Adriana Gadioli Tarone

Pascale Goupy

Christian Ginies

Mario Roberto Marostica

Claire Dufour
email: claire.dufour@inrae.fr

Adriana Gadioli Tarone

Mario Roberto Marostica Junior

Advanced characterization of polyphenols from Myrciaria jaboticaba peel and lipid protection in in vitro gastrointestinal digestion

Keywords: anthocyanins, hydrolyzable tannins, lipid oxidation, in vitro digestion, bioaccessibility, ultrasound-assisted extraction

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

Introduction

Jabuticaba (Myrciaria jaboticaba (Vell.) O. Berg, also known as Plinia cauliflora (Mart.) Kausel), is a typical Brazilian berry from the Myrtaceae family. Fruits, presenting a dark violet peel and a white gelatinous pulp at maturity, are consumed in natura but also largely after processing.

Because of its stiffness and astringent taste, the peel (up to 35% of the fruit weight) is not consumed leading to wastes rich in phenolic compounds, mainly anthocyanins, flavonols, and hydrolyzable tannins (ellagitannins, gallotannins) (Neves, Stringheta, Gomez-Alonso, & Hermosin-Gutierrez, 2018;[START_REF] Morales | Nonfermented and fermented jabuticaba (Myrciaria cauliflora Mart.) pomaces as valuable sources of functional ingredients[END_REF][START_REF] Plaza | Characterization of antioxidant polyphenols from Myrciaria jaboticaba peel and their effects on glucose metabolism and antioxidant status: A pilot clinical study[END_REF][START_REF] Pereira | Polyphenol and Ellagitannin Constituents of Jabuticaba (Myrciaria cauliflora) and Chemical Variability at Different Stages of Fruit Development[END_REF][START_REF] Wu | Metabolite profiling of jaboticaba (Myrciaria cauliflora) and other dark-colored fruit juices[END_REF]. These studies suggest the contribution of galloyl and hexahydroxydiphenoyl (HHDP) groups to jabuticaba hydrolyzable tannins although full identification and quantification of these tannins remain elusive owing to a large structural diversity and a lack of standards. Anthocyanins are present in high concentrations (0.14-3.2 g/100 g dry peel), mainly as cyanidin and delphinidin glucosides [START_REF] Albuquerque | Jabuticaba residues (Myrciaria jaboticaba (Vell.) Berg) are rich sources of valuable compounds with bioactive properties[END_REF][START_REF] Quatrin | Characterization and quantification of tannins, flavonols, anthocyanins and matrix-bound polyphenols from jaboticaba fruit peel: A comparison between Myrciaria trunciflora and M. jaboticaba[END_REF][START_REF] Plaza | Characterization of antioxidant polyphenols from Myrciaria jaboticaba peel and their effects on glucose metabolism and antioxidant status: A pilot clinical study[END_REF][START_REF] Alezandro | Comparative study of chemical and phenolic compositions of two species of jaboticaba: Myrciaria jaboticaba (Vell.) Berg and Myrciaria cauliflora (Mart.) O. Berg[END_REF][START_REF] Peixoto | Simulation of in vitro digestion coupled to gastric and intestinal transport models to estimate absorption of anthocyanins from peel powder of jabuticaba, jamelão and jambo fruits[END_REF].

Phenolic compounds appear to contribute to the in vitro antioxidant, antimicrobial, anti-proliferative and anti-inflammatory properties described for jabuticaba extracts [START_REF] Albuquerque | Jabuticaba residues (Myrciaria jaboticaba (Vell.) Berg) are rich sources of valuable compounds with bioactive properties[END_REF][START_REF] Leite-Legatti | Jaboticaba peel: Antioxidant compounds, antiproliferative and antimutagenic activities[END_REF]. Moreover, jabuticaba fruit, tea and peel improve plasma lipid profile and insulin sensitivity, reduce lipid peroxidation in plasma and brain, and increase antioxidant enzyme activity in various organs of animal models for diabetes and obesity [START_REF] Lenquiste | Jaboticaba peel powder and jaboticaba peel aqueous extract reduces obesity, insulin resistance and hepatic fat accumulation in rats[END_REF][START_REF] Alezandro | Jaboticaba (Myrciaria jaboticaba (Vell.) Berg), a Brazilian grape-like fruit, improves plasma lipid profile in streptozotocin-mediated oxidative stress in diabetic rats[END_REF]. A human study by [START_REF] Plaza | Characterization of antioxidant polyphenols from Myrciaria jaboticaba peel and their effects on glucose metabolism and antioxidant status: A pilot clinical study[END_REF] supported the preventive role of jabuticaba peel in metabolic diseases decreasing insulin and glucose levels. Acute and chronic human supplementations with anthocyanin-rich foods and extracts are clearly in favor of a health benefit in vascular protection [START_REF] Fairlie-Jones | The effect of anthocyanin-rich foods or extracts on vascular function in adults: a systematic review and meta-analysis of randomised controlled trials[END_REF]. Of further interest, cardiometabolic risk biomarkers such as LDL-cholesterol, blood pressure or flow-mediated-dilation responded differently to ellagitannin-rich foods (pomegranate and nuts) and anthocyanin-containing products (berries and grape products) [START_REF] Garcia-Conesa | Meta-analysis of the effects of foods and derived products containing ellagitannins and anthocyanins on cardiometabolic biomarkers: analysis of factors influencing variability of the individual responses[END_REF]. Oxidative stress is a key factor of inflammation and antioxidant phenolic compounds are expected to interfere with the production of reactive oxygen species even in locations such as the artery wall.

Only anthocyanin glucosides are bioavailable and the gastric mucosa was found to play an important role in absorption [START_REF] Peixoto | Simulation of in vitro digestion coupled to gastric and intestinal transport models to estimate absorption of anthocyanins from peel powder of jabuticaba, jamelão and jambo fruits[END_REF]. Other anthocyanin glycosides and ellagitannins are not absorbed in the upper gastrointestinal tract and are further metabolized in the colon by the microbial flora. Ellagitannins mainly yield urolithins [START_REF] Quatrin | Bioaccessibility and catabolism of phenolic compounds from jaboticaba (Myrciaria trunciflora) fruit peel during in vitro gastrointestinal digestion and colonic fermentation[END_REF] while anthocyanins are cleaved to small phenolic acids such as protocatechuic acid and phloroglucinaldehyde [START_REF] De Ferrars | The pharmacokinetics of anthocyanins and their metabolites in humans[END_REF]. The resulting microbial metabolites are partly deoxygenated and subjected to phase II metabolism resulting in a partial masking of residual hydroxyl groups. Bioavailable forms of anthocyanins and ellagitannins are thus less likely to play an antioxidant role in the circulation compared to their native forms.

As a matter of fact, the gastrointestinal tract has been proposed as a major site for diet-related oxidative stress and antioxidant activity of plant polyphenols [START_REF] Gobert | Fruits, vegetables and their polyphenols protect dietary lipids from oxidation during gastric digestion[END_REF]. Red meat iron, as provided by the Western diet, can trigger the oxidation of dietary polyunsaturated lipids leading to the formation of 4-hydroxyalkenals. These reactive lipid oxidation products are absorbable and further recovered in lipoproteins. LDL covalent modification is a key event in atherogenesis.

Besides, the presence of cytotoxic and genotoxic 4-hydroxynonenal (4-HNE) in feces and plasma was correlated to the development of colonic preneoplastic lesions and atherosclerotic plaques in rodents fed with diets enriched in heme iron and polyunsaturated lipids. Interestingly, polyphenolrich red wine and pomegranate extracts were found to decrease heme-induced luminal peroxidation and the promotion of colonic lesions [START_REF] Bastide | Red wine and pomegranate extracts suppress cured meat promotion of colonic mucin-depleted foci in carcinogen-induced rats[END_REF]. On the other hand, both apple puree and extract inhibited the formation of 4-HNE in feces and plasma and largely reduced aortic plaque formation [START_REF] Bolea | Digestive n-6 lipid oxidation, a key trigger of vascular dysfunction and atherosclerosis in the Western diet: protective effects of apple polyphenols[END_REF].

Because anthocyanins display a large spectrum of coloring properties, they are nowadays widely used as natural food additives in dairy products and beverages. Ultrasound-assisted extraction of the jabuticaba peel and further extract stabilization by encapsulation are currently investigated to take advantage of polyphenol nutritional and sensory properties (Tarone, da Silva, Cazarin & Marostica Junior, 2021). Besides, peel extracts were shown to display antioxidant and antimicrobial activities in sausages [START_REF] Baldin | Microencapsulated jabuticaba (Myrciaria cauliflora) extract added to fresh sausage as natural dye with antioxidant and antimicrobial activity[END_REF].

The first objective of this work was to assess the efficiency of high-intensity ultrasound for polyphenol extraction from dried jabuticaba peel by comparison with solvent extraction using full structural elucidation and quantification by UPLC/DAD/ESI-MS n . Next, the ultrasonic extract was compared to pure cyanidin-3-O-glucoside for its capacity to inhibit lipid oxidation in the in vitro gastrointestinal digestion of a Western type diet. Anthocyanin recovery was further evaluated.

Materials and Methods

Materials

Cyanidin-3-O-glucoside (C3G), gallic acid, ellagic acid and quercetin 3-O-glucoside were purchased from Extrasynthese (Genay, France The dried jabuticaba peel powder (DJPP) was packed in plastic bags under vacuum and stored at -20 °C until use.

Dried jabuticaba peel extraction by ultrasound (DJP-US)

DJPP prepared as above (1 g) was added into a falcon with 25 mL of 50% ethanol in ultrapure water (v/v) and sonicated using a 13-mm ultrasonic probe (Unique, Desruptor, 800 W, 19 kHz, Indaiatuba, Brazil) at an ultrasound intensity of 3.7 W/cm 2 for 3 min. The probe contact height within the dispersion was 40 mm and an ice bath was used (temp. < 35 ºC). The solvent was removed in vacuum at 37 °C. The extraction was made in triplicate. The dry residues were reconstituted in the same volume of ultrapure water before freeze-drying and storage at -20 °C.

Dried jabuticaba peel extraction by a conventional solvent method (DJP-CM)

In a 2 mL microtube with conical bottom, 100 mg (for analysis of phenolic compounds) or 10 mg (for anthocyanin analysis) of DJPP were placed with balls of zirconium and 1.5 mL of extraction solvent (70% ethanol in ultrapure water (v/v) containing 1% of formic acid). After shaking at 1200 rpm for 45 s in a 1600 MiniG ® SPEX Sample Prep (New Jersey, USA), the microtube was centrifuged at 16000 g and 4 °C for 5 min. The supernatant was collected and the residue was extracted again twice in the same conditions. Supernatants were pooled, the solvent was evaporated in vacuum at 37 °C and the sample was stored at -20 °C until analysis. The extraction was made in triplicate.

Hydrolysis of phenolic compounds

The hydrolysis of the phenolic extracts was conducted according to Garcia-Villalba et al.

(2015) with some modifications. DJP-US or DJPP (100 mg) was put into a glass tube with 3.34 mL of ultrapure water and 1.66 mL of 37% HCl. The tube was vortexed for 1 min and incubated at 90 °C for 24 h. After cooling to room temperature, the pH was adjusted to 2.5 with 5 M NaOH and the volume adjusted to 6 mL with water in a graduated syringe. After centrifugation (5 min, 16000 g and 4 °C), the supernatant was recovered and filtered through a Minisart RC4 filter before injection onto UPLC/DAD/ESI-MS n . The residual pellet was vortexed twice with 1 mL of DMSO/MeOH (50:50, v/v) for 2 min and centrifuged as above. The mixed supernatants were filtered through a Minisart RC4 filter before analysis. All samples were prepared in triplicate.

Identification and quantification of phenolic compounds and anthocyanins

Freshly reconstituted solutions of DJP-US and DJP-CM extracts were prepared in 98 vol. of 1% formic acid in ultrapure water (v/v) and 2 vol. of 1% formic acid in acetonitrile (v/v) at concentrations of 100 mg/mL and 1 mg/mL for UPLC/DAD/ESI-MS n analysis of phenolic compounds and anthocyanins, respectively. Separation and mass conditions are detailed in the Supplementary Material.

The quantification was performed using 5 to 10 point-calibration curves with C3G, quercetin-3-O-glucoside, gallic acid and ellagic acid. All the standards were prepared in methanol except C3G in methanol acidified with 0.1% HCl (v/v). Gallic acid was quantified with its own standard and gallic acid derivatives were quantified as gallic acid equivalent at 280 nm; ellagic acid was quantified with its own standard and ellagic acid derivatives as ellagic acid equivalent at 370 nm; quercetin derivatives and myricetin derivatives as quercetin-3-O-glucoside equivalent at 370 nm, and anthocyanins as C3G equivalent at 520 nm.

Simulated static in vitro gastrointestinal digestion

Preparation of the simulated digestion fluids and antioxidants

The simulated gastric (SGF) and intestinal (SIF) fluids were made up as described in [START_REF] Minekus | A standardised static in vitro digestion method suitable for food -an international consensus[END_REF]. Enzymes were prepared prior to use. Porcine pepsin was prepared as an 8.08 mg/mL solution in SGF for a final concentration in the gastric phase of 1000 U/mL. Porcine pancreatin was prepared as a 94 mg/mL solution in SIF for a final concentration in the intestinal phase of 100 U trypsin/mL. The porcine bile extract was prepared as a 20.85 mg/mL solution in SIF for a final concentration of 5 mg/mL (ca. 10 mM) in bile salts. CaCl2 (H2O)2 (0.1 mM), HCl (0.1 M)

and NaOH (0.25 M) were prepared in ultrapure water. DJP-US (1 mg or 5 mg) was dissolved prior to use in 1 mL of SGF to bring respectively 14.88 µg (US-1) or 74.4 µg (US-5) of total anthocyanins with 89% of C3G and 11% of delphinidin-3-O-glucoside (D3G). C3G-1 and C3G-5

were prepared by adding 21 and 104 µL of C3G at 0.859 mg/mL in methanol acidified with 0.1% HCl (v/v) to 1979 and 1896 µL of SGF, respectively.

Preparation of oil-in water emulsions

The physical state of lipids during digestion was simulated by a 12.5% oil-in-water emulsion stabilized by egg yolk L-α-phosphatidylcholine (PL). In a 60 mL short-necked glass bottle, 5 g of sunflower oil were added to 100 mg of PL previously dispersed in 35 mL of SGF under magnetic stirring. The mixed oil and aqueous phases were then homogenized using a rotor stator homogenizer (Silent Crusher M-01, Heidolph) at 24000 rpm for 2 min. The resulting coarse emulsion was sonicated on ice for 8 periods of 30 s with rest intervals of 30 s and an amplitude of 60% (Q700, QSonica, 20 kHz). See Suppl. Material for emulsion characterization.

Lipid oxidation in in vitro gastrointestinal digestion

The physicochemical conditions for in vitro static gastrointestinal digestion were from [START_REF] Minekus | A standardised static in vitro digestion method suitable for food -an international consensus[END_REF] with slight modifications. First, 12 mL of the fine emulsion were placed in a 50 mL round-bottom flask containing a magnetic stirrer. For the gastric phase, 1 mL of pepsin, 50 µL of 0.1 mM CaCl2, 1 mL of antioxidant solution (US-1, US-5, C3G-1 or C3G-5) or 1 mL of SGF for the blank were added. If necessary, the pH was adjusted to 5 with 0.1 M HCl or 0.25 M NaOH.

The first sample was collected (G0). Lipid oxidation was initiated by adding 2.5 mL of 200 µM MbFe III prepared in SGF (using ε =7700 L.mol -1 .cm -1 at 525 nm [START_REF] Bolea | Lipid protection by polyphenol-rich apple matrices is modulated by pH and pepsin in in vitro gastric digestion[END_REF] to reach a final concentration of 30 µM. Bottom flasks were protected by punched parafilm and incubated in an oven at 37 °C under stirring at 280 rpm during 60 min with sampling at 30 and 60 min. After 60 min, the pH was adjusted to 3 with 0.1 M HCl and the digestion was carried on another 60 min (sampling at 90 and 120 min). After 2 hours of gastric digestion, pancreatin (6.5 mL), bile (6 mL), and CaCl2 (75 µl) were then added to simulate the intestinal phase. pH was adjusted to 6.5 with 0.25 M NaOH and sampling continued for the next 2 h. Experiments were run at least in triplicate.

Determination of lipid oxidation products

Digesta samples (200 µL) were diluted in a microtube with 1000 µL of a 2-propanol/hexane

(2:3, v/v) mixture, vortexed and centrifuged (10 min, 16 200 g, 4 °C). The upper hexane phase (ca. 600 µL) was collected and 1200 µL of hexane were added to the lower phase, vortexed and centrifuged as above. Pooled hexane phases were evaporated under nitrogen and taken up in 2 mL of 2-propanol to yield extract S1. S1 was immediately used for measurement of conjugated dienes (CD) then stored at -20 °C until the determination of 4-hydroxy-2-nonenal (4-HNE).

2.4.4.1 Measurement of lipid-derived conjugated dienes (CD)

After further dilution of S1 (300 μL) in 2-propanol (1700 μL), the concentration in CD was determined by measuring the absorbance at 234 nm (HP 8453 diode-array spectrometer) using a molar absorption coefficient of 27 000 M -1 cm -1 for conjugated linoleyl hydroperoxides.

Measurement of 4-hydroxy-2-nonenal

The secondary lipid oxidation product 4-HNE was derivatized with DNPH before quantification in the MRM mode in HPLC-MS (See Supplementary Material).

Determination of anthocyanin bioaccessibility

Anthocyanin bioaccessibility was defined as the content in free C3G and D3G in the aqueous phase of the digesta. Digesta samples (600 µL) were collected and placed in a microtube with 50 or 75 µL of 0.1 M HCl for gastric and intestinal samples, respectively. After centrifugation (16 200 g

for 10 min at 4 °C), the aqueous phase was removed via syringe, filtered (Phenex RC 0.45 µm) and stored at -20 °C until anthocyanin analysis by UPLC/DAD/ESI-MS n as described in section 2.3.

Statistical analyses

All the results are expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) with repeated measures was performed to test the effect of variation factors. If significant effects were found at a 95% confidence level, ANOVA was followed by a Tukey-Kramer post hoc test to identify differences among groups (XLStat software (2020), Addinsoft, Paris, France).

Results and discussion

Profile and content in phenolic compounds and anthocyanins of dried jabuticaba peel by UPLC/DAD/ESI-MS n analyses

In order to assess the effect of ultrasound on polyphenol extraction, the extracts produced by ultrasound-assisted extraction and conventional solvent extraction of dried jabuticaba peel were compared through their profiles in phenolic compounds and anthocyanins (Fig. 1 & Fig. S1 in Suppl. Mat.). For both extracts, the same 58 phenolic compounds were tentatively identified (Table 1). Maximum absorption wavelength, molecular ion and fragmentation pattern in MS were used in the absence of standards for structure assessment.

Hydrolyzable tannins

Hydrolyzable tannins are polyesters between a sugar moiety and hydroxylated benzoic acid derivatives. In the DJP-US and DJP-CM extracts, gallic acid and ellagic acid were the main contributors. Gallic acid was not found free but esterified by a hexose unit which could be glucose according to [START_REF] Pereira | Polyphenol and Ellagitannin Constituents of Jabuticaba (Myrciaria cauliflora) and Chemical Variability at Different Stages of Fruit Development[END_REF]. Galloylhexose (1) displays a parent ion at m/z 331 with major fragment ions at m/z 271 and 169, which are typical of galloylglucose fragmentation [START_REF] Quatrin | Characterization and quantification of tannins, flavonols, anthocyanins and matrix-bound polyphenols from jaboticaba fruit peel: A comparison between Myrciaria trunciflora and M. jaboticaba[END_REF] [START_REF] Quatrin | Characterization and quantification of tannins, flavonols, anthocyanins and matrix-bound polyphenols from jaboticaba fruit peel: A comparison between Myrciaria trunciflora and M. jaboticaba[END_REF][START_REF] Pereira | Polyphenol and Ellagitannin Constituents of Jabuticaba (Myrciaria cauliflora) and Chemical Variability at Different Stages of Fruit Development[END_REF][START_REF] Plaza | Characterization of antioxidant polyphenols from Myrciaria jaboticaba peel and their effects on glucose metabolism and antioxidant status: A pilot clinical study[END_REF], camu-camu (Fracassetti et al., 2013) and pomegranate [START_REF] Fischer | Identification and quantification of phenolic compounds from pomegranate (Punica granatum L.) peel, mesocarp, aril and differently produced juices by HPLC-DAD-ESI/MSn[END_REF][START_REF] Garcia-Villalba | Validated method for the characterization and quantification of extractable and nonextractable ellagitannins after acid hydrolysis in pomegranate fruits, juices, and extracts[END_REF]. Casuarictin was formerly identified by NMR in Myrciaria cauliflora by [START_REF] Pereira | Polyphenol and Ellagitannin Constituents of Jabuticaba (Myrciaria cauliflora) and Chemical Variability at Different Stages of Fruit Development[END_REF]. Fast elution, close retention times but different fragmentation schemes are observed for 26 and 27 suggesting that these compounds could be casuarinin and casuarictin [START_REF] Plaza | Characterization of antioxidant polyphenols from Myrciaria jaboticaba peel and their effects on glucose metabolism and antioxidant status: A pilot clinical study[END_REF]. Di-HHDPdigalloylhexose (55) displays a parent ion at m/z 1087 with a major fragment ion at m/z 917 (loss of gallic acid) as well as a fragment ion at m/z 749 (loss of hexahydroxydiphenic acid). This compound is tentatively reported for the first time for the jabuticaba species. Finally, HHDPtrigalloylhexose ( 41) is characterized by a parent ion at m/z 937 and displays losses of gallic acid (m/z 767), HHDP (m/z 635), gallic acid + HHDP (m/z 465) and formation of ellagic acid (m/z 301) [START_REF] Plaza | Characterization of antioxidant polyphenols from Myrciaria jaboticaba peel and their effects on glucose metabolism and antioxidant status: A pilot clinical study[END_REF][START_REF] Fracassetti | Ellagic acid derivatives, ellagitannins, proanthocyanidins and other phenolics, vitamin C and antioxidant capacity of two powder products from camu-camu fruit (Myrciaria dubia)[END_REF]. They were all quantified except 55.

HHDP-galloylgluconic acid (3), HHDP-digalloylgluconic acids (8, 12) and di-HHDPgalloylgluconic acids (13, 18, 19 and 31) are tentatively reported for the first time in the jabuticaba species although they were previously described [START_REF] Tanaka | Tannins and relatedcompounds.117. Isolation and characterization of 3 new ellagitannins, lagerstannins A, B and C, having a gluconic acid core, from Lagerstroemia-speciosa (L) pers[END_REF] proposed to be di-HHDP-galloylgluconic acid, totally differs from that reported for DHHDP-HHDP-galloylglucose in pomegranate [START_REF] Calani | Ultra-HPLC MSn (poly)phenolic profiling and chemometric analysis of juices from ancient Punica granatum L. cultivars: a nontargeted approach[END_REF].

Isomeric nonahydroxyterphenic acid dilactones (14 and 30) were detected as previously found in jabuticaba [START_REF] Wu | Metabolite profiling of jaboticaba (Myrciaria cauliflora) and other dark-colored fruit juices[END_REF] and camu-camu [START_REF] Fracassetti | Ellagic acid derivatives, ellagitannins, proanthocyanidins and other phenolics, vitamin C and antioxidant capacity of two powder products from camu-camu fruit (Myrciaria dubia)[END_REF]. They This fragmentation pathway is consistent with structures such as vescalagin, castalagin, and αor βalnusiins as reported in jabuticaba [START_REF] Albuquerque | Jabuticaba residues (Myrciaria jaboticaba (Vell.) Berg) are rich sources of valuable compounds with bioactive properties[END_REF][START_REF] Quatrin | Characterization and quantification of tannins, flavonols, anthocyanins and matrix-bound polyphenols from jaboticaba fruit peel: A comparison between Myrciaria trunciflora and M. jaboticaba[END_REF]. According to Hydrolyzable tannins 51, 53, 56 and 57 are thus clearly tergalloyl derivatives.

Anthocyanins

Two main anthocyanins, delphinidin-3-O-glucoside (D3G) and cyanidin-3-O-glucoside (C3G) were identified, as described before in the jabuticaba peel (Figure 1B). D3G (24) displayed a parent ion in the positive mode at m/z 465 with a major fragment ion at m/z 303 (loss of a hexose moiety) [START_REF] Plaza | Characterization of antioxidant polyphenols from Myrciaria jaboticaba peel and their effects on glucose metabolism and antioxidant status: A pilot clinical study[END_REF][START_REF] Mena | Rapid and Comprehensive Evaluation of (Poly)phenolic Compounds in Pomegranate (Punica granatum L.) Juice by UHPLC-MSn[END_REF].

Others

No caffeoylquinic acids and flavan-3-ols ((epi)catechin, type-B procyanidin dimers, (epi)catechin gallate, gallo(epi)catechin) could be evidenced in jabuticaba peel by search of their parent ions. Thioacidolysis was additionally conducted on both peel extract and peel powder indicating the absence of flavan-3-ol oligomers in these materials.

Polyphenol quantification by UPLC/DAD

Quantification was possible for 32 and 27 compounds out of the 58 tentatively assigned for the DJP-US and DJP-CM extracts, respectively (Table 2).

Gallic acid derivatives are the largest class of compounds found for both extracts. They comprise single and mixed esters containing at least a galloyl or HHDP group. The DJP-US extract presented the highest amount in gallic acid derivatives (9.2 ± 0.3 mg/g DJP), differing statistically from the DJP-CM extract (4.9 ± 0.7 mg/g DJP). Contents between 3.5 and 8.2 mg/g DJP were previously

reported using different extraction methods and standards [START_REF] Albuquerque | Jabuticaba residues (Myrciaria jaboticaba (Vell.) Berg) are rich sources of valuable compounds with bioactive properties[END_REF][START_REF] Quatrin | Characterization and quantification of tannins, flavonols, anthocyanins and matrix-bound polyphenols from jaboticaba fruit peel: A comparison between Myrciaria trunciflora and M. jaboticaba[END_REF][START_REF] Plaza | Characterization of antioxidant polyphenols from Myrciaria jaboticaba peel and their effects on glucose metabolism and antioxidant status: A pilot clinical study[END_REF]. Additionally, the DJP-CM extract presented a statistically higher level of flavonol derivatives (0.32 ± 0.02 mg/g DJP) than the DJP-US extract (0.24 ± 0.01 mg/g DJP). Both [START_REF] Quatrin | Characterization and quantification of tannins, flavonols, anthocyanins and matrix-bound polyphenols from jaboticaba fruit peel: A comparison between Myrciaria trunciflora and M. jaboticaba[END_REF] and [START_REF] Plaza | Characterization of antioxidant polyphenols from Myrciaria jaboticaba peel and their effects on glucose metabolism and antioxidant status: A pilot clinical study[END_REF] found similar amounts (0.6 mg/g DJP) with solvent and pressurized hot water extractions. Finally, our work did not reveal any differences for the overall contents in phenolic compounds whatever the extraction method used. Quantification differences between classes can be explained by the behavior of specific polyphenols towards ultrasound. The acoustic cavitation produced by ultrasound promotes a very efficient cell wall disruption. This may have considerably increased the transfer of galloyl, HHDP and tergalloyl hydrophobic esters to the liquid phase.

Similar levels in ellagic acid

However, this process promotes the creation of shear forces that generate critical temperature and pressure, locally generating hydroxyl radicals which cause the degradation of sensitive compounds such as anthocyanins and flavonols [START_REF] Wang | Degradation behavior of polyphenols in model aqueous extraction system based on mechanical and sonochemical effects induced by ultrasound[END_REF].

Acid hydrolysis of DJP and DJP-US extract

Acid hydrolysis of the dried jabuticaba peel and ultrasonic extract was conducted to release the phenolic depsides present as O-hexosides. Because of the low water solubility of the released phenolic compounds, an additional step of pellet washing by MeOH/DMSO (1:1) was performed as proposed by [START_REF] Garcia-Villalba | Validated method for the characterization and quantification of extractable and nonextractable ellagitannins after acid hydrolysis in pomegranate fruits, juices, and extracts[END_REF]. Identification and quantification of the compounds after acid hydrolysis are presented in Table 3 while the chromatographic profiles are in Figure 1BC and Suppl. Mat.

Gallic acid (1) was not present in the jabuticaba peel and appeared in large amounts after acid hydrolysis. Digalloylhexose (2) presented a similar MS pattern as compound 4 before hydrolysis (Table 1) with a parent ion at m/z 483 and fragment ions at m/z 331, 313 and 169. Its resistance to acid hydrolysis points to the presence of C-glycosidic linkages. The dehydrated form of gallic acid C-hexoside (3) was identified with a parent ion at m/z 313 and a fragment at m/z 169 for gallic acid. A parent ion at m/z 181 and fragments at m/z 137 and 109 as for the standard allowed the identification of dihydroxyphenylpropionic acid (4).

Newly formed compounds 14 and 15 were tentatively assigned as valoneic acid dilactone and sanguisorbic acid dilactone, respectively. Sanguisorbic acid dilactone presented a parent ion at m/z 469 and fragments at m/z 425, 301 and 299 as observed from sanguisorbic acid dilactone obtained from hydrolysis of strawberry [START_REF] Mattila | Determination of free and total phenolic acids in plant-derived foods by HPLC with diode-array detection[END_REF]. Additionally, valoneic acid dilactone eluted earlier than sanguisorbic acid dilactone and presented fragments at m/z 425, 407 and 301 as in pomegranate [START_REF] Garcia-Villalba | Validated method for the characterization and quantification of extractable and nonextractable ellagitannins after acid hydrolysis in pomegranate fruits, juices, and extracts[END_REF]. Contents in valoneic acid dilactone were 3 to 4-fold higher than contents in sanguisorbic acid dilactone in both DJP and the DJP-US extract.

No gallagic acid dilactone was found to form after 24 h of hydrolysis in contrast to pomegranate peel (García-Villalba et al., 2015). Compounds 7, 11, 15, 33, 38 and 52 (Table 1) were without ambiguity assessed as HHDP-tergalloylhexoses rather than gallagylgalloylhexoses and amounted to 0.55 mg/g of DJP after conventional extraction and 1.03 mg/g after ultrasound extraction. Therefore, hexose acylated with the gallagyl group as in pedunculagin III, punicalagin and punicalin remains typical of pomegranate ellagitannins (García-Villalba et al., 2015). Similarly, 51 and 57 should be HHDP-galloyltergalloylhexoses while 53 and 56 ditergalloylhexoses.

Additionally, various C-hexosides of valoneic/sanguisorbic acid dilactone or tergallic acid formed in tiny amounts and could not be quantified in this work (7, 9, 11, 12 and 13) (Table 2). Castalagin and vescalagin are expected to release tergalloyl-C-glucose after acid hydrolysis while alnusiin will give valoneic acid dilactone.

Ellagic acid ( 16) was the most intense peak from pellet washing. Four HHDP-ellagic acid derivatives were identified (5, 6, 8, 10) with parent ions at m/z 783 and fragmentation indicating the loss of HHDP/ellagic acid ([M-302-H] -at m/z 481 and fragment at m/z 301). One of the substituent must be ellagic acid rather than HHDP as these compounds absorb between 365 and 375 nm. They could be C-hexosides rather than O-hexosides as they are resistant to acid hydrolysis and are likely arising from a rearrangement of the five di-HHDP hexosides detected at different retention times in peel (Table 1).

Most abundant hydrolysis compounds are in decreasing order: gallic acid C-hexoside (3), ellagic acid ( 16), gallic acid (1) and valoneic acid dilactone ( 14). Gallic acid amounted to 11.4 mg/g in DJP and 6.3 mg/g after ultrasound extraction. The content in ellagic acid increased upon hydrolysis from 1.1 to 16.4 mg/g for DJP and from 1.1 to 10.9 mg/g after ultrasound treatment.

These values agree well with that found by [START_REF] Alezandro | Comparative study of chemical and phenolic compositions of two species of jaboticaba: Myrciaria jaboticaba (Vell.) Berg and Myrciaria cauliflora (Mart.) O. Berg[END_REF] after acid hydrolysis of DJP (22.5 mg/g DJP). Largely lower contents in gallic acid (0.4 and 1.5 mg/g DJP, respectively)

and ellagic acid (0.3 and 2.8 mg/g DJP, respectively) were found by [START_REF] Quatrin | Characterization and quantification of tannins, flavonols, anthocyanins and matrix-bound polyphenols from jaboticaba fruit peel: A comparison between Myrciaria trunciflora and M. jaboticaba[END_REF][START_REF] Inada | Screening of the chemical composition and occurring antioxidants in jabuticaba (Myrciaria jaboticaba) and jussara (Euterpe edulis) fruits and their fractions[END_REF] after polyphenol extraction and subsequent alkaline and acid hydrolyses of the residue supporting the degradation of the oxidizable trihydroxyphenyl moiety in basic conditions.

Overall, hydrolytic products of ellagitannins amounted to 70.3 mg/g DJP (no extraction) and 42.4 mg/g DJP after ultrasound extraction. This difference was likely due to incomplete extraction by EtOH:water (1:1) of phenolic compounds which are rather hydrophobic or bound to cell wall polysaccharides and proteins. These amounts are markedly higher than those obtained by summing individual compounds from UPLC analysis (10.3 and 6.0 mg/g DJP after ultrasound and solvent extractions, respectively, and subtraction of anthocyanins and flavonols in Table 2). Acid hydrolysis, which relies on the titration of two standards ellagic acid and gallic acid, appears by far more accurate than UPLC in the absence of individual standards. Subsequent washing of the pellet with MeOH/DMSO provided one third of the hydrolysis products. It is worth noting that hydrophobic molecules such as ellagic acid and valoneic acid dilactone were mainly recovered using this additional step whereas sanguisorbic acid dilactone was only soluble in this solvent system.

Inhibition of lipid oxidation by the DJP-US extract and cyanidin-3-O-glucoside in in vitro gastrointestinal digestion

The lipid oxidation initiated by metmyoglobin (MbFe III ) of a 10% oil-in-water emulsion stabilized by egg yolk phospholipids was used to simulate the digestion of a Western type diet rich in ω-6 polyunsaturated lipids [START_REF] Gobert | Fruits, vegetables and their polyphenols protect dietary lipids from oxidation during gastric digestion[END_REF]. Initial gastric digestion is characterized by a fast rise in pH after meal intake. Then pH almost linearly decays returning to a basal pH of 2 after gastric emptying. Although static modeling cannot reproduce pH kinetics, two one hour-periods were sequentially run in this work, one at pH 5 and the other at pH 3. Conjugated dienes (CD), consisting mainly of the lipid-derived hydroperoxides formed in the propagation step of the lipid oxidation process, were followed as primary markers. Secondary marker 4-hydroxy-2-nonenal (4-HNE) was selected as a specific end-product of the oxidation of ω-6 lipids.

Lipid oxidation in the absence of antioxidant

In the control experiment, CD and 4-HNE accumulated in a similar linear pattern yielding 28 µmoles of CD/g of lipids and 86 nmoles of 4-HNE/g of lipids after 2 hours of gastric digestion (Fig. 2AB). The low 4-HNE/CD ratio of 3 to 1000 observed at this stage can be explained by the formation of various other secondary oxidation products such as short-chain aldehydes, epoxides and alcohols as well as by the high reactivity of 4-HNE. Electrophilic 4-HNE reacts rapidly with nucleophilic cysteine, histidine and lysine residues in proteins as already evidenced in pig gastric digesta [START_REF] Delosiere | Quantification of 4-hydroxy-2-nonenal-protein adducts in the in vivo gastric digesta of mini-pigs using a GC-MS/MS method with accuracy profile validation[END_REF]. Increase of pH at 6.5 and addition of bile and pancreatin induced an apparent drop in lipid oxidizability in the early intestinal phase. A CD faster degradation or an incomplete extraction of lipids from the mixed micelles by the 2-propanol/hexane system could explain this apparent CD loss. As to 4-HNE, it disappeared almost totally. Then, CD and 4-HNE exhibited different patterns in the intestinal step. CD kept accumulating reaching 40 µmoles/g lipids at the end of the intestinal step. In contrast to the concomitant formation of 4-HNE and CD in the gastric phase, free 4-HNE only weakly accumulated reaching 7.5 µmoles per g lipids after 240 min.

Additional proteins and enzymes from bile and pancreatin may have reacted with continuously formed 4-HNE.

Lipid oxidation in the presence of the DJP-US extract and cyanidin-3-O-glucoside

The DJP-US extract and C3G were evaluated as inhibitors of lipid oxidation at similar anthocyanin concentrations although anthocyanins in the ultrasonic extract were constituted by 89% of C3G and 11% of D3G. Five-fold higher levels in anthocyanins were brought through US-5 and C3G-5 compared to US-1 and C3G-1.

In the gastric step, the addition of C3G slowed down CD accumulation in a concentrationdependent manner with a significant effect (p = 0.05) for both levels between 90 and 120 min. After 2 h of gastric digestion, CD inhibition was of 52% and 65% for C3G-1 and C3G-5, respectively.

Interestingly, US-1 and US-5 extracts afforded a largely higher protection with inhibition rates of 87% and 100%, respectively. This difference was also outlined for the accumulation of 4-HNE. As a matter of fact, C3G-1 and C3G-5 significantly inhibited 4-HNE formation between 90 and 120 min while being significantly less antioxidant than the two concentrations used for the DJP-US extract. Inhibition rates were 28% and 55% for C3G-1 and C3G-5 while 91% and 97% for US-1 and US-5. With pure C3G, 30 and 60 min-long lag phases were evidenced and found consistent with remaining C3G during these periods (Fig. 3).

After 30 min of intestinal digestion, the DJP-US extracts appeared unexpectedly unable to inhibit the accumulation of CD with apparent rates almost faster than for control. In contrast, the less antioxidant C3G-1 and C3G-5 kept their inhibitory action throughout the intestinal phase. This unexpected increase in CD with the DJP-US extracts may be ascribed to the extraction by the 2propanol/hexane mixture of amino acids or small peptides released through proteolysis of the jabuticaba peel extract and absorbing at 234 nm. This apparent loss of activity has also been observed for apple puree and extract (unpublished data). Epicatechin used for comparison strongly inhibited CD accumulation as evidenced here with C3G. Primary lipid oxidation products should be assessed with a different method because of the possible interferences at 234 nm from complex plant products.

As observed earlier for control, no clear accumulation of 4-HNE occurred in the intestinal step in agreement with the likely fast reaction of this marker with digestive proteins at a higher pH.

Bioaccessibility of anthocyanins

Only anthocyanins could be recovered during the digestion of phospholipid-stabilized emulsions under oxidative stress. Other phenolic compounds from the DJP-US extracts were present at too low levels to be quantified.

In the gastric step, pure C3G disappeared very fast from the aqueous phase since only 14% were recovered for the highest concentration at 30 min and 0% for the lowest concentration. By contrast, C3G from US-1 and US-5 was largely present at the end of the gastric step with recovery rates of 21% and 72%. Less abundant D3G could only be recovered from US-5 at a rate of 22%. This lower recovery is mainly due to the higher oxidizability of the 1,2,3-trihydroxyphenyl moiety of delphinidin when compared to that of the 1,2-dihydroxyphenyl nucleus of cyanidin. Similarly, [START_REF] Quatrin | Bioaccessibility and catabolism of phenolic compounds from jaboticaba (Myrciaria trunciflora) fruit peel during in vitro gastrointestinal digestion and colonic fermentation[END_REF] showed a higher recovery of C3G (85%) than D3G (71%) at the end of the gastric step for a jabuticaba peel powder.

The 5 min-long transition from the gastric to the intestinal phase led to a complete disappearance of C3G from US-1 and D3G from US-5. C3G from US-5 dropped to 26% during this short period before totally disappearing. As sample acidification was conducted before anthocyanin quantification, the decays observed suggest a degradation of the flavylium nucleus as a result of both antioxidant action and unfavorable near neutral conditions.

Discussion

Lipid oxidation and inhibition in gastrointestinal digestion

Chemical reactions and physicochemical interactions occurring between bolus constituents should be unraveled to shed some light on the bioavailability of plant secondary metabolites and their health benefit directly in the gastrointestinal tract. Digestion modeling allows to take into account both human food and physiological conditions of digestion. An average Western adult daily consumes 100 to 150 g of triglycerides and 2 to 10 g of phospholipids. After gastric antral contractions, dietary lipids are recovered in the emulsion form with most of the lipid droplets in the 1-50 µm diameter range [START_REF] Lorrain | Chemical modeling of heme-induced lipid oxidation in gastric conditions and inhibition by dietary polyphenols[END_REF]. A 12.5% oil-in water emulsion stabilized by egg yolk PL and characterized by a single mode at 14.8 ± 0.3 µm and a D(3,2) at 1.09 ± 0.18 µm was thus prepared as food model. While simulated fluids, enzymes (1000 U pepsin/mL, 100 U trypsin/mL) and bile levels were as proposed by [START_REF] Minekus | A standardised static in vitro digestion method suitable for food -an international consensus[END_REF], the gastric pH was more physiologically simulated with a pH jump from 5 to 3 after one hour.

The heme iron form of red meat, metmyoglobin, was added at a 30 µM level in the gastric step simulating the consumption of 85 g of beef steak (chyme volume of 1 L). Metmyoglobininitiated oxidation of polyunsaturated fatty acid led to the formation of lipid-derived conjugated dienes and 4-HNE as primary and end markers, respectively (Fig. 2AB). The change in pH from 5 to 3 appeared to increase both rates of accumulation. As a matter of fact, the proteolysis of metmyoglobin by pepsin has been evidenced at pH 5 although this process is faster at lower pHs [START_REF] Bolea | Lipid protection by polyphenol-rich apple matrices is modulated by pH and pepsin in in vitro gastric digestion[END_REF]. The resulting micro-metmyoglobin can accelerate lipid oxidation likely due to a facilitated access for lipids to the iron center. Then, pH lowering leads to the formation of an unfolded form of metmyoglobin which appeared to be stable during at least 30 min at pH 3. The release of hematin from proteolyzed unfolded metmyoglobin at pH 3, although suggested by some authors, is still unclear by now. Whatever the heme iron form, lipid oxidation proceeded at both gastric pHs suggesting that this deleterious process is susceptible to degrade essential ω-6 lipids in vivo.

Thus, it is critical to identify protective food that, when co-ingested, could limit lipid oxidation in the GIT and the onset of cardiovascular diseases in a longer term. In this work, the effect of a jabuticaba peel rich in anthocyanins and hydrolyzable tannins was compared to that of a pure anthocyanin, C3G. At identical anthocyanin contents, the DJP-US extracts more extensively inhibited the accumulation of CD and 4-HNE than C3G-1 and C3G-5. This difference was particularly significant at 90 and 120 min of gastric digestion for 4-HNE (p < 0.05) and at 60, 90 and 120 min for CD (Fig. 2AB). C3G-1 and C3G-5 were found to inhibit the accumulation of the primary marker CD in a concentration-dependent manner while totally inhibiting that of 4-HNE during lag phases that correlate with the presence of C3G in the digesta. After the disappearance of C3G, 4-HNE formed at rates that are lower than observed for control suggesting an additional antioxidant effect of the degradation products of C3G. In the pH range of 2 to 4 or in oxidative conditions at pH 7, cyanidin glycosides were reported to be degraded into protocatechuic acid, phloroglucinaldehyde and 3,5,7-trihydroxycoumarin among others [START_REF] Dangles | The chemical reactivity of anthocyanins and its consequences in food science and nutrition[END_REF].

Protocatechuic acid, which is also a metabolite from quercetin oxidation, could contribute to the antioxidant capacity of C3G as previously demonstrated for quercetin [START_REF] Lorrain | Chemical modeling of heme-induced lipid oxidation in gastric conditions and inhibition by dietary polyphenols[END_REF].

The almost total inhibition of lipid oxidation in the presence of US-5 is consistent with the high rate of remaining anthocyanins, found to be 72% for C3G and 22% for D3G, after the gastric step (Fig. 3). The potent capacity of the DJP-US extract can also be attributed to the additional presence of hydrolyzable tannins and flavonols which constitute 84% and 0.5% of the phenolic pool of the jabuticaba peel extract (evaluation after acid hydrolysis). All of these compounds display the typical 1,2-dihydroxy-and 1,2,3-trihydroxyphenyl moieties that are critical to the reducing capacity of phenolic compounds.

In the intestinal phase at pH 6.5, the formation of lipid-derived conjugated dienes was demonstrated for the first time suggesting that the heme iron form maintains its prooxidant activity towards triacylglycerols and their lipolytic products. Those are mainly monoacylglycerols and free fatty acids solubilized in mixed micelles with biliary salts. Although this work did not allow to evidence the accumulation of 4-HNE because of its rapid scavenging by peptides and proteins, primary lipid oxidation products are known to be unstable in intestinal conditions. Indeed, the in vitro digestion of a thermodegraded sunflower oil generated hexanal and nonanal as the major alkenals while 2heptenal and 2-octenal were recovered in higher rates than 4-HNE [START_REF] Goicoechea | Use of an in vitro digestion model to study the bioaccessibility of 4-hydroxy-2-nonenal and related aldehydes present in oxidized oils rich in omega-6 acyl groups[END_REF].

Moreover, [START_REF] Kanazawa | Dietary hydroperoxides of linoleic acid decompose to aldehydes in stomach before being absorbed into the body[END_REF] identified uncleaved epoxyketones, 9-oxononanoic acid and hexanal in the intestinal lumen of rats fed with linoleyl hydroperoxides. 4-HNE is absorbable and can be recovered under various metabolized forms [START_REF] Keller | Twin peaks": Searching for 4hydroxynonenal urinary metabolites after oral administration in rats[END_REF]. By covalently reacting with the ApoB protein of LDL, 4-HNE is involved in the formation of atherogenic oxLDL particles. Furthermore, 4-HNE appears to play a role in the development of colonic preneoplastic lesions [START_REF] Bastide | Red wine and pomegranate extracts suppress cured meat promotion of colonic mucin-depleted foci in carcinogen-induced rats[END_REF].

Bioaccessibility and reactivity of anthocyanins and hydrolyzable tannins

Diet-induced oxidative stress led to the fast disappearance of C3G during gastric digestion.

Anthocyanins can play an antioxidant role in metmyoglobin-initiated lipid oxidation by reducing the ferrylmyoglobin initiating species rather than the propagating peroxyl radicals [START_REF] Goupy | Antioxidant activity of wine pigments derived from anthocyanins: hydrogen transfer reactions to the DPPH radical and inhibition of the heme-induced peroxidation of linoleic acid[END_REF]. Interestingly, hydrolyzable tannins, which are present in a 5fold excess compared to anthocyanins in the DJP-US extract, had the ability to slow down their degradation. Hydrolyzable tannins were constituted for half by HHDP-galloylhexose 2, ellagic acid 42, HHDP-digalloylhexoses 29 and 37, tetragalloylhexose 46, pentagalloylhexose 50, HHDPtergalloylhexose 38, and di-HHDP-hexoses 10 and 17, in decreasing order. This composition agrees well with results from [START_REF] Plaza | Characterization of antioxidant polyphenols from Myrciaria jaboticaba peel and their effects on glucose metabolism and antioxidant status: A pilot clinical study[END_REF] and [START_REF] Quatrin | Characterization and quantification of tannins, flavonols, anthocyanins and matrix-bound polyphenols from jaboticaba fruit peel: A comparison between Myrciaria trunciflora and M. jaboticaba[END_REF]. The possible interaction of these high-molecular weight compounds with the iron-oxo center of ferrylmyoglobin is suggested by the superior antioxidant activity of the extracts. Furthermore, ellagitannins and gallotannins are prone to degradation in in vitro gastrointestinal digestion, yielding mainly gallic acid and ellagic acid for a jabuticaba peel powder [START_REF] Quatrin | Bioaccessibility and catabolism of phenolic compounds from jaboticaba (Myrciaria trunciflora) fruit peel during in vitro gastrointestinal digestion and colonic fermentation[END_REF]. Their stability was mostly affected by intestinal conditions which also led to the total disappearance of anthocyanins while only lightly affecting flavonols. A matrix effect should also be taken into consideration when investigating extracts and peel powders [START_REF] Quatrin | Bioaccessibility and catabolism of phenolic compounds from jaboticaba (Myrciaria trunciflora) fruit peel during in vitro gastrointestinal digestion and colonic fermentation[END_REF][START_REF] Peixoto | Simulation of in vitro digestion coupled to gastric and intestinal transport models to estimate absorption of anthocyanins from peel powder of jabuticaba, jamelão and jambo fruits[END_REF]. Binding to cell wall polysaccharides and proteins is expected to slow polyphenol diffusion out of the matrix increasing thus their life time.

Conclusion

Jabuticaba peel hydroalcoholic extracts exhibit high potency in limiting the formation of deleterious lipid hydroperoxides and aldehydes arising from diet-induced oxidation of essential ω-6 lipids in the gastrointestinal tract. This inhibitory capacity can be ascribed to high levels in antioxidant anthocyanins (1% DM) and hydrolyzable tannins (7% DM), thus pointing to promising future uses for jabuticaba extracts as food ingredient or colorant.

Conflicts of interest

There are no conflicts of interest to declare.

Gastric Step Intestinal Step Gastric Step Intestinal

Step

B A

Gastric Step Intestinal

Step

  presented a parent ion at m/z 469 and a major fragment ion at m/z 425 (loss of CO2) and differed remarkably by their retention times. Compound 14 was not further fragmented and it was thus assigned as tergallic acid dilactone owing to likely strong C-C bonds between gallic acid units. Additional fragmentation for 30 revealed fragments at m/z 407, 301 and 167. Fragment m/z 301 corresponds to ellagic acid and is in favor of an additional labile ether linkage between 2 aromatic units, as found for valoneic acid dilactone in pomegranate[START_REF] Garcia-Villalba | Validated method for the characterization and quantification of extractable and nonextractable ellagitannins after acid hydrolysis in pomegranate fruits, juices, and extracts[END_REF]. Two compounds (20 and 23), in trace amounts, presented a parent ion at m/z 631 and a major fragment ion at m/z 451. The loss of 180 amu suggests the presence of a hexose moiety linked through a C-or O-glycosidic linkage to a tergalloyl group as found in jabuticaba[START_REF] Quatrin | Characterization and quantification of tannins, flavonols, anthocyanins and matrix-bound polyphenols from jaboticaba fruit peel: A comparison between Myrciaria trunciflora and M. jaboticaba[END_REF] and in hydrolyzed pomegranate(García-Villalba et al., 2015).[START_REF] Pereira | Polyphenol and Ellagitannin Constituents of Jabuticaba (Myrciaria cauliflora) and Chemical Variability at Different Stages of Fruit Development[END_REF] identified 4,6-O-tergalloyl-D-glucose (Cauliflorin) by NMR in jabuticaba.Six compounds with a parent ion at m/z 933 were identified although only 3 of them could be quantified. Fragment ions at m/z 451 and 631 are observed for four compounds (15, 33, 38 and 52) corresponding respectively to the tergalloyl group and the loss of HHDP from the parent ion.

  [START_REF] Pereira | Polyphenol and Ellagitannin Constituents of Jabuticaba (Myrciaria cauliflora) and Chemical Variability at Different Stages of Fruit Development[END_REF] who secured by NMR experiment the structures of two these compounds, vescalagin was only present in peel of Myrciaria cauliflora whereas vescalagin and castalagin were both present in seeds and pulp. The two other compounds (7, 11) eluted faster and display common fragment ions at m/z 915 (-H2O), 613 and 569, as observed for vescalagin and castalagin standards[START_REF] Tavares | Comprehensive study of the phenolic composition of the edible parts of jambolan fruit (Syzygium cumini (L.) Skeels)[END_REF]. Standards are thus required for the unequivocal attribution of these different HHDP-tergalloylhexoses.High-molecular weight compounds likely bearing a tergalloyl or a gallagyl group appeared in trace amounts. They are reported for the first time in the jabuticaba species. Isomers 51 and 57 display m/z at 1085 and 542, corresponding to singly and doubly charged ions, respectively, and a fragment ion at m/z 633 which could be HHDP-galloylhexose. Isomers 53 and 56 displayed m/z at 1083 and 541 (doubly charged ion) and yielded m/z 631 upon fragmentation as found for tergalloylhexose. It is worth noting that HHDP-galloyltergalloylhexose (MW 1086) exhibits the same molecular weight as digalloylgallagylhexose and ditergalloylhexose (MW 1084) as HHDPdigallagylhexose. However, only dilactones of valoneic acid and sanguisorbic acid (tergalloyl derivatives) were quantified after acid hydrolysis and no gallagic acid dilactone could be recovered.

  C3G (28) ([M] + at m/z 449 and 287 in MS 2 ) was identified by comparison with the standard compound. Minor pelargonidin-O-hexoside (32, [M] + at m/z 433 and 271 in MS 2 ) and peonidin-O-hexoside (35, [M] + at m/z 463 and 301 in MS 2 ) were also identified as previously in jabuticaba peel by[START_REF] Quatrin | Characterization and quantification of tannins, flavonols, anthocyanins and matrix-bound polyphenols from jaboticaba fruit peel: A comparison between Myrciaria trunciflora and M. jaboticaba[END_REF].3.1.3 FlavonolsMyricetin deoxyhexoside (43; m/z 463), quercetin hexosides (45, 47; m/z 463) and quercetin deoxyhexoside (54; m/z 447) display daughter ions at m/z 317 or 301, allowing to assign their aglycone part (Figure1C). For quercetin pentosides (48, 49), their parent and fragment ions at m/z 433 and m/z 301 as well as their maximal absorption in the range 350-360 nm were not sufficient to discriminate them from ellagic acid pentoside. However, they yielded in MS n subsequent fragmentation at m/z 273, 257, 179 and 151 which are typical of the benzopyrane moiety of flavonols as evidenced for quercetin (58)[START_REF] Neves | Flavonols and ellagic acid derivatives in peels of different species of jabuticaba (Plinia spp.) identified by HPLC-DAD-ESI/MSn[END_REF]

  were quantified in both DJP-US and DJP-CM extracts (1.1 mg/g DJP) and these contents are in the same range as those found by[START_REF] Inada | Screening of the chemical composition and occurring antioxidants in jabuticaba (Myrciaria jaboticaba) and jussara (Euterpe edulis) fruits and their fractions[END_REF] (1.8 mg/g DJP),[START_REF] Plaza | Characterization of antioxidant polyphenols from Myrciaria jaboticaba peel and their effects on glucose metabolism and antioxidant status: A pilot clinical study[END_REF] (1.4 mg/g DJP) and[START_REF] Quatrin | Characterization and quantification of tannins, flavonols, anthocyanins and matrix-bound polyphenols from jaboticaba fruit peel: A comparison between Myrciaria trunciflora and M. jaboticaba[END_REF] (0.5 mg/g DJP) although without ellagic acid as standard for the latter two groups.Anthocyanins, quantified as both C3G and D3G, are the second most abundant group. Contents in anthocyanins were found to be significantly lower for the DJP-US extract (7.8 ± 0.1 mg/g DJP) than for the DJP-CM extract (10.2 ± 1.2 mg/g DJP).[START_REF] Plaza | Characterization of antioxidant polyphenols from Myrciaria jaboticaba peel and their effects on glucose metabolism and antioxidant status: A pilot clinical study[END_REF] and[START_REF] Albuquerque | Jabuticaba residues (Myrciaria jaboticaba (Vell.) Berg) are rich sources of valuable compounds with bioactive properties[END_REF] found higher amounts of anthocyanin derivatives (32.2 and 24.5 mg/g DJP, respectively) while[START_REF] Quatrin | Characterization and quantification of tannins, flavonols, anthocyanins and matrix-bound polyphenols from jaboticaba fruit peel: A comparison between Myrciaria trunciflora and M. jaboticaba[END_REF][START_REF] Peixoto | Simulation of in vitro digestion coupled to gastric and intestinal transport models to estimate absorption of anthocyanins from peel powder of jabuticaba, jamelão and jambo fruits[END_REF] and[START_REF] Alezandro | Comparative study of chemical and phenolic compositions of two species of jaboticaba: Myrciaria jaboticaba (Vell.) Berg and Myrciaria cauliflora (Mart.) O. Berg[END_REF] found relatively similar or lower amounts (11.5, 1.7, and 3.5 mg/g DJP).

Figure 1 .Figure 2 :

 12 Figure 1. Chromatographic profile of the phenolic compounds for the DJP-US extract before hydrolysis at 280 nm, 370 nm and 530 nm (A) and after hydrolysis at 280 nm and 370 nm (B -Supernatant and C -Pellet washing).

Figure 3 :

 3 Figure 3: Bioaccessibility of C3G and D3G during the in vitro simulated gastrointestinal digestion of phospholipid-stabilized emulsions in the presence of metmyoglobin (30 µM) and the DJP-US extract or pure C3G. Values represent mean ± SD (n = 3).

  Jabuticaba (Myrciaria jaboticaba (Vell.) O. Berg) fruits were kindly donated by "Indústria e Comércio Lagoa Branca Ltda", located at Boa Vista II Farm, in White House city(São Paulo, Brazil) under the authorization of the Brazilian Management System of National Genetic Heritage and Traditional Associated Knowledge (#A72354F) (August 2016). The species used in this work was assessed by comparison with species deposited in the herbarium of the Agronomic Institute of Campinas, Santa Elisa farm, numbers 48093 and 48094.Twenty kg of fresh fruits were washed, manually peeled and the peel was dried in a stove with air circulation (Marconi, Piracicaba, SP,

	2.2. Jabuticaba peel extracts
	2.2.1. Jabuticaba peel processing
	4-Hydroxy-2-
	nonenal and 4-hydroxy-2-nonenal-D3 were purchased from Bertin Pharma (Montigny le
	Bretonneux, France). Commercial sunflower oil, from Auchan (lot No. A07611), was stored at -20
	°C after purchase. L-α-phosphatidylcholine from dried egg yolk (PL) (P3556) was from Fluka
	(Buchs, Switzerland). Oil and PL compositions are detailed in the Supplementary Material.

). Methanol, acetonitrile, hexane and 2-propanol were HPLC-MS grade from Fisher Scientific (Illkirch, France); formic acid was HPLC-MS grade from Merck (Darmstadt, Germany). Ultrapure water (resistivity 18.2 MΩ/cm at 25 °C) was obtained with a Millipore OPak 2 (Millipore Corporation, Bedford, MA, USA).

Horse heart myoglobin (M1882, type II), pepsin from porcine gastric mucosa (P6887, 2829 U/mg according to

[START_REF] Minekus | A standardised static in vitro digestion method suitable for food -an international consensus[END_REF]

), porcine pancreatin (P7545, trypsin activity 2.8 U/mg according to

[START_REF] Minekus | A standardised static in vitro digestion method suitable for food -an international consensus[END_REF]

), porcine bile extract (B8631) and 2,4-dinitrophenylhydrazine (DNPH) were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Brazil) at 40 °C for 72 h until constant weight. The dried peel (ca. 1 kg) was transformed in a fine powder with an electrical mill (Marconi, MA 630/1, Piracicaba, SP, Brazil) and sifted (mash 20).

  [START_REF] Plaza | Characterization of antioxidant polyphenols from Myrciaria jaboticaba peel and their effects on glucose metabolism and antioxidant status: A pilot clinical study[END_REF][START_REF] Wu | Metabolite profiling of jaboticaba (Myrciaria cauliflora) and other dark-colored fruit juices[END_REF] reported 2 isomers in jabuticaba peel whileSilva et al.

	(2016) isolated pedunculagin from jabuticaba seed and Garcia-Villalba et al. (2015) found one
	isomer in pomegranate.
	A larger group of hydrolyzable tannins is constituted by mixed esters containing both galloyl
	and HHDP groups. Quantified HHDP-galloylhexose isomers (2, 5, 16, 25) display a parent ion at
	m/z 633 and a major fragment ion at m/z 301 indicating the loss of HHDP. HHDP-digalloylhexose
	isomers (21, 29, 37) were evidenced as previously described in Myrciaria species (Quatrin et al.,
	2019; Plaza et al., 2016; Fracassetti, Costa, Moulay, & Tomas-Barberan, 2013). They present a
	parent ion at m/z 785 and fragment ions at m/z 633 and 483 resulting from the loss of a galloyl or a

. Di-(4, 22; m/z 483), tri-(34; m/z 635), tetra-(44, 46; m/z 787) and pentagalloylhexoses (50;

m/z 939) were recovered as several isomers, as found in jabuticaba

[START_REF] Quatrin | Characterization and quantification of tannins, flavonols, anthocyanins and matrix-bound polyphenols from jaboticaba fruit peel: A comparison between Myrciaria trunciflora and M. jaboticaba[END_REF]

and in pomegranate

[START_REF] Garcia-Villalba | Validated method for the characterization and quantification of extractable and nonextractable ellagitannins after acid hydrolysis in pomegranate fruits, juices, and extracts[END_REF]

.

Ellagic acid was found free (42; m/z 301) and bound to a hexose moiety

[START_REF] Pereira | Polyphenol and Ellagitannin Constituents of Jabuticaba (Myrciaria cauliflora) and Chemical Variability at Different Stages of Fruit Development[END_REF][START_REF] Plaza | Characterization of antioxidant polyphenols from Myrciaria jaboticaba peel and their effects on glucose metabolism and antioxidant status: A pilot clinical study[END_REF][START_REF] Morales | Nonfermented and fermented jabuticaba (Myrciaria cauliflora Mart.) pomaces as valuable sources of functional ingredients[END_REF][START_REF] Fischer | Identification and quantification of phenolic compounds from pomegranate (Punica granatum L.) peel, mesocarp, aril and differently produced juices by HPLC-DAD-ESI/MSn[END_REF]

. Ellagic acid pentoside (39) displays a parent ion at m/z 433 and a major fragment at m/z 301 indicating the loss of a pentose unit. The hexahydroxydiphenoyl (HHDP) group related to ellagic acid appears in five isomeric di-HHDP-hexosides (6, 9, 10, 17, 40). Two of them, 10 and 17, could be quantified. They display a parent ion at m/z 783 and major fragments at m/z 481 and 301 indicating the loss of a HHDP group except 40 which gave a dehydrated form first (m/z 765). Previous studies

[START_REF] Quatrin | Characterization and quantification of tannins, flavonols, anthocyanins and matrix-bound polyphenols from jaboticaba fruit peel: A comparison between Myrciaria trunciflora and M. jaboticaba[END_REF]

HHDP group. They were all quantified. For di-HHDP-galloylhexoses (26, 27, 36), with a parent ion at m/z 935, two different fragmentation patterns are observed. Isomer 26 fragments to yield a major ion at m/z 917 (-H2O) while the two others yielded m/z 633 in agreement with the loss of a HHDP group. Isomeric casuarictin, stachyurin, potentillin and casuarinin have been tentatively identified in jabuticaba

  in pomegranate and Lagerstroemia speciosa. The parent ions at m/z 649, 801 and 951 differ by 16 amu from those found for HHDP-galloylhexose (m/z 633), HHDP-digalloylhexose (m/z 785) and di-HHDPgalloylhexose (m/z 935). Major fragment ions at m/z 605, 757 and 907 indicate the loss of CO2which is in agreement with the presence of a carboxylic acid function. HHDP (m/z 301) could be observed in MS 3 for 3 while the loss of HHDP was observed in the MS 2 fragmentations of 13, 18, 19 and 31 with m/z 605 resulting from the major fragment at m/z 907. They proved to be in smaller amounts compared to related hexose derivatives. Additionally, the fragmentation of compound 31,

Table 1 .

 1 Phenolic compounds identified by UPLC/DAD/ESI-MS n in dried jabuticaba peel extracts obtained by ultrasound technology and conventional solvent extraction.

	N°	RT (min)	λmax (nm)	[M -H] -/ [M] + (m/z)*	MS² fragments (m/z)	Tentative identification
	1	1.1	277	271-169	Galloylhexose a
	2	1.3	270	301-275-249	HHDP-galloylhexose (1) abh
	3	1.6	266	605-481-479-425	HHDP-galloylgluconic acid c
	4	1.6	266	331-313-169	Digalloylhexose (1) ad
	5	2.1	264	301-275-249	HHDP-galloylhexose (2) abh
	6	2.1	264	765-721-507-481-419-341-301-275 Di-HHDP-hexose (1) abdhj
	7	2.2	282	915-871-613-569	HHDP-tergalloylhexose (1) defg
	8	2.2	282	783-757-633	HHDP-digalloylgluconic acid (punigluconin) (1) cd
	9	2.5	260	481-301-275	Di-HHDP-hexose (2) abdhj
	10	2.7	260	481-301-275	Di-HHDP-hexose (3) abdhj
	11	3.1	260	915-631-613-569	HHDP-tergalloylhexose (2) adefg
	12	3.1	260	757-633	HHDP-digalloylgluconic acid (punigluconin) (2) cd
	13	3.1	260	907-

783-605-481-405-347-301

		4.7	260	631	613-571-451-407-351	Valoneic (sanguisorbic) acid dilactone C-hexoside or Tergalloyl-O-hexose (Cauliflorin) e or Tergalloyl-C-hexose a (1)
		5.0	270	785	633-483-419-331-301-275	HHDP-digalloylhexose (1) abdfj
		5.1	272	483	331-271-169	Digalloylhexose (2) ad
		5.1	272	631	613-571-451-407-351	Valoneic (sanguisorbic) (Cauliflorin) e or Tergalloyl-C-hexose a (2) acid dilactone C-hexoside or Tergalloyl-O-hexose
		5.4	526	465*	303*	Delphinidin-3-O-glucoside abh
		5.6	260	633	615-463-301	HHDP-galloylhexose (4) abhj
		5.7	260	935	917-873-855-573	Di-HHDP-galloylhexose (1) (Casuarinin/Stachyurin/Casuarictin/Potentillin) abdefhj
		5.9	265	935	917-873-783-659-633-571-301	Di-HHDP-galloylhexose (2) (Casuarinin/Stachyurin/Casuarictin/Potentillin) abcdefh
		6.2	514	449*	287*	Cyanidin-3-O-glucoside (std) abh
		6.3	270	785	633-483-419-301-249	HHDP-digalloylhexose (2) abdfj
		6.3	372	469	451-425-407-301-167	Valoneic acid dilactone cfh
		6.5	260	951	907-783-605	Di-HHDP-galloylgluconic acid (4)
		6.9	516	433*	271*	Pelargonidin-O-hexoside a
		7.0	260	933	763-631-481-451-425-301	HHDP-tergalloylhexose (4) adefj
		7.2	264	635	617-483-423-271	Trigalloylhexose a
		7.4	524	463*	301*	Di-HHDP-galloylgluconic acid (1) Peonidin-O-hexoside a
	14	3.5 7.7	370 260	935	425 917-765-633-615-481-451-301	Tergallic acid dilactone (1) Di-HHDP-galloylhexose (3) (Casuarinin/Stachyurin/Casuarictin/potentillin) abdf
	15	3.6 7.9	260 264	785	631-571-451-425-301 633-483-301	HHDP-tergalloylhexose (3) adefj HHDP-digalloylhexose (3) abdfj
	16	3.8 8.0	260 260	933	615-481-301-275 631-481-451-301	HHDP-galloylhexose (3) abh HHDP-tergalloylhexose (5) adefj
	17	4.2 8.3	260 355	433	481-421-301-275 301-287-273-209	Di-HHDP-hexose (4) abdhj Ellagic acid pentoside abdfh
	18	4.4 8.3	260 -	783	907-783-605-481-425-361-299 765-613-465-451-419-301	Di-HHDP-galloylgluconic acid (2) Di-HHDP-hexose (5) abdh
	19	4.6 8.3	260 -	937	907-783-605-481-425-361-299 785-767-741-635-465-419-301-275 HHDP-trigalloylhexose bfhj Di-HHDP-galloylgluconic acid (3)

Acknowledgements

Mario R. Marostica Junior is grateful to CNPQ for financial support (301108/2016-1). Adriana G.

Tarone thanks CNPQ for the Ph.D. assistantship (140942/2016-5). Authors acknowledge the São Paulo Research Foundation (FAPESP) for the grant (2015/50333-1) and INRAE. This work was

supported in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brasil (CAPES) -Finance Code 001. Values represent mean ± SD (n = 3). Sum of phenolic compounds is obtained from the different columns on the left (UPLC). DJP: dried jabuticaba peel; Different letters indicate a significant difference between both extracts at p < 0.05. The main fragment in MS 2 is given in boldface, the minor fragments are given in normal font and the ultra-minor fragments are given in italics. Values represent mean ± SD (n = 3). Sum of phenolic compounds is the addition of Supernatant and Pellet. Std: identified with authentic standard. -: Means below quantification limit or not present. a Quatrin et al., 2019; b Garcia-Villalba et al., 2015; c Fischer et al., 2011; d Fragmentation as for standard; e Morales et al., 2016; f Neves et al., 2018.


Eliane Bsaibess

Fabian Delorme

Isabelle Monot-Laffez

Fabien Giovannelli

Ultra-low thermal conductivity in scheelite and A-deficient scheelite ceramics

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

Materials exhibiting a low thermal conductivity are the key components in several areas such as thermal barrier coatings (TBCs) or thermoelectric materials. TBCs have been the subject of considerable research over the past few decades, driven by the demands for improved efficiency and substantially higher operating temperatures required for future gas turbine applications [1][2][3]. To date, extensive efforts have been dedicated to explore insulator materials with low thermal conductivity. Considering its high temperature stability and low thermal conductivity (below 3 W m -1 K -1 [4], the (6-8)wt% yttria stabilized zirconia (YSZ) [5,6] has been widely used as bond and top coat for thermal barrier coatings systems. With continuous improvements, several TBCs materials have been proposed to replace YSZ, such as rare earth zirconates [7,8], pyrochlore oxides [9], magnetoplumbite [10,11], perovskite [START_REF] Dietrich | Ceramic Engineering and Science Proceedings[END_REF], garnet [START_REF] Padture | [END_REF] and metal phosphate [14]. Such materials have been extensively investigated showing a low thermal conductivity ranging from 1.2 to 2 W m -1 K -1 in the temperature range from 25 °C to 700 °C. Moreover, several studies have revealed the potential of lanthanum molybdate (La 2 Mo 2 O 9 ) as promising candidates for TBC material having lower thermal conductivity (0.95 W m -1 K -1 at 1273 K) than the current TBC materials [4,[15][16][17][18]. As Winter and Clarke pointed out, the low thermal conductivity may be attributed to the presence of several cation sublattices, large mean atomic mass and high concentration of structural oxygen vacancies [4]. For thermoelectric applications, an efficient material should exhibit high power factor and low thermal conductivity. Therefore, tremendous efforts have been made to reduce thermal conductivity of semiconductors by nanostructuring, composite and defect chemistry approaches [19][20][21][22][23]. However, for high temperature applications, stability of nanostructures may be an issue [23], and the thermoelectric community is actively looking for new materials with low intrinsic thermal conductivity [24][25][26][27][28][29][30][31]. Recently, Liu et al. have predicted very low thermal conductivity of scheelite type structure [32]. Scheelite oxides with general formula ABO 4 consists of strong units of BO 4 tetrahedra, week units of AO 8 as well as the anisotropy in A-O and B-O bonds that allow them having low thermal conductivity and mechanical properties which eventually can fulfil the requirements for next generation TBC materials. Various molybdenum-and tungsten-based scheelites (Ca, Sr, Ba) (Mo, W) O4 have been reported, by Liu et al. [32], predicting good intrinsic quasi-ductility and ultra-low thermal conductivity , ranging in 0.556 -1.2 W m -1 K -1 which is lower than common thermal insulating materials such as YSZ and pyrochlore oxides. Interestingly, in this study, results from previous experimental evidence and simulations models are successfully conducted. It is found that experimental result of thermal conductivity of BaWO 4 (1.63 W m -1 K -1 at 563 K) is consistent with theoretical predictions of Debye-Callaway model, thereby offering promising potential in exploring such materials for TBC applications. Among the compositions studied by Liu et al. [32], BaMoO 4 thermal conductivity is expected to reach value below 1 W m -1 K -1 according to the Debye-Callaway model. Another approach applied for reducing thermal conductivity consists in introducing defects on the cationic sublattice. Several studies reported that A-site deficiency in perovskite structures yields to lower thermal conductivity values [33,34]. Furthermore, Brixner et al. [35] have previously evidenced that it is easy to bring high defect concentrations at the A site of scheelites, and shown that vacancies can be as high as 1/3 in some rare-earth molybdate systems such as La 0.67 0.33 MoO 4 (also written as La 2 Mo 3 O 12 ) where stands for the vacancy site [35]. Assuming that such model predictions and experimental investigations are potentially valuable for improving TBC materials, our contribution in this work consists in studying structural and thermal properties of barium molybdate (BaMoO 4 ) belonging to scheelite family and the defect scheelite-type structure La 2 Mo 3 O 12 . Both ceramics were elaborated by solid state reaction method. A systematic study of the thermal conductivity of both samples was investigated from room temperature to 1000 K and differences were discussed. BaMoO 4 and La 2 Mo 3 O 12 ceramic samples were fabricated by conventional solid-state reaction starting from a stoichiometric mixture of the corresponding component oxides: BaCO 3 (99.9%), MoO 3 (99.9%) and La 2 O 3 (99.9%), purchased from ChemPur company. MoO 3 and La 2 O 3 reactants were preheated before weighing in air atmosphere respectively at 300 °C and 1000 °C overnight. The precursors were then grounded in planetary ball mill of tungsten carbide for 5 minutes at 300 rpm (Retsch PM 100). The mixed powders were then calcined in an alumina crucible, in a muffle furnace at 850 °C for 8 h at a heating rate of 5 °C min -1 in air, to ensure the single-phase formation. The calcined powders were then reground with few drops of polyvinyl alcohol (PVA) solution (2 wt% in water) and cold-pressed into pellets of about 13 mm in diameter. Finally, BaMoO 4 and La 2 Mo 3 O 12 pellets were heated in an alumina crucible at a heating rate of 5 °C min -1 , up to 650 °C for 2 h to drive off the PVA and then sintered respectively at 1100 °C and 980 °C for 4 h each. The density was determined from the weight and the calculated volume of the pellets.

The presence of tetragonal scheelite crystal structure and its phase purity were confirmed by X-ray diffraction (XRD) measurements [Bruker D8 advance diffractometer with CuKα radiations λ 1.5418 ¦ A]. XRD patterns were collected with a scanning speed of 2.4°(2Θ)/min with a step size of 0.02°over an angular range from 20°to 80°at room temperature. The microstructures of the samples were examined using scanning electron microscopy (TESCAN -MIRA 3 SEM) operating in backscattered electron (BSE) emission mode. The elementary composition was also analysed by Energy Dispersive X-ray (EDX) coupled to SEM with an acceleration voltage of 20 kV and a current of 20 pA. For both samples, a gold coating was deposited in order to reveal the surface and provide a conductive path for electrons.

The laser flash method (Netzsch LFA 457 instrument) was adopted for the measurement of the thermal diffusivity. Samples were coated with a thin layer of graphite in order to improve absorption of the laser light and then fixed in the holders of the LFA apparatus (6 × 6 mm). The measurements were carried out from 400 to 1000 K at 100 K intervals in a primary vacuum after three N 2 purges. In order to guarantee the reliability of the testing data, thermal diffusivity value was recorded three times at each temperature for averaging.

Specific heat capacity of both samples was evaluated by Differential Scanning Calorimeter (DSC, Netzsch STA 449 F3 Jupiter) analysis. The powdered samples of about 50 mg were spread evenly in a platinum crucible and heated continuously up to 1000 K under nitrogen atmosphere with a heating rate of 20 K min -1 . In conjunction with the DSC analysis, a Netzsch dilatometer (402 CL) was used to measure the thermal expansion evolution of the sintered La 2 Mo 3 O 12 pellet as a function of the temperature. Measurements were conducted during a single heating cycle from 400 to 1100 K at a heating rate of 10 K min -1 .

Based on the obtained experimental data of the thermal diffusivity (D), the heat capacity (C p ), and the density (ρ), one can deduce the thermal conductivity coefficient (κ) of both samples as follows:

κ = D × C p × ρ (1)
The as-sintered pellets were white in colour. The theoretical density of pure BaMoO 4 and La 2 Mo 3 O 12 is 4.738 and 4.865 g cm -3 , respectively [36,37]. The maximum achieved relative density is ≈ 95% and ≈ 92% of theoretical value for BaMoO The measured heat capacities of BaMoO 4 and La 2 Mo 3 O 12 in the temperature range of 400 K to 1000 K are shown in figure 3. For BaMoO 4 , our data are consistent with the Calvet micro-calorimeter measurements of Singh et al. [38] and they also join smoothly and have similar evolution with measurements made by Saha et al. [39] and Kubaschewsi et al. [START_REF] Kubaschewsi | Materials Thermochemistry 6th ed[END_REF]. For those studies, the reported heat capacity data varies from about 0.4 J g -1 K -1 across 300 K to 0.7 J g -1 K -1 at 1800 K. The heat capacity of La 2 Mo 3 O 12 have been measured for the first time. A peak at around 850 K can be observed. It indicates that material undergoes a reversible ferroelastic phase transition behavior from monoclinic (P2 1 /a) phase at low temperatures to orthorhombic (Pnca) phase at high temperatures. Accordingly, Sleight et al. [START_REF] Sleight | [END_REF] have reported that A 2 (MoO 4 ) 3 compounds with monoclinic crystal structure at room temperature show a transition between 25 and 600 °C.

The small magnitude of the heat effect accompanying the phase transition in A 2 M 3 O 12 samples could be based on the rotation of polyhedral network of the lattice [42,43].

As La 2 Mo 3 O 12 shows phase transition around 850 K, the dilatometric thermal expansion behavior was also analysed in order to evaluate the impact of phase transition on the dilatation during heat treatment up to 1100 K with a rate of 10 K min -1 . Results reveal that the as-sintered La 2 Mo 3 O 12 exhibits a positive thermal expansion coefficient. This latter is approximately linear and does not display discontinuities. Therefore, the phase transition from a monoclinic to an orthorhombic form is not reflected. The La 2 Mo 3 O 12 compound exhibited a thermal expansion coefficient (α L ) ranging from 10 to 9 × 10 -6 K -1 at temperatures from 400 to 1100 K, indicating that α L remains almost constant all along the temperature range. This result is consistent with previous results obtained on La 2 Mo 3 O 12 indicating an average linear expansion coefficient of about 6.9 × 10 -6 K -1 at 1000 K [44]. Therefore, this phase transition does not hinder the possibility to use La 2 Mo 3 O 12 compound as a TBC.

Using the above-mentioned parameters, the thermal conductivity was calculated by mean of equation 1. The temperature dependence of the thermal conductivity (κ) of both La 2 Mo 3 O 12 and BaMoO 4 sintered ceramics is illustrated in figure 4. BaMoO 4 thermal conductivity decreases from 0.8 W m -1 K -1 at 400 K with increasing temperature and reaches 0.65 W m -1 K -1 at 650 K and then increases up to 1.15 W m -1 K -1 . The La 2 Mo 3 O 12 thermal conductivity is almost constant starting from 0.59 W m -1 K -1 at 400 K to 0.62 W m -1 K -1 at 1000 K. As for measurement uncertainty, the overall average value of the relative standard uncertainty on the thermal conductivity was estimated to be ±11% [45]. Furthermore, in an attempt to exclude the effect of porosity, thermal conductivity values were normalized to represent values for 100% dense materials (κ dense ) using Maxwell's relation [4] as follow:

κ dense = κ measured × 1 1 -1.5Φ (2)
where Φ is the porosity. The corrected data, obtained with 100% dense material show a thermal conductivity of 0.87 and 0.65 W m -1 K -1 at 400 K for BaMoO 4 and La 2 Mo 3 O 12 , respectively.

As shown in figure 4, for BaMoO 4 sample, one can see that κ decreases from ≈ 0.87 W m -1 K -1 at 400 K with increasing temperature and reaches 0.7 W m -1 K -1 at 650 K. The values of κ are reasonably consistent with theoretical prediction based on Debye-Callaway model (κ 1473 =0.974 W m -1 K -1 ) for the same composition [32], except for low temperature below 600 K. This discrepancy could originate from additional defects formed during the synthesis and sintering processes. As predicted, the A +2 B +6 O 4 scheelites structure possesses low thermal conductivity. The low thermal conductivity may be attributed to the structural feature of a BO 4 tetrahedron and the anisotropy in A -O and B -O bonds. However, thermal conductivity increases for temperatures higher than 700 K. This could be related to the enhanced radiative thermal conductivity. Indeed, the thermal diffusivity measured by laser flash method includes both the lattice and radiative contributions [46]. At low temperature, as the pellets were coated with carbon black as reported previously, radiation effect could be neglected and temperature dependence of thermal conductivity accords well with the 1/T law. However, at higher temperature as the carbon coating layer was gradually removed, and the pellet's color turns to white, the radiative effect arises indicating extensive internal optical scattering which was also reported in previous studies [4,47].

In the case of La 2 Mo 3 O 12 , the thermal conductivity value is almost constant starting from 0.65 W m -1 K -1 at 400 K to 0.72 W m -1 K -1 at 1000 K. Noticeably, in comparison to previous researches on La 2 Mo 2 O 9 , for which the experimentally reported thermal conductivity varies from 0.82 W m -1 K -1 at 400 K to 0.95 W m -1 K -1 at 1000 K [4,15,17,18], the A-deficiency of La in La 2 Mo 3 O 12 can significantly lower the thermal conductivity value as already reported for perovskite structure [33,34]. As expected, by introducing vacancies on the scheelite structure an important decrease on thermal conductivity of about ≈ 23% compared to BaMoO 4 is obtained as a result of phonon scattering by point defects [33,34,47]. An ultra-low thermal conductivity of about 0.65 -0.7 W m -1 K -1 is obtained in the range of 400 -1000 K. As far as the authors know, to date, this is the lowest thermal conductivity value reported for a dense crystalline oxide.

In conclusion, stoichiometric and cation deficient scheelite ceramic materials, BaMoO 4 and La 2 Mo 3 O 12

were prepared by a conventional solid-state route and sintered respectively at 1100 °C and 980 °C for 4 h in air. Dense ceramic bodies (95 and 92%, respectively) have been obtained. Structural and thermal properties of scheelite structures were studied. BaMoO 4 compound exhibited a low thermal conductivity, below 1 W m -1 K -1 in the range 400-800 K. We reported an ultra-low thermal conductivity of ≈0.65 W m -1 K -1 for A-site deficiency La 2 Mo 3 O 12 compound, in the range of 400 -1000 K. As far as the authors know, this is the lowest thermal conductivity value reported for a dense crystalline oxide. Therefore, scheelite-type structure provides a highly promising route to further improve thermal barrier coating materials. Relative to thermoelectric properties, low thermal conductivity is a first step, but electric transport properties should be improved by substitutions, doping or oxygen non-stoichiometry. This will be the subject of further studies.

4 and La 2 Figure 1 :

 21 Figure 1: XRD patterns of the obtained BaMoO 4 and La 2 Mo 3 O 12 sintered ceramics at room temperature: (*) monoclinic La 2 Mo 3 O 12 , (•) La 2 Mo 2 O 9 and (•) tetragonal BaMoO 4 The scanning electron microscopic study of both fractured surfaces of BaMoO 4 and La 2 Mo 3 O 12 sintered ceramics are shown in figure 2. It is observed from the micrographs a dense and fine microstructure. The grain sizes are estimated from SEM micrographs. For both samples, the SEM images exhibit grain size ranging from 7 to 15 µm. The average grain size is circa 11 µm and 9 µm for BaMoO 4 and La 2 Mo 3 O 12 , respectively.

Figure 2 :

 2 Figure 2: BSE-SEM micrograph of the fractured surfaces of BaMoO 4 and La 2 Mo 3 O 12 sintered ceramics

Figure 3 :

 3 Figure 3: Mean heat capacity of BaMoO 4 and La 2 Mo 3 O 12 as function of temperature

Figure 4 :

 4 Figure 4: Calculated (solid line) and experimental (dash line) thermal conductivity of La 2 Mo 3 O 12 and BaMoO 4 sintered ceramics as a function of temperature. The vertical lines represents the total error bars.

  The average grain size is circa 11 µm and 9 µm for BaMoO 4 and La 2 Mo 3 O 12 , respectively. Some voids and pores can be observed for La 2 Mo 3 O 12 and BaMoO 4 ceramics, which is consistent with the relative density of about 92% and 95% respectively. EDX analyses or backscattered electrons images were unable to identify La 2 Mo 2 O 9 impurity.

	BaMoO 4	La 2 Mo 3 O 12
	95%	92%
	20 µm	20 µm

Acknowledgements

The authors are grateful to Tatiana Chartier for technical support.