domain: chim sdv sdv.mhep

text:
Inés Castro

Christiane André-Barrès

Nicolas Fabre

Stéphane Massou

Michel Sauvain

Denis Castillo Pareja

Dr Valérie Jullian
email: valerie.jullian@ird.fr

Affiliation

Cordiasecosides G-J, 9,10-Seco-29-norcycloartane glycosides isolated from Cordia lutea and their antibacterial activities

Keywords: Cordia lutea, Boraginaceae, Helicobacter pylori, antibacterial, secocycloartane, traditional medicine

Four undescribed secocycloartane monoglycosides (1-4) were isolated from an ethanolic extract of the dry flowers of Cordia lutea Lam. Their structural assignment is based on NMR and MS analysis. Their stereochemistry is confirmed by molecular modelling studies using DFT-NMR calculations done for compound 3. In vitro antibacterial activity of the four compounds was moderate on Helicobacter pylori (MIC = 15.6 µg/mL), and much weaker on Staphylococcus aureus, Pseudomonas aeruginosa or Escherichia coli (MIC > 125 µg/mL). Toxicity evaluated against RAW 264.7 cells was weak (IC50 values ranging from 24 to 41 µM i.e 15 to 24 µg/mL), but in the same range as anti-Helicobacter activity.

Introduction

In the course of our investigation on traditional Peruvian medicine, we have already described Cordiasecosides A-F, six 9,10-Seco-29-norcycloartanes isolated from an ethanolic extract of the dried flowers of Cordia lutea Lam. (Boraginaceae), together with their antibacterial activity [1]. These flowers are sold on traditional medicinal plant markets in Peru and are a highly valued remedy against liver and digestive disfunctions [2]. This research was undertaken because we evidenced the flowers crude extract activity against Helicobacter pylori (MIC = 500 µg/mL). After isolation of Cordiasecosides A-F, some active fractions remained to be investigated. We wish to describe here Cordiasecosides G-J (1-4) (Fig. 1), establish their structure thanks to analysis of their mass and NMR data, and comparison of the latter with computed ones. We will also evaluate their antibacterial activity on H. pylori, and compare it with antibacterial activity on Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and toxicity against RAW 264.7 cells to evaluate selectivity.

Materials and Methods

Chemicals

Ethanol (96%) was from Laboratoire ALKOFARMA E.I.R.L. Petroleum ether, cyclohexane, dichloromethane, ethyl acetate, and methanol were of analytical or HPLC grade and bought from Fisher Scientific SAS. Merck Silica gel 60 (15-40 µm) was used for medium pressure column chromatography (MPLC). Analytical thin-layer chromatography was carried out on precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm); detection was performed under UV 254 nm and after spraying with 1% vanillin/10% H2SO4 in ethanol.

General experimental procedures

MPLC was performed on a Büchi pump manager C-615 connected to a Büchi pump module C-601 with glass columns of various sizes. Optical rotations were determined with a JASCO P2000 digital polarimeter. IR spectra were recorded on a Perkin-Elmer Frontier FT-IR in ATR mode. The NMR spectra were recorded on a Bruker Avance 500 MHz instrument, fitted with cryoprobe, in CDCl3 (δH 7.28 and δC 77.1) and methanol-d4 (δH 3.33 and δC 49.1). Spectra were calibrated using CDCl3 as reference. UHPLC/MS analysis was performed using a UHPLC Ultimate 3000 system (Dionex) controlled by Chromeleon Xpress 6.8 software (Dionex), coupled with an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). All spectra were acquired and processed using Thermo Xcalibur 3.0 software (Thermo Fisher Scientific). Semipreparative 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 column.

Plant material

Extraction and isolation

Nine hundred grams of dry flowers of C. lutea were proceeded as previously described to afford a methanolic extract (6 g) [1]. This extract was processed by successive MPLC on silica gel as described in supplementary material. Briefly, 6 fractions were obtained. Fractions 3 (762 mg), 4 (983 mg), and 5 (384 mg) showed activity against H. pylori. Fractions 3 and 4 were submitted to successive MPLC on silica gel to give a mixture (31.6 mg) which was further separated by several injections of 3 mg on semipreparative HPLC eluted with H2O-CH3CN at 3 mL/min and the gradient as follows: 0 min 50 H2O/50 CH3CN, 18-23 min 0 H2O/100 CH3CN, 30 min 50 H2O/50 CH3CN to isolate as white, amorphous solids Cordiasecoside G (compound 1, 4.5 mg) whose purity was estimated at 89% on 1 H NMR spectra and Cordiasecoside H (compound 2, 4.5 mg) whose purity was estimated at 82% on 1 H NMR spectra.

Fraction 5 was pooled with other fractions of similar TLC profiles obtained during the purification of fraction 4. This fraction (528 mg) was submitted to successive MPLC on silica gel to give a mixture (41.8 mg), which was further separated by successive injections of 3 mg on semi-preparative HPLC eluted with H2O-CH3CN at 3 mL/min and the gradient as follows: 0 min 30 H2O/70 CH3OH, 10-15 min 0 H2O/100 CH3OH, 25 min 30 H2O/70 CH3OH to isolate as white, amorphous solids Cordiasecoside I (compound 3, 5.1 mg) whose purity was estimated at 89% on 1 H NMR spectra and Cordiasecoside J (compound 4, 3.3 mg) whose purity was estimated at 85% on 1 H NMR spectra. Strains were maintained as described [1]. The minimum inhibitory concentration (MIC) was determined using the broth microdilution method in 96-wells culture plates. Each well contains a bacterial inoculum of 50 µL at 2.10 6 CFU/mL, and 50 µL of a solution of compounds at concentrations ranging from 3.9 to 250 µg/mL. Positive controls for H. pylori are clarithromycin and amoxicillin (purity> 95%, Sigma-Aldrich) and tetracycline for others bacteria (purity> 95%, Sigma-Aldrich). Plates were incubated at 37°C 24h for E. coli, P. aeruginosa and S. aureus, and 72h under microaerophilic condition for H. Pylori.

Cordiasecoside

Plates were then examined visually and read at 660 nm to determine MIC. Tests were performed in triplicate.

RAW cells have been cultivated and used for cytotoxic assay as described [1]. Briefly, cells were seeded in 96-wells culture plates (100 µL of culture at 5.10 4 cells/mL) and incubated for 24 h at 37°C and 5% CO2.

Then, culture medium was removed and 100 µL of medium containing compounds at 4 different concentrations were added. Positive control was doxorubicin (102%>purity>98%, sigma-aldrich). Plates were incubated again for 48 h, and medium was replaced by 100 µL of RPMI and 20 µL of MTT solution (5 mg/mL). After 4 h of incubation, formazan crystals were dissolved with 100 µL of a solution of isopropanol (50%) and sodium dodecyl sulfate (10%). Plates were then read at 550 nm. The 50% inhibitory concentration (IC50) was determined based on 2 independent experiments using the GraphPad Prism software.

Results and discussion

Compounds 1-4 were purified from the ethanol extract of the dried flowers of C. lutea. Further analysis of 2D NMR spectra confirmed that the two compounds had similar structures (Fig. 2), and allowed the assignment of all 1 H and 13 C NMR signals (Tables 1 and2). The two methyl singlets at δH 0.70 (s, H3-18) and 0.76 (s, H3-30) were identified and their HMBC correlations allowed to assign C- This planar structure was identical to that already described for Cordiasecoside C. However, there are obvious differences between the 1 H and 13 C NMR spectra (see sup. info.) in the A and B rings, suggesting changes in the configuration of some carbons in this part of the molecule.

Examination of NOESY spectra of compound 1 gave some indications (Fig. 3). The multiplicity bond. We proposed that compound 1 is the C-10 epimer of Cordiasecoside C (Fig. 1).

Fig 3. Key NOESY for compound 1

Besides clear signals depicted in figure 3, some other NOESY correlations were ambiguous or difficult to interpret due to signal overlaps. Therefore, we wanted to gather more data to confirm the proposed structures for compounds 1-4. For this purpose, we used DFT quantum calculation based on the protocol described by Lodewyk and coworkers to calculate 1 H and 13 C chemical shifts for compound 3 [START_REF] Lodewyk | Computational Prediction of 1 H and 13 C Chemical Shifts: A Useful Tool for Natural Product, Mechanistic, and Synthetic Organic Chemistry[END_REF]. DFT calculation were also previously used to establish the structure of cimipodocarpaside, a 9,10seco-cycloartane isolated from black cohosh (Actaea racemosa). [START_REF] Jamróz | Novel and unusual triterpene from Black Cohosh. Determination of structure of 9,10-seco-9,19-cyclolanostane xyloside (cimipodocarpaside) by NMR, IR and Raman spectroscopy and DFT calculations[END_REF] Relevant conformers were modelled by DFT, using Gaussian 09 at the B3LYP/6-31+G(d,p) level. Frequencies calculations were performed on the optimized geometries at 298 K, showing all positive frequencies and allowing evaluation of the enthalpies of the minima.

The contribution of each conformer in the constitution of the overall spectrum was based on previous Boltzmann conformational analysis in the chloroform continuum.

13

C NMR and 1 H chemical shift calculations were then performed using the GIAO NMR method with B3LYP/6-31G(d) and using the chloroform polarizable continuum model (SMD) on optimized geometries at the B3LYP/6-31+G(d,p) level. Isotropic shielding constants (σ) for the 13 C nucleus and 1 H were transformed in chemical shifts (δ) using linear regression analysis of the calculated chemical shifts versus the experimental ones. Detailed calculations are consigned in Supporting Information. With the chosen method, the root mean square deviation (RMSD) is 1.39 ppm for 13 C chemical shift and 0.12 ppm for the 1 H, indicating a good match between simulated and experimental spectra [START_REF] Lodewyk | Computational Prediction of 1 H and 13 C Chemical Shifts: A Useful Tool for Natural Product, Mechanistic, and Synthetic Organic Chemistry[END_REF][START_REF] Jamróz | Novel and unusual triterpene from Black Cohosh. Determination of structure of 9,10-seco-9,19-cyclolanostane xyloside (cimipodocarpaside) by NMR, IR and Raman spectroscopy and DFT calculations[END_REF]. For the protons with measurable coupling constants, calculated and experimental values are also in agreement (see table 1 and7S and in the same range as their antibacterial activity. Compounds 1-4 are therefore moderately active against H. pylori, compared to other bacteria, and have a low selectivity index compared to RAW cells.

Given the limited quantity of compounds 1-4 present in the plant and their moderate activity, it is unlikely that they alone are responsible for the bioactivity and function of the medicinal plant Cordia lutea.

Some food or traditional herbal medicines have a strong reputation for the treatment of dyspepsia or gastric ulcer, like broccoli sprouts (Brassica oleracea) or licorice (Glycyrrhiza species) [START_REF] Fahey | Dietary amelioration of Helicobacter infection[END_REF]. It has been shown that the isothiocyanate sulforaphane presents as its glucosinolate precursor (glucoraphanin) in broccoli sprouts, displayed a median MIC value of 2 µg/mL on 48 clinical isolates and three reference strains [START_REF] Fahey | Sulforaphane inhibits extracellular, intracellular, and antibiotic-resistant strains of Helicobacter pylori and prevents benzopyrene-induced stomach tumors[END_REF]. Flavonoids isolated from licorice extract displayed MIC ranging from 6.25 to 50 µg/mL [START_REF] Fukai | Anti-Helicobacter pylori flavonoids from licorice extract[END_REF], and a standardized licorice extract is marketed as food supplement against dyspepsia [START_REF] Asha | In vitro anti-Helicobacter pylori activity of a flavonoid rich extract of Glycyrrhiza glabra and its probable mechanisms of action[END_REF] In a recent review about phytochemicals active against H. pylori reporting 27 active compounds, berberine isolated from Hydrastis canadensis L. was the most active with a MIC of 0.78 µg/mL, and 10 compounds displayed MIC between 1 and 10 µg/mL [START_REF] Salehi | Phytochemicals in Helicobacter pylori Infections: What Are We Doing Now[END_REF]. Our own search in literature showed that the most active compounds isolated from plants displayed MIC values between 0.1 and 10 µg/mL. The 2methoxy-1,4-naphthoquinone isolated from Impatiens balsamica showed MIC between 0.1 and 0.6 µg/mL, depending on the H. pylori strain [START_REF] Wang | In Vitro Activity of 2-methoxy-1,4naphthoquinone and Stigmasta-7,22-diene-3β-ol from Impatiens balsamina L. against Multiple Antibiotic-Resistant Helicobacter pylori[END_REF]. Two naphtoquinones and one anthraquinone isolated from Tabebuia impetiginosa displayed MIC between 2 and 8 µg/mL, in the same range as naphtoquinone plumbagine (MIC = 4 µg/mL) [START_REF] Park | Antibacterial activity of Tabebuia impetiginosa Martius ex DC (Taheebo) against Helicobacter pylori[END_REF].

This showed that Cordiasecosides activity against H. pylori was weaker than these of natural compounds like naphtoquinone, but compared with these of compounds isolated from licorice, a traditional remedy against gastric ulcer.

Conclusion

In this work, we continued our exploration of the chemical content of C. lutea, a highly valued traditional remedy in northern Peru against hepatic and digestive dysfunction. We report here 4

undescribed Cordiasecosides, which are the C-10 epimers of previously published compounds. All

Cordiasecosides displayed a similar antibacterial profile, with a moderate antibacterial activity against

  Dried flowers from C. lutea were bought in a local market in Trujillo, Peru, on May 2017. The botanist Severo Baldeón (National Herbarium of the San Marcos University, Lima, Peru) confirmed the determination of the plant. A voucher specimen was deposited at the National Herbarium of the San Marcos University in Lima, Peru under register N° 032-2017-USM-MHN.

Fig. 1

 1 Fig. 1 Cordiasecosides G-J isolated from C. lutea

12 (

 12 δc 37.5), C-13 (δc 44.8), C-14 (δc 49.3), C-15 (δc 33.4) and C-17 (δc 51.4). The 1 H-1 H COSY correlation between H2-12 (δH 2.02 and 2.08, m) and the olefinic methine proton signal at δH 5.45 enabled to assign H-11. The methyl doublet at δH 0.90 (d, 6.3 Hz, H3-21) was attributed thanks to its HMBC correlation with C-17. Analysis of successive COSY correlations from H3-21 allowed the attribution of H-20, H2-22, and H-23. Analysis of HMBC correlations of C-23 (δc 70.8) and HMBC and COSY correlation of H-25 (hept, 6.8 Hz) allowed to complete the attribution of the sidechain. The carbon signal at δc 85.7, bearing a proton at δH 3.15 (td, 10.6, 4.5 Hz) is typical of the C-3 position of a triterpene monomethylated in C-4. Analysis of the HMBC and COSY correlations of H-3 allowed the attribution of positions 2, 4, and 28. H-5 (δH 1.19, m) was attributed thanks to its correlation with H3-28 (δH 0.95, d, 6.8 Hz). HMBC correlation of the anomeric proton at δH 4.25 (d, 6.6 Hz, H-1') with C-3 (δc 85.7) confirmed the position of the pentosyl moiety at C-3. Comparison of 1 H and 13 C data of the pentosyl moiety with those of Cordiasecoside C allowed its identification as a xylosyl unit. The two doublets at δH 1.94 (d, 13.4 Hz) and 2.65 (d, 13.3 Hz) are typical of the H2-19 protons of a 9,10-secocycloartane skeleton. The HMBC correlations of H-19a allowed to assign C-8 (δc 44.6), C-9 (δc 135.4) and C-10 (δc 70.1), respectively. The COSY correlations of H-5 (δH 1.19) and H-8 (δH 2.24) with signals at δH 1.53 and δH 1.64 respectively allowed the attribution of H2-6 and H-7b.

Fig 2 .

 2 Fig 2. COSY and HMBC correlations for compound 1

  of 1 H signal of H-3 (δH 3.15, td 10.6, 4.5 Hz) is typical of an axial proton coupling with two axial and one equatorial protons, as in Cordiasecoside C, indicating an axial H-4. Therefore, the strong NOESY signal between H-4 (δH 1.68) and H-19b (δH 2.65, d, 13.4) indicates an axial position for the C-10-C-19

,

  Measured for H-3 : td, J = 10.5, 4.3 Hz ; calculated for H-3 : J = 9.7, 8.5, 4.7 Hz ; measured for H-19 : d, J = 13.4 Hz ; calculated for H-19 : J = -15.4 Hz ; measured for H-23 : d, J = 10.3 Hz calculated for H-23 : J = 9.1 Hz). The main NOESY effects observed are also in agreement with calculated interatomic distances. This confirmed the proposed structure for compound 3, and therefore compounds 1-4.

H

  . pylori, and a low selectivity index compared to RAW cells. This class of original compounds could be used as biomarkers for characterization and quality control of C. lutea flowers sold as traditional remedy. I.C. thanks the Institut de Recherche pour le Développement (IRD) for the award of a grant "Allocations de Recherche pour une Thèse au Sud" (ARTS). The authors thank the logistic support of the LMI-LaVi (UPCH-IRD, Lima, Peru) and their directors: Pr. Rosario Rojas (UPCH) and Dr. Michel Sauvain (IRD). The authors are grateful to Pierre Lavedan, Marc Vedrenne and Caroline Toppan (NMR platform of the Institut de Chimie de Toulouse, ICT) for NMR analyses. The authors are indebted to the personnel of Universidad Nacional Mayor de 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 and research permits (No 146-2016-SERFOR/DGGSPFFS).

G (1): white

	solid; Rf 0.50, silica gel 60 F254, EtOAc/MeOH (90:10); [α] 𝐷 20 -8 (c 0.2,
	CH3OH), IR (ATR mode) max 3410, 2955, 2925, 1374, 1040 cm -1	.	1 H and 13 C NMR: see Tables 1 and
	2. HRESIMS (neg. ion mode) m/z 635.4158, [M + HCOO] -(calcd. for C36H59O9 -635.4165).
	Cordiasecoside H (2): white solid; Rf 0.50, silica gel 60 F254, EtOAc/MeOH (90:10); [α] 𝐷 20 -8 [c 0.2,
	CH2Cl2/CH3OH (2/1)], IR (ATR mode) max 3410, 2955, 2925, 1374, 1090 cm -1	.	1 H and 13 C NMR: see

Tables

1 and 2

. HRESIMS (neg. ion mode) m/z 649.4317, [M + HCOO] -(calcd. for C36H59O9 -649.4321).

Cordiasecoside I (

3): white solid; Rf 0.36, silica gel 60 F254, EtOAc/MeOH (90:10); [α] 𝐷 20 +7 (c 0.2, CH3OH), IR (ATR mode) max 3410, 2950, 2871, 1374, 1050 cm -1 . 1 H and 13 C NMR: see Tables 1 and 2. HRESIMS (neg. ion mode) m/z 635.4159, [M + HCOO] -(calcd. for C36H59O9 -635.4165). Cordiasecoside J (4): white solid; Rf 0.36, silica gel 60 F254, EtOAc/MeOH (90:10); [α] 𝐷 20 + 5 [c 0.2, CH2Cl2/CH3OH (2/1)], IR (ATR mode) max 3410, 2925, 2865, 1374, 1055 cm -1 . 1 H and 13 C NMR: see Tables 1 and 2. HRESIMS (neg. ion mode) m/z 649.4316, [M + HCOO] -(calcd. for C36H59O9 -649.4321). 2.5. Antibacterial activity and cytotoxicity evaluation on RAW cells 264.7 Antibacterial activity and cytotoxicity evaluation on RAW cells 264.7 were assessed as previously described [1]. Briefly, the Helicobacter pylori strain (CIP 103995) was obtained from Pr. Christine Roques (Laboratoire de Génie Chimique, UMR CNRS 5503, Université Toulouse 3). Other bacterial reference strains were from ATCC (E. Coli ATCC 25922, P. aeruginosa ATCC 27853, S. aureus ATCC 25923).

Table 1 1 H NMR spectroscopic data (500 MHz, CDCl3 -CD3OD 9:1) for compounds 1-4 (δ in ppm, J in Hz)

 1

	Position	1 Cordiasecoside G	2 Cordiasecoside H	3 Cordiasecoside I	4 Cordiasecoside J
	1	1.58 m	1.57 a	1.57 m	1.57 a
		1.64 a	1.65 a	1.65	1.64
	2	1.46 m	1.43 m	1.45 m	1.44 m
		2.02 a	2.03 a	2.04 a	2.04 a
	3	3.15 td (10.6, 4.5)	3.15 td (10.6, 4.5)	3.14 td (10.5, 4.3)	3.13 td (10.5, 4.4)
	4	1.68 a	1.68 a	1.67 a	1.67 a
	5	1.19 a	1.18 a	1.18 a	1.18 a
	6	1.53 a	1.53 a	1.54 a	1.53 a
		1.53	1.53	1.54	1.53
	7	1.39 m	1.40 m	1.41 m	1.40 m
		1.64 a	1.64 a	1.64 a	1.63 a
	8	2.24 m	2.24 m	2.24 m	2.24 m
	11	5.45 m	5.46 m	5.45 m	5.44 m
	12	2.02 a	2.01 a	2.03 a	2.01 a
		2.08 m	2.08 m	2.09 m	2.08 m
	15	1.29 a	1.29 a	1.29 a	1.29 a
	16	1.29 a	1.29 a	1.29 a	1.29 a
		1.86 m	1.90 m	1.87 m	1.88 m
	17	1.55 a	1.53 a	1.55 a	1.53 a
	18	0.70 s	0.69 s	0.69 s	0.69 s
	19	1.94 d (13.4)	1.94 d (13.3)	1.93 d (13.4)	1.93 d (13.5)
		2.65 d (13.4)	2.65 a	2.64 d (13.3)	2.64 a
	20	1.68 a	1.66 a	1.67 a	1.64 a
	21	0.90 d (6.3)	0.89 d (6.2)	0.90 d (6.3)	0.89 d (6.3)
	22	1.08 m	0.99 a	1.09 m	0.99 a
		1.50 a	1.57 a	1.50 a	1.57 a
	23	4.09 d (10.3)	4.10 d (9.8)	4.09 d (10.3)	4.10 d (10.3)
	24 1	4.80 s	5.45 q (7.0)	4.80 s	5.46 q (7.0)
		4.99 s		4.99 s
	24 2		1.60 d (6.9)		1.60 d (7.0)
	25	2.17 hept (6.8)	2.68 a	2.16 hept (6.8)	2.67 a
	26	1.02 d (6.8)	0.98 d (7.1)	1.02 d (6.9)	0.97 d (7.2)
	27	1.00 d (6.8)	1.05 d (7.0)	0.99 d (6.8)	1.05 d (7.1)
	28	0.95 d (6.3)	0.95 d (6.3)	0.94 d (6.3)	0.94 d (6.5)
	30	0.76 s	0.76 s	0.76 s	0.76 s
	1'	4.32 d (6.8)	4.32 d (6.9)	4.25 d (6.6)	4.25 d (6.6)
	2'	3.30 dd (8.4, 6.8)	3.30 dd (8.4, 6.8)	3.57 dd (8.6, 6.5)	3.57 dd (8.6, 6.5)
	3'	3.39 m	3.38 m	3.52 dd (8.6, 3.3)	3.52 dd (8.6, 3.3)
	4'	3.53 ddd (9.3, 8.0, 4.9) 3.53 ddd (9.1, 8.0, 4.9)	3.80 m	3.80 m
	5'	3.20 dd (11.7, 9.2)	3.20 dd (11.7, 9.2)	3.46 dd (12.6, 2.0)	3.47 dd (12.6, 1.9)
		3.89 dd (11.7, 4.9)	3.89 dd (11.7, 4.9)	3.85 dd (12.6, 3.3)	3.85 dd (12.5, 3.4)

a Signals overlapped

Table 2 . 13 C NMR spectroscopic data (125 MHz, CDCl3 -CD3OD 9:1) for compounds 1-4 (δ in ppm)

 2 Mass spectra analysis in ESI positive mode showed also a similar fragmentation pattern with consecutive loss of two molecules of water and neutral loss of 132, indicating the loss of a pentosyl unit.

	Position	Type	1 Cordiasecoside G	2 Cordiasecoside H	3 Cordiasecoside I	4 Cordiasecoside J
	1	CH2	39.3	39.3	39.3	39.3
	2	CH2	30.3	30.3	30.3	30.3
	3	CH	85.7	85.7	85.6	85.6
	4	CH	37.1	37.0	37.1	37.1
	5	CH	50.1	50.1	50.1	50.1
	6	CH2	21.4	21.4	21.4	21.4
	7	CH2	18.9	18.9	18.9	18.9
	8	CH	44.6	44.6	44.5	44.6
	9	C	135.4	135.4	135.4	135.4
	10	C	70.1	70.1	70.1	70.1
	11	CH	129.7	129.8	129.7	129.8
	12	CH2	37.5	37.5	37.5	37.5
	13	C	44.8	44.8	44.8	44.8
	14	C	49.3	49.3	49.3	49.3
	15	CH2	33.4	33.4	33.4	33.4
	16	CH2	27.6	27.6	27.6	27.6
	17	CH	51.4	51.5	51.4	51.5
	18	CH3	14.8	14.8	14.8	14.8
	19	CH2	43.2	43.2	43.2	43.2
	20	CH	33.0	33.2	33.0	33.2
	21	CH3	18.0	18.0	18.0	18.0
	22	CH2	43.6	44.8	43.7	44.8
	23	CH	70.8	68.9	70.8	68.9
	24	C	160.4	149.3	160.4	149.3
	24 1	CH	105.5	118.1	105.5	118.1
	24 2	CH3	-	12.9	-	12.9
	25	CH	30.4	27.9	30.5	27.9
	26	CH3	22.3	21.1	22.3	21.2
	27	CH3	23.2	21.2	23.2	21.2
	28	CH3	14.8	14.8	14.8	14.8
	30	CH3	17.9	17.9	18.0	18.0
	1'	CH	104.6	104.6	104.6	104.6
	2'	CH	73.1	73.1	71.5	71.5
	3'	CH	75.5	75.5	72.7	72.7
	4'	CH	69.5	69.5	67.7	67.7
	5'	CH2	64.8	64.8	65.2	65.1
	a : Signals overlap

Acknowledgements

This conclusion is coherent with the fact that the major shifts in 13 C NMR spectra between compound 1 and Cordiasecoside C are observed for positions 6, 7, 11 and 19. Furthermore, configurations of C-3, C-5 and C-8 do not vary in the cycloartanes family, and is imposed by biogenesis [START_REF] Dewick | Medicinal natural products[END_REF][START_REF] Azimova | Natural Compounds: Cycloartane Triterpenoids and Glycosides[END_REF]. There is also an example of a similar epimerization in literature: two pairs of glycosylated 9,10secocycloartanes which are hydroxylated and epimers in C-10 have already been described in Cimicifuga foetida [START_REF] Chen | Cycloartane Triterpenoids and Their Glycosides from the Rhizomes of Cimicifuga foetida[END_REF]. 9,10-secocycloartanes bearing a hydroxyl group at C-10 with the same relative configuration were also isolated from Astragalus membranaceus [START_REF] Kuang | Secocycloartane Triterpenoidal Saponins from the Leaves of Astragalus membranaceus Bunge[END_REF][START_REF] Kuang | Huangqiyenins G -J, Four New 9,10-Secocycloartane (=9,19-Cyclo-9,10-secolanostane) Triterpenoidal Saponins from Astragalus membranaceus Bunge Leaves[END_REF].

Compound 2 was isolated as a white solid. Its molecular formula was established as C36H60O7 by negative HRESIMS (m/z 649.4317, [M + HCOO] -) corresponding to 7 indices of hydrogen deficiency.

1 H, 13 C and 2D NMR spectra were highly similar to those of 1. The only differences in the 1 H spectrum is the replacement of the two singlets at δH 4.80 and δH 4.99 (H2-24 1 ) by a methyl doublet at δH 1.60 (d, 6.9 Hz) and a quadruplet at δH 5.45 (q, 7.0 Hz). In the 13 C spectrum of compound 2, an additional methyl signal at δc 12.9 appeared. This indicates the replacement of a H-24 1 proton by a methyl group. This is confirmed by the HMBC correlation observed between δH 1.60 (d, 6.9 Hz) attributed as H3-24 2 , and the olefinic carbons signals at δc 118.1 (C-24 1 ) and 149.3 (C-24). NOESY correlation between H-24 1 (δH 5.45, q, 7.0 Hz), and H-23 (δH 4.10, d, 9.8 Hz) indicates the E configuration of the Δ 24(24 1 ) double bond.

Therefore, compound 2 is identified as the C-10 epimer of Cordiasecoside D.

Conflict of Interest

The authors declare no conflict of interest


Amir Shahdin
email: amir.shahdin@isae.fr

Laurent Mezeix

Christophe Bouvet

Joseph Morlier

Yves Gourinat

Fabrication and Mechanical Testing of a New Sandwich Structure with Carbon Fiber Network Core

Keywords: Sandwich Fiber Network Materials, Mechanical Testing, Vibration Testing, Damping

The aim is the fabrication and mechanical testing of sandwich structures including a new core material known as fiber network sandwich materials. As fabrication norms for such a material do not exist as such, so the primary goal is to reproduce successfully fiber network sandwich specimens. Enhanced vibration testing diagnoses the quality of the fabrication process. These sandwich materials possess low structural strength as proved by the static tests (compression, bending), but the vibration test results give high damping values, making the material suitable for vibro-acoustic applications where structural strength is of secondary importance e.g., internal panelling of a helicopter.

BACKGROUND AND MOTIVATION

Characterization of fiber network sandwich materials both statically and dynamically presents a new challenge for the domain of mechanical testing. The determination of the sandwich material behavior under crushing loads and the measurements of the ductile fracture limits is normally done with the help of compression tests [START_REF] Hayman | Advanced Mechanical Testing of Sandwich Structures[END_REF]2]. Typically, cores are the weakest part of sandwich structures and they fail due to shear. Understanding the shear strength properties of sandwich core plays an important role in the design of sandwich structures subjected to flexural loading [START_REF]Standard test method for flexural properties of flat sandwich constructions[END_REF][START_REF] Sendlein | Characterization of Face Sheet/Core Shear Fracture of Composite Sandwich Beams[END_REF]. Therefore, 3-point bending tests are often performed to find the flexural and shear rigidities of sandwich beams [START_REF] Brauch | Bending of Sandwich Beams with Transversly Flexible Core[END_REF][START_REF] Lingaiah | Strength and Stiffness of Sandwich Beams in Bending[END_REF][START_REF] Soares | Testing of Sandwich Structures with Cork Agglomerate Cores[END_REF]. The vibration characteristics of sandwich materials have drawn much attention recently. The dynamic parameters of a structure i.e. natural frequency, damping and mode shapes are determined with the help of vibration testing which provides the basis for rapid inexpensive dynamic characterization of composite structures [START_REF] Gibson | Modal Vibration Response Measurements for Characterization of Composite Materials and Structures[END_REF]. D.J Ewins [START_REF] Ewins | Modal Testing Theory and practice[END_REF] gave a detailed overview of the vibration based methods. A wide amount of literature is present related to vibration testing of composite sandwich structures [START_REF] Hwu | Vibration Suppression of Composite Sandwich Beams[END_REF][START_REF] Hu | Free Vibration of Delaminated Composite Sandwich Beams[END_REF][START_REF] Mead | Loss factors and resonant frequencies of encastre damped sandwich beams[END_REF][START_REF] Nilsson | Prediction and Measurement of some Dynamic Properties of Sandwich Structures with Honeycomb and Foam Cores[END_REF][START_REF] Renji | Experimental Modal Densities of Honeycomb Sandwich Panels at High Frequencies[END_REF][START_REF] Renji | Loss Factors of Composite Honeycomb Sandwich Panels[END_REF][START_REF] He | Vibration and damping analysis of multi-span sandwich beams with arbitrary boundary conditions[END_REF]. The equations that explain the dynamic behavior of sandwich beams are described extensively in the literature and notably in the references [START_REF] Yan | Governing Equations for Vibrating Constrained-Layer Damping Sandwich Plates and Beams[END_REF][START_REF] Mead | A Comparison of some Equations for the Flexural Vibration of Damped Sandwich Beams[END_REF].

The importance of material damping in the design process has increased in recent years as the control of noise and vibration in high precision, high performance structures and machines has become more of a concern. At the same time, composites sandwich researchers have focused more attention on damping as a design variable [START_REF] Wang | Damping of Honeycomb Sandwich Beams[END_REF][START_REF] Li | Effects of thickness and Delamination on the Damping in Honeycomb-Foam Sandwich Beams[END_REF]. One way of increasing damping in sandwich materials is by putting a viscoelastic layer as core between the two laminates [START_REF] Hao | Vibration and Damping Analysis of a Sandwich Beam Containing a Viscoelastic Constraining Layer[END_REF][START_REF] Rao | Dynamic Analysis and Design of Laminated Composite Beams with Multiple Damping Layers[END_REF]. Jueng and Aref reported that sandwich structures with combined honeycomb-foam cores have higher damping than those with individual honeycomb or solid viscoelastic cores [START_REF] Jung | A Combined Honeycomb and Solid Viscoelastic Material for Structural Damping Application[END_REF].

These advancements have led to the need for developing materials possessing better damping characteristics. New materials like fiber network materials can be used as potential dampers and sound absorbers in specific applications like the inner paneling of a helicopter, where structural strength is not the primary requirement. Fiber network materials are made from natural materials (wool, cotton etc) as well as artificial ones (carbon, steel, glass etc) and are quickly becoming of widespread use as sound absorbers [START_REF] Garai | A Simple Empirical Model of Polyester Fiber Materials for Acoustic Applications[END_REF]. Bonded metal fibers network materials offer advantages for use as heat exchanger [START_REF] Golosnoy | Optimisation of Metallic Fiber Network Materials for Compact Heat Exchangers[END_REF] or insulation [START_REF] Zhang | Experimental and Theoretical Studies on High-Temperature Thermal Properties of Fibrous Insulation[END_REF]. These materials possess low relative density, high porosity and are cost-effective. Sandwich structures normally consist of two thin skins separated by a thick core. Traditional core materials are usually honeycomb, foam or balsa. Recently, a novel type of sandwich has been developed with bonded metallic fibers as core material [START_REF] Gustavsson | Formable Sandwich Construction Material and Use of the Material as Construction Material in Vehicles, Refrigerators, Boats, etc[END_REF][START_REF] Markaki | Ultra Light Stainless Steel Sheet Material[END_REF][START_REF] Markaki | Mechanics of Thin Ultra-Light Stainless Steel Sandwich Sheet Material, Part I. Stiffness[END_REF][START_REF] Dean | Energy absorption during projectile perforation of lightweight panels with metallic fibre cores[END_REF][START_REF] Zhou | Mechanical Properties of Fibrous Core Sandwich Panels[END_REF]. This material presents attractive combination of properties like high specific stiffness, good damping capacity and energy absorption. Fiber network materials with carbon fibers have been also studied for using as core material [START_REF] Mezeix | A New Sandwich Structured Composite with Entangled Carbon Fibers as Core Material[END_REF]. Entangled cross-linked carbon fibers present many advantages as core materials i.e., open porosity, multifunctional material or the possibility to reeve electric or control cables on core material. L. Mezeix et al. studied the mechanical behavior of fiber network materials in compression [START_REF] Mezeix | Experimental Data and Modelling of Entangled Fibers and Entangled Cross-Linked Fibers During Compression[END_REF]. Mechanical testing has also been carried out on specimens made of wood fibers [START_REF] Castéra | Comportement Physico-mécanique des Matériaux Fibreux Cellulosiques considéré comme des Milieux Aléatoires[END_REF], glass fibers [START_REF] Baudequin | Identification des Mécanismes Physiques mis en jeu lors de la reprise d'épaisseur de la Laine de Verre[END_REF] and various matted fibers [START_REF] Poquillon | Experimental Data about Mechanical Behavior during Compression Tests for Various Matted Fibres[END_REF]. There are also some works in the literature related to 3D modeling of wood based fibrous networks based on X-ray tomography and image analysis [START_REF] Faessel | 3D Modelling of random cellulosic fibrous netwroks based on X-ray tomography and image analysis[END_REF]. Although few works can be found in the scientific literature devoted to the mechanical testing of sandwich fiber network materials, and in fact no scientific literature can be found related to the vibration testing of sandwich fiber network materials or even simple fiber network materials.

Therefore, the main motivation of this work is to develop a process in order to fabricate sandwich fiber network specimens, and to test them both statically (compression and bending tests) and dynamically (vibration test). Vibration testing is used to diagnose the quality of the fabrication process and to verify the potential damping capabilities of these sandwich specimens.

MATERIALS AND FABRICATION PROCESS

Core Materials

The core of the sandwich test specimens used in this article consists of carbon fibers that are made of a yarn of standard carbon filaments. The specifications and materials properties are presented in Table 1. The fibers are provided by the company Toho-Tenax. For the cross-linking of carbon fibers, epoxy resin is used. The epoxy resin SR 8100 and injection hardener SD 8824 are used provided by SICOMIN. All the test specimens presented in the article are carefully weighed using METTLER balance. A better vaporization is achieved if the resin is heated up to 35°C before being sprayed on the carbon fibers. This allows the mixture of resin and hardener to become less viscous.

Skin Materials

Resin-containing carbon-fiber/epoxy prepreg of T700/M21 is used to fabricate the skin materials. The material is supplied by Hexcel composites, the physical properties are set out in Table 1. The skin materials are made of unidirectional pre-impregnated plies of type T700/M21 [START_REF] Hexply | Data Sheet[END_REF]. The upper and lower skins consist of four plies each with a stacking sequence of [0/90/90/0] [START_REF] Aminanda | Experimental Analysis and Modelling of the Crushing of Honeycomb Cores[END_REF]. The skin properties can be further viewed in Table 1.

Fabrication Process

The fabrication of fiber network specimens is often a tedious and complex process. As these types of materials are still mostly in the research phase, so as such standard fabrication processes do not exist. The fabrication procedure used in this article is the one developed by Mezeix et al. [START_REF] Mezeix | Experimental Data and Modelling of Entangled Fibers and Entangled Cross-Linked Fibers During Compression[END_REF]. Objective of this fabrication process is to ensure that these test specimens can be successfully reproduced with a relative efficiency, having the same composition. For this purpose, vibration testing is used to diagnose the quality of the fabrication process. The three types of test specimens used in this article are shown in Figure 1 and their specifications are provided in Table 2.

Sandwich Network Fiber Specimens

Figure 1 Core Material and Sandwich Test Specimens (compression, bending and vibration)

For the test specimens used in this article, approximately 900 g of fibers of 10mm length are cut manually. The carbon fibers are then treated in a solution of dichloromethane for 24 hours and are then cleaned for 2 hours in methanol. A cut fiber filament along with a bunch of fibers after chemical treatment is shown in Figure 1. These uncoated carbon fibers are then separated by a blast of compressed air. The mixture of resin and hardener is then sprayed on the separated carbon fibers by a spray paint gun. In case of larger test specimens, the volume of carbon fibers is large, so with the current technology it is not easy to spray the resin equally on the carbon fibers. Therefore, sandwich test specimens fabricated from the same mold differ a little in weight due to the inhomogeneous distribution of the manually sprayed resin (Table 4). The fibers vaporized by the resin are then placed in the mold between the two skins of unidirectional composites. In order to polymerize the fiber network sandwich specimens, the mold is then heated in an oven up to 180°C for two hours in a press [START_REF] Hexply | Data Sheet[END_REF]. First the small mold is used to fabricate a single vibration test specimen with a fiber core density of 100 kg/m 3 . This specimen showed that 100 kg/m 3 fiber core density is relatively insufficient for a volume of 480 x 50 x 10 mm 3 , as there are places in the core of the sandwich beam that lack sufficient quantity of fibers. So for the next vibration test specimen, a fiber core density of 150 kg/m 3 is chosen. As compared to the previous specimen (fiber core density of 100 kg/m 3 ), the specimen with 150 kg/m 3 fiber core density has a far better fiber distribution. Finally a vibration test specimen with a fiber core density of 200 kg/m 3 is fabricated by using the same small mold. It is seen that a fiber density of 200 kg/m 3 for the core is on the higher side and it is not possible to close the mold properly. So for the fabrication of the next batch of specimens, a fiber core density of 150 kg/m 3 is chosen. The composition of the vibration test specimen having a fiber core density of 150 kg/m 3 fabricated from the small mold is presented in Table 3. Total Weight of Specimen 120 g 95 g 120 g

In the next step a large mold is used to produce multiple test specimens. The aim of using the large mold is to produce multiple test specimens possessing the same characteristics.

The fiber core density is kept 150 kg/m 3 as discussed previously. The specifications, the quantity and the types of test specimens that can be extracted from the large mold are mentioned in Table 2. First the large mold is used to fabricate test specimens having the same composition as that of the single vibration test specimen fabricated from the small mold.

These four vibration test specimens fabricated from the large mold are referred to as heavy specimens in the article. The average composition of the four heavy vibration test specimens is presented in Table 3. Next, the same large mold is used to produce relatively lighter specimens. They have approximately 25 g less mixture of resin and hardener than the previously produced heavier specimens. These specimens shall be referred to as light specimens in future discussions (Table 3). The ratio of the mixture of resin and hardener in the heavy specimens is approximately 2.5 times more than that of the light specimens. The weight comparison of the vibration test specimens fabricated from the small and large mold is presented in Table 4. The difference in weights observed between the specimens is due to the uneven distribution of the manually sprayed resin.

The difference in resin between the heavy and light specimens can also be observed with the help of a scanning electron microscope (SEM). Figure 2 shows SEM observations on heavy and light specimens. In case of the heavy specimen (Figure 2a), the resin can be seen all along the length of the fibers and is seldom in the form of droplets. On the other hand, for the light specimen (Figure 2b), droplets of resin can be seen at the fiber joints but the presence of resin along the fiber lengths is negligible.

Scanning Electron Microscope (SEM) Tests

Figure 2 SEM observations (a) heavy specimen (b) light specimen

EXPERIMENTAL PROCEDURE Compression Tests

The quasi-static compressive response of the sandwich fiber network test specimens are measured in 100 kN Instron machine. The aim is to calculate the elastic modulus. Both light and heavy compression test specimens are tested. The specimen size chosen for the compression tests is 30 x 30 x 11 mm. The test specimens are placed between the moveable and the fixed plate as shown in Figure 3. The displacement of the machine is used to measure the displacement of the test-specimen. As the entangled materials are not very rigid, therefore a relatively small force is applied to compress the entangled sandwich materials. As the force is small, so it is safe to assume that the displacement of the machine is same as the displacement of the specimen. The applied velocity of v o = 2 mm/min corresponds to a nominal strain rate of ε& = 3x10 -3 /s at the beginning of the test. The maximum applied load is 20 kN corresponding to 22.2 MPa. To analyze the experimental results, the following definitions for the true strain and stress are used.

Strain,         = o h h ln ε (1) Stress, S F = σ (2)
where h is the height during compression, h o is the initial height of the sample, S is the area during compression. S o is the initial area and F is the applied force. In our case, the area S varies very little during the compression tests, so it is assumed that S = S o . No lateral displacement of the test-specimen is observed during the compression tests and the section remains constant. So it can be concluded that there are no barreling effects.

Compression Test

Figure 3 Test Specimen between the fixed and moveable plate during compression test

Bending Tests

In order to measure the shear modulus of the test specimens, three point bending tests are carried out on the heavy and light bending test specimens on a 10 kN Instron machine. As discussed previously, these specimens are fabricated from the large mold and have 150 kg/m 3 fiber core density. The dimensions of these test specimens are 140 x 20 x 11 mm and the distance between the two supports is 80 mm, the choice of which is based on the three-point bending tests carried out in detail in reference [START_REF] Aminanda | Experimental Analysis and Modelling of the Crushing of Honeycomb Cores[END_REF]. The applied velocity is v o = 1 mm/min and the maximum applied load is 100 N. Round steel bars or pipes are used as supports having a diameter of 6 mm which is not less than one half the core thickness (5 mm) and not greater than 1.5 times the sandwich thickness as per ASTM standards [START_REF] Soares | Testing of Sandwich Structures with Cork Agglomerate Cores[END_REF]. The three point bending test is shown in Figure 4.

Bending Test

Figure 4 Test specimen between the three supports during 3 point bending test For the analysis of a sandwich beam under three-point bending, consider a sandwich beam of width b and length l, comprising of two identical face sheets of thickness t f and core of thickness t c . Also h is the spacing of the mid plane of the face sheets (h = t c + t f ), as shown in Figure 5. where E s is the elastic modulus of the skins and G c is the shear modulus of the core. The maximum deflection δ is calculated experimentally by the three-point bending test, the only unknown is the shear modulus G c which is calculated by putting Equation ( 5) in Equation (3).

Schematic of a Sandwich Beam under Three-Point Bending Test

Equations [START_REF]Standard test method for flexural properties of flat sandwich constructions[END_REF][START_REF] Sendlein | Characterization of Face Sheet/Core Shear Fracture of Composite Sandwich Beams[END_REF][START_REF] Brauch | Bending of Sandwich Beams with Transversly Flexible Core[END_REF] are only valid for the beginning of the bending tests when the deflections are relatively small.

Vibration Tests

The experimental equipment used to obtain the vibration test results discussed in this paper is shown in Figure 6. The experimental set-up is that of a free-free beam excited at its center, based on Oberst method [START_REF] Wojtowicki | New Approach for the Measurements of Damping Properties of Materials using Oberst Beam[END_REF]. The Oberst method states that a free-free beam excited at its center has the same dynamical behavior as that of a half length cantilever beam. The test specimen is placed at its center on a B&K force sensor (type 8200) which is then assembled on a shaker supplied by Prodera having a maximum force of 100 N.

Compression Test Results

Figure 8 Compression stress/strain curves for the heavy and light fiber network sandwich specimens (150kg/m 3 fiber density). The zoomed view shows the linear-elastic phase used for calculating the elastic modulus From the graph (Figure 8), it can be noticed that there is a good reproducibility of compression tests carried out on the two types of fiber network sandwich specimens. In case of the heavy specimens, compression curves move from the right to the left and the densification starts for smaller strains (100 % for the heavy specimens). The extra resin in the heavy specimens results in better cross-linking of the fibers and increases the initial stiffness of the material which is evident by the linear-elastic phase which is quite prominent (zoomed view in Figure 8). The heavy specimens show a linear-elastic behavior up to around 0.8 MPa.

The calculation of the elastic modulus is based on the linear-elastic part of the curves as shown in the zoomed view in Figure 8. Even if the stress-strain curves shown in Figure 8 are highly non-linear, there is a linear-elastic part in case of the heavy specimens. The elastic modulus is calculated by measuring the slope of this linear-elastic part as explained in the ASTM standard in reference [START_REF]Standard test method for Young's Modulus, Tangent Modulus, and Chord Modulus[END_REF]. The average elastic modulus for the two heavy specimens is around 1.4 MPa. However in case of the light specimens, one has to compress longer in order to achieve densification (strain around 160 %) as they are less rigid as compared to the heavy specimens due to the lack of resin. The linear elastic phase is non-existent in case of the light specimens so the calculation of the elastic modulus is not possible. Unloading has not been performed to reveal the effects of plasticity because it is not in the scope of this work.

The authors are mainly interested in the linear-elastic part in order to calculate the elastic modulus which shall be used in future for the modeling of the entangled sandwich beams.

Three Point Bending Test Results

Figure 9 Force-deflection curves measured under three-point bending tests for the two types of fiber network sandwich specimens. The appearance of the first damage in the skins is indicated

The aim of the bending tests is to calculate the out of plane shear modulus G c of the fiber network sandwich materials with the help of the Equations (3-5) as discussed before. It can be seen from the force-deflection curves in Figure 9 that despite the complex nature of the fabrication process, the results of the three heavy as well as the three light specimens show a relatively good correlation. In case of the heavy specimens, the first damage in the skin appears around the 4-5 mm mark, whereas for the lighter specimens the first damage signs are after 10 mm. In addition, lesser force is required in the case of the light specimens to crack the skin. The average shear modulus for the heavy specimens is 10 MPa, and for the light specimens is 4 MPa. The values of the elastic and shear modulli are considerably low from a structural strength point of view. It is important to underline here that these types of sandwich specimens are designed for specific applications only. The main idea behind the fabrication of these sandwich specimens is to have a material having good damping characteristics even if it does not possess high structural strength.

The aim of the vibration testing is to diagnose the variability of the fabrication process and to extract the global modal parameters i.e., natural frequencies and damping ratios. The modal parameters for the first four bending modes are studied. Firstly, vibration tests are carried out on the vibration test specimen fabricated from the small mold and then on the four light and heavy specimens fabricated from the large mold. The modal parameters extracted from 41 high quality frequency response functions with the help of Polymax algorithm integrated in the LMS data acquisition system, are presented in Table 4. As discussed before, the difference in weight between each of the four heavy and light specimens is due to the fact that with the currently available fabrication technology it is difficult to spray the resin equally on the fibers. This difference in weight is also evident in the natural frequencies of these specimens (Table 4). The heavy specimen 5 and light specimen 6 have comparatively high natural frequencies due to them being relatively heavier in weight than the rest.

Keeping in view the difficulty level of the fabrication process, a major aim of this work is to successfully reproduce multiple sandwich fiber network specimens having the same characteristics. For this reason, the intent is to reproduce four vibration test specimens identical to Specimen 1 (150 kg/m 3 fiber core density and 43g of epoxy resin) in a large mold.

The comparison of the modal parameters of Specimen 1 with those of the heavy specimens (Specimen 2-5) show that large sandwich specimens with fiber network core can be reproduced with a relative efficiency. The natural frequency of the first mode for Specimen 1 is 225 Hz (Table 4), where as for the heavy specimens (Specimens 2-5) the average natural frequency for the first mode is 211 Hz with a standard deviation of 18 Hz. The main reason for this dispersion is the uneven spray of resin on the fibers. So vibration tests have been used

here as a diagnostic tool in order to verify the quality of the fabrication process.

On the other hand, the light specimens (Specimens 6-9) possess better damping characteristics because lesser amount of resin increases the friction between the carbon fibers in the sandwich core (weakening the cross-linking between the fibers), which leads to higher values of damping ratios (Table 4). It can be said that the vibration tests verify the results previously found by the compression and the bending tests, which also underline the softness of the light specimens. The vibration tests results show that the heavy specimens (Specimens 1-5) being more dense are less dissipative in nature thus possessing lower damping characteristics whereas, the light specimens (Specimens 6-9) having higher damping values are more dissipative in nature.

This observation can be further explained by studying the comparison of the sum of frequency response functions (FRF) for Specimen 2 (Heavy) and Specimen 7 (Light) in Figure 10. The resonance peaks for the first four bending modes for each specimen are also highlighted.

An interesting global approach is developed next in order to compare the average vibratory level of the specimens. The average vibratory level is calculated by taking the average of the amplitude (in dB) of the sum of the frequency response function for each specimen. The sum of the frequency response functions can be compared for all the vibration test specimens in this article, as same number of measurement points (41) has been chosen i.e.

symmetricity has been respected for all the specimens. It can be seen in Figure 10, that the resonance peaks of the heavy specimen are more pointed and possess higher amplitude than the light specimen. The resonance peaks of the light specimens are comparatively rounded specially after 800Hz. The average vibratory level (AVL) is also compared for the two types of specimens, and it can be seen that the light specimens possess on average a 15 dB lower amplitude than the heavy ones.

Comparison of the Sum of Frequency Response Functions

Figure 10 Comparison of the Sum of Frequency Response Function (FRF) for a light (Specimen 2) and a heavy specimen (Specimen 7) having 150 kg/m 3 fiber core density.

From Figure 10, it can be inferred that the difference in the natural frequencies between the heavy and light specimens signifies a loss in rigidity, whereas the change in vibratory level signifies a reduction in the amplitude of vibration. The percentage change in frequency and the change in vibrational level between the four heavy and four light vibration test specimens (150kg/m 3 fiber core density) for the first four bending modes are calculated with the help of Equations ( 6) and [START_REF] Soares | Testing of Sandwich Structures with Cork Agglomerate Cores[END_REF] and the resulting values are laid out in Table 5.

Change in frequency

(∆f) = ) ( ) ( ) ( k f k f k f H L H - (6)
where f H (k) is the average frequency for the four heavy specimens (Specimens 2-5) for the k th mode and likewise f L (k) is the average frequency for the four light specimens (Specimens 6-9)

for the k th mode

Change in vibratory level

(∆a) = ) ( ) ( k a k a L H - (7)
where a H (k) is the average amplitude in dB for the four heavy specimens for the k th mode and a L (k) is the average amplitude in dB for the four light specimens for the k th mode Table 5 shows that the average change in frequency for the first four bending modes between the light and heavy specimens is approximately 33 %. As the decrease in natural frequency signifies a loss of rigidity, therefore it can be concluded that the lighter specimens are less rigid than the heavy ones.

CONCLUSION

The aim of this article is to fabricate a sandwich material that possesses high damping characteristics for specific applications like the inner paneling of a helicopter cabin, even if the structural strength of this material is on the lower side. For this reason, a new type of sandwich material with fiber network materials as core and unidirectional carbon preimpregnated plies as skins is fabricated. The advantage of the vibration testing is to diagnose the variability of the fabrication process and to verify experimentally the potentially good damping characteristics of the fiber network sandwich specimens. The static test results show that the structural strength of these sandwich specimens is quite low but this is admissible because the aim of these types of materials is not structural strength. The vibration tests on these specimens showed that the lighter specimens possess five times more damping and on the average a 15 dB lesser vibratory level than the heavier ones. Despite the complexity of the fabrication process, large fiber network sandwich specimens are relatively successfully reproduced.

In future, these types of sandwich test specimens shall be fabricated with glass fibers as core material with a view to improve the strength of the material. These sandwich specimens shall be compared with classical sandwich materials with honeycomb and foam as cores in order to compare their static and dynamic properties. In addition, impact tests shall be carried out on these sandwich materials to study their impact toughness with the help of modal parameter shifts. Furthermore, the authors plan to model the entangled sandwich beam with measured core properties in order to calculate the natural frequencies to compare with the test result. As entangled sandwiches are a new brand of sandwich materials, therefore such comparisons will provide a verification of the measured core properties.

Figure 5

 5 Figure 5 Schematic of a sandwich beam under three point bending showing geometrical parameters

Figure 6 Figure 7 Figure 8

 678 Figure 6 Diagram of the experimental set-up

Table 4 .

 4 Modal parameters comparison for the light and heavy vibration test specimens fabricated from the small and large mold (150 kg/m 3 fiber core density)

Table 1 . Core and skin properties of the sandwich fiber network material Core Properties

 1

	Type of Fibers	HTS-5631 Elastic Modulus	240 GPa
	Length of Fibers	10mm	Diameter of Fibers	7µm
		Skin Properties
	Type	T700/M21 No of Plies in the Upper Skin 4
	Stacking Sequence	[0/90/90/0] No of Plies in the Lower Skin 4
	Thickness of Ply	0.125mm	Thickness of Upper Skin	0.5mm
	Density of Laminate 1580 kg/m 3 Thickness of Lower Skin	0.5mm

Table 2 . Types of Test Specimens and Molds Specimen Types and Specifications

 2

		Compression	Bending	Vibration
		Tests	Tests	Tests
	Length (mm)	30	140	480
	Width (mm)	30	20	50
	Core Thickness (mm)	10	10	10
	Skin Thickness (mm)	1	1	1
	Mold Types, Specifications and Usage
	Type of Molds	Dimensions	No and Type of Specimens
		(mm 3 )
	Small	510 x 65 x 11 01 Vibration Test Specimen
	Large	510 x 250 x 11 02 Compression Test Specimens
			03 Bending Test Specimens
			04 Vibration Test Specimens

Table 3 . Composition of vibration test specimens having fiber core density of 150 kg/m 3 fabricated from the small and large mold

 3

		Small Mold	Large Mold
	Density	150 kg/m 3	150 kg/m 3 (Light)	150 kg/m 3 (Heavy)
	Weight of Fibers	39 g	39 g	39 g
	Weight of Resin & Hardener	43 g	18 g	43 g
	Weight of Skin (Upper + Lower)	38 g	38 g	38 g

Table 5 . Change in frequency and vibrational level for the light and heavy vibration test specimens for the first four modes Mode 1 Mode 2 Mode 3 Mode 4

 5 % Change in Frequency (∆f) 30.1 % 45.7 % 34.3 % 23.1 % Change in Vibratory Level (∆a) 10.1 dB 13.7 dB 17.9 dB 17 dB

ACKNOWLEDGMENTS

The authors gratefully thank Professor B. Castanié and Samuel Rivallant from Université de Toulouse for his technical support during the experimental work and also to project students J. B. Lecaillon and A. de Nazelle from Saint-Cyr l'Ecole for their help during the fabrication process.