Inés Castro

Nicolas Fabre

Sandra Bourgeade-Delmas

Nathalie Saffon

Chloé Gandini

Michel Sauvain

Denis Castillo

Geneviève Bourdy

Valérie Jullian

Structural Characterization and Anti-infective Activity of 9,10-Seco-29-norcycloartane Glycosides Isolated from the Flowers of the Peruvian Medicinal Plant Cordia lutea

published or not. The documents may come

ABSTRACT: Six new secocycloartane glycosides (1-6) were isolated from the ethanol extract of the flowers of Cordia lutea Lam. (Boraginaceae) on the basis of bioassay-guided fractionation. Their structures were determined by the application of NMR and MS data analyses together with X-ray crystallographic analyses for compounds 1 and 2. Compounds 1 -6 represent the first examples of 9,10-seco-29-norcycloartane glycosides. These compounds showed significant in vitro anti-Helicobacter pylori activity, and no activity against either Escherichia coli or Pseudomonas aeruginosa. Significant activity was observed for 5 and 6 against Staphylococcus aureus. All compounds displayed weak cytotoxicity against RAW 264.7 cells. The in vitro antileishmanial and antiplasmodial activities of 1-6 were also evaluated.

The genus Cordia (Boraginaceae) encompasses more than 300 species of trees and shrubs distributed in the tropical areas of Africa, America, and Asia, several of which possess different ethnopharmacological properties. [1][2][3] In Peru, the genus Cordia is represented by approximately 30 species, among which Cordia lutea has significant medicinal use.

The dried flowers of C. lutea, also known as "flor de overo", "overall" or "membrillejo", are commercialized and widely used in Northern Peru for the treatment of liver diseases, kidney pain, and gastrointestinal disorders. [START_REF] Mostacero-León | Asamblea Nacional de Rectores[END_REF] However, there have only been two reported phytochemical investigations of C. lutea. Mayevych and coworkers described some ubiquitous flavonoids and lipidic compounds and verified the absence of pyrrolizidine alkaloids in the whole plant. [START_REF] Mayevych | [END_REF] A second study reported the LC-MS analysis of a C. lutea flower extract. 6 This extract showed characteristic signals for the reported products, but some minor compounds remained unidentified. Furthermore, we found interesting activity for the ethanol extract of C. lutea dried flowers against the bacteria Helicobacter pylori, a microaerophilic gram-negative bacterium that has been associated with severe gastrointestinal disorders in humans such as chronic gastritis, peptic ulcers, MALT-type lymphoma, and gastric cancer. The prevalence of infection is considered to be above 70% in developing countries, while it ranges from 20 to 50% in developed countries. Antimicrobial resistance is an important global health problem that leads to therapeutic failure. 7 The limited knowledge about the chemical composition of the flowers of C. lutea, together with this interesting biological activity, prompted us to isolate the compounds responsible for the antibacterial effect.

Cycloartane-type triterpenoids and their glycosides have been described as major constituents of and are responsible for the biological activities of Cimifuga and Actae species (Ranunculaceae) and Astragalus species (Leguminosae). [8][9][10][11] Collectively, they represent approximately 900 structures. [START_REF] Azimova | Natural Compounds: Cycloartane Triterpenoids and Glycosides[END_REF] 9,10-Secocycloartanes comprise a small group of cycloartane triterpenoids featuring a seven-membered B ring. 9,[START_REF] Hao | [END_REF][14] Recently, an unusual 9,10secocycloartane triterpenoid was isolated for the first time from black cohosh (Actaea racemosa), a widely accepted herbal medicine used in the United States and Europe for the treatment of climacteric symptoms. 8 Herein is the first report of new glycosides derived from 4-methyl-9,10secocycloartane triterpenoids, cordiasecosides A-F, 1-6, isolated from the dried flowers of C. lutea. The antibacterial, antileishmanial, and antiplasmodial activities of these compounds were evaluated, as well as the cytotoxicity activity against RAW 264.7 macrophages.

RESULTS AND DISCUSSION

Compounds 1-6 were isolated from the ethanol extract of the flowers of C. lutea by bioguided fractionation. Their structures were assigned via the analysis of spectroscopic data, including 2D NMR techniques. 4.81, br s 5.47, q (6.9) 4.81, br s 5.47, q (6.9) 4.81, br s 5.47, q (6.9) + HCOO] -) and showed seven indices of hydrogen deficiency. The IR spectrum showed an absorption band for hydroxy groups at 3385 cm -1 .

The J-modulated [START_REF] Hao | [END_REF] C NMR (Table 1) and HSQC data of compound 1 showed 36 carbon signals, 30 of which were assignable to a triterpenoid skeleton and six to a hexosyl moiety. These carbons could be resolved as seven methyls, 10 methylenes, 14 methines, an oxygenated tertiary, and four quaternary carbons. The 13 C NMR spectrum showed four olefinic carbons at δ C 136.7 (C-9), 124.0 (C-11), 160.4 (C-24), and 105.5 (C-24 1 ).

Additionally, the 13 C NMR spectrum of 1 showed two oxygen-bearing carbons at δ C 71.8 (C-10) and 70.7 (C-23) as well as six additional carbon signals, which were in agreement with those previously described for a rhamnopyranosyl moiety. 15 All these data indicated that one index of hydrogen deficiency was due to the sugar moiety and that two indices of hydrogen deficiency were due to two double bonds. Thus, a tetracyclic core is consistent with the remaining four indices of hydrogen deficiency.

The 1 H NMR spectrum ( The 1 H NMR spectrum also showed two diagnostic methylene proton signals for H 2 -

19. Although they are typically present in the extremely upfield region in the 29norcycloartane skeleton, these proton signals were present as doublets at δ H 2.21 and 2.52 with a large geminal coupling constant (15.0 Hz), indicating that compound 1 is a 9,10-seco-29-norcycloartane rhamnoside. 9,18 As expected from its [START_REF] Hao | [END_REF] C NMR data, the 1 H NMR data (Table 2) also displayed signals for an olefinic methine proton at δ H 5.27 (br s, H-11) and two terminal olefinic methylene protons at δ H 4.81 (br s, H-24 1 ) and 5.00 (t, J = 1.0 Hz, H-24 1 ). The locations of the two double bonds (Δ 9 (11) , Δ The attachment of the rhamnopyranose moiety to C-3 was confirmed via the HMBC cross peak between the anomeric proton H-1' and C-3. The coupling constant of the anomeric proton at δ H 4.74 (d, J = 1.5 Hz, H-1') indicated an anomeric α-configuration of the rhamnopyranosyl unit. 15 The presence of an axial (α)-oriented H-3 methine signal at δ H 3.00 (dt, J = 4.6, 10.7

Hz) was deduced from its coupling constants, which suggested a diaxial vicinal interaction between H-3 and H-4. 17,20,21 This implied the presence of an α-oriented H 3 -28. NOESY correlations (Figure 3) between H-3 and H 3 -28 confirmed this assumption.

Further examination of the NOESY spectrum showed key correlations between H-5 and H-19a, H-11 and H-19 b , H-8 and H 3 -18, H 3 -18 and H-20, and H-17 and H 3 -28.

Comparison of these key correlations with those in the literature indicated that H- The structural skeleton of compound 1 resembles that of podocarpaside D, for which a putative biosynthetic pathway has been published. 18 Interestingly, when comparing the [START_REF] Hao | [END_REF] determined via Mosher's method the (23S) absolute configuration in a steroid with the same side chain, and showed that in this case, H-23 appeared as a triplet with a coupling constant of 6.7 Hz. 23,24 The latter was assigned as C-24 2 based on the 1 H-1 H COSY correlation between the methyl at δ H 1.62 (d, J = 6.9 Hz, H 3 -24 2 ) and H-24 1 and HMBC correlations between H 3 -24 2 and C-24

and between H-24 1 , C-23, C-24 2 , and C-25 (Figure 2). The NOESY correlations (Figure 3) between H-23 and H-24 1 and between H 3 -24 2 1 and2), as well as the 2D NMR spectra, were highly similar to those of 1, except for the signals of the sugar moiety. Thus, 3 and 5 are both analogues of 1 bearing a different sugar moiety. In the [START_REF] Hao | [END_REF] putative biosynthetic pathway for the formation of this type of secocycloartane glycoside has been proposed. 18 However, in addition to the formation of the seven-membered B ring, the cycloartane skeleton undergoes sterol-like 4α-demethylation and modification of the C-24 side chain by the introduction of a methyl group (compounds 1, 3, and 5) or two methyl groups (compounds 2, 4, and 6). These types of biosynthetic modifications have already been described for the bioformation of citrostadienol from cycloartenol during phytosterol biosynthesis. 26 Fractions of different polarities were assessed on an in vitro culture of the bacteria Helicobacter pylori during the first stages of the bioguided fractionation of the EtOH extract of C. lutea (MIC = 500 µg/mL). The CH 2 Cl 2 extract (MIC = 125 µg/mL) was significantly In conclusion, we report here a family of compounds isolated for the first time from C. lutea flowers, which presented interesting in vitro anti-H. pylori activity, but also show some toxicity. Despite their lack of selectivity, the identification and characterization of these potential biomarkers paves the way for future pharmacological and quality control studies of this herbal remedy. diffractometer equipped with a CMOS area detector. Data for compound 2 were collected at 106(2) K on a Bruker-AXS APEX II QUAZAR diffractometer equipped with a 30 W aircooled microfocus source, using MoKα radiation ( = 0.71073 Å). The data were integrated with SAINT, and an empirical absorption correction with SADABS was applied. [START_REF] Sadabs | Program for data correction[END_REF] The structure was resolved by using the intrinsic phasing method (SHELXT-2015) [START_REF] Sheldrick | [END_REF] and all nonhydrogen atoms were refined anisotropically using the least-squares method on . 31 The H atoms were refined isotropically at calculated positions using a riding model, except for the oxygen atoms. When possible, these H atoms were located by a difference Fourier map. For 1, the quality of the data did not allow us to locate all of the hydrogens on the oxygen atoms of the solvent molecules (H 2 O). The crystal was found to be nonmerohedrally twinned (matrix: 0 0 1, 0 -1 0, 1 0 0), and the data generated were used in the final refinement (refined BASF = 0.1988). [START_REF] Spek | Twin Rot Mat)[END_REF] For 2, some residual electron density due to the presence of disordered solvent was difficult to modelize. Therefore, the SQUEEZE function of PLATON 33 was used to eliminate the electron density contribution in the solvent region from the intensity data, and the solvent-free model was employed for the final refinement.

EXPERIMENTAL SECTION

Crystallographic data for the structures reported in this paper have been deposited with the Cambridge Crystallographic Data Centre with accession numbers CCDC 1891236 (1) and CCDC 1915222 (2). Copies of the data can be obtained, free of charge, upon application to the CCDC, 12 Union road, Cambridge CB2 1EZ, UK (fax: +44-(0) 1223-336033 or email: deposit@ccdc.cam.ac.uk). Crystal data for 1: 2(C36H60O7 H2O ,Mr = 677.84, monoclinic, space group P2 1 , , , , 34 The same procedure was accomplished with a mixture (4. Antiplasmodial Activity. The FcB1 strain of P. falciparum (chloroquine-resistant strain) was maintained in RPMI 1640 medium and 5% human serum (EFS, Toulouse, France). Parasitized RBCs were maintained in 25 cm 2 culture flasks in a controlled atmosphere. As a control, a stock solution of chloroquine was dissolved in culture medium (stock solution: 1 mg/mL). For the drug assays, serial drug dilutions in triplicate (100 µL/well) were performed in RPMI medium (final concentration of DMSO was 0.5%) and added to P. falciparum culture media (2% hematocrit, 1% parasitemia, 100 µL/well) in a 96well culture plates. Forty-eight hours after the beginning of incubation, the plates were washed three times with PBS and resuspended in 150 µL of PBS. Then, 100 μL of a lysis buffer containing 2× SYBR Green (Thermo Fisher) was added to 100 μL of each dilution of the parasitized RBCs + drugs in a dark 96-well plate and incubated in a dark place at room temperature for 2 h. Subsequently, the fluorescence values were determined using a fluorescence plate reader at 485 nm and 518 nm excitation and emission wavelengths, supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) were distributed in 96-well culture plates (Corning) and incubated at 37 °C and 5% CO 2 for 24 h. The medium was replaced by 100 µL of medium with compounds at different concentrations and incubated at 37 °C and 5% CO 2 for 48 h. The medium was replaced with RPMI-1640, and 20 μL of 5 mg/mL MTT was added to each well followed by incubation for 4 h at 37 °C. Formazan crystals were solubilized by adding 100 μL of a solution containing 50% isopropanol and 10% sodium dodecyl sulfate (IPA/SDS, pH 5.4), and the absorbance was measured at 550 nm. Controls for cell growth (without treatment) in the maximum DMSO concentration (0.1% for all assays)

, Z = 4, T = 193(2) K, V = 3864.2(2) Å 3 , , F (000)
and the fraction absorbance controls of all concentrations and medium controls were also included. The positive control doxorubicin (Sigma) was also included. The concentration of each fraction or pure compound that inhibited cell viability by 50% compared to the control (IC 50 ) was determined by dose-response curve fitting using the GraphPad Prism software (version 7 for Windows, GraphPad Software).

ASSOCIATED CONTENT Supporting Information. HRESIMS for 1-6, 1 H, [START_REF] Hao | [END_REF] C, HSQC, COSY, HMBC and NOESY NMR spectra for 1-6, UHPLC/MS chromatograms of samples and standards allowing the

Figure 1 .

 1 Figure 1. Structures of compounds 1-6.

Figure 2 .

 2 Figure 2. Key COSY (─) and HMBC (→) correlations for 1 and part of the structure of 2.

Figure 3 .

 3 Figure 3. Key NOESY correlations for compounds 1 and 2.

Figure 4 .

 4 Figure 4. Molecular views of compounds 1 and 2. The thermal ellipsoids represent a 50%

24(24 1 ))

 1 were supported by the HMBC cross peaks between H 2 -19, C-8, C-9, and C-11; between H 2 -24 1 , C-23, and C-25; and between H-25, C-23, and C-24 (Figure 2). The position of the oxymethine signal at δ H 4.11 (d, J = 10.2 Hz, H-23) was deduced via the presence of vicinal 1 H-1 H COSY correlations between H-20, H 2 -22, and H-23. Further HMBC correlations between H-25, C-23, and C-24 confirmed the location of the hydroxy group at C-23 (Figure 2). Similarly, the presence of an oxygenated tertiary carbon at C-10 was supported by HMBC correlations between H 2 -19, C-1, C-5, and C-10. The presence of two hydroxy groups and a rhamnosyl moiety were consistent with the HRESIMS data recorded in positive mode (Figure S2, Supporting Information). The ion at m/z 569 is attributable to the loss of two water molecules from the protonated molecule [M + H] + . The ion at m/z 423 is consistent with a consecutive neutral loss of the rhamnosyl unit, that is, [M + H -2H 2 O -146] + .19

  5 and H b -19 (δ H 2.21, d, J = 15.5 Hz) were α-oriented and H a -19 (δ H 2.52, d, J = 15.0 Hz) was β-oriented.Although the Flack parameter [0.2(2), CuKα radiation] was too large to be used to determine the absolute configuration, the relative configuration of compound 1, including the sugar moiety, was determined by X-ray crystallography (Figure4), which showed the  orientation of the 10-hydroxy group, and the relative configuration at C-23. Furthermore, the structure and absolute configuration of the monosaccharide moiety was identified as Lrhamnose after hydrolysis, derivatization, and UHPLC/MS analysis of a mixture containing the same amount of 1 and 2 (rt of sample: 19.73 min, rt of L-rhamnose standard: 19.73 min, rt of D-rhamnose standard: 11.80 min).22 This allowed definition of the structure of cordiasecoside A (1) as (23R)-3β,10β,23-trihydroxy-24-methyl-9,10-seco-29-norcycloart-9(11),24(24 1 )-diene-3-O-α-L-rhamnopyranoside.

  C NMR data of 1 with those of the structurally related 9,10-secocycloartane glycosides cimifoetidanolides A, B, C, and D, the chemical shifts of C-1, C-5, and C-10 (δ C 40.1, 52.4, 71.8) are closer to those of cimifoetidanolides A and C (δ C 41.0, 53.1, 72.0), with a 10β-hydroxy group, than to those of cimifoetidanolides B and D (δ C 32.7, 57.4, 74.6), which are their corresponding C-10 epimers. 9 Therefore, comparison of the C-1, C-5, and C-10 chemical shifts could be used to determine the C-10 configuration in similar compounds. Furthermore, regarding the C-23 absolute configuration, Ma et al. and Cheng et al.

  and H-25 established the E configuration of the Δ 24(24 1 ) double bond. The monosaccharide unit was identified as L-rhamnose, and singlecrystal X-ray diffraction analysis using MoKα radiation permitted the determination of the relative configuration only. The multiplicity and coupling constant of H-23 (d, J=10.0 Hz) are consistent with the (23R) absolute configuration. Consequently, the structure of cordiasecoside B (2) was defined as (24E,23R)-24-ethyl-3β,10β,23-trihydroxy-9,10-seco-29norcycloart-9(11),24(24 1 )-diene-3-O-α-L-rhamnopyranoside. Compounds 3 and 5 were isolated as white powders and showed a molecular ion in the negative mode HRESIMS at m/z 635.4167 [M + HCOO] -, which corresponded to the molecular formula C 35 H 58 O 7 . Positive mode HRESIMS data of 3 and 5 (Figures S20 and S38, Supporting Information) showed two ions at m/z 555 and 423, attributable to the loss of two water molecules from the protonated molecule [M + H] + , followed by the neutral loss of 132 units, that is, [M + H -2H 2 O -132] + , which may correspond to a pentosyl unit. The 1 H and 13 C NMR data of 3 and 5 (Tables

5 and 6 ,Compounds 4 Compounds 1 - 6

 56416 C NMR spectra of 3 and 5, five signals belonging to a carbohydrate moiety were observed, thus confirming the presence of a pentosyl moiety. The 2D 1 H-1 H COSY and HMBC correlations, as well as comparison with literature data, permitted the identification of a xylosyl unit for compound 3 and an arabinosyl moiety for compound 5.25 The NMR data of the xylosyl unit ( 1 H and 13 C) corresponding to C-2 ' , C-3 ' , and C-4' were distinguishable from those of the arabinosyl unit. This was confirmed by the TLC R f values of the monosaccharides obtained after acid hydrolysis of 3 and 5. Hydrolysis, derivatization, and analysis by UHPLC/MS of a mixture containing equivalent amounts of 3 and 4, and a mixture containing equivalent amounts of allowed the identification of D-xylose and L-arabinose, respectively, by comparing the retention times observed for the samples (16.61 min and 16.99 min) with those observed for D-and L-xylosyl standards (16.64 min, 15.86 min) and L-and D-arabinosyl standards (16.98 min, 17.32 min).22 The coupling constants of the anomeric protons in the 1 H NMR spectra of 3 (δ H 4.33, d, J = 6.6 Hz) and 5 (δ H 4.26, d, J = 6.4 Hz) indicated that the D-xylosyl and L-arabinosyl units were in the β and α configurations, respectively.25 These stereochemical assignments were confirmed by NOESY correlations between H-1' and H-3 in both compounds 3 and 5.Finally, comparison of the NOESY correlations of 3 and 5 with those of 1 indicated the same relative configuration. We assumed that the absolute configurations of their seconorcycloartane skeleton were the same as in compound 1. Hence, the structure of cordiasecoside C (3) was defined as (23R)-3β,10β,23-trihydroxy-24-methyl-9,10-seco-29norcycloart-9(11),24(241 )-diene-3-O-β-D-xylopyranoside and cordiasecoside E (5) as (23R)-3β,10β,23-trihydroxy-24-methyl-9,10-seco-29-norcycloart-9(11),24(24 1 )-diene-3-O-α-Larabinopyranoside. and 6 had the same molecular formula, C 36 H 60 O 7 , based on their negative mode HRESIMS data (m/z 649.4323, [M + HCOO] -). The 1 H and 13 C NMR spectra of 4 showed a general resemblance to those of 3 except for the presence of an additional methyl group (C-24 2 ), as previously described for 2. Cordiasecoside D (4) was therefore characterized as (24E,23R)-24-ethyl-3β,10β,23-trihydroxy-9,10-seco-29-norcycloart-9(11),24(24 1 )-diene-3-O-β-D-xylopyranoside. Similarly, when comparing the 1 H and 13 C NMR data of 5 and 6, a methyl group linked to the double bond of the side chain was also identified. Therefore, cordiasecoside F (6) was characterized as (24E,23R)-24-ethyl-3β,10β,23-trihydroxy-9,10-seco-29-norcycloart-9(11),24(24 1 )-diene-3-O-α-Larabinopyranoside. represent the first secocycloartane compounds isolated from the genus Cordia. They were identified as monoglycosylated triterpenoids with two nuclei in common and attached to rhamnopyranosyl, xylopyranosyl and arabinopyranosyl moieties. A

5 and 6 . 6 .

 566 7 mg) containing equivalent amounts of compounds 3 and 4 and a mixture (4.7 mg) containing equivalent amounts of compounds These two last hydrolyzed mixtures were analyzed by TLC over silica gel (CHCl 3 -MeOH-H 2 O, 8:5:1) by comparison with authentic samples. The following sugars were detected: xylose for compounds 3 and 4 and arabinose for compounds 5 and 6. Furthermore, the absolute configuration of the sugar moiety was determined as previously described by Wang et al., with slight modifications. 22 Briefly, derivatization of the monosaccharide enantiomers with L-cysteine methyl ester and phenylisothiocyanate converted them into diastereoisomeric arylthiocarbamates, which were separated and detected by UHPLC/MS analysis. The three hydrolyzed mixtures (2 mg) were dissolved in 120 µL of a solution of Lcysteine methyl ester in pyridine (0.3 M), and incubated for 1 h at 90°C, then 160 µL of a solution of phenyl isothiocyanate in pyridine (0.69 M) was added, and the mixture was incubated for 1 h at 90 °C. The reaction mixture was allowed to cool and diluted 20-fold with CH 3 CN and filtered before UHPLC/MS analysis. The injection volume was 6 µL. Each sugar standard (2 mg of L-rhamnose, D-rhamnose, L-xylose, D-xylose, L-arabinose, and D-arabinose) was subjected to the same protocol, with 240 µL of the L-cysteine methyl ester solution and 320 µL of the phenyl isothiocyanate solution. The reaction mixtures were diluted 60-fold with CH 3 CN and filtered before UHPLC/MS analysis. The injection volume was 3 µL. The obtained solutions were analyzed with an ACQUITY UPLC BEH C 18 column (100 × 2.1 mm, 1.7 µm), Waters. The column temperature was 35 °C, and the flow rate was 0.3 mL/min. Eluent A was a mixture of water and formic acid (0.05%). Eluent B was a mixture of CH 3 CN, MeOH, and iPrOH (50/25/25 v/v) with 0.05% formic acid. The gradient consisted of linear mixtures of A and B, with 14% of eluent B at t = 0, reaching 30% at t = 22 min, and 90% at t = 25 min. Then, the return to initial conditions was achieved at t = 28 min and maintained for 2 min before the next injection. Analysis of the positive extracted ion chromatograms at m/z 403 and 417 (corresponding to the [M + H] + adduct for the arylthiocarbamates obtained from arabinose, xylose and rhamnose) obtained from the samples and standards allowed for the identification of the sugar moieties in compounds 1-The observed retention times were 19.73 min, 16.61 min, and 16.99 min for the samples obtained from 1 and 2, 3 and 4, and 5 and 6, respectively.

  respectively. The fluorescence (after subtraction of the background fluorescence for the nonparasitized RBCs) values were plotted, and the IC 50 values were determined by linear regression analysis. Cell Viability Assay. The evaluation of cell viability was measured using the MTT assay according to Mosmann (1983) with slight modifications. Murine macrophage-like RAW 264.7 cells (5 10 4 cells/mL) in 100 µL of RPMI-1640 medium (Thermo Scientific)

Table 1 . 13 C NMR Spectroscopic Data (125 MHz, CDCl 3 -Methanol-d 4 9:1) for Compounds 1-6 (δ in ppm)

 1

	Position	Type	1	2	3	4	5	6
	1	CH2	40.1	40.1	40.1	40.1	40.1	40.1
	2	CH2	28.7	28.7	29.1	29.1	29.1	29.1
	3	CH	85.2	85.2	85.6	85.6	85.5	85.5
	4	CH	39.6	39.5	39.6	39.6	39.6	39.6
	5	CH	52.4	52.3	52.5	52.5	52.5	52.5
	6	CH2	31.9	31.9	31.8	31.8	31.8	31.8
	7	CH2	26.5	26.5	26.5	26.5	26.5	26.5
	8	CH	48.4	48.4	48.4	48.3	48.3	48.3
	9	C	136.7	136.5	136.6	136.6	136.6	136.6
		C	71.8	71.8	71.7	71.7	71.7	71.7
		CH	124	124.1	124.1	124.1	124	124
		CH2	38.3	38.3	38.3	38.3	38.3	38.3
		C	44	44	44.1	44.1	44.1	44.1
		C	48.7	48.7	48.7	48.7	48.7	48.7
		CH2	32.9	32.9	32.9	32.9	32.9	32.9
		CH2	27.9	27.9	27.8	27.9	27.8	27.9
		CH	51.5	51.5	51.4	51.5	51.4	51.5
		CH3	15.7	15.7	15.7	15.7	15.7	15.7
		CH2	50.5	50.5	50.5	50.5	50.5	50.5
		CH	33	33.2	33	33.2	33	33.2
		CH3	17.9	17.9	17.9	17.9	17.9	17.9
		CH2	43.4	44.8	43.7	44.8	43.7	44.8
		CH	70.7	68.9	70.9	68.9	70.8	68.9
		C	160.4	149.3	160.4	149.3	160.4	149.3
	24 1	CH	105.5	118.1	105.5	118.1	105.5	118.1
	24 2	CH3	-	12.9	-	12.9	-	12.9
		CH	30.5	27.9	30.5	27.9	30.5	27.9
		CH3	23.2	21.2	23.2	21.2	23.2	21.2
		CH3	22.4	21.2	22.3	21.2	22.3	21.2
		CH3	15.5	15.5	15.4	15.3	15.4	15.4
		CH3	16.6	16.5	16.5	16.5	16.5	16.5
	1'	CH	102.2	102.2	104.6	104.7	104.5	104.6
	2'	CH	70.8	70.9	72.8	72.9	71.3	71.3
	3'	CH	71.3	71.4	75.1	75.1	72.6	72.6
	4'	CH	72.9	72.9	69.5	69.5	67.5	67.6
	5'	CH/CH2 a	68.2	68.1	64.6	64.6	64.8	64.9
	6'	CH3	17.1	17.1	-	-	-	-

a: CH for 1-2, CH 2 for 3-6.

Table 2 . 1 H NMR Spectroscopic Data (500 MHz, CDCl 3 -Methanol-d 4 9:1) for Compounds 1-6 (δ in ppm)

 2

	Position	1	2	3	4	5	6
	1	1.48 a	1.48 a	1.48 a	1.48 a	1.48 a	1.48 a
	2	1.74, m	1.74, m	1.75, m	1.76, m	1.74, m	1.74, m
		1.83, m	1.83, m	1.88, m	1.88, m	1.90, m	1.91, m
	3	3.00, td (10.7, 4.6) 3.00, td (10.7, 4.6)	3.06, td (10.8, 4.6)	3.06, td (10.8, 4.6)	3.05, td (10.8, 4.6) 3.05, td (11.0, 4.6)
	4	1.40, m	1.40, m	1.48 a	1.48 a	1.48 a	1.48 a
	5	0.95, m	0.95, m	0.95, m	0.95, m	0.95, m	0.95, m
	6	1.17, m	1.17, m	1.19, m	1.18, m	1.19, m	1.18, m
		2.09, m	2.09, m	2.10, m	2.11, m	2.10, m	2.10, m
	7	1.30 a	1.29 a	1.30 a	1.29 a	1.29 a	1.29 a
		1.48 a	1.48 a	1.48 a	1.48 a	1.48 a	1.48 a
	8	2.15 a	2.15, d (10.9)	2.15 a	2.16, d (10.8)	2.15 a	2.16, d (11.2)
		5.27, br s	5.27, br s	5.27, br s	5.27, br s	5.27, br s	5.26, br s
		1.96, br s	1.96, br s	1.96, br s	1.96, br s	1.96, br s	1.96, br s
		1.31 a	1.32 a	1.30 a	1.32 a	1.31 a	1.31 a
		1.31 a	1.32 a	1.30 a	1.32 a	1.31 a	1.31 a
		1.89, m	1.89, m	1.87, m	1.88, m	1.87, m	1.88, m
		1.48 a	1.48 a	1.48 a	1.48 a	1.48 a	1.48 a
		0.76, s	0.76, s	0.76, s	0.75, s	0.75, s	0.75, s
		2.21, d (15.5)	2.21, d (15.6)	2.21, d (15.5)	2.21, d (15.6)	2.21, d (15.6)	2.21, d (15.7)
		2.52, d (15.0)	2.52, d (15.2)	2.52, d (15.2)	2.52, d (15.5)	2.52, d (15.3)	2.52, d (15.1)
		1.70, m	1.65, m	1.68, m	1.65, m	1.68, m	1.65, m
		0.91, d (6.3)	0.91, d (6.3)	0.91, d (6.4)	0.91, d (6.3)	0.91, d (6.4)	0.91, d (6.4)
		1.09, m	0.99 a	1.09, m	0.99 a	1.09, m	0.99 a
		1.53, m	1.57, m	1.51, m	1.57, m	1.51 a	1.57, m
		4.10, d (10.2)	4.12, d (10.0)	4.10, d (10.4)	4.12, d (10.3)	4.10, d (10.4)	4.12, d (10.4)
	24 1

Table 2

 2

	) of 1 displayed methyl signals associated with a 29-
	norcycloartane skeleton including two angular methyl groups at δ H 0.76 (s, H 3 -18) and 0.61
	(s, H

3 -30)

and four secondary methyl groups at δ H 0.91 (d, J = 6.3 Hz, H-21), 1.01 (d, J = 6.8

Hz, H-26), 1.03 (d, J = 6.8 Hz, H-27) and 0.90 (d, J = 6.4 Hz, H-28). These signals suggested the presence of a single methyl substituent at C-4 as demonstrated for cycloeucalenol, a biosynthetic intermediate of 4-methylphytosterols.

16,17

Table 4 . Anti-infective Activities of Compounds 1-6

 4

	No.	H. pylori CIP 103995 MIC 72 h (µg/mL)	S. aureus ATCC 25923 MIC 24 h (µg/mL)	E.coli ATCC 25922 MIC 24 h (µg/mL)	P. aeruginosa ATCC 27853 MIC 24 h (µg/mL)	P. falciparum FcB1 IC50 (µM) a	L. infantum IC50 (µM) a	RAW Cells 267.7 CC50 (µM) b
	1	31.3	>125	>125	>125	55.0 ± 6.5	6.7 ± 1.6	35.1 ± 4.5
	2	31.3	>125	>125	>125	61.3 ± 1.7	5.2 ± 1.3	21.9 ± 1.5
	3	15.6	62.5	>125	>125	>150	5.0 ± 0.8	24.2 ± 3.9
	4	15.6	>125	>125	>125	46.9 ± 4.3	5.2 ± 1.3	30.3 ± 3.9
	5	15.6	31.3	>125	>125	52.4 ± 9.4	7.7 ± 3.0	26.6 ± 1.7
	6	15.6	31.3	>125	>125	53.7 ± 4.9	6.3 ± 1.3	19.6 ± 1.7
	PC	Clarithromycin 0.01	Tetracycline 0.5	Tetracycline 2	Tetracycline 16	Chloroquine 0.15	Amph. B c 0.07	Doxorubicin 0.80

a IC50: 50% inhibitory concentration. b CC50: 50% cytotoxic concentration. PC: positive control. c Amphotericin B

  chromatographed with an authentic standard of rutin. The aqueous residue was extracted sequentially with CH 2 Cl 2 . The CH 2 Cl 2 extract (14 g) was suspended in 800 mL of MeOH/H 2 O (90/10) and extracted with petroleum ether (4 800 mL). Evaporation under reduced pressure of the upper and lower phase gave rise to 6 g and 8 g of residue, respectively. Each of the organic extracts (CH 2 Cl 2 , MeOH, and petroleum ether), the aqueous residue, and the yellow precipitate were tested in the H. pylori assay. The MeOH fraction was shown to have the greatest activity against the H. pylori strain. O CH 3 CN (50:50) at 3 mL/min to isolate 5 (15 mg) and 6 (12 mg) as white amorphous solids and other minor fractions.

	Experimental Procedures. Melting points were determined using an Electrothermal IA 9200 melting point apparatus. Optical rotations were determined with a JASCO P2000 digital polarimeter. Infrared (IR) spectra were obtained on a Perkin Elmer Frontier FT/IR spectrophotometer. The NMR spectra were recorded on a Bruker Avance 500 MHz instrument with samples diluted in CDCl 3 (δ H 7.28 and δ C 77.1) and CD 3 OD (δ H 3.33 acquired and processed using LCQ Xcalibur 3.0 software (Thermo Fisher Scientific). ESI parameters were tuned as follows: capillary temperature was fixed at 300 °C, sheath gas flow rate and auxiliary gas flow rates were, respectively, at 55 and 10 arbitrary units. The source voltage was at 3.5 kV, and the source current was 100 µA. Capillary and tube lens voltages were set to 50 and 120 V, respectively. A mass range of 100 to 2000 m/z with a resolution of 15000 [full width at half maximum (FWHM) of 400 m/z] was applied. All MS data were first acquired in full MS scanning mode followed by MS/MS data dependent in CID fragmentations at 35 arbitrary energy units. Merck Silica gel 60 (15-40 µm) was used for medium pressure column chromatography (MPLC). Analytical thin-layer chromatography was achieved on precoated silica gel plates (Merck, Kieselgel 60 F 254 , 0.25 mm) using UV 254 nm and a 1% vanillin/10% H 2 SO 4 reagent in EtOH for visualization. Semipreparative high-performance liquid chromatography (HPLC) was carried out using a LaChrom Merck Hitachi system consisting of a LaChrom L-7100 Pump, a L-7455 DAD, a D-7000 interface and employing a Phenomenex Luna 5 µm C18(2) 100 Å, 250 10 mm 2 column. Extraction and Isolation. The air-dried and powdered flowers of C. lutea (900 g) were extracted with 95% EtOH (4 9 L) at room temperature for 24 h to give 372 g of dry The MeOH extract (6 g) was fractionated by successive MPLC (silica gel 60) eluted with EtOAc containing increasing proportions of MeOH (200 mL 0%, 200 mL 10%, 200 mL 20%, 150 mL 30%, 60 mL 40% and 60 mL 50%) with the collection of 10 mL fractions. Fraction 3 (762 mg) was chromatographed over silica gel 60 and eluted with EtOAc MeOH (9.5:0.5) to afford six subfractions. Subfraction 3-3 (235 mg) was fractionated over silica gel and eluted with CH 2 Cl 2 MeOH (9.5:0.5) to afford a mixture (78 mg) that was further separated by semipreparative RP C 18 HPLC eluted with H 2 O CH 3 CN (20:80) at 3 mL/min to isolate 1 (15 mg) and 2 (8 mg) as white amorphous solids. Fraction 4 (983 mg), was chromatographed over silica gel 60 and eluted with EtOAc MeOH (9:1) to afford three subfractions. Subfraction 4-3 (280 mg) was fractionated over silica gel 60 and eluted with CH 2 Cl 2 MeOH (9.5:0.5) to afford a mixture (96 mg) that was further separated by preparative RP C 18 HPLC eluted with H 2 O CH 3 CN (20:80) at 3 mL/min to isolate 3 (15 Cordiasecoside A (1): colorless needles (MeOH); mp 253-254 °C; +2 (c 1, MeOH); IR υ max 3385, 2922, 2873, 1374, 1045 cm -1 ; 1 H and 13 C NMR data, see Tables 1 and 2; HRESIMS m/z 649.4324 [M + HCOO] -Δppm = 0.4 (calcd for C 37 H 61 O 9 -, 649.4321). Cordiasecoside B (2): white amorphous solid; +3 [c 0.5, CH 2 Cl 2 -MeOH (9:1)]; IR υ max 3408, 2925, 2868, 1374, 1045 cm -1 ; 1 H and 13 C NMR data, see Tables 1 and 2; HRESIMS m/z 663.4481 [M + HCOO] -Δppm = 0.5 (calcd for C 38 H 63 O 9 -, 663.4478). Cordiasecoside C (3): white amorphous solid; +14 [c 0.4, CH 2 Cl 2 -MeOH (9:1)]; IR υ max 3418, 2920, 2871, 1374, 1040 cm -1 ; 1 H and 13 C NMR data, see Tables 1 and 2; HRESIMS m/z 635.4167 [M + HCOO] -Δppm = 0.5 (calcd for C 36 H 59 O 9 -, 635.4165). Cordiasecoside D (4): white amorphous solid; +15 [c 0.3, CH 2 Cl 2 -MeOH (9:1)]; IR υ max 3418, 2917, 2871, 1374, 1037 cm -1 ; 1 H and 13 C NMR data, see Tables 1 and 3; HRESIMS m/z 649.4323 [M + HCOO] -Δppm = 0.3 (calcd for C 37 H 61 O 9 -, 649.4321). Cordiasecoside E (5): white amorphous solid; +26 [c 0.4, CH 2 Cl 2 -MeOH (9:1)]; IR υ max 3418, 2920, 2871, 1374, 1050 cm -1 ; 1 H and 13 C NMR data, see Tables 1 and 2; HRESIMS m/z 635.4170 [M + HCOO] -Δppm = 0.8 (calcd for C 36 H 59 O 9 -, 635.4165). Cordiasecoside F (6): white, amorphous solid; +24 [c 0.4, CH 2 Cl 2 -MeOH (9:1)]; IR υ max 3413, 2920, 2871, 1374, 1050 cm -1 ; 1 H and 13 C NMR data, see Tables 1 and 3; HRESIMS m/z 649.4323 [M + HCOO] -Δppm = 0.3 (calcd for C 37 H 61 O 9 -, 649.4321). X-ray Crystal Structure Analysis and Crystallographic Data of compounds 1 and 2. Compounds 1 and 2 were crystallized by slow evaporation from MeOH at room and δ an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). All spectra were residue (yield 33% w/w). Each 80 g of the dried residue was dissolved in 800 mL of distilled mg) and 4 (12 mg) as white amorphous solids and other minor fractions. Fraction 5 (618 mg) temperature. X-ray crystallographic diffraction data were measured for 1 with Cu Κ
	radiation (wavelength	) at 193(2) Κ on a Bruker-AXS D8 Venture

C 49.1) with a 9/1 ratio. UHPLC/MS analysis was performed using a UHPLC Ultimate 3000 system (Dionex) controlled by Chromeleon Xpress 6.8 software (Dionex), coupled with Plant Material. Flowers from C. lutea Lam. were purchased from a local market in Trujillo, Peru on May 2017. The plant was authenticated by the botanist Severo Baldeón. A voucher specimen was deposited at the National Herbarium of the San Marcos University in Lima, Peru under register No 032-2017-USM-MHN.

water, and the extracted solution was left for 12 h at 4 °C. The extracted solution was centrifuged for 10 min at 5000 rpm to recover 48 g of a yellow precipitate, which was co-Based on TLC analysis, the eluted fractions were pooled into six fractions (1-6), each of which was tested in the H. pylori assay. Fractions 3 (762 mg), 4 (983 mg), and 5 (618 mg) all showed activity against the H. pylori strain.

was chromatographed over silica gel 60 and eluted with CH 2 Cl 2 MeOH (9.5:0.5) to afford a mixture (128 mg) that was further separated by preparative RP C 18 HPLC eluted with H 2

  After extraction with CH 2 Cl 2 (3 × 5 mL), the aqueous layer was repeatedly evaporated to dryness with MeOH until it reached a neutral pH.

	650.90, orthorhombic, space group P2 1 2 1 2 1 ,	,	,
		, Z = 4, T = 106(2) K, V = 3901.6(9) Å 3 ,	, F (000) =
	1432, Dc = 1.108 mg/m 3 . A total of 81970 reflections were collected in the range of
			, with 6597 independent reflections	; completeness
	to	was 98.8%; final	indices	,	; absolute
	structure parameter -0.2(7).
		Acid Hydrolysis and Identification of the Sugar Moieties. A mixture (4.6 mg)
	containing equivalent amounts of compounds 1 and 2 was refluxed in 2N aqueous CF 3 COOH
	(3 mL) for 2 h at 100 °C.
						=
	1476, Dc = 1.165 mg/m 3 . A total of 61058 reflections were collected in the range of
			, with 12462 independent reflections	;
	completeness to	was 98.8%; final indices	,	;
	absolute structure parameter 0.2(2). Crystal data for 2: C37H62O7 H4O (+ solvent), Mr =

  °C under the microaerophilic conditions of 5% O 2 and 10% CO 2 for 72 h. Bacterial growth was confirmed by Gram staining, urease, catalase and oxidase tests. Cultures of the other reference strains were performed in Mueller-Hinton agar. Samples were dissolved in dimethyl sulfoxide (DMSO) to an initial concentration of 100 mg/mL and further dissolved in BHI medium supplemented with 10% fetal bovine serum (FBS) for H. pylori and Mueller-Hinton broth for E. coli, S. aureus and P. aeruginosa strains. The minimal inhibitory concentration (MIC) was determined by the broth microdilution method in 96-well culture plates. Fifty microliters of the extracts, fractions or compounds at concentrations ranging from 3.9 to 250 µg/mL (using two-fold serial dilutions) were added to each well and incubated with 50 µL of the bacterial inoculum at a final concentration of 1 × 10 6 CFU/mL. Clarithromycin at 0.0012 to 0.04 µg/mL (H. pylori) and tetracycline at 0.125 to 64 µg/mL (E. at a density of 2 × 10 6 parasites/mL in sterile 96-well culture plates with various concentrations of compounds dissolved in DMSO (final concentration of DMSO of less than 0.5% v/v), in duplicate. Appropriate controls, DMSO and amphotericin B, were added to each set of experiments. After a 48 h incubation period at 37 °C, each plate-well was then microscopically examined to detect any precipitate formation. To estimate the luciferase activity of the axenic amastigotes, 80 µL of each well was transferred to white 96-well plates, Steady Glow® reagent (Promega) was added according to the manufacturer's instructions, and plates were incubated for 2 min. The luminescence was measured in a Microbeta luminescence counter (PerkinElmer). The inhibitory concentration 50% (IC 50 ) was defined as the concentration of drug required to inhibit the metabolic activity of L. infantum amastigotes by 50% compared to the control. IC 50 values were calculated by nonlinear regression analysis processed on dose response curves, using TableCurve 2D V5 software. IC 50 values reported represent the mean value.

	Following incubation, the plates were examined visually and spectrophotometrically at 660
	These retention times were in agreement with those observed for L-rhamnose (19.73 min), D-nm, from which the lowest concentration showing complete inhibition of growth was
	xylose (16.64 min), L-arabinose (16.98 min), compared to D-rhamnose (11.80 min), L-xylose recorded as the MIC, according to the Clinical and Laboratory Standards Institute, CLSI
	(15.86 min), D-arabinose (17.32 min) (see Supporting Information). (2007). Tests were performed in triplicate.
	MIC Determination. H. pylori CIP 103995 (reference strain) was obtained from Antileishmanial Activity. L. infantum promastigotes (MHOM/MA/67/ITMAP-263,
	Prof. Christine Roques (Laboratoire de Génie Chimique, UMR CNRS 5503, Département CNR Leishmania, Montpellier, France, expressing luciferase activity) in log-phase growth
	BioSym, Faculté de Pharmacie, Université Paul Sabatier). Reference strains E. coli ATCC were centrifuged at 900 g for 10 min. The supernatant was removed carefully and replaced by
	25922, P. aeruginosa ATCC 27853 and S. aureus ATCC 25923 were also tested. Stock the same volume of RPMI 1640 complete medium at pH 5.4 and incubated for 24 h at 24 °C.
	cultures of the strains were stored at -80 °C in brain heart infusion broth (BHI) (BBL) The acidified promastigotes were incubated for 24 h at 37 °C in a ventilated flask.

containing 10% (w/v) glycerol until use. The H. pylori strain was grown in BHI agar (BBL) supplemented with Skirrow (Oxoid), isovitalex (BD), amphotericin B and 10% sheep blood at 37 coli, S. aureus and P. aeruginosa) were used as positive controls. The plates containing E. coli, S. aureus and P. aeruginosa were incubated for 24 h at 37 °C, while the plates containing H. pylori were incubated for 72 h at 37 °C under microaerophilic conditions.

Promastigotes were transformed into amastigotes. The effects of the tested compounds on the growth of L. infantum axenic amastigotes were assessed as follows. L. infantum amastigotes were incubated

determination of the glycosidic moieties of 1-6, and the checkCIF files for X-ray analysis of 1 and 2 are available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENTS

This research was supported by the Institut de Recherche pour le Développement of France (IRD) through The Allocations de Recherche pour une Thèse au Sud (ARTS) grant awarded to Castro I. The authors thank as well the logistic support of the LMI-LaVi (UPCH-IRD). The authors are grateful to Pierre Lavedan, Marc Vedrenne and Caroline Toppan from the NMR platform of the Institut de Chimie de Toulouse (ICT) for NMR analyses. Previous funding from the Peruvian Council of Science, Technology and Innovation (CONCYTEC) is acknowledged. The authors are indebted to the personnel of Universidad Nacional Mayor San Marcos, Lima, Peru, (UNMSM) herbarium for plant determination and the Servicio Nacional Forestal y de Fauna Silvestre (SERFOR) for issuing the C. lutea collecting permit (No 146-2016-SERFOR/DGGSPFFS).

active against H. pylori; thus, it was further partitioned between MeOH (MIC = 62.5 µg/mL) and petroleum ether (MIC > 1000 µg/mL). Cordiasecosides A-F were responsible for the in vitro anti-H. pylori activity of the MeOH extract (Table 4). In a previous study, the anti-H. pylori activity of plant extracts or fractions was classified as follows: strong (MIC < 10 µg/mL), strong-moderate (MIC = 10-100 µg/mL) and weak-moderate (MIC = 100-1000 µg/mL). 27 Cordiasecosides C, D, E, and F exhibited moderately strong activity (MIC = 25 µM) comparable to the activity of cordiasecosides A and B (MIC = 50 µM). To gain information on their antibacterial selectivity against H. pylori, the cordiasecosides were also tested against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa.

Interestingly, the cordiasecosides were inactive against E. coli, and P. aeruginosa, which are Gram negative bacteria, as H. pylori, and only cordiasecosides E and F displayed significant activity on S. aureus, a Gram positive bacteria. Therefore, cordiasecosides displayed some selectivity against H. pylori.

All isolated compounds were also tested for their anti-parasitic activity against axenic amastigotes of Leishmania infantum and the chloroquinine-resistant FcB1 strain of Plasmodium falciparum (Table 4). They displayed weak antiplasmodial activity with IC 50 values ranging from 46.9 to 61.3 µM. All isolated compounds showed a moderate activity against the axenic amastigotes of L. infantum, with IC 50 values ranging from 5.0 to 7.7 µM.

To compare the anti-infective potential of cordiasecosides A-F with their toxicity on healthy mammalian cells, and evaluate their selectivity, the cytotoxicities of cordiasecosides A-F were tested against a murine macrophage cell line (RAW 264.7) using the MTT assay (Table 4). They displayed IC 50 values ranging from 19.6 to 35.1 µM, which indicated weak cytotoxicity (IC 50 > 10 µM), but also low selectivity. 9,28

AUTHOR INFORMATION

Corresponding Author

*Tel: +33-562256886. E-mail: valerie.jullian@ird.fr ORCID Valérie Jullian: 0000-0002-1675-9105

Notes

The authors declare no competing financial interests.


Mounia Farah

Jacqueline Saliba

Frédéric Grondin

Ahmed Loukili

Multi-Scale Methods for the Analysis of Creep-Damage Coupling in Concrete

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

Introduction

In civil engineering, the control of creep has a major role in concrete structure design. Indeed, the viscoelasticity of concrete leads to delayed deformations which could be at the origin of cracking in structures [OMA 09]. So, these delayed strains have an impact on their safety margin and life time, especially when they are subjected to high loads. For example, underground concrete structures and other infrastructures undergo, during the construction phase, load due to soil pressure, which leads to important creep deformations.

According to several studies, the viscoelastic behavior of concrete is strongly linked to the main hydrates of the cement paste (C-S-H) which could deform under a constant load [BAZ 88]. The magnitude of these deformations is very significant in the cement paste of hardening concrete [BRI 11]. Also, some theories suggested that micro-cracks nucleate and grow when concrete is subjected to a high sustained loading [BAR 05, BAZ 97b, ROS 94], which makes the concrete weaker. Thus, it is important to understand the interaction between viscoelastic deformation and damage in order to design reliable civil engineering structures with reference to delayed strains and residual strength.

The study of tensile creep has shown different effects on the behavior of concrete with sometimes some contradiction. On one hand, creep may relax internal tensile stresses generated by restrained shrinkage, by increasing the deformation capacity to the failure and consequently reducing the risk of potential cracking in concrete [ALT 03, BIS 00]. On the other hand, the main mechanism of tensile creep is due to the micro-cracks development, which could be responsible of the decrease of the residual strength and the modification of concrete's properties [COO 74,ROS 94]. In order to enhance the current knowledge of creep mechanisms of concrete, it is interesting to study the creep response and phenomena occurring at the component level. Concrete can be properly represented by three phases in the microstructure: cement paste, aggregate and the interfacial transition zone (ITZ) between them [XIA 13, ZIM 85].

To examine this, an experimental investigation on mortar and concrete beams has been realized to study how aggregates influence the mechanical properties and damage behavior of concrete under creep. The acoustic emission (AE) technique was applied simultaneously to monitor damage evolution during the failure flexural tests of concrete and mortar beams. This non-destructive technique proves to be very effective, especially to check and measure micro-cracking that takes place inside a structure under a mechanical loading [ALA 14, DEN 06, SAL 12a, SAL 12b, SAL 12c].

The use of classical creep models [BAZ 89, DES 99] to simulate the macroscopic viscoelastic deformation of concrete structures requires the calibration of viscoelastic parameters based on experimental measurements. This leads to select values allowing to find the measured strain. However, the part of the "hidden" damage in the experimental response is neglected. It appears therefore necessary to determine the viscoelastic parameters of concrete components by another method in order to take into account the part of damage [SAL 13]. The coupling of a linear viscoelastic model with a damage model at the mesoscopic scale may allow for the characterization of damage evolution during creep and its influence on the global behavior.

The following section presents experimental observations for flexural creep tests on concrete under high level loadings. Analyses were made with the AE technique. Then, in section 7.3, models are presented to show the coupling between the viscoelasticity and damage at different scales. A study at early ages is performed to show the competiveness between the sustained load and the strength increase.

Experimental methods for the identification of creep-damage coupling in concrete

State-of-the-art of creep analysis of cementitious materials

Deformation and fracture of concrete are associated with very complicated progressive failure. Creep strains are directly related to stresses and micro-cracks in concrete. Thus, understanding the behavior of concrete requires t h e d e t a i l e d assessment of creep and the part of damage. For stress levels below 40% of the compressive strength, creep strains are proportional to stress and creep coefficients can be described using a formulation independent of the stress. It is assumed that linear visco-elasticity takes place and the instantaneous mechanical behavior of concrete remains elastic. For high stress levels, this linearity is lost and the creep coefficient is no longer independent of the stress. Micro-cracks, initiated by the local stress, develop and interact with the viscoelasticity [MAZ 02]; deviation of the linearity of the mechanical response may occur and nonlinear creep occurs [BAZ 97b, FRE 58, MAS 95, MAZ 02]. Finally, for stress levels above 70% of the maximum strength, cracks propagate quickly and lead to failure (tertiary creep). In fact, micro-cracking may initiate at the moment of load application and begins to grow to form a time-dependent crack path [BAZ 92, MEY 69]. The effects of tensile creep could be divided into beneficial and detrimental effects due on one hand to the relaxation of the stresses generated by autogenous shrinkage and thermal dilation at early ages [ALT 03, BIS 95] and on the second hand to micro-cracking [COO 74]. In most cases, structures are loaded by efforts of low intensity, relatively to material strength (<30% of the strength). For these loading levels, maintained constant, it is considered that concrete is not damaged and creep remains linear [GAR 09, RAN 13, SAL 12b]. Usually, creep mechanisms are associated with the hydrated cement paste and can be considered as material properties. Micro-cracking is an apparent creep mechanism that can also contribute to the observed deformation and is responsible of the permanent deformation.

A number of studies investigated the effects of creep loading history on the failure mechanisms, including its influence on strength, stiffness and fracture energy. Most of these studies tried to quantify the effect of creep on the behavior of concrete mainly by measuring the residual strength 7.1) at high stress levels (70% and 85% of the maximal strength F max ). Concrete specimens were mixed with Portland cement CPA-CEMII 42.5, crushed limestone aggregate distributed in fine sand, with a maximum size of 5 mm and a density of 2570 kg/m 3 and crushed gravel of size 5-12.5 mm with a density of 2620 kg/m 3 . A super plasticizer agent (Glenium 27) has been added for the workability shows the mix quantities of constituent materials. The influence of creep on the residual strength and fracture energy has been studied by submitting the beams loaded previously under flexural creep to further loadings until failure (Figure 7.2). This figure gives the results of aging and loaded concrete beams on basic creep at 70% and 85% of the maximal load (BC0, BC70 and BC85), respectively, the aging and loaded mortar beams on basic creep (MBC0, MBC70 and MBC85). Results show that basic creep displacement is more important for mortar beams; the lower displacement of concrete beams could be due to the presence of aggregates that restrained creep deformation. In fact, the aggregates follow a linear elastic behavior and do not creep. The deformation reported to the total volume is then reduced with the decrease of the relative volume of the cement paste. In addition, the larger the applied stress, the more important the kinetics and magnitude of creep displacement are. Even after 1 year of high rate loading, beams subjected to basic creep did not reach tertiary creep. This may be due to the limited time of loading considering the low kinetic of crack evolution. In order to deepen our knowledge of creep damage coupling on long-term and better evaluate the heterogeneities effect, the kinetic of creep was accelerated by submitting mortar and concrete beams to drying where damage was continuously assessed by the AE technique.

Case of the desiccation creep

Desiccation creep tests were realized at 50% of F max in order to avoid the premature rupture of concrete (DC50). In addition, in order to quantify the rate of desiccation creep, combined basic-desiccation creep tests were realized in parallel. For that, concrete beams were loaded in basic creep for 1 month and then subjected to desiccation (BDC50). At the moment when BDC50 was exposed to drying, creep displacement increased very fast at the beginning then decreased proportionally to the rate of shrinkage to join later the creep displacement of DC50 (Figure 7.4). The effect of desiccation creep on the evolution of the fracture properties of concrete beams has been characterized by submitting concrete beams loaded previously at 50% of F max and unloaded beams subjected to the same drying conditions (DC0) to further loading up to rupture. The flexural strength of unloaded concrete beams did not seem to be affected by desiccation in comparison with BC0, however a slight decrease of the flexural modulus is observed, that can be linked to a decrease of Young's modulus (Figures 7.2(b) and 7.4(b)). This is in agreement with the results found in the literature, where most of the authors noticed a decrease in the Young's modulus after desiccation due to shrinkage restrained by aggregates [BUR 05]. However, the desiccation effect on the residual strength has shown many contradictions: while some researchers did not observe any direct effect [BRO 78], others have reported a decrease [HAN 68, KAN 98] and an increase of the residual strength in flexion [DEL 91]. Aditionally, a decrease in Young's modulus, the maximal strength and the fracture energy has been observed for the beams subjected to desiccation creep indicating an important damage (Figure 7.4(b)). Thus, even with a low level of loading, the coupling between creep and damage is more important in desiccation creep.

AE monitoring of creep tests for concrete and mortar beams

AE activity during desiccation creep tests

The kinetic of desiccation creep at 70% (Figure 7.4) is more important in comparison with that of basic creep (Figure 7.2). In addition, while concrete beams loaded at 70% (DC70) failed after few days, mortar beams loaded at 70% of F max (MDC70) did not fail even after more than 4 months of loading showing a more important viscous behavior considering the more important amplitude and kinetic evolution. At this level another issue arises due to the influence of desiccation on the mechanical properties. In fact, the surfaces exposed to drying present a non-uniform desiccation (humidity gradient) which induces a non-uniform shrinkage and thus presents a structural effect due to tensile stresses at the surfaces that are superposed to the flexural stresses [BAZ 82, BIS 02]. The evolution of damage under creep seems to be different in mortar and concrete beams and thus the presence of aggregates modifies the fracture behavior. The AE activity was analyzed during the distinct phases of creep, corresponding to the evolution of creep displacement into three regimes which generate different signals covering different amplitude ranges (Figure 7.5). The cumulated AE energy shows a good proportionality with creep displacement and the different phases of creep are revealed. During primary creep, the kinetic of creep displacement and AE energy decreases, with AE hits of high amplitude and an important emission of AE energy. During secondary creep, the rate of the AE activity is quasi-constant with a stable emission of AE energy. During tertiary creep in concrete beams, the AE activity increases quickly in correlation with creep displacement with the emission of high energetic signals. This phase corresponds to the coalescence and the fast propagation of micro-cracks where the events begin to concentrate in the cracking zone generating a localized crack. Due to desiccation, the neutral axis of the beam moves upward causing an additional curvature and leading to the failure of concrete beams [BAZ 85, BAZ 94].

The emission of weak amplitude signals is less important during desiccation creep in mortar beams and seems to correspond to micro-cracks at the ITZ in concrete beams. In addition, the AE activity is more important in concrete beams with a more important AE energy and high amplitude signals due to a m ore important damage. This may be due to the higher porosity in concrete due to the presence of aggregate which generates a more active process of humidity diffusion within concrete micro-pores and macro-pores. It could also be due to the presence of ITZ in concrete which is generally weaker than the matrix [ZIM 85] and constitutes a zone of stress concentration. In fact, this zone contains a more important part of free water in concrete responsible of the important shrinkage when it is free to escape

Assessment of damage mechanisms occurring under creep

The quantitative understanding of the physical processes and the microstructural phenomena that control the fracture behavior on the macroscopic scale under creep is poor. It appeared necessary to take the analysis further to obtain information about the physical mechanisms, the origin of the AE signatures. For this, a multi variable analysis of the recorded acoustic signals was proposed to discriminate the damage mechanisms in the material according to the typical AE signals and their apparition chronology [SAL 14].

For the classification process of the monitored AE data under creep, the nonsupervised method K-means associated with a principal component analysis (PCA) and the dendrogram can be used [KOS 03]. The K-means method allows for obtaining a non-supervised classification in n class representative of the n damage mechanisms in the case of multidimensional data. The PCA method is achieved in order to improve the visualization of the classification result by reducing the data dimension [OJA 89]. In the non-supervised method each signal can be associated to a pattern composed of multiple relevant features, extracted from the random AE waveforms (amplitude, energy, rising time, average frequency, etc.). Two clusters are distinguished for basic creep and three for desiccation creep. The hits number by amplitude recorded is averaged to create an AE hits amplitude distribution for each cluster and is fitted with a statistical Laplace-Gauss model. Figure 7.6 shows the experimental distribution and the corresponding statistical law of the AE hits amplitude for each class. The first cluster can be associated to micro-cracking at the matrix-aggregate interface [ROS 89]. The second class can be associated to micro-cracking in the matrix. Based on the variation of the AE parameters, the third cluster could correspond to signals due to drying as liquid transfer in concrete [CHO 06] and cavitation [LUR 09]. Note that the differences between ranges of amplitude for the classes obtained here could be different for others specimen geometries and recording system parameters. The ranges have to be considered as relative differences.

Summary

The experimental tests showed that these deformations, restrained by the presence of aggregates, lead to the creation of micro-cracking in the cement paste, which could be detrimental for the mechanical behavior [SAL 12a]. The experimental methods for the characterization of micro-mechanisms at this scale is limited to the use of the acoustic emission technique [SAL 12a, SAL 13]. The multiscale modeling from the hydrates scale to the structure scale seems to be relevant for this characterization.

Numerical methods for the analysis of damage during creep

State-of-the-art of the creep modeling of cement-based materials

Several creep-damage theoretical models have been proposed in the literature. The modeling of the interaction between the viscoelasticity and damage has been performed by coupling linear creep models with damage models [BEN 08, CHA 05, MAZ 03, OMA 04, OZB 01, REV 07, TOR 08], or by using plasticity [RUI 07] and viscoelastic-viscoplastic [BER 03] models in series in order to reproduce the nonlinear behavior of concrete. These approaches are interesting because they allow us to directly define the damage threshold by taking into account the influence of the viscoelastic behavior. These models suggest an extension for the writing of the equivalent strain to obtain a triaxial state by equivalence to a uniaxial state and are defined by the positive eigenvalues of the strain tensor. The creep strains are a part of the total strains and they are weighted by introducing an intensity factor β in the equivalent strain [MAZ 03]:

ε = 〈 ̿ 〉 : 〈 ̿ 〉 + 〈 ̿ 〉 : 〈 ̿ 〉 [ 7 . 1 ]
where . + represents the positive part of the strains fields, e ε the elastic strain tensor and v ε the viscoelastic strain tensor. β is used to control the viscoelastic effects on the damage of the matrix and can be characterized by experimental creep tests.

However, most of these models are based on empirical relations applied at the macroscopic scale. Coupling between creep and damage is mostly realized by adding some parameters to take into account the microstructure effects. With the same idea of introducing damage due to creep, some models were developed by considering the effect of creep on crack opening and softening mechanisms. Three approaches were considered: the concept of activation energy [BAZ 97b], the introduction of creep models in a cohesive model to control the crack width [BAR 05, CAR 97] and finally the combination of micromechanical models for the softening behavior of concrete and creep models [BAR 04]. However, Le et al.

[LE 07] have shown how the microscopic retardation and relaxation times of the viscoelastic matrix of a heterogeneous material control the creep behavior of the homogenized material.

Most of the macroscopic models require the calibration of a large number of parameters or the use of artificial parameters for weighting the viscoelasticity proportion in the damage evolution. Artificial parameters are generally used to take into account implicitly the interaction between the phases of concrete under loading. This interaction can be modeled with a good consideration of the microstructure and a good approach to creep mechanisms involved. Mesoscopic modeling for concrete has shown a particular interest in the analysis of interactions between the cementitious matrix (cement paste, mortar) and aggregates [DUP 09, GRA 10, GRO 07, LOP 08, NGU 11]. The multi-scales approach was found to be very useful for the evaluation of material characteristics affected by the components' material characteristics. Thus, the mesoscale modeling presents many advantages in the understanding of the fracture process and the corresponding effect of concrete heterogeneities. The behavior of concrete is modeled at the mesoscopic scale by coupling a linear viscoelastic model, defined by Kelvin chains and a damage model [SAL 13].

In addition, several researchers have developed models coupling the viscoelastic behavior and the hydration degree of cementitious materials [BRI 12, DES 99, DES 00, ROS 93]. These models have been developed to simulate the behavior of structures taking into account the implicit effects of the microstructure; which needs the calibration of several parameters. The development of new modeling techniques has allowed evolving the solidification theory into an explicit representation of creep-hydration coupling. Pichler and Lackner [PIC 08] showed that it is possible to assimilate the creep compliance to the mean effective compliance of the phases constituting the material. This result has been confirmed further using a numerical approach [TRA 11].

The study of cement paste morphology is therefore of particular interest in the evaluation of concrete creep [SAN 09]. In addition, the explicit description of the microstructure with inclusions generated randomly allows to localize the mechanical fields and reduces the number of empirical relations [GRO 07, GRO 11].

Creep-damage modeling

Problem formulation of the coupling between creep and damage

A volume V of concrete is formed by two media: a matrix defined by the medium V m and inclusions defined by the medium V i . With the objective to model the damage due to creep of concrete, phases have a damage viscoelastic behavior law. A constant load F is applied on one of the surface boundary of V 1 () Γ with the unit normal vector . This load implies local displacements fields ( ), local strain fields ̿ ( ) and local stress fields ( ) in each point of V. The non-linear viscoelastic problem is written as follow: where C ( ) is the initial stiffness of the material phases considered isotropic and linear elastic and C ( , ̿ ( ) the stiffness of the damaged material. For the isotropic version of the model, the relation between the effective stress and the total stress is given by Fichant, La Borderie.and Pijaudier-Cabot [FIC 99]:

( ) =0 ∀ ∈ [ 7 . 2 ] ( ) = , ̿ ( ) ∶ ̿ ( ) -̿ ( ) ∀ ∈ [ 7 . 3 ] ε ( ) = (∇ ( ) +∇ ( ) ∀ ∈ [ 7 . 4 ] F = .
σ = (1 -) [7.8]
where d is the scalar value of the isotropic damage that depends only on the equivalent strain calculated according to the elastic strain tensor ε =( ̿ ( ) -̿ ( ))

ε = ̿ : ̿ [ 7 . 9 ]
The evolution of the damage variable, due to external mechanical loads, is an exponential form:

( )

0 0 1e x p d td e q eq dB ε εε ε  =- -  [7.10]
where t B represents a damage parameter to control the slope of the strain softening constitutive relation in function of the width h of the element and 0 d ε the strain threshold. Damage increases when the equivalent strain eq ε is higher than the threshold strain 0 d ε linked to the fracture energy f G as follow [MAT 10]:

0 2 f td t t G f f hB ε =+ [7.11]
where h is the element size and t f the tensile strength.

The viscoelastic strain is defined by several Kelvin chains. With those elements, the stress history does not have to be stored for the calculation of creep strain [BEN 08]. For a Kelvin unit i, the basic strain evolution is given by:

η ̿ ( ) + ̿ ( ) = ( ) [7.12]
where () v t ε represents the derivative of the elementary basic creep strains, v k the stiffness and v η the viscosity of the Kelvin unit.

The creep process occurs only in the undamaged part of the material and the effective stresses are linearized for each time step. The local total viscoelastic strains are obtained by solving analytically the differential equations and are expressed as:

̅ = ̅ -̅ = ̅ + + [7.13]
where ̅ is the viscoelastic strain vector at time-step number n and ,, vvv abc depend upon material parameters [REV 07]. The total viscoelastic strain is deduced from the sum of all the elementary viscoelastic strains.

E ( ) = ∑ ̿ ( ) [7.14]
The material volume is modeled by a digital concrete model which takes into account the "real" aggregate size distribution of concrete [GRO 07, MOU 02]. The volume of concrete is defined by a matrix, consisting of the mortar, in which aggregates of different diameters are placed, from the largest to the smallest, according to a random process. Details are given in Grondin et al. [GRO 07]. This approach requires defining two behavior laws for the mortar and aggregates. Each phase is characterized by a set of physical and geometrical parameters: the volume fraction, the unit size and physico-mechanical properties. A specific algorithm has been developed to make a spatial and random distribution of these phases on the basis of a finite element grid.

Simulation of tensile creep of concrete

Concrete is modeled by a two-phase material consisting of aggregates embedded in the mortar matrix. So, the mechanical properties of mortar and aggregates have to be determined separately. Creep strain for aggregates is considered equal to zero and the mechanical characteristics are obtained from the literature. The parameters (failure and creep properties) adopted for the simulations are identified by experimental direct tensile tests on mortar and concrete (Figure 7.7). Tensile tests are conducted first and then mortar and concrete specimens are loaded in creep at 70% and 85% of their maximum load. Concrete and mortar specimens with the same constituents and the same water-to-cement ratio should be used. In order to quantify the damage occurring in concrete under creep, the AE technique is also used to assess the state of the material during loading and thus could be used to validate numerical models.

The considered mesoscale for modeling the behavior of concrete is the scale at which the material can be observed as a set of coarse aggregates embedded in a mortar matrix. Here, coarse aggregates are inclusions of a size greater than 5 mm following the experimental aggregate size distribution while mortar matrix is a mixture of finer aggregates and the cement paste. The aggregates' volume represents 37% of the total volume of concrete. The stability of results depending on the size of the specimen and the maximal diameter of the inclusion was demonstrated in a recent study [GRO 07] and was respected here with dimensions equal to 4 times the highest inclusion diameter (L/Dmax > 4) and finite element size equal to 0.8 times the smallest inclusion diameter. These results show that the mechanical parameters selected for mortar and aggregates are valid and that the mesoscopic modeling can reproduce well the concrete failure. The tensile strength varies slightly with the random distribution of the aggregates. However, no effect was observed on the pre-peak part of the loaddisplacement curves where specimens were loaded in creep.

The imposed constant load is equal to 70% and 85% of the maximal strength of concrete determined by the fracture tests. Numerical simulations reproduce well the experimental creep tests (Figure 7.9). The numerical creep displacement obtained with the digital concrete model is lower than creep displacement for mortar specimens which is expected as the aggregates do not creep and restrain the viscoelastic strain of the matrix. In experiments, specimens loaded at 85% fail after a few days. However, the crack is localized at the end of the specimen which may be due to stress concentration at both extremities (Figure 7.10). In addition, while concrete specimens failed, mortar specimens did not show any damage. Thus, the .11 shows the damage localization in concrete specimens loaded at 70% and 85% after 15 days. Specimens loaded at 70% show little damage localized at the mortar-aggregates interface. The same result is obtained for the specimen loaded at 85%, however the damage intensity is more important. This damage is due to the load application at the beginning and the increase with creep. These results are confirmed by the AE measurements. The number of hits increases sharply during the first period which corresponds to the instantaneous deformation in correlation with the load application. Then, the number of hits increases slightly with time for the specimen loaded at 70% of the maximal strength during secondary creep. For the specimen loaded at 85%, an exponential increase of the number of AE hits is observed during tertiary creep, reached due to the high initial damage at loading. AE hits are related to the release of elastic energy similarly to the strain energies released by damaged elements. So, it could be reasonable to assume, as an approximation, that the number of AE hits is proportional to the number of damaged elements [GRE 15, SAL 16, ZHU 02, ZHU 10].

Simulation of the flexural creep tests of concrete

Numerical simulations are also performed on concrete submitted to a three-point bending test and compared to experimental results analysed in the previous section [SAL 12a]. The loading is applied as an incremental vertical displacement of a rigid plate (linear elastic law) fixed at the middle of the top of the beam (Figure 7.12). A regular mesoscopic mesh related to the middle part of the concrete specimen is generated and two homogeneous concrete blocks are attached to the left and right end of the beam with progressively larger mesh to avoid stress concentration. The numerical simulations were conducted under plane stress conditions. The parameters determined by the direct tensile tests are used for the mesoscopic mesh. For the homogeneous part, the parameters are determined by the direct tensile tests on concrete specimens. A fracture test is performed to determine the maximal flexural strength and to validate the choice of the mechanical properties defined for mortar and aggregates (Figure 7.13). The results reproduced well the experimental load-CMOD curves. The proposed model is also suitable for the computation of creep damage in bending. Creep compliance is assumed to be equal in compression and in tension which is in accordance with experimental evidence for mature concrete [BRO 78]. A constant load is applied to the plate at 70% and 85% of the maximal strength. Deflection-time curves are plotted in Figure 7.13 and exhibit good agreement with the experimental data. The macroscopic behavior of concrete under creep loading is characterized by a progressive degradation of the material stiffness. This behavior is caused by the growth and coalescence of micro-cracks at the mortar-aggregates interfaces due to strain incompatibilities (Figure 7.15). That means that the mechanical properties of concrete are gradually and locally degraded under creep. Further, it is assumed that the concrete specimen collapses when a long continuous crack develops at the bottom of the specimen and at the same time the maximum axial strains of the elements along the main crack are higher than the corresponding ultimate strain.

A predictive creep modeling

The multi-scale approach

For modeling the creep behavior of concrete, three scales have been considered: microscopic, mesoscopic and macroscopic (Figure 7.16). The simulation of the creep of concrete needs the knowledge of the compliance of its matrix (mortar); at a lower scale, the knowledge of the compliance of the mortar's matrix (cement paste); and at the lowest scale, the knowledge of the cement paste's matrix. The compliance of these matrices are calculated by a homogenization approach by solving the viscoelastic problem presented in the next section. At the lowest scale, the viscoelasticity of the cement paste is considered strictly dependent on that of its matrix. The cement paste is formed by a matrix constituted of C-S-H, pores and inclusions formed by the other hydrated phases (V hyd = [CH, Ettringite, gypsum, C 3 AH 6 , FH 3 ]) and the residual cement clinkers ([C 3 S, C 2 S, C 3 A, C 4 AF]). With the advancement of the hydration process, the clinker volume fraction decreases while the main hydrate's (C-S-H) and V hyd volume fractions increase significantly. The hydration process which determines the component's volume in the microstructure is based on the Arrhenius' law:

= ( ) [7.15]
where and T i represent the normalized affinity and the characteristic hydration time, respectively, and the hydration coefficient of the clinker i. The hydration process leads to the dissolution of clinkers and to the formation of hydrates with a volume defined by Berthollet [BER 03] and [GRO 10]:

( ) = ∑ ( ) =1 , [7.16]
where represents the residual clinkers volume in the cement paste, ( ) the new formed hydrates volume, M the molar mass [g.mol -1 ], ρ the mass density [g.cm -3 ] Macro Meso Micro

Concrete Mortar Hydrates

and n the mole. The index k represents the products (clinkers), l the reactants and c the cement. Along the hydration process, the volume fraction of the main hydrates increases and that of the pores into the cement paste's matrix (f p ) decreases (water is consumed and the voids are filled with formed hydrates). The calculated evolutions of the C-S-H and fp for three w/c ratios (0.4, 0.5 and 0.57) are given in Figure 7.17. We observe that the evolution of the sum of the residual clinkers and the other hydrates is not important during the hydration process from 24h.

So, in accordance with the assumption of the restraining effect of the elastic phases on the C-S-H, the modeling at this scale was simplified by considering only one type of particle with a unique size for the inclusions. Their elastic characteristics are then calculated by applying the self-consistent scheme [GRO 10] with the intrinsic elastic characteristics of the different hydrates (V hyd ) and clinkers [BER 03].

Figure 7.17. Left: porosity and C-S-H hydrate evolution with age for different w/c ratios (black: porosity; grey: C-S-H). Right: evolution of hydration degree and V hydclinker with age (black: V hyd -clinker; grey: hydration degree)

The choice of scales is made based on the definition of the representative elementary volume (REV). Theoretically, the REV must be large enough to represent the specimen or structure and small enough to take into account the effects of heterogeneities. In the finite elements methods, the dimensions of the REV must take in consideration the independence of the calculated effective values regarding the chosen mesh. To ensure this independence as well as the volume stability, the REV dimensions must be greater than or equal to four times the diameter of the largest inclusions and the elements dimensions must be less than or equal to 0.8 times the smaller diameter inclusions [GRO 07].

The homogenization problem

The creep model allows the calculation of the average elementary strain 〈 ̿ ( , )〉 and the average stress 〈 ( , )〉 at each step t p of the time discretization interval {t 0 =0, t 1 , t p ., t n =t max }. At each time t', the resolution of the problem is equivalent to solving an elastic homogenization problem on the biphasic REV. The tensor connecting the two fields is constant at the time (t p ) of the calculation, such as:

〈 ̿ ( , )〉 = ̿ ̿ ( ): 〈 ( , )〉 [7.17]
The integral over timeJ ̿ ̿ (t ) gives the final shape of the creep compliance. Considering only the time-dependent stress, the applied stress is expressed by the following relationship:

〈σ (t, y )〉 =H(t)〈σ 〉 [7.18]
with H(t)the Heaviside function as:

H(t) =0 si t=0 H(t) =1 si t>0
Summing over time the components of constant elementary tensors, the formula [7.17] becomes: The viscoelastic strain is modeled by a generalized Kelvin model with three chains (Figure 7.18), thus the creep compliance can be written as a function of the model parameters:

∑〈 ε (t ,y )〉 = ∑ J (t )
J (t) = ( ) + ∑ ( )
(1 -e ) [7.23] This approach is based on the knowledge of the viscoelastic coefficients giving J h (t). For this, it is necessary to calculate these coefficients from the properties of C-S-H in the presence of porosity; however, this latter evolves with age. A first calculation is then proposed to define the viscoelastic coefficients J h (t).

Simulation of tensile creep of concrete at early ages

Concrete structures are brittle in tension so the tensile behavior of concrete has to be characterized as a priority. Constant tensile loads were applied and only the effective part of the compliance in the loading axis was considered, which gives the predominant deformation for the tensile load. The other components showed negligible values. So, the homogenization calculations were performed only in 2D. At the concrete scale, the calculations were performed on slim cylindrical concrete specimens (11 x 45 cm 2 ) and the plane stresses (2D) conditions were retained.

By considering that in mature concrete the evolution of the capillary porosity does not influence the creep deformation, the viscoelastic properties of the concrete components can be assessed for a constant capillary porosity. Therefore, an inverse approach was applied to calculate the intrinsic creep parameters of the cement paste matrix. A creep displacement measured on a mature concrete [SAL 13] with a w/c ratio of 0.57 has been fitted. First of all, the hydration model has been validated by comparing the calculated and the measured hydration degree of the cement paste used in the concrete mixture. At each scale, the viscoelastic coefficients of the matrices were calibrated to adjust the homogenized compliance of the material. At the first step, the viscoelastic coefficients of mortar J m (t) were adjusted from experimental results on the tensile creep compliance of concrete [SAL 13] to obtain the best fitting compliance of concrete given by J c hom (Figure 7.20). In order to limit the number of calibrated parameters, some of the elastic parameters were calculated by the hydration model. The parameters used at the three scales are summarized in [FAR 14].

For the calculation of the viscoelastic parameters of the cement paste matrix ( ) the formula of Ricaud and Masson [RIC 09] was used to link the viscoelastic bulk coefficients of C-S-H ( ) with the pores content:

k =k ( ) [7.24]
For a mature concrete, the calculation of hydration gives a pore volume fraction equal to 27% in the cement paste which corresponds to 36% in the cement paste matrix. So we obtained:

=63.3 , =33.8 , = 27.4 . For the simulation of aging creep, the evolution of V hyd and the residual clinker volume fraction with hydration have been taken into account in the generation of the inclusions in the mesh at the cement paste scale. The percentage of inclusions decreases along the hydration process to transform into the matr i x . T h u s , t h e evolution of the microstructure has been taken into account in the calculation by attributing the matrix properties to the transformed inclusions. This finite element method gives an approximation of the evolution of the microstructure in order to take into account explicitly the interactions between the matrix (C-S-H + pores) and inclusions during creep tests.

At the cement paste scale, the inverse of the relation [7.24] has been applied to calculate the viscoelastic parameters of the matrix to define J h (t). The more the w/c ratio increases the more becomes low and the more the loaded material creeps; when the age increases, the porosity decreases and the bulk coefficients increase showing the solidification of the material.

In order to avoid the damage risk in the cement paste and in the mortar, the applied load is kept constant throughout the creep test. In this paper, a constant load of 30% of the tensile strength has been applied at the age of 24h during 28 days. The tensile strengths of the cement paste and mortar at the loading age have been calculated by solving the problem in which the viscoelastic strain is maintained equal to zero and for a monotonic load applied on the bottom of the specimen until the failure. The tensile strengths and the constant load applied are presented in At the concrete scale, creep-damage coupling is taken into account by solving the nonlinear viscoelastic problem. The tensile strengths of concretes at 24h calculated for a monotonic loading for the w/c ratios of 0.4, 0.5 and 0.57 are equal to: 1.06 MPa, 1.05 MPa and 1 Mpa (respectively). Five creep loads have been simulated: tensile creep tests with 30%, 70% and 90% of ft and compressive creep tests with 70% and 90% offc (Figure 7.22). For a stress-to-strength ratio of 30% (Figure 7.23), the most important strains are localized at the interface between the aggregates and the matrix. In these areas, the strains are important but remain smaller than the threshold strain and concrete does not damage. If equivalent loads are applied on mortars and cement pastes, the damage appears. Thus, on one hand, the aggregates result in deformations of mortar at the concrete scale and, on the other hand, they support some of the applied creep load and contribute to minimizing the effect of the latter on the deformations of mortar.

It is admitted that the matrix-aggregates interaction can lead to damage and increase the apparent deformation. At the concrete scale, the results show that the deformation of the matrix (mortar) does not lead to damage because it is restrained by aggregates and the interactions are not sufficient to create damage at their interfaces. At the cement paste and mortar scales, the deformations are not restrained by aggregates. This is the reason that the deformations are so high at these scales compared to those observed at the concrete scale (Figure 7.21).

The calculations at the cement paste and mortar scales have to be used only for the determination of the viscoelastic properties without any damage. If damage is considered at these scales, the effective creep compliance will not give the real deformation at the concrete scale. So, these results validate the choice of a linear viscoelastic problem for the calculation of the effective viscoelastic properties of the concrete's components. The compliances are the same for the two tests and equal to that obtained for the tensile test at 70% of ft (Figure 24). The damage fields are similar for the two compressive tests, with a little difference in the highest values (0.85 for 90% and 0.72 for70%), but very different from the tensile test at 70%. It can be explained by the values of the compressive loads: for the test at 70%, respectively 90%, the compressive load applied at 24h is equal to 3.01 MPa, respectively 3.87 MPa. By considering a linear approximation of the Poisson effect at 24h, the tensile stress response in the perpendicular axis of the load axis is equal to 0.75 MPa for 70%, respectively 0.97 MPa for 90%. So, these stresses represent 75% and 97% of ft which is equal to 1 MPa at 24h. The tensile test performed with a load of 90% of ft confirms this analysis with a damage reaching locally the value of 0.98. The difference in the compliance for this test is due to a high damage since 24h. In the compressive tests, the material absorbs a part of the stress energy before a restitution into a tensile strain. That is why in the compressive tests the damage is lower than in the tensile test at 90%. These results show the relevance of calculating the compressive and the tensile creep independently for the case of high loading.

Conclusion

Using the AE method to characterize local damage and to analyze the physical mechanisms underlying the creep of concrete seems to be a relevant method. A good correlation was observed between creep displacement and variation of the AE parameters. As a part of the investigation, quantitative analysis of the acoustic emission data was applied and characteristics of individual micro-mechanisms were evaluated and associated to two clusters for basic creep and three clusters for desiccation creep. In order to better understand the micro-cracking during creep, a mesoscopic model was first developed. It was based on the coupling between a viscoelastic behavior and damage. The model needs parameters which can be characterized experimentally by calibration. The influence of the microstructure was taken into account by explicit interactions between the concrete matrix and inclusions. This model allows us to understand the physical mechanisms behind the failure of concrete under constant loading. Under tensile creep, damage induced at the moment of load application increases due to strain incompatibilities between mortars and aggregates and causes a strength decrease. Similar observations were obtained with the acoustic emission technique. In the case of low loading, damage can also increased without any influence of the load application amplitude, but it is due to the viscoelastic strain which reaches threshold damage. It was shown by the multi-scale model.

Figure 7 . 1 .

 71 Figure 7.1. Geometry of concrete and mortar beams for flexural creep tests

Figure 7 . 2 .

 72 Figure 7.2. Top: basic creep displacement at 70% and 85% of Fmax. Bottom: load-CMOD curves for loaded and unloaded mortar and concrete beams. For a color version of the figure, see www.iste.co.uk/millet/advances.zip

Figure 7 . 3 .

 73 Figure 7.3. Evolution of cumulative events per grid: a) aging specimen, b) creep specimen. For a color version of the figure, see www.iste.co.uk/millet/advances.zip

Figure 7 . 4 .

 74 Figure 7.4. Top: combined desiccation and basic-desiccation creep displacement at 50% of F max of concrete beams. Bottom: load-CMOD curves for loaded and unloaded beams.

Figure 7 . 5 .

 75 Figure 7.5. AE activity during desiccation creep tests for mortar (top) and concrete (bottom) beams loaded at 70% of F max : correlation between the cumulated AE energy and the hits amplitude.

  [XIA 13]. Stress concentrations generated in those zones are accentuated by the strain incompatibility between the aggregates and the cement paste due to desiccation shrinkage [BIS 02, IDI 11, WON 09] that increases with drying, the volume and the aggregates' diameter [GRA 10, WON 09]. The localization of micro-cracks in the ITZ controls the behavior of concrete under creep and may be responsible of fracture [SAL 12a]. In fact, micro-cracking initiated due to the applied constant load begins to growth and form a crack path leading to the failure of concrete beams [BAZ 92, NGA 81, ROS 94]. In mortar beams, stresses related to the load and shrinkage are more diffuse. Aggregates play thus an important role in the development of micro-cracks in the ITZ and increase the risk of rupture of concrete under creep due to different mechanisms of stress concentration and redistribution [SAL 13].

Figure 7 . 6 .

 76 Figure 7.6. Experimental and statistical AE hits amplitude distributions for each cluster

  is the secant stiffness tensor depending on the local strains and damage, ̿ ( ) the local viscoelastic strain fields and 2 Γ the boundary of V on which macroscopic displacements conditions are applied. The chosen damage model is the isotropic model developed by Fichant, Pijaudier-Cabot and La Borderie [FIC 97]. It allows to represent the unilateral effect and to obtain objective results independently of the length of the finite elements by controlling the fracture energy. This model is a simplified version of the microplane model of Bazant and Ozbolt [BAZ 90] and is based on the relation between the total stress and the effective stress σ ( ) of the material defined by: σ = (y ) ∶ ε (y ) or σ = ( ): ( , ̿ ( ): ( ) [ 7 . 7 ]

Figure 7 . 7 .

 77 Figure 7.7. AE transducers location on the concrete specimen in the tensile tests

Figure 7 .

 7 Figure 7.8 shows a good correlation between the calculated load-displacement evolution and the experimental measurements. The comparison is made only prepeak because the tensile tests were performed by the force control and did not allow post-peak part.

  associated with the presence of the ITZ which is characterized by weak mechanical properties [GRO 14].

Figure 7 . 8 .Figure 7 . 9 .Figure 7 . 10 .

 7879710 Figure 7.8. Comparison between the calculated and measured loaddisplacement of concrete in tension. For a color version of the figure, see www.iste.co.uk/millet/advances.zip

Figure 7 . 11 .

 711 Figure 7.11. Damage localization after 15 days and acoustic emission activity in concrete specimens loaded at 70% and 85% of the maximal strength. For a color version of the figure, see www.iste.co.uk/millet/advances.zip

Figure 7 .

 7 Figure 7.12. Meso-macro meshing and boundary conditions of concrete beams for the three-point bending test simulation. For a color version of the figure, see www.iste.co.uk/millet/advances.zip

Figure 7 . 14 .

 714 Figure 7.14. Deflection-time curves of concrete beams in flexion at 70% and 85% of the maximal strength.

Figure 7 .

 7 Figure 7.15. Damage and crack opening of concrete beams in flexion at 70% and 85% of the maximal strength.

Figure 7 .

 7 Figure 7.16. Three observation scales of concrete

Figure 7 . 18 .

 718 Figure 7.18. Generalized Kelvin model

Figure 7 . 19 .

 719 Figure 7.19. Numerical resolution procedure of multi-scale method

Figure 7 .

 7 Figure 7.20. Above: comparison between the measured compliance of concrete and that calculated with the calibrated viscoelastic coefficients of mortar (Jc(hom)). Below: comparison between the measured hydration degree and that calculated for acement paste with a w/c ratio of 0.57

  Based on the evolution of the viscoelastic parameters of the cement pastes' matrices, the effective tensile creep compliances of the cement pastes have been calculated by homogenization for different w/c ratios. Then, the effective compliances of the cement pastes were defined as viscoelastic parameters of the m o r t a r s ' m a t r i c e s f o r t h e c a l c u l a t i o n o f t h e c r e e p a t t h e m o r t a r scale (Figure7.21). Finally, the effective compliances of mortars were defined as viscoelastic parameters of the concretes' matrices for the calculation of creep at the concrete scale (Figure7.19).

Figure 7 . 21 .

 721 Figure 7.21. Evolution of the of the cement pastes (left) and mortars (right) with age for different w/c ratios.

Figure 7 . 22 .

 722 Figure 7.22. Evolution of creep compliances of concrete submitted to different loads

Figure 7 . 23 .

 723 Figure 7.23. Localization zones of deformations after creep loading in concrete: w/c = 0.4; w/c = 0.5; w/c = 0.57.

Figure 7 . 24 .

 724 Figure 7.24. Damage in concrete for different loads: compressive load at 70% of f c , compressive load at 90% of fc, tensile load at 70% of ft, tensile load at 90% of ft.

. Measurement of the residual strength of concrete after creep 7

  [CAR 97, COO 74, DEN 06, LIN 87, OMA 09, SHA 70]. Recently, in order to quantify the part of damage occurring in concrete under creep, the AE technique was used [ROS 12, SAL 12a]. This technique is a non-destructive testing method increasingly u s e d i n t h e determination of structural changes in relation with local phenomena [GRA 07, HAI 05, MIH 96, OTS 00]. .2.2.1. Case of the basic creep Basic creep tests were first realized on mortar and concrete beams under flexural creep loading (Figure

	7.2.2

Table 7

 7

.1. Concrete mixture proportions

Table 7

 7

	.2.
	w/c Stress (MPa) Cement paste Mortar	0.4 0.45 0.97	0.135 0.291	0.5 0.38 0.7	0.114 0.21	0.57 0.35 0.6	0.105 0.18
	Table 7.2. Tensile strengths of cement pastes and mortars and applied creep stresses

Mounia FARAH, Jacqueline SALIBA, Frédéric GRONDIN and Ahmed LOUKILI.