carbon balance were the highest with CaO, La2O3 and MgO. Moreover, the presence in the plasma zone of each metallic oxides, tested as powder of calibrated size, limits, to a small extent, the formation of CO through methane partial oxidation. The reactivity was linked to the permittivity of the oxides: the lower the permittivities, the higher the conversions for both CO2 and CH4. A more detailed study on MgO catalyst revealed that CH4 conversion increased with the temperature, whereas selectivities to oxygenated compounds decreased. This second effect was correlated, via sample characterization, to the presence of surface hydroxyl groups. The higher the surface -OH groups, the better the methanol selectivity.

  Conversion (%) of CH4 and CO2 = 100  mole of CH4 (or CO2) converted/ mole of CH4 (or CO2) in the feed The selectivity was calculated based on carbon atoms: Selectivity to CnHy (%) = 100  n  mole of CnHy / (mole of CH4 converted + mole of CO2 converted) Selectivity to CO (%) = 100  mole of CO / (mole of CH4 + mole of CO2) converted Yield in H2 (%) = 100 mole of H2 /2*(mole of CH4) introduced Carbon Balance: CB (%) = 100 (mole of CO +∑ n  mole of CnHy)/ (mole of CH4 converted + mole of CO2 converted)

Figure 1

 1 Figure 1: Reactor scheme

Figure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 7 :Figure 8 :

 2345678 Figure 2: Voltage and current waveforms for a) -Al2O3 and b) γ-Al2O3, grain size: 355-650µm, P=8W, total flow: 40mL.min -1 , CO2/CH4=2, He: 75%

Table 1 :

 1 Properties of the selected oxides: surface area, pore volume before and after reaction under plasma (1 hour, grain size: 355-650µm, P=8W, total flow: 40mL/min, CO2/CH4=2, He: 75%) number of surface acid and basic sites.

	Oxide	S BET (m 2 /g) Before plasma After plasma	Pore volume (cm 3 /g) Before plasma After plasma	Dielectric constant εr* (800 Hz) εr *(1 MHz)	εr**[ref.]
	BaO	<1	<1	nd	nd	4.2	4.0		33 [25]
	La2O3	<1	<1	nd	nd	6.6	5.1		25 [25]
	ZnO	1.4	1.1	0.004 0.003	10.7	4.3		~5 [22]
	CaO	4.5	2.0	0.01	0.01	2.8	2.7		11 [25]
	Al2O3	6.1	5.5	0.01	0.01	96	3.8	9-10 [16,21, 25]
	MgO	9.5	9.0	0.01	0.01	18.2	4.3	9-30 [22,25]
	γAl2O3	65	61	0.2	0.2	3.1	2.9	9-10 [16,21, 25]
									58-100 to
	TiO2	77	69	0.13	0.12	2903	3.0		>1000
								[16,18,19, 25]
	CeO2	79	73	0.2	0.2	21.1	6.4		52 [23]
	nd: not determined
	* this study
	** from literature

Table 2 :

 2 Capacitance of the dielectric and effective capacitance at 13.5kV

Table 3 :

 3 Selectivity to products for a series of oxide, 1 hour, grain size: 355-650µm, P=8W,

		Alumina			C dielectric (pF)		C effective (pF)
		γ-Al2O3			9			22
		α -Al2O3			24			61
	total flow: 40mL/min, CO2/CH4=2, He: 75%
	Oxide	H2 yield (%)	CO	C2H4 C2H6 C3H8 MeOH C3H6O CH2O C2H4O	H2/CO	CB (%)
	Quartz wool	9.4	80	0.4	14	0.9	0.6	0.4	1.7	0.4	0.36	98
	CaO	11	81	0.2	11	1.2	1.0	0.5	1.5	0.2	0.35	97
	La2O3 9.4	76	0.4	12	1.4	0.7	0.7	1.6	0.3	0.38	93
	MgO	11	74	0.3	11	1.2	1.0	0.6	1.3	0.2	0.38	89
	γ-Al2O3 12	70	0.3	8.8	1.2	0.4	0.6	0.9	0.2	0.37	83
	α-Al2O3 9.4	69	1.0	11	0.7	0.5	0.6	1.4	0.3	0.43	85
	TiO2	1.8	66	0	11	0	0	0	0	0	0.38	77
	CeO2	8.0	64	0	8.8	0	0	0	0	0	0.40	73
	BaO	8.1	63	0.5	10	0.7	0.6	0.4	1.7	0.3	0.38	78
	ZnO	6.7	62	0	13	0	0	0	0	0	0.50	75

Table 4 :

 4 Influence of temperature on product yield (average value, reaction time: 30 minutes), MgO grain size: 355-650µm, P=8W, total flow: 40mL/min, He: 75%, ratio CO2/CH4 =2.

	MgO	Conversion (%)		Selectivity (%)		CB
	T (°C)	CO2	CH4	CO	C2H6	CH3OH	C2H5OH	(%)
	25	11	20	66	10.0	1.5	0.8	74
	100	11	22	68	12.0	1.7	1.2	83
	200	12	24	61	13.0	1.6	1.3	76
	300	13	28	60	15.0	0.2	0.2	72

Table 5 :

 5 TGA analysis after reaction under plasma, grain size: 355-650µm, P=8W, total flow: 40mL/min, He: 75%, ratio CO2/CH4 =2.

			Weight loss (%)
	Oxide	Temperature	(TGA analysis)
			-OH	-CO3
	MgO	RT	3.6	0.3
		100°C	2.0	0.1
		200°C	1.1	0.2
		300°C	0.8	0.2

Acknowledgements

The authors grateful acknowledge the ANR for the financial support of the PRC program VALCO2PLAS and the financial support from the European Union (ERDF) and "Région Nouvelle Aquitaine"


Thomas Migkos

Jana Pourová

Marie Vopršalová

Cyril Auger

Valérie Schini-Kerth

Přemysl Mladěnka
email: mladenkap@faf.cuni.cz

Prof Assoc Přemysl Mladěnka

Relaxation of the Coronary Artery Through the Calcium Channel and cGMP-dependent Pathway

Keywords: blood vessels, cardiovascular, ex vivo, glycitein, isoflavonoid

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

Introduction

Cardiovascular diseases remain the leading cause of mortality and morbidity worldwide. Among them, ischemic coronary disease and stroke are major culprits of mortality. Hence, vascular function can be considered one of the main targets when developing innovative drugs focused on both the treatment and prevention of cardiovascular disorders.

Indeed, isoflavonoids appear to be related to a lower incidence of myocardial and cerebral infarctions [START_REF] Kokubo | Association of dietary intake of soy, beans, and isoflavones with risk of cerebral and myocardial infarctions in Japanese populations: the Japan Public Health Center-based (JPHC) study cohort I[END_REF], reduced arterial stiffness [START_REF] Pase | The effects of dietary and nutrient interventions on arterial stiffness: a systematic review[END_REF], and blood pressure normalization [START_REF] Sagara | Effects of dietary intake of soy protein and isoflavones on cardiovascular disease risk factors in high risk, middle-aged men in Scotland[END_REF]. Due to their structural similarity to endogenous estrogens, they have affinity to estrogen receptors [START_REF] Setchell | Soy isoflavonesbenefits and risks from nature's selective estrogen receptor modulators (SERMs)[END_REF] and are known as phytoestrogens. Their main natural sources are legumes and herbs, such as soybean (Glycine max), red clover (Trifolium pratense), milkvetch (Astragalus sp.), Prunus, Pueraria, and Ononis sp. [START_REF] Li | Formononetin induces vasorelaxation in rat thoracic aorta via regulation of the PI3K/ PTEN/Akt signaling pathway[END_REF][START_REF] Tseng | Calycosin and formononetin induce endothelium-dependent vasodilation by the activation of large-conductance Ca(2+)-activated K(+) channels (BKCa)[END_REF][START_REF] Yeung | Puerarin, an isoflavonoid derived from Radix puerariae, potentiates endothelium-independent relaxation via the cyclic AMP pathway in porcine coronary artery[END_REF][START_REF] Choi | Mechanisms of phytoestrogen biochanin A-induced vasorelaxation in renovascular hypertensive rats[END_REF]. Isoflavonoid intake in humans is also increasing due to the use of a variety of dairy products, processed food, and food supplements derived from the aforementioned plants [START_REF] Socas-Rodriguez | Multiresidue determination of estrogens in different dairy products by ultra-high-performance liquid chromatography triple quadrupole mass spectrometry[END_REF][START_REF] Krizova | Changes in isoflavones concentrations in cheese during processing and ripening[END_REF]. Most of these species are traditionally used in Chinese medicine, among other things, for the treatment of cardiovascular diseases [START_REF] Yeung | Puerarin, an isoflavonoid derived from Radix puerariae, potentiates endothelium-independent relaxation via the cyclic AMP pathway in porcine coronary artery[END_REF]. The lower incidence of cardiovascular disorders in East Asian populations, compared to those from Western countries, has been also attributed to the higher consumption of isoflavonoids, particularly soy isoflavones [START_REF] Torregrosa | Pharmacological profile of phytoestrogens in cerebral vessels: in vitro study with rabbit basilar artery[END_REF]. Despite these encouraging epidemiological observations, most of the conducted human studies have investigated the effect of isoflavone-rich diets,

ABSTR AC T

The dietary intake of flavonoids seems to be inversely related to cardiovascular mortality. The consumption of isoflavonoids is increasing in the general population, especially due to the use of food supplements and a variety of isoflavonoid-rich foods. However, detailed studies on the vascular influence of individual pure isoflavonoids are mostly missing. For this study, 16 isoflavonoids were initially screened for their vasorelaxant properties on rat aortas. The 2 most potent of them, biochanin A and glycitein, were further tested for the mechanism of action on porcine coronary arteries. They both induced an endothelium independent vascular relaxation, with EC 50 below 6 and 17 µM, respectively. Biochanin A, but not glycitein, was able to block the vasoconstriction caused by KCl, CaCl 2 , serotonin, and U46619 in a dose-dependent manner. Another series of experiments suggested that the major mechanism of action of biochanin A was the inhibition of L-type calcium channels. Moreover, biochanin A in relatively small concentrations (2-4 µM) interfered with the cGMP, but not cAMP, pathway in isolated coronary arteries. These results indicate that some isoflavonoids, in particular biochanin A, are able to have vasodilatory effects in micromolar concentrations, which is of potential clinical interest for the management of cardiovascular pathologies.

Biochanin A, the Most Potent of 16 Isoflavones, Induces Relaxation of the Coronary Artery Through the Calcium Channel and cGMP-dependent Pathway plant extracts, and mixtures of isoflavonoids [START_REF] Kokubo | Association of dietary intake of soy, beans, and isoflavones with risk of cerebral and myocardial infarctions in Japanese populations: the Japan Public Health Center-based (JPHC) study cohort I[END_REF][START_REF] Pase | The effects of dietary and nutrient interventions on arterial stiffness: a systematic review[END_REF][START_REF] Sagara | Effects of dietary intake of soy protein and isoflavones on cardiovascular disease risk factors in high risk, middle-aged men in Scotland[END_REF]. The data on the effects of individual purified isoflavonoids themselves are hence largely missing, with some exceptions [START_REF] Teede | Isoflavones reduce arterial stiffness: a placebo-controlled study in men and postmenopausal women[END_REF]. Additionally, ex vivo and animal vascular studies have been performed for a limited number of compounds [START_REF] Tseng | Calycosin and formononetin induce endothelium-dependent vasodilation by the activation of large-conductance Ca(2+)-activated K(+) channels (BKCa)[END_REF][START_REF] Torregrosa | Pharmacological profile of phytoestrogens in cerebral vessels: in vitro study with rabbit basilar artery[END_REF][START_REF] Wu | Formononetin, an isoflavone, relaxes rat isolated aorta through endothelium-dependent and endothelium-independent pathways[END_REF][START_REF] Sun | Activation of largeconductance calcium-activated potassium channels by puerarin: the underlying mechanism of puerarin-mediated vasodilation[END_REF], and even less information is available in respect to coronary vessels [START_REF] Figtree | Plant-derived estrogens relax coronary arteries in vitro by a calcium antagonistic mechanism[END_REF][START_REF] Lee | Genistein reduces agonistinduced contractions of porcine coronary arterial smooth muscle in a cyclic AMP-dependent manner[END_REF][START_REF] Xu | Structure-activity relationships of flavonoids for vascular relaxation in porcine coronary artery[END_REF].

With the present study, we strove to directly compare the vascular effects of 16 pure isoflavones, several of which were tested for the first time, and to further elucidate the mechanism of action of the most active ones, biochanin A and glycitein. Within this framework, we also challenged the hypothesis that biochanin A functions as a calcium channel blocker.

Results

In the first series of experiments, a screening of the vasodilatory effects of 16 isoflavonoids (▶ Table 1) on norepinephrine precontracted rat aortic rings was performed. All glycosides, namely genistin, daidzin, glycitin, ononin, tectoridin, and puerarin, had clearly very low vasorelaxant effects, which were comparable to that of the control containing only the solvent (Fig. 1S, Supporting Information) and hence much lower than that of their corresponding aglycones (▶ Fig. 1, Fig. 2S, Supporting Information). In particular, biochanin A and glycitein appeared the most potent vasodilators, having EC 50 below 30 µM, followed in ascending order by genistein, isoformononetin, formononetin, tectorigenin, calycosin, cladrin, prunetin, and daidzein, with EC 50 ranging from 30 to 110 µM (▶ Table 2).

Thereafter, the 2 most potent isoflavonoids, biochanin A and glycitein, were selected for further mechanistic experiments. Similarly to rat aorta, as is also the case with coronary arteries, both compounds induced dose-dependent vasorelaxation. The effect was not significantly modified by the absence of the vascular endothelium (▶ Fig. 2, Fig. 3S, Supporting Information) in any of the artery models with a small exception of higher concentration of glycitein in coronary rings. Interestingly, the EC 50 of biochanin A on coronary arteries (5.5 ± 0.6 µM) was lower than that on rat aortas (25.4 ± 2.2 µM) and that of glycitein (16.2 ± 1.0 µM). For glycitein, the difference between EC 50 s for the 2 types of vessels was smaller (EC 50 s = 27.5 ± 2.8 µM and 16.2 ± 1.0 µM on aortas and coronary arteries, respectively). Since the effect on coronary arteries was achieved in lower concentrations, and given the similarities between porcine and human hearts [START_REF] Lelovas | A comparative anatomic and physiologic overview of the porcine heart[END_REF], this model was used for further testing.

To further elucidate the mechanism(s) of the selected isoflavonoids, endothelium-denuded coronary arteries pretreated with biochanin A (3, 10, and 30 µM) or glycitein (10, 15, and 30 µM) were exposed to increasing concentrations of 4 different vasocontricting stimuli. Contractions induced by both KCl and CaCl 2 were dose-dependently inhibited by biochanin A. In the highest dose, biochanin A equally blocked 73 % of the maximal vasoconstriction caused by both inducers (▶ Fig. 3 a). On the other hand, glycitein only caused an approximate 25 % reduction of the maximal effect of KCl and did not significantly alter the contraction induced by the maximal concentration of CaCl 2 . However, 30 µM of glycitein partly blocked the effect of lower concentrations of CaCl 2 (▶ Fig. 3 b). Similarly, biochanin A demonstrated higher potency against receptor-mediated contractions induced by serotonin or thromboxane-analogue U46619. In the highest concentration, it blocked 88 % and 56 % of their maximal effects, respectively (▶ Fig. 3 a). On the contrary, only the highest used concentration of glycitein (30 µM) managed to block, at least partially (38 %), the maximal effect of serotonin, as well as decreased the contractions induced by low concentrations of U46619 (▶ Fig. 3 b).

▶ Table 1 The chemical structures of the 16 tested isoflavonoids.

Isoflavonoid

H O C H 3 O-G l c H H O H ononin H H O -G l c H H O C H 3 tectoridin OH OCH 3 O-G l c H H O H puerarin H H O H G l c H O H Glc: glucose.
Due to the higher potency of biochanin A, subsequent series of mechanistic experiments were performed with this isoflavonoid. Since biochanin A blocked constrictions induced by different inducers, intracellular calcium signalling could be the target of this compound. Hence, porcine coronary arteries were pre-treated with different concentrations of biochanin A and then exposed to increasing concentrations of Bay K8644, an activator of L-type calcium channels. Under these conditions, biochanin A dose dependently blocked the contractions (▶ Fig. 4). In preliminary experiments in rat aorta, biochanin A in a concentration of 30 μΜ also inhibited the contractions induced by phenylephrine in the absence of extracellular calcium (Fig. 4S, Supporting Information).

In a separate series of experiments, sodium nitroprusside and forskolin were used to relax precontracted porcine coronary artery rings without endothelium, by raising the intracellular levels of cyclic GMP and cyclic AMP, respectively. Co-treatment of the vessels with low concentrations of biochanin A (2-4 μΜ) led to a significant potentiation of the sodium nitroprusside-induced relaxation (▶ Fig. 5 a), decreasing its EC 50 from 24.2 ± 4.5 nM to 9.4 ± 6.3 nM. However, no significant influence on the effect of forskolin was observed (▶ Fig. 5 b), indicating that biochanin A might stimulate the cGMP, but not the cAMP, related vasodilatory pathway.

A notable step within the cGMP pathway, activated by nitric oxide, is the conversion of GTP to cGMP, by soluble guanylyl cyclase. The presence of ODQ (10 -6 Μ), a selective inhibitor of this enzyme, delayed the onset of the vasorelaxant response to biochanin A in endothelium-denuded coronary rings (▶ Fig. 6 a). The EC 50 shifted from 5.1 ± 0.8 to 6.8 ± 0.5 µM, while the achieved maximal relaxation was not affected. Additionally, in order to determine whether the production of prostanoids is involved in the mechanism of action of biochanin A, coronary artery rings without endothelium were incubated with indomethacin (10 -5 Μ) prior to the administration of the isoflavonoid. However, the inhibition of cyclooxygenase did not cause any alteration in the vasorelaxant effect (▶ Fig. 6 b).

Discussion

Isoflavonoids have always attracted scientific interest because of their potentially positive effects on human beings. The studies testing their vasorelaxant activity have typically involved 1, 2, or several isoflavonoids. Due to various experimental approaches, the comparison is difficult, if even possible at all. To the best of our knowledge, this is the first comparative investigation of the vascular effects of 16 isoflavonoids. Six of them, namely isoformononetin, cladrin, tectorigenin, tectoridin, ononin, and glycitin, had never been tested as vasodilators before. Interestingly, isoformononetin, tectorigenin, and cladrin appeared more potent than daidzein in this study. Previous articles showed different potencies of several isoflavones on various arteries [START_REF] Li | Formononetin induces vasorelaxation in rat thoracic aorta via regulation of the PI3K/ PTEN/Akt signaling pathway[END_REF][START_REF] Yeung | Puerarin, an isoflavonoid derived from Radix puerariae, potentiates endothelium-independent relaxation via the cyclic AMP pathway in porcine coronary artery[END_REF][START_REF] Torregrosa | Pharmacological profile of phytoestrogens in cerebral vessels: in vitro study with rabbit basilar artery[END_REF][START_REF] Figtree | Plant-derived estrogens relax coronary arteries in vitro by a calcium antagonistic mechanism[END_REF][START_REF] Lee | Genistein reduces agonistinduced contractions of porcine coronary arterial smooth muscle in a cyclic AMP-dependent manner[END_REF][START_REF] Kim | Prunetin relaxed isolated rat aortic rings by blocking calcium channels[END_REF][START_REF] Mishra | Endothelium-dependent relaxation of rat aorta and main pulmonary artery by the phytoestrogens genistein and daidzein[END_REF][START_REF] Sun | Vasorelaxant and antihypertensive effects of formononetin through endothelium-dependent and -independent mechanisms[END_REF][START_REF] Wu | Calcium channel blocking activity of calycosin, a major active component of Astragali radix, on rat aorta[END_REF]. Small discrepancies between the aforementioned studies might derive from the use of different animal, breed, or tissue models or may be due to different vasoconstrictors used to precontract the tested arteries. In our experiments, porcine coronary arteries appeared to be more sensitive than rat aortas to both biochanin A and glycitein, but this difference was more pronounced in the case of biochanin A. Summing up the available studies, biochanin A was always a more potent vasodilator than other isoflavonoids when tested on KCl or UTP precontracted rabbit coronary and basilar arteries [START_REF] Torregrosa | Pharmacological profile of phytoestrogens in cerebral vessels: in vitro study with rabbit basilar artery[END_REF][START_REF] Figtree | Plant-derived estrogens relax coronary arteries in vitro by a calcium antagonistic mechanism[END_REF]. There is only 1 partial exception: formononetin reached the level of the activity of biochanin A in phenylephrine precontracted rat aorta [START_REF] Wu | Formononetin, an isoflavone, relaxes rat isolated aorta through endothelium-dependent and endothelium-independent pathways[END_REF].

Concerning the structure-activity relationship, our results suggest that the methoxylation of 4′ carbon of the isoflavonoid structure increases vasorelaxant potency, since biochanin A and formononetin demonstrate higher potency than the corresponding structures of genistein and daidzein respectively, which lack the specific methoxylation. Moreover, genistein appears more active than daidzein and glycitein more active than tectorigenin. The only structural difference between these 2 pairs of compounds is the presence of 5-hydroxyl group in the cases of genistein and tectorigenin. Hence this substituent has different impact on the vasodilatory effect. The presence of a 3′-methoxy group does not improve the effect, as can be deduced by making a comparison of calycosin with cladrin. The presence of a 6-or 7-methoxy group can increase or decrease the potency, apparently depend-ing on other substitutions. For example, in the case of tectorigenin, the methoxy group at C6 decreased the potency vs. genistein, while in the case of glycitein, the additional methoxy group at C6 increased the potency vs. daidzein. The probable reason for this paradoxical effect may lie in additional mechanisms of action, in particular genistein is known to be a potent inhibitor of tyrosine kinases [START_REF] Wijetunge | Tyrosine kinase inhibitors block calcium channel currents in vascular smooth muscle cells[END_REF]. Additionally, in all cases, the presence of sugar moiety at any position of the isoflavonoid core abolished or significantly decreased the efficacy.

Based on this initial screening, we selected the 2 most active compounds, glycitein and biochanin A. Quite surprisingly, although glycitein is present in relatively high amounts in soy foods and some dairy products [START_REF] Socas-Rodriguez | Multiresidue determination of estrogens in different dairy products by ultra-high-performance liquid chromatography triple quadrupole mass spectrometry[END_REF], little is known about its vasoactive properties. In fact, it has been mainly tested as a part of mixed isoflavone preparations in the past [START_REF] Yamaguchi | Effects of phytoestrogens on acetylcholine-and isoprenaline-induced vasodilation in rat aorta[END_REF]. Therefore, it was unexpected that glycitein is a potent vasodilator, with an EC 50 comparable to that of biochanin A. Its effect was endothelium-independent, but the inhibition of contraction by KCl, CaCl 2 , serotonin, and U46619 was achieved in high concentrations and was only partial. Due to this, we concentrated only on the more active biochanin Α in next experiments.

In the case of biochanin A, more data showing its vasorelaxing properties are available. These include rabbit coronary and basilar arteries [START_REF] Torregrosa | Pharmacological profile of phytoestrogens in cerebral vessels: in vitro study with rabbit basilar artery[END_REF][START_REF] Figtree | Plant-derived estrogens relax coronary arteries in vitro by a calcium antagonistic mechanism[END_REF], as well as rat aortas [START_REF] Choi | Mechanisms of phytoestrogen biochanin A-induced vasorelaxation in renovascular hypertensive rats[END_REF][START_REF] Wang | Mechanisms underlying biochanin A-induced relaxation of the aorta differ between normotensive and hypertensive rats[END_REF]. Biochanin A induces dose-dependent and endothelium-independent relaxation. This study confirmed these outcomes in rat aortas and newly documented it in porcine coronary arteries as well.

Although these aforementioned studies confirmed the effects of biochanin, the precise mechanism of action was unknown. Before this study was initiated, there was rather only negative evidence reporting that biochanin A did not affect the production of nitric oxide or prostaglandins [START_REF] Choi | Mechanisms of phytoestrogen biochanin A-induced vasorelaxation in renovascular hypertensive rats[END_REF][START_REF] Wang | Mechanisms underlying biochanin A-induced relaxation of the aorta differ between normotensive and hypertensive rats[END_REF]. The only known mechanism of action was a partial effect of biochanin A on the activation of ATP-sensitive and large-conductance Ca 2+ -activated K + channels [START_REF] Choi | Mechanisms of phytoestrogen biochanin A-induced vasorelaxation in renovascular hypertensive rats[END_REF][START_REF] Wang | Mechanisms underlying biochanin A-induced relaxation of the aorta differ between normotensive and hypertensive rats[END_REF]. Since these effects cannot fully explain the ability of this compound to evoke complete or almost complete relaxation of the tested vessels, the mechanism of action was further sought. To investigate it, 4 different vasoconstrictive impulses were tested initially. In all these cases, biochanin A dose dependently blocked or markedly diminished their effects in endothelium-denuded coronary arteries.

Both CaCl 2 and potassium-induced depolarization lead to extracellular Ca 2+ influx through the L-type voltage gated Ca 2+ channels [START_REF] Nobe | Distinct pathways of Ca(2+) sensitization in porcine coronary artery: effects of Rho-related kinase and protein kinase C inhibition on force and intracellular Ca(2+)[END_REF]. Serotonin and U46619 cause vasoconstriction through 5-HT 2A and thromboxane A 2 Gq protein-coupled receptors, respectively [START_REF] Nobe | Distinct pathways of Ca(2+) sensitization in porcine coronary artery: effects of Rho-related kinase and protein kinase C inhibition on force and intracellular Ca(2+)[END_REF][START_REF] Miyata | Sarpogrelate, a selective 5-HT2A serotonergic receptor antagonist, inhibits serotonininduced coronary artery spasm in a porcine model[END_REF]. Considering that the effects of both are inhibited by biochanin A, we presumed that this isoflavonoid rather acts on downstream contraction pathways than on the receptor level. Biochanin A (1-10 µM) also managed to inhibit the contractions induced by phenylephrine, another agonist on Gq proteincoupled receptors, in rat aortas [START_REF] Wang | Mechanisms underlying biochanin A-induced relaxation of the aorta differ between normotensive and hypertensive rats[END_REF]. Similarly, in this study, biochanin A caused the vasodilation of norepinephrine precontracted vessels in the same experimental model (▶ Fig. 1). We hypothesized that the inhibition was mediated at the level of L-type channels, because these channels are involved in Gq protein-initiated vasoconstriction [START_REF] Nobe | Distinct pathways of Ca(2+) sensitization in porcine coronary artery: effects of Rho-related kinase and protein kinase C inhibition on force and intracellular Ca(2+)[END_REF] and thus their activation seems to be a common step between the ion and receptor-mediated pathways. For this reason, we decided to confirm this assumption experimentally for the first time. The achieved results indicate that biochanin A blocks the effect of Bay K8644 and thus the influx of calcium through L-type channels into vascular smooth muscles in concentrations above 10 μΜ. Although this seems to be the major mechanism of action, looking at the vasorelaxant curves of biochanin A, it is apparent that this compound also has vasodilatory effects in lower concentrations in coronary arteries (▶ Fig. 2 a).

For this reason, other mechanisms of action, which might be relevant in lower concentrations, were also considered. Indeed, low concentrations (2-4 μΜ) of biochanin A potentiated the relaxation induced by cumulative doses of sodium nitroprusside. Moreover, biochanin A-induced relaxation was delayed in the presence of a soluble guanylyl cyclase inhibitor (ODQ). Such potentiating effect can be limited to coronary vasculature since it was not observed in rat aortic rings [START_REF] Choi | Mechanisms of phytoestrogen biochanin A-induced vasorelaxation in renovascular hypertensive rats[END_REF], and [START_REF] Torregrosa | Pharmacological profile of phytoestrogens in cerebral vessels: in vitro study with rabbit basilar artery[END_REF] reported that soluble guanylyl cyclase inhibitors (ODQ, NS2028) did not affect the biochanin A-induced dilation of rabbit basilar arteries. In addition to the aforementioned differences in vascular beds or potential interspecies differences, the discrepancy may also depend on the use of a high concentration of the inhibitors (10 -5 Μ) by the afore-mentioned researchers. Collectively, the presented data reveal that biochanin A activates the cGMP pathway in units of micromolar concentrations, while in slightly higher concentrations, it acts as an L-type calcium channel blocker (▶ Fig. 7). From this point of view, it is interesting that biochanin A imitates the mechanism of action of estradiol [START_REF] White | Estrogen relaxes coronary arteries by opening BKCa channels through a cGMP-dependent mechanism[END_REF][START_REF] Han | 17 beta-Estradiol inhibits Ca2+ influx and Ca2+ release induced by thromboxane A2 in porcine coronary artery[END_REF]. Our preliminary results (Fig. 4S, Supporting Information) in rat aorta indicate that an inhibiting effect on calcium release from intracellular stores might be also involved when higher concentrations of the compound are used.

It can be concluded that isoflavonoids started to exhibit direct vasorelaxant activity in tens of µM, while even lower concentrations of biochanin A significantly potentiated the effect of sodium nitroprusside. Such results are of potential clinical interest, since concentrations up to 2 µM might be feasible in human blood circulation, after eating a special diet or being administered food supplements rich in isoflavones [START_REF] Setchell | Bioavailability of pure isoflavones in healthy humans and analysis of commercial soy isoflavone supplements[END_REF][START_REF] Setchell | Bioavailability, disposition, and dose-response effects of soy isoflavones when consumed by healthy women at physiologically typical dietary intakes[END_REF]. Indeed, biochanin A is present in chickpeas (Cicer arietinum) [START_REF] Wu | Germination dramatically increases isoflavonoid content and diversity in chickpea (Cicer arietinum L.) seeds[END_REF], alfalfa (Medicago sativa) [START_REF] Hloucalova | Determination of phytoestrogen content in fresh-cut legume forage[END_REF], peanuts (Arachis hypogaea), almonds (Prunus dulcis), walnuts (Juglans regia), and pecans (Carya illinoinensis) [START_REF] Chukwumah | Changes in the phytochemical composition and profile of raw, boiled, and roasted peanuts[END_REF][START_REF] Kuhnle | Phytoestrogen content of beverages, nuts, seeds, and oils[END_REF], and broadly used food supplements derived from red clover (Trifolium pratense) contain relatively high amounts of the isoflavone too [START_REF] Setchell | Bioavailability of pure isoflavones in healthy humans and analysis of commercial soy isoflavone supplements[END_REF]. This study provided evidence about the vasorelaxant of action of glycitein and deeper insight into the mode of action of biochanin A.

Materials and Methods

Tested compounds

Calycosin (purity: 99 %), cladrin (98 %), and isoformononetin (99 %) were purchased from Phytolab. Biochanin A (≥ 99 %), daidzin (≥ 99 %), formononetin (≥ 99 %), genistin (≥ 99 %), glycitein (≥ 95 %), glycitin (≥ 95 %), ononin (≥ 99 %), prunetin (≥ 95 %), and puerarin (≥ 99 %) were purchased from Extrasynthese and daidzein (≥ 98 %), genistein (≥ 98 %), tectoridin (≥ 98 %), and tectorigenin (≥ 98 %) from Sigma-Aldrich.

Chemicals

Urethane, norepinephrine bitartrate (NE), phenylephrine hydrochloride, sodium nitroprusside, acetylcholine iodide, bradykinin acetate salt, nifedipine, indomethacin, serotonin (5-HT), forskolin, ethylenediaminetetraacetic acid (EDTA), dimethyl sulfoxide (DMSO), KCl, and CaCl 2 .2H 2 O were purchased from Sigma-Aldrich, [1H- [START_REF] Kokubo | Association of dietary intake of soy, beans, and isoflavones with risk of cerebral and myocardial infarctions in Japanese populations: the Japan Public Health Center-based (JPHC) study cohort I[END_REF][START_REF] Pase | The effects of dietary and nutrient interventions on arterial stiffness: a systematic review[END_REF][START_REF] Setchell | Soy isoflavonesbenefits and risks from nature's selective estrogen receptor modulators (SERMs)[END_REF]

Experiments on rat aortas and porcine coronary arteries

The experiments were carried out on aortas excised from male Wistar Han rats, obtained from Charles River or on pig hearts collected from a local slaughterhouse. The initial screening experiments followed commonly used methods (see supplementary

Determination of the mechanism of the vasorelaxant effect

The arterial rings were constricted with U46619 (6-16 nM) or NE (10 μΜ), and cumulative doses of isoflavonoids (0.1-100 μΜ) or vehicle (DMSO, 0.001-1 %) were applied. The next dose was always added after stabilization of the relaxant response to the previous dose. Enzyme inhibitors (ODQ, 10 -6 Μ/indomethacin, 10 -5 Μ, dissolved in DMSO and 5 % aqueous sodium carbonate solution respectively) were added in the chamber 30 min prior to U46619 application when needed. The addition of sodium nitroprusside (10 µM) induced the maximal (100 %) relaxation of the arterial rings at the end of each experiment.

Inhibition of contractile responses

For the determination of the inhibition of contractile responses, coronary endothelium-denuded rings were exposed to 3 different concentrations (3 or 15, 10, and 30 µM) of the selected isoflavonoids for 30 min before the cumulative administration of increasing concentrations of either KCl, 5-HT, U46619, or CaCl 2 in the presence of 40 mM KCl.

Inhibition of the effect of Bay K8644

Endothelium-denuded coronary rings were treated with 4 different concentrations (3, 10, 20, and 30 µM) of biochanin A in the presence of 15 mM KCl and protected from intense light. Then, Bay K8644, an L-type calcium channel activator, was applied cumulatively (10 -10 -10 -6 Μ) to induce contraction of the smooth muscles.

Effect on relaxation induced by activators of cyclic nucleotide synthesis

Endothelium-denuded coronary artery rings were contracted with U46619 (6-16 nM) and then exposed to a low concentration of isoflavonoid (2-4 µM), so that relaxation to about 15-20 % of the maximum amount was induced. Subsequently, concentration-dependent relaxation was caused by the administration of cumulative doses (1 nM-1 µM) of either sodium nitroprusside or forskolin, which activate guanylyl and adenylyl cyclase, respectively.

Statistical analysis

Data were presented as mean ± S. E. M. The number (n) of different experiments was always mentioned. The EC 50 value corresponded to the concentration of the isoflavonoid inducing 50 % of the vasorelaxant response, determined by GraphPad Prism 7 (GraphPad Software, Inc.). The mean values were compared by analyzing the variance followed by the Sidak posthoc test employing GraphPad Prism 7. The difference was considered to be significant when a p-value less than 0.05 was found. Concentration-relaxation curves were compared using 95 % confidence intervals, plotted by the above-mentioned software.

Supporting information

Detailed description of the experimental procedures, as well as the graphs depicting the effect of the isoflavonoids of lower potency and the mechanism of action tested in rat aorta are available as Supporting Information.

▶

  Fig.1Concentration-response curves showing the vasorelaxant effect of the most potent isoflavonoid aglycones in endothelium-intact rat aortic rings, precontracted by norepinephrine (10 -5 M). Relaxation is expressed as percentages of the sodium nitroprusside-induced relaxation of the vessels, n = 5. A concentration-response curve to the solvent was used as the negative control, and the final concentration of DMSO in the organ bath is indicated below the x-axis.

Fig. 2 ▶

 2 Concentration-response curves, with 95 % confidence intervals, showing the vasorelaxant effect of biochanin A (a) and glycitein (b) in endothelium-intact (E+, blue line) or -denuded (E-, red line) coronary artery rings, precontracted by U46619 (10 -8 M). Relaxation is expressed as percentages of the initial basal tension of the rings; data are shown as the mean ± SEM, n = 6. A concentration-response curve to the solvent was used as the negative control, and the final concentration of DMSO in the organ bath is indicated below the x-axis. Fig. 3 Concentration-dependent effects of biochanin A (a) or glycitein (b) on the contraction of coronary arteries induced by KCl, CaCl 2 , serotonin (5-HT), and U46619. Porcine coronary artery rings were pretreated with indicated concentrations of isoflavonoids before the addition of cumulative doses of the inducer. * p < 0.05, ** p < 0.01, *** < 0.001 vs. control, n = 6.

▶

  oxadiazolo-[4, 3-a]quinoxalin-1-one] (ODQ) from Enzo Life Sciences, U46619 from Cayman Chemical Company, and Bay K8644 from Axon Medchem BV. NaCl, NaHCO 3 , and D-glucose were provided by PENTA s. r. o., KCl and Fig. 4 Concentration-dependent effects of biochanin A on vessel contraction induced by the activation of L-type channels. Porcine coronary artery rings were pretreated with indicated concentrations of biochanin A before the addition of cumulative doses of calcium channel activator BAY K8644. * p < 0.05, ** p < 0.01, *** < 0.001 vs. control, n = 5. MgSO 4 .7H 2 O by Erba Lachema s. r. o., and KH 2 PO 4 by Dr. Kulich Pharma s. r. o. Krebs solution had the following composition in mM: NaCl 119, KCl 4.7, CaCl 2 .2H 2 O 1.25, KH 2 PO 4 1.18, MgSO 4 .7H 2 O 1.17, NaHCO 3 25, and D-glucose 11 (pH 7.4).

Fig. 5 ▶Fig. 6

 56 The effect of biochanin A on relaxation induced by sodium nitroprusside (a) and forskolin (b). After exposure of endothelium-denuded coronary artery rings to biochanin A (2-4 µM, able to cause 20 % of relaxation), relaxation was induced by the addition of cumulative doses of either nitroprusside or forskolin. Relaxation is expressed as percentages of the initial basal tension of the rings. * a difference at p < 0.05 between corresponding values, n = 5. The influence of inhibition of guanylyl cyclase (a) and cyclooxygenase (b) on biochanin A-induced vasorelaxation. Endothelium-denuded coronary artery rings were incubated with ODQ (a, 10 -6 M) or indomethacin (b, 10 -5 M) before cumulative doses of biochanin A were administered. Relaxation is expressed as percentages of the initial basal tension of the rings. * indicates a difference (p < 0.05) between the absence and presence of the inhibitor, n = 6. A concentration-response curve to the solvent was used as the negative control, and the final concentration of DMSO in the organ bath is indicated below the x-axis. data for details). The study (reg. No. MSMT-7041/2014-10) was approved on March 7, 2014 by the Ministry of Education, Youth and Sports and conformed to The Guide for the Care and Use of Laboratory Animals published by the U. S. National Institutes of Health (8th edition, revised 2011, ISBN-13: 978-0-309-15400-0).

▶Fig. 7

 7 Scheme depicting the proposed mechanism of action of biochanin A on smooth muscle cells. In higher concentrations, biochanin A can also likely have an effect on the release of calcium from sarcoplasmic reticulum (not shown). cGMP = cyclic guanosine monophosphate, DAG = diacylglycerol, GC = guanylate cyclase, GTP = guanosine triphosphate, IP 3 = inositol 1,4,5-triphosphate, IP 3 R = inositol 1,4,5-triphosphate receptor, MLC = myosin light chain, MLCK = myosin light chain kinase, MLCP = myosin light chain phosphatase, ODQ = 1H-[1, 2, 4]oxadiazolo-[4,3-a]quinoxalin-1-one, P = phosphate group, PKC = protein kinase C, PLC = phospholipase C, ROCK = rho-associated protein kinase, SR = sarcoplasmic reticulum, 5-HT = 5-hydroxytryptamine.

  EC 50 of the vasodilatory response to isoflavonoid aglycones in rat thoracic aortic rings.

	Relaxation (%)
		-7	-6	-5	-4	-3
				Concentration (log M)
	DMSO:	0.001%	0.01%	0.1%	1%	2%
				Isoflavonoid	EC 50 (μΜ)	95 % confidence
							interval (μΜ)
				Biochanin A		25.44	21.41 to 30.24
				Glycitein		27.51	22.36 to 33.84
				Genistein		32.09	29.40 to 37.26
				Isoformononetin	51.19	44.46 to 58.93
				Formononetin	59.53	56.76 to 62.43
				Tectorigenin	62.40	56.10 to 69.42
				Calycosin		68.38	58.65 to 79.71
				Cladrin		72.30	64.63 to 80.87
				Prunetin		74.14	48.25 to 113.92
				Daidzein		112.09	82.46 to 152.37
				The data are derived from 5 experiments.

▶ Table

2

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Migkos T et al. Biochanin A, the … Planta Med Original Papers Downloaded by: Université de Strasbourg. Copyrighted material.

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Acknowledgements

This work was supported by the EFSA-CDN project (grant number: CZ.02.1.01/0.0/0.0/16_019/0000841) co-funded by the ERDF and Charles University (grant number: SVV 260 414). English language was revised by the agency "theBESTtranslation".

Conflict of Interest

The authors declare that they have no conflict of interest.